June 16–19, 2018, Fiera Milano Congressi, Milan Italy

Sponsorship: Publication of this supplement was sponsored by the European Society of Human Genetics. All content was reviewed and approved by the ESHG Scientific Programme Committee, which held full responsibility for the abstract selections.

Disclosure Information In order to help readers form their own judgments of potential bias in published abstracts, authors are asked to declare any competing financial interests.

Contributions of up to EUR 10 000.- (Ten thousand Euros, or equivalent value in kind) per year per company are considered “Modest”. Contributions above EUR 10 000.- per year are considered “Significant”.

P01 Reproductive Genetics/Prenatal Genetics

P01.01AInternal analytical verification of a targeted microarray-based cell-free DNA test for 22q11.2 deletion

F. R. Grati, L. Marcato, B. Malvestiti, S. Crippa, L. Martinoni, V. Zanatta, S. Saragozza, B. Grimi, F. Maggi, G. Simoni

TOMA, Advanced Biomedical Assays S.p.A., Busto Arsizio, Italy

Objectives: Laboratories are required to verify their assays determining that the test is being performed correctly, even if kit/software are CE-IVD marked with suitable performance specifications or a new test is implemented using a technology that is already well established in a source/reference laboratory. This study presents the internal analytical verification after the implementation of 22q11.2DS cfDNA test by a targeted microarray-based technology in an independent decentralized European laboratory.

Methods: Analytical sensitivity: 25 samples with 22q11.2DS were tested (2 maternal plasma and 23 simulated pregnancy samples). Deletions spanned through the A-D 22q11.2 LCRs, sizes ranged from 2.37 and 2.89Mb and simulated fetal fractions ranged from 7 to 39%. Analytical specificity: 423 prospectively ascertained maternal plasma samples with no known diagnosis of fetal/maternal 22q11.2DS were tested. This study was approved by the laboratory IRB. All samples were de-identified before study.

Results:Analytical sensitivity was 96.00%(95%CI:80.46-99.28) (24/25) and specificity was 99.76%(95%CI:98.67-99.96) (422/423) not statistically different from that reported in a previous clinical validation/verification (OR 0.12;95%CI:0.01-0.97 and OR 0.54;95%CI:0.07-4.31). The specificity must be considered a lower-bound estimate as the sample classified as “false positives” may be a true positive from an undiagnosed mother or affected fetus.

Conclusions: We have verified the internal analytical performances of a targeted microarray-based cfDNA test for 22q11.2 deletions inside the typical 3Mb region in the decentralized laboratory match the performance specification of the source laboratory. The low FPR, below 0.5%, for this cfDNA test expansion is critical when testing low-risk population as it highly impacts PPV.

F.R. Grati: D. Speakers Bureau/Honoraria (speakers bureau, symposia, and expert witness); Modest; Roche. F. Consultant/Advisory Board; Modest; Roche. L. Marcato: None. B. Malvestiti: None. S. Crippa: None. L. Martinoni: None. V. Zanatta: None. S. Saragozza: None. B. Grimi: None. F. Maggi: E. Ownership Interest (stock, stock options, patent or other intellectual property); Significant; TOMA Advanced Biomedical Assays S.p.A. G. Simoni: E. Ownership Interest (stock, stock options, patent or other intellectual property); Significant; TOMA Advanced Biomedical Assays S.p.A.

P01.013CPotential role of ACKR3/CXCR7 duplication in pregnancy loss

I. Amarillo, V. Wesevich, M. Smith, D. Gray

Washington Univ School of Medicine in St. Louis, St. Louis, MO, United States

Introduction: The atypical chemokine receptor 3 (ACKR3) is highly expressed in vascularized structures including the placenta and umbilical cord. Aberrant expression of ACKR3/ligands resulting in dysregulation of trophoblast-endometrial interaction has been implicated in recurrent pregnancy loss, intrauterine growth restriction, preterm labor, and preeclampsia. Copy number variations (CNV’s) overlapping with ACKR3 have been documented in patients with variable findings including neurodevelopmental defects (NDD’s), and in 0.01% of the healthy population (DGV). No CNV’s involving ACKR3 have been reported in pregnancy loss. Here we present our novel data from our retrospective chromosome microarray analysis (CMA) associating duplications of ACKR3 with spontaneous abortions (SAB’s) and intrauterine fetal demise (IUFD).

Materials and Methods: Retrospective CMA was performed from ~4,700 samples to identify CNV’s overlapping with ACKR3 (aka CXCR7; 2q37.3), of which ~350 were products of conception (POC).

Results:This study revealed 11 CNV’s overlapping with ACKR3. Nine gains were detected from six SAB’s and three IUFD (7 males, 2 females) ranging from small intervals (n = 4, 36.8-65.5 kb) to trisomy 2 (n = 4) and tetrasomy 2 (n = 1). These gains account for 0.19% of all CMA cases and 2.6% of all POC cases. A 610 kb gain and a 6.1 Mb loss were observed from blood samples of two patients with NDD’s.

Conclusions: This study is the first to report rare duplications of ACKR3 in SAB’s and IUFD, providing additional information to existing genomics data. Findings from this study may aid in enhanced understanding of ACKR3’s role in pregnancy, genetic counseling and management of pregnancy loss.

I. Amarillo: None. V. Wesevich: None. M. Smith: None. D. Gray: None.

P01.05A Aneuploidy rate in 530 miscarriage cases

R. Raynova1, S. Andonova1, S. Bichev1, I. Bradinova1, V. Dimitrova2, M. Tzankova2, S. Savova2, A. Savov1

1University Hospital of Obstetrics and Gynecology, National Genetic Laboratory, Sofia, Bulgaria, 2University Hospital of Obstetrics and Gynecology, Sofia, Bulgaria

Introduction: Spontaneous loss of pregnancy before the fetus reaches viability is the most frequent pregnancy complication. The term miscarriage includes all pregnancy losses from the time of conception until 20 weeks of gestation. According to published data about 50% of first-trimester pregnancy losses are the consequence of fetal chromosomal abnormalities. Most of these abnormalities are numerical (86%).

Materials and Methods: A total of 570 spontaneous miscarriage cases were collected between 2002 and 2017, referred to our laboratory from all over the country. DNA was extracted from placental or fetal tissue. Samples that did not contain tissue, appropriate for DNA analysis (7%) were excluded. QF-PCR focused on chromosomes 13, 18, 21, X and Y was performed on 530 samples. One hundred of them were additionally analyzed for aneuploidies involving chromosomes 15, 16 and 22.

Results: Aneuploidy was found in 117 (22% of the cases). Thirty-five of them (29.9%) were with triploidy, 27 (23%) - with trisomy 21, 24 (20%) - with monosomy X, 17(14%) - with trisomy 13 and 14(11.9%) - with trisomy 18. Additionally, trisomy 15 was found in 2 cases, trisomy 16 - in 4 and trisomy 22 in 3 cases out from 100.

Conclusions:QF-PCR analysis, being rapid and cost-efficient proved to be useful for genetic analysis of miscarriage samples. Although the vast majority of chromosomal aneuploidies in miscarriages are de novo, such results provide valuable information for the clinicians and genetic counselors and for the couple.

R. Raynova: None. S. Andonova: None. S. Bichev: None. I. Bradinova: None. V. Dimitrova: None. M. Tzankova: None. S. Savova: None. A. Savov: None.

P01.06B Clinical implementation of a custom oligonucleotide array-CGH. Experience in the largest cohort of Spanish prenatal samples (>3400 samples) Clinical implementation of a custom oligonucleotide array-CGH. Experience in the largest cohort of Spanish prenatal samples (&gt3400 samples)

O. Villa1, M. Viñas1, P. Muñoz1, L. Vila1, C. Hernando1,2, N. Fornés1, S. Cano1, A. Zurano1, M. García-Aragonés1, L. Pérez-Jurado1,3,4, L. Armengol1

1qGenomics Laboratory, Esplugues de Llobregat, Spain, 2Laboratori Citogenètica Molecular. Servei de Medicina Genètica i Molecular. Instituto Pediátrico de Enfermedades Raras (IPER). Hospital Sant Joan de Déu, Barcelona, Spain, 3Universitat Pompeu Fabra, Hospital del Mar - IMIM, and CIBER de Enfermedades Raras (CIBERER), Barcelona, Spain, 4Women’s and Children’s Health Network and University of Adelaide, Adelaide, Australia

Introduction: The implementation of genomic array in prenatal routines, when accompanied by pre- and post-test genetic counselling, has demonstrated its utility by fulfilling the longstanding need for a diagnostic test with a higher resolution and higher diagnostic yield than its predecessor, the conventional karyotype.

Materials and methods: Array CGH was performed in 3.438 prenatal samples (2.604 amniotic fluids, 728 corions and 106 fetal samples), using a custom 60K oligonucleotide-based microarray (qChip® CM) designed to maximize the detection of clinically relevant copy-number alterations, and minimize the detection of variants of unknown significance (VOUS). As a general rule, VOUS with unclear phenotypic effect according to current knowledge, and some susceptibility variants are not reported.

Results: We identified a total of 247 pathogenic or probably pathogenic alterations (detection rate: 7.18%) and 60 VOUS (1.74%). As expected, the greatest pathogenic detection rate (7.66%, 202/2637) was among fetuses with ultrasound anomalies, while detection rate was 5.61% (45/801) in ecografically normal gestations (2.24%, 18/801 only with altered maternal serum screning). The vast majority of the VOUS were inherited from a non-affected parent (89.65%) and could be reclassified as most likely benign.

Conclusions: Our series reinforces the clinical utility of prenatal microarray testing: it nearly doubles the diagnostic yield of conventional karyotype (110/247 with variants <10Mb), with no significant increase in the frequency of VOUS that could interfere in decision making. In our experience, we highlight the importance of implementing aCGH in prenatal routines (for all gestations with an indication of invasive fetal sampling).

O. Villa: A. Employment (full or part-time); Significant; qGenomics Laboratory. M. Viñas: None. P. Muñoz: A. Employment (full or part-time); Significant; qGenomics Laboratory. L. Vila: A. Employment (full or part-time); Significant; qGenomics Laboratory. C. Hernando: A. Employment (full or part-time); Significant; qGenomics Laboratory. N. Fornés: A. Employment (full or part-time); Significant; qGenomics Laboratory. S. Cano: A. Employment (full or part-time); Significant; qGenomics Laboratory. A. Zurano: A. Employment (full or part-time); Significant; qGenomics Laboratory. M. García-Aragonés: A. Employment (full or part-time); Significant; qGenomics Laboratory. L. Pérez-Jurado: A. Employment (full or part-time); Significant; qGenomics Laboratory. L. Armengol: A. Employment (full or part-time); Significant; qGenomics Laboratory.

P01.07C Targeted exome sequencing for mutation detection in rare autosomal recessive disorders in families with recurrent undiagnosed fetal anomalies

L. Quteineh1, M. Guipponi1,2, A. Godhino3, E. Hammar1, T. Nouspikel1, L. Lemmens1, J. M. Pellegrinelli4, M. Abramowicz1, J. L. Blouin1,2, S. Fokstuen1

1Service of Genetic Medicine, University Hospitals of Geneva, Geneva, Switzerland, 2Department of Genetic Medicine and Development, University of Geneva, Geneva, Switzerland, 3Gynecology and obstetric private clinic, Geneva, Switzerland, 4Department of gynecology and obstetrics, University Hospitals of Geneva, Geneva, Switzerland

Introduction: Prenatal ultrasound allows the detection of fetal malformation syndromes which often remain without conclusive diagnosis. In case of a recurrent fetal phenotype an autosomal recessive disorder is suspected.

Materials and Methods: We report on 2 families with recurrent fetal anomalies:

  • -Family A: Consanguineous couple from Afghanistan, who experienced 2 terminated pregnancies due to severe brain malformation with hydrocephalus and absence of cerebellar vermis.

  • -Family B: Non-consanguineous couple from Kosovo who experienced 2 perinatal deaths after pregnancies marked by third trimester polyhydramnios and fetal akinesia. Both newborns presented at post-mortem examination distal joints flexion contractures.

We performed on stored fetal DNA whole exome sequencing (WES) with targeted bioinformatic analysis. In family A, we analyzed genes associated with brain malformation and in family B genes known to cause arthrogryposis and fetal akinesia.

Results: In family A, we found a novel nonsense homozygous mutation in LARGE1 causing a rare form of congenital muscular dystrophy-dystroglycanopathy with brain and eye anomalies (OMIM: 613154) and in family B a novel nonsense homozygous mutation in GLDN causing lethal congenital contracture syndrome 11 (OMIM: 617194). In both families, Sanger sequencing confirmed homozygosity in the proband and in the second affected fetus, and also confirmed carrier status in both parents. Based on these results, we were able to offer invasive prenatal testing for both families.

Conclusions: Targeted WES is an effective tool for mutation detection in rare autosomal recessive disorders causing recurrent undiagnosed fetal phenotypes, allowing accurate recurrence risk counseling and early prenatal diagnosis for future pregnancies.

L. Quteineh: None. M. Guipponi: None. A. Godhino: None. E. Hammar: None. T. Nouspikel: None. L. Lemmens: None. J.M. Pellegrinelli: None. M. Abramowicz: None. J.L. Blouin: None. S. Fokstuen: None.

P01.08D Whole exome sequencing in non-obstructive azoospermia allows the identification of a high-risk subgroup of infertile men for undiagnosed Fanconi Anemia, a cancer-prone disease

C. Krausz1, A. Riera-Escamilla2, C. Chianese2, D. Moreno-Mendoza2, O. Rajmil2, M. Bogliolo3, I. Blanco4, E. Ars2, E. Ruiz-Castañé2, J. Surrallés3

1Department of Experimental and Clinical Biomedical Sciences “Mario Serio”, Centre of Excellence DeNo, Florence, Italy, 2Andrology Department, Fundació Puigvert, Universitat Autònoma de Barcelona, Instituto de Investigaciones Biomédicas Sant Pau (IIB-Sant Pau), Barcelona, Spain, 3Genetics Department and Biomedical Reseach Institute, Hospital de Sant Pau, Center for Biomedical Research on Rare Diseases (CIBERER), and Department of Genetics and Microbiology, Universitat Autònoma de Barcelona, Barcelona, Spain, 4Hospital Germans Trias i Pujol, Badalona, Spain

Background: The etiology of non-obstructive azoospermia (NOA) remains unknown in about 40% of cases and a genetic origin is likely to be involved in idiopathic NOA. Genes implicated in stem cell proliferation and DNA repair may cause isolated NOA or be responsible for syndromic diseases, such as Fanconi Anemia (FA). In about 10% of FA cases the diagnosis is delayed until adulthood when a malignant tumor is diagnosed.

Methods: Whole-Exome Sequencing (WES) in an idiopathic NOA patient (index case) with consanguineous parents. Sanger sequencing of the FANCA gene in the brother of the index case and in 27 selected NOA patients. DEB-induced chromosome breakage test.

Results:a rare pathogenic homozygous FANCA variant (c.2639G>A) was identified in the index case, affected by Sertoli Cell only syndrome. The patient’s brother (also with NOA) carried the same genotype. The two brothers did not manifest overt anemia, though chromosomal breakage test confirmed FA. In 27 selected NOA patients with similar testicular phenotype and borderline/mild hematological alterations revealed one additional SCOS patient with compound heterozygosis in FANCA (c.3788_3790delTCT;c.3913C>T).

Conclusions: we identified a specific subgroup of NOA patients with mild or borderline hematological alterations presenting high frequency of occult FA (7.1%). This discovery have important clinical implications: the screening for FANCA mutations in such patients may allow the identification of undiagnosed FA; it corroborates previous epidemiological observations reporting a higher risk of morbidity (including cancer) and a lower life expectancy in infertile men in respect to fertile, normozoospermic men.

Funding: Instituto Carlos III (FIS/FEDER-PI14/01250)

C. Krausz: None. A. Riera-Escamilla: None. C. Chianese: None. D. Moreno-Mendoza: None. O. Rajmil: None. M. Bogliolo: None. I. Blanco: None. E. Ars: None. E. Ruiz-Castañé: None. J. Surrallés: None.

P01.09A Antenatal presentation of Bardet-Biedl syndrome: the question of phenotype-genotype correlations

L. Mary1, K. Chennen2,3, C. Stoetzel2, E. Alanio-Detton4, C. Antal5,6, T. Attie-Bitach7,8, P. Bouvagnet9, R. Bouvier10, A. Buenerd11, D. Carles12, A. Delezoide13, L. Devisme14, B. Gilbert-Dussardier15, F. Guimiot13, P. Khau Van Kien16, P. Loget17, J. Martinovic18, M. Perez19, F. Petit20, L. Pinson21, C. Rooryck-Thambo22, O. Poch3, H. Dollfus2,23,24, E. Schaefer2,23, J. Muller1,2

1Laboratoire de Diagnostic Génétique, Hôpitaux Universitaires de Strasbourg, Strasbourg, France, 2Laboratoire de Génétique médicale, UMR_S INSERM U1112, IGMA, Faculté de Médecine FMTS, Université de Strasbourg, Strasbourg, France, 3Complex Systems and Translational Bioinformatics, ICube, University of Strasbourg, CNRS, Illkirch, France, 4: Gynécologie-obstétrique, centre de dépistage anténatal, hôpital Maison-Blanche, CHU de Reims, Reims, France, 5Institut d'Histologie, Icube, UMR7357, Université de Strasbourg, Strasbourg, France, 6Service de Pathologie, UF6349 Fœtopathologie, Hôpitaux Universitaires de Strasbourg, Strasbourg, France, 7INSERM U1163, Institut IMAGINE, Paris, Paris, France, 8Service d'Histologie-Embryologie-Cytogénétique, Hôpital Necker-Enfants Malades, Assistance Publique-Hôpitaux de Paris, Paris, France, 9Laboratoire de Cardiogénétique, Malformations cardiaques congénitales, Hôpitaux Civils de Lyon, Lyon, France, 10Anatomie et Cytologie Pathologiques, Hôpital Edouard Herriot, Hôpitaux Civils de Lyon, Lyon, France, 11Département de pathologie, centre hospitalier Est, Hôpitaux Civils de Lyon, Lyon, France, 12Service d'Anatomie-Cytologie Pathologique, Centre Hospitalier Universitaire de Bordeaux, Bordeaux, France, 13Service de Biologie du Développement, Hôpital Robert Debré, Assistance Publique-Hôpitaux de Paris, Paris, France, 14Service de Pathologie, Hôpital Jeanne de Flandres, Centre Hospitalier Régional Universitaire de Lille, Lille, France, 15Service de Génétique Médicale, Centre Hospitalier Universitaire de Poitiers, Poitiers, France, 16Unité de Génétique Médicale et Cytogénétique, Centre Hospitalier Universitaire de Nîmes, Nîmes, France, 17Service d'Anatomie Pathologique, Hôpital Pontchaillou, Université Rennes 1, Rennes, France, 18Unité de Fœtopathologie, Hôpital Antoine Béclère, Assistance Publique-Hôpitaux de Paris, Clamart, France, 19Unité de fœtopathologie, Service de Génétique Médicale, Centre Hospitalier Universitaire de Montpellier, Montpellier, France, 20Clinique de Génétique Guy Fontaine, Centre Hospitalier Régional Universitaire de Lille, Lille, France, 21Département de Génétique Médicale, Centre Hospitalier Régional Universitaire de Montpellier, Montpellier, France, 22Laboratoire Maladies rares, génétique et métabolisme, Centre Hospitalier Universitaire de Bordeaux, Bordeaux, France, 23Service de Génétique Médicale, IGMA, Hôpitaux Universitaires de Strasbourg, Strasbourg, France, 24Centre des Affections Rares en Génétique Ophtalmologique, FSMR SENSGENE, Hôpitaux Universitaires de Strasbourg, Strasbourg, France

Introduction:Bardet-Biedl syndrome (BBS) is an emblematic ciliopathy associating retinal dystrophy, obesity, postaxial polydactyly, learning disabilities and renal dysfunction. Before birth, enlarged/cystic kidneys as well as polydactyly revealed by ultrasound (US) are the usual hints to consider this diagnosis in absence of familial history. However, these symptoms are not specific of BBS, raising the problem of differential diagnoses and prognosis. Molecular diagnosis during pregnancies remains a timely challenge for this heterogeneous disease (21 known BBS genes). We report a large cohort of BBS fetuses to better characterize antenatal phenotype-genotype correlations.

Materials and Methods: Prenatal US and/or autopsic data from 74 interrupted fetuses with putative BBS diagnosis were collected.

Results:Using targeted Next Generation Sequencing, we established a molecular diagnostic in 52 cases mainly in BBS genes (47 cases) following the classical gene distribution, but also in other ciliopathy genes (5 cases). Polydactyly (81%, of postaxial localization only) and renal cysts (72%) were the most prevalent symptoms in BBS-mutated fetuses. However, autopsy revealed polydactyly missed by US in 44% of cases. Hydrometrocolpos, evocative of BBS, was found in 3 cases. Ductal plate anomalies, hepatic portal fibrosis, cardiovascular or central nervous system anomalies were rare (6, 4 and 6 cases respectively).

Conclusion: Polydactyly and renal anomalies are confirmed as major prenatal manifestations for BBS. Polydactyly must be carefully controlled in case of apparent isolated renal anomalies. The use of prenatal “fast track” NGS in case of enlarged/cystic kidneys and/or polydactyly has a high utility for diagnosis and prognosis for improved parental information.

L. Mary: None. K. Chennen: None. C. Stoetzel: None. E. Alanio-Detton: None. C. Antal: None. T. Attie-Bitach: None. P. Bouvagnet: None. R. Bouvier: None. A. Buenerd: None. D. Carles: None. A. Delezoide: None. L. Devisme: None. B. Gilbert-Dussardier: None. F. Guimiot: None. P. Khau Van Kien: None. P. Loget: None. J. Martinovic: None. M. Perez: None. F. Petit: None. L. Pinson: None. C. Rooryck-Thambo: None. O. Poch: None. H. Dollfus: None. E. Schaefer: None. J. Muller: None.

P01.10B Different pattern of concordance rate in birth defects according to the zygosity in twins

Y. Jung1, S. Lee1, S. Oh2, C. Park1, J. Park1, J. Jun1

1Seoul National University College of Medicine, Seoul, Korea, Republic of, 2Seoul Metropolitan Government Seoul National University Boramae Medical Center, Seoul, Korea, Republic of

Objective: The pathogenesis of birth defects is multifactorial, and comparing the concordance rate of birth defects according to zygosity in the twin can help to understand the genetic and environmental impacts of the occurrence of birth defects. The objective of this study was to determine the concordance rate of birth defects in central nervous system (CNS) and cardiovascular system (CV), according to the zygosity.

Method: Twins born at Seoul National University Hospital were examined. Zygosity was confirmed by sex, chorionicity, and DNA analysis of umbilical cord blood. PCR amplified short tandem repeat (STR analysis) was conducted with the DNA of cord blood.

Result:The risk of birth defects in CNS and CV of the second twin (F2) was increased when birth defects was present in the first twin (F1) (table). However, higher concordance rates of CNS birth defects were observed in MZ than in DZ [probandwise concordance rate (%, 95% CI): 50.00 (15.70-84.30) in MZ vs. 0.00 (0.00-23.16) in DZ, p < 0.01], whereas the concordance rate of CV birth defects was not different [probandwise concordance rate (%, 95% CI): 26.67 (14.60-41.94) in MZ vs. 20.29 (11.56-31.69) in DZ, p = NS]

Conclusion: The concordance rate according to the zygosity was different between CNS and CV birth defects. It may be speculated that CNS birth defects are highly genetically affected, whereas CV birth defects are affected by the environment.

Birth defects in the 2nd twin


Birth defects in the 1st twin (-)

Birth defects in the 1st twin (+)


Central nervous system

0.5% (12/2375)

25% (2/8)


- Monozygotic

0.4% (3/681)

66.7% (2/3)


- Dizygotic

0.5% (9/1694)

0% (0/5)


Cardiovascular system

1.9% (45/2327)

23.2% (13/56)


- Monozygotic

2.4% (16/661)

26.1% (6/23)


- Dizygotic

1.7% (29/1666)

21.2% (7/33)


Y. Jung: None. S. Lee: None. S. Oh: None. C. Park: None. J. Park: None. J. Jun: None.

P01.11C Dizygotic sex-discordant IVF/ICSI twins with blood but not tissue chimerism

S. Andonova1, S. Hadjidekova2,3, R. Staneva2,3, M. Mihova3, B. Zaharova1, A. Savov1

1National Genetic Laboratory, UHOG "Maichin dom", Sofia, Bulgaria, 2Department of Medical Genetics, Medical University of Sofia, Sofia, Bulgaria, 3Woman Health Hospital "Nadezhda", Sofia, Bulgaria

Introduction:Blood-chimerism in dizigotic twins is very rare condition with presence of two genetically distinct cell lines in one individual that are derived from two separate zygotes. It can occur through intrauterine transfer of hematopoietic cells between the fetuses via vascular anastomoses. Here we present a case of IVF/ICSI pregnancy initially defined as monozygotic; during second trimester a sex-discordance between twins was noted.

Materials and Methods: Cytogenetic analysis of whole blood lymphocytes was performed twice - after delivery and at age of 6 months of the children. DNA was subsequently extracted from buccal cells and from suspensions of cultivated lymphocytes used for karyotyping. DNA profiling was performed with a special attention on markers located on X and Y-chromosomes.

Results: A demonstrable blood chimerism was detected by karioyping and DNA analysis after birth in both twins. Karyotypes for twin 1 (healthy female) and twin 2 (healthy male) at time of birth were chi46,XY[78]/46,XX[22] and chi46,XY[69]/46,XX[31], respectively. After 6 months the karyotypes detected were chi46,XY[87]/46,XX[13] and chi46,XY[86]/46,XX[14], respectively. DNA data from cultivated lymphocytes was in accordance with the cytogenetic results and confined the blood chimerism. This phenomenon was not detected by DNA analysis of buccal cells of both twins.

Conclusions: An efficient use of both cytogenetic and molecular analysis techniques in this case of blood but not buccal cells chimerism was demonstrated. When XX/XY chimerism is detected in blood cells, a careful monitoring of reproductive organs of the twins is recommended. In this case no genital anomalies are detected up to now.

S. Andonova: None. S. Hadjidekova: None. R. Staneva: None. M. Mihova: None. B. Zaharova: None. A. Savov: None.

P01.12D Comprehensive Carrier Screening using a combination of NGS panel, ddPCR and RPA

F. Vogel, K. Brüsehafer, S. Kishore, K. Kandaswamy, M. Weiss, G. Oprea, A. Rolfs, P. Bauer

Centogene AG, Rostock, Germany

Introduction: Carrier screening is a genetic test used to determine if a healthy person is a carrier of a recessive genetic disease. The goal of carrier screening is to help individuals understand their risks of having a child with a genetic disorder and review the range of options available to guide pregnancy and family planning.

Method & test design rationale: Autosomal and X-linked recessive disorders were selected based on the following criteria: Early onset and high-severity disorders, high carrier frequency, availability of treatment and effect on quality of life. Based on this, a NGS panel of 331 genes was designed assessing CCDS +/-20 bases and relevant deep intronic mutations from HGMD® and Centogene’s proprietary variant database CentoMD®. An in-house developed pipeline provides CNV calling on NGS data. Technically challenging but relevant risk genes (FMR1, SMN1, CYP21A2) are analyzed by additional assays based on ddPCR, RPA and Sanger. Adult-onset conditions and X-linked genes in males are not analyzed.

Results: Routine NGS processing covers ≥99% of targeted bases at ≥20 reads. For a comprehensive carrier evaluation, ”pathogenic”, ”likely pathogenic” and strong VUS (class 3.1) are reported. Couples are offered complete screening for partner one and check for partner two genes with actionable variants.

Conclusions: CentoScreen® offers a comprehensive carrier screening to accurately determine genetic risks. It can be especially used in couples from regions with high consanguinity as well as ethnicities with high incidence of certain genetic diseases even without any family history of genetic disease to understand their genetic risks.

F. Vogel: A. Employment (full or part-time); Significant; Centogene AG. K. Brüsehafer: A. Employment (full or part-time); Significant; Centogene AG. S. Kishore: A. Employment (full or part-time); Significant; Centogene AG. K. Kandaswamy: A. Employment (full or part-time); Significant; Centogene AG. M. Weiss: A. Employment (full or part-time); Significant; Centogene AG. G. Oprea: A. Employment (full or part-time); Significant; Centogene AG. A. Rolfs: A. Employment (full or part-time); Significant; Centogene AG. P. Bauer: A. Employment (full or part-time); Significant; Centogene AG.

P01.13A CarrierTest - one year experience with expanded preconception carrier screening

L. Dohnalová, F. Lhota, Z. Vilímová, F. Zembol, I. Soldatova, M. Bittóová, M. Koudová, D. Stejskal

Centre for Medical Genetics and Reproductive Medicine GENNET, Prague, Czech Republic

We have designed a NGS (next-generation sequencing) amplicon-based panel testing 835 key mutations of 77 genes which can influence reproductive health of prospective parents or can cause recessive disorders in offspring. We have developed a bioinformatic pipeline using local installation of Ensembl genomic database for annotation and SQL server variant database for data handling and clinical reporting. To replace MLPA and fragmentation analysis methods we developed coverage analysis-based CNV detection of frequent large deletions of SMN1 and CFTR genes. A software tool developed for the application generates report semi-automatically. Results are grouped according to clinical impact: (1) mutations in genes associated with severe recessive disorders in offspring (e.g. SMN1, CFTR, GJB2 genes), (2) mutations in set of genes predisposing to blood hypercoagulation- trombophilic profile (F2, F5, MTHFR, ANXA5- M2 haplotype), (3) ovarian response to FSH (FSHR polymorphism).

We analysed 3238 samples In the year 2017: 1296 couples in IVF programme (2592 individuals), 354 gamete donors (F 339, M 15) and 141 individuals with a reproductive disorder without compatibility testing (F 88, M 53). The most frequent occurrence of carriers was recorded in the commonly investigated genes (SMN1, CFTR, GJB2), but also in other genes (e.g. BTD, MEFV, ABCA4, SERPINA1, ACADS, DHCR7, PAH, AR). 28 couples with reproductive risks were identified, who were offered preimplantation genetic testing of monogenic diseases (PGT-M). So far we have done IVF with PGT-M in four cases (AR, ABCA4, CFTR genes) and invasive prenatal diagnosis in two cases after a spontaneous conception (SERPINA1 and PAH genes).

L. Dohnalová: None. F. Lhota: None. Z. Vilímová: None. F. Zembol: None. I. Soldatova: None. M. Bittóová: None. M. Koudová: None. D. Stejskal: None.

P01.14B Male infertility and deafness caused by homozygous deletion of CATSPER2 and STRC on 15q15.3

A. Röpke1, C. Brenker2, M. Hoffmann1, C. Krallmann2, S. Kliesch2, T. Strünker2, F. Tüttelmann1

1Institute of Human Genetics, University of Münster, Münster, Germany, 2Centre of Reproductive Medicine and Andrology (CeRA), University Hospital Münster, Münster, Germany

We report on three brothers and one sister born to non-consanguineous parents. The sister and two of the brothers suffer from hearing loss. In addition, the affected brothers, but not the sister, are infertile. Although semen analyses yielded normal sperm concentrations, motility, and morphology (= normozoospermia), the sperm failed to fertilize the oocytes in vitro. However, both brothers conceived a child after ICSI. The sister and the unaffected brother each conceived children spontaneously. Conventional chromosomal analysis of the affected brothers demonstrated apparently normal karyotypes, whereas array-CGH revealed a homozygous loss of approximately 45 kb on chromosome 15q15.3. Homozygous microdeletions in 15q15.3 lead to the deafness-infertility syndrome (OMIM 611102) that is characterized by prelingual hearing loss and infertility in males but not in females. This homozygous loss was also demonstrated in the sister. Both parents were heterozygous for this deletion. The deletion encompasses the genes CKMT1B, STRC and CATSPER2. CKMT1B is translated to a mitochondrial creatine kinase. STRC encodes stereocilin, a protein required for the function ofthe outer hair cells in the inner ear. CATSPER2 encodes a subunit of the sperm-specific CatSper Ca2+-channel complex. CatSper controls the intracellular Ca2+ concentration and, thereby, the swimming behavior of sperm. Functional analysis of sperm from one of the affected brothers demonstrated a lack of functional CatSper channels - this deficit explains the infertility and IVF failure (Brenker et al., 2018 https://doi.org/10.1073/pnas.1717929115). This work was carried out within the frame of the DFG Clinical Research Unit ‘Male Germ Cells: from Genes to Function‘ (CRU 326).

A. Röpke: None. C. Brenker: None. M. Hoffmann: None. C. Krallmann: None. S. Kliesch: None. T. Strünker: None. F. Tüttelmann: None.

P01.15C Positive predictive value (PPV) estimates for cell-free DNA based screening and choice of confirmatory invasive procedure: experience of a large Italian referral prenatal diagnostic laboratory

F. R. Grati1, B. Grimi1, L. Branca1, L. Marcato1, B. Malvestiti1, K. Bajaj2, S. J. Gross3, J. C. P. B. Ferreira4,5,6, G. Simoni1, F. Maggi1

1TOMA, Advanced Biomedical Assays S.p.A, Busto Arsizio, Italy, 2Department of Obstetrics & Gynecology, NYC Health + Hospitals/Jacobi, Bronx, NY, NY, United States, 3Icahn School of Medicine at Mount Sinai, New York, NY, United States, 4Genomed SA, Warsaw, Poland, 5Hospital Central de Maputo, Maputo, Mozambique, 6Faculdade de Medicina da Universidade Eduardo Mondlane, Maputo, Mozambique

Introduction: The aim of this study is to determine the PPV for common trisomies and SCAs using cfDNA screening, based on karyotype results obtained following a high-risk cfDNA testing result.

Materials and Methods: Initial CfDNA screening was ordered by the referring physicians and performed by a variety of technologies. One-hundreds and eighteen confirmatory samples were sent to our lab following a high risk cfDNA test result. PPV stratified by maternal age was calculated for maternal age-dependent aneuploidies.

Results: PPV for T13, T18, T21 was 23.1%, 66.7%, 91.9%, respectively. PPV for SCAs was 7.7% for 45,X, 50% for 47,XXX, 30.8% for 47,XXY and 50% for 47,XYY. In women <35y and ≥35y the PPV for T21, T18 and T13 was, 92%(81,97)*, 57%(25,84)*, 33.3%(12,65)*, and 90(60,98)*, 100%(34,100)*, 0%(0,49)*, respectively. Most of prenatal confirmations were performed on amniocytes (81/116;69.8%); this trend was more evident when the high-risk result involved a SCA (28/33;84.8%) versus a trisomy (54/84;64.3%) (OR: 3.1111;95%CI: 1.1-8.9). However, CVS shows a higher PPV (28/30,93.3%) for non-mosaic trisomies than AF (37/54,68.5%) (OR: 6.43;95%CI: 1.4-30.2).

Conclusions: The higher rate of confirmatory AF after a SCA high-risk result probably indicates that referring clinicians are aware of the proneness of sex chromosomes to generate confined placental mosaicism, therefore causing a decreased PPV when using CVS for SCAs. A possible hypothesis for the higher PPV of CVS for non-mosaic trisomies might be the performance of an ultrasound-scan before the choice of confirmatory invasive procedure, whereby amniocentesis is preferred when no ultrasound anomalies are identified. * 95th% CI

F.R. Grati: None. B. Grimi: None. L. Branca: None. L. Marcato: None. B. Malvestiti: None. K. Bajaj: None. S.J. Gross: None. J.C.P.B. Ferreira: None. G. Simoni: E. Ownership Interest (stock, stock options, patent or other intellectual property); Significant; TOMA Advanced Biomedical Assays S.p.A. F. Maggi: E. Ownership Interest (stock, stock options, patent or other intellectual property); Significant; TOMA, Avanced Biomedical Assays, S.p.A.

P01.16D Genome-wide cell-free fetal DNA screening in routine non-invasive prenatal testing practice: a prospective study on over 38.000 clinical cases

F. Fiorentino, S. Bono, F. Pizzuti, A. Polverari, S. Duca, M. Faieta, M. Baldi, M. Sessa, L. Diano, F. Spinella

Genoma Group, Rome, Italy

Introduction: Conventional cell-free fetal DNA (cfDNA)-based non-invasive prenatal testing (NIPT) focuses on detection of common aneuploidies, leaving a gap of ~17% of clinically relevant chromosomal abnormalities that would go undetected. Genome-wide NIPT would greatly expand the range of chromosomal rearrangements detectable. In this study, we expanded conventional cfDNA-based NIPT to cover the entire genome in a large general population of pregnant women, in order to assess the incidence of chromosomal abnormalities not detectable by traditional NIPT.

Method:38.095 pregnant women undergoing genome-wide cfDNA-based NIPT were enrolled in the study. Sequencing data were analyzed using algorithms for common fetal aneuploidies, aneuploidies and subchromosomal aberrations. Clinical outcomes were obtained in 37.804 pregnancies.

Results:Clinically relevant chromosomal abnormalities were detected in 630 (1.7%) pregnancies, 560 (1.6%) of which were confirmed by invasive prenatal diagnosis. In 500/560 cases common aneuploidies were involved, 30/560 were rare autosomal trisomies (RAT) and 30/560 were segmental imbalances. 60 fetal conditions would have otherwise been overlooked if only a conventional NIPT had been performed. The specificity for common aneuploidy, RAT and segmental aneuploidies was 99.92%, 99.98%, and 99.98%, respectively; the sensitivity was 100%.

Conclusion:The results of this study demonstrate that genome-wide cfDNA analysis represents an enhanced screening tool for prenatal detection of chromosomal abnormalities, allowing identification of clinically relevant imbalances that are not detectable by conventional cfDNA testing. This screening provides improved detection rate as compared to conventional NIPT, with no appreciable decrease in specificity. These findings provide substantial evidence for the feasibility of introducing genome-wide NIPT into routine prenatal diagnosis practice.

F. Fiorentino: None. S. Bono: None. F. Pizzuti: None. A. Polverari: None. S. Duca: None. M. Faieta: None. M. Baldi: None. M. Sessa: None. L. Diano: None. F. Spinella: None.

P01.17A Microarray analysis in fetuses with a high risk combined test

L. Ronzoni1, N. Persico2, A. Sajeva1, R. Silipigni3, S. Guerneri3, D. Alberico2, S. Boito2, I. Fabietti2, M. Disegni3, F. Lalatta1

1Clinical Genetic Unit, Fondazione IRCCS Ca' Granda, Ospedale Maggiore Policlinico, Milan, Italy, 2Department of Obstetrics and Gynaecology 'L. Mangiagalli', Fondazione IRCCS Ca' Granda, Ospedale Maggiore Policlinico, Milan, Italy, 3Laboratory of Medical Genetics, Fondazione IRCCS Ca' Granda, Ospedale Maggiore Policlinico, Milan, Italy

Introduction: to investigate the incremental yield of detecting submicroscopic chromosomal abnormalities by genomic microarray compared to karyotyping in high risk fetuses after combined testing.

Materials and Methods: A total of 250 fetuses, with a high risk after combined test, were tested by conventional CVS karyotyping. If the short-term cytogenetic analysis appeared to be normal, women were informed about CGH-array analysis. If the woman refused the CGH-array, a karyotype analysis was carried out using the long-term culture method.

Results: chromosomal abnormalities were detected in 58 fetuses (23%), particularly in pregnancies complicated by ultrasound abnormalities. In 192 fetuses (77%), the karyotype, after short-term analysis, was normal. All these women were counselled: 74% agreed to proceed with CGH-array analysis while26% refused. In these cases, karyotyping was completed with long-term culture methods confirming the chromosomal normality. Only 19% of the women with a fetus with an increased NT (> 3.5 mm) or ultrasound abnormalities, but with a normal karyotype, refused CGH-array compared to 28% of the women with a high risk after combined test principally due to altered biochemistry. Submicroscopic chromosomal abnormalities were detected only in two cases (1.5%). One was an incidental finding with the detection of a microdeletion causative of dystrophinopathy in a female fetus. In the other case, it was a pathogenic 22q11.21 microduplication in a fetus with a NT < 3.5 mm.

Conclusions:CGH-array analysis, performed only after a multidisciplinary counselling, should also be part of the investigation in fetuses with biochemical high risk after a combined test.

L. Ronzoni: None. N. Persico: None. A. Sajeva: None. R. Silipigni: None. S. Guerneri: None. D. Alberico: None. S. Boito: None. I. Fabietti: None. M. Disegni: None. F. Lalatta: None.

P01.18B Prevalence of submicroscopic chromosome aberrations in pregnancies without increased risk for structural chromosome aberrations - a systematic review of the literature

M. I. Srebniak1, M. Joosten1, M. F. C. M. Knapen1, L. R. Arends1, M. Polak2, S. van Veen1, A. T. J. I. Go1, D. Van Opstal1

1Erasmus MC, Rotterdam, Netherlands, 2Erasmus University, Rotterdam, Netherlands

Objectives: To establish the frequency of pathogenic submicroscopic chromosome aberrations in fetuses that are not at increased risk for unbalanced structural chromosome aberrations, a systematic literature search was performed. The aim was to determine whether high resolution testing for submicroscopic aberrations is beneficial in a general pregnant population.

Methods: On 3rd June 2016 Embase and PubMed databases were systematically searched for all relevant articles on prevalence of pathogenic submicroscopic CNVs in fetuses tested due to advanced maternal age or parental anxiety. Relevant full text articles were analyzed and based on the extracted data the prevalence of submicroscopic CNVs was calculated.

Results: Meta analysis was conducted in a pooled cohort of 10,614 fetuses based on the 10 largest studies (N > 300) of a total of 19 relevant studies. In 0.84%, 95%CI [0.55%, 1.30%] of fetuses a submicroscopic pathogenic aberration was detected prenatally. The onset/penetrance of the submicroscopic findings was studied in 10,314 fetuses out of 8 papers that presented aberrant cases with all necessary details. The prevalence of early onset syndromic disorders due to a submicroscopic aberration was calculated to be 1:270, based on 0.37%, 95%CI [0.27%, 0.52%] cases where aberrations were specified.

Conclusions: This systematic review shows that a significant proportion of fetuses in a general pregnant population carry a submicroscopic pathogenic CNV. Based on these figures all women should be informed on their individual risk for all pathogenic chromosome aberrations and not only for common trisomies.

M.I. Srebniak: None. M. Joosten: None. M.F.C.M. Knapen: None. L.R. Arends: None. M. Polak: None. S. van Veen: None. A.T.J.I. Go: None. D. Van Opstal: None.

P01.20D Chromothriptic events in healthy people: pay attention to ''innocent'' insertional translocations

N. E. KURTAS1, L. Zumerle2, L. Leonardelli2, U. Giussani3, A. Pansa3, L. Cardarelli4, V. Bertini5, E. Errichiello1, M. Delledonne2, O. Zuffardi1

1Department of Molecular Medicine, University of Pavia, Pavia, Italy, 2Department of Biotechnologies, University of Verona, Verona, Italy, 3Laboratorio di Genetica, Ospedali Riuniti di Bergamo, Bergamo, Italy, 4Laboratorio Analisi CITOTEST, Padova, Italy, 5Laboratory of Medical Genetics, Azienda Ospedaliero-Universitaria Pisana, S. Chiara Hospital, Pisa, Italy

Chromothripsis is characterized by extensive genomic rearrangements consisting in multiple deletions and disordered orientation of the rearranged portions of one or few chromosomes. By whole genome sequencing (WGS) in three unrelated families, we demonstrated that in one parent of each family a balanced chromothripsis was present causing a genomic imbalance in the index case consisting in a deletion and a non-contiguous duplication within 3q22.1-q26.31 in case 1, a simple two-way reciprocal translocation t(6;14) in case 2, and a complex rearrangement involving chromosomes 6, 7 and 15 in case 3. It is noteworthy that a parental chromothripsis at the origin of the proband’s imbalance was far from predictable in two of them, in which the proband’s rearrangement was at first interpreted as de novo in case 1 and consisting in a simple translocation in case 2. In all parents-of-origin a small size fragment of the shattered chromosome was inserted into an additional chromosome. Our findings strongly indicate that (i) both simple and complex unbalanced rearrangements, can in fact be recombinant chromosomes derived by a balanced chromothripsis present in one healthy parent; (ii) WGS investigations in the parent may reveal unexpected genomic complexity that are impossible to foresee by conventional and molecular cytogenetic approaches; (iii) insertional translocations cannot anymore be considered three breakpoints events but rather are the spy of more complex rearrangements. Our partially novel and partially confirmatory data call for WGS as first tier genomic analysis in order to properly evaluate any possible risk for chromosome imbalances at following pregnancies.

N.E. Kurtas: None. L. Zumerle: None. L. Leonardelli: None. U. Giussani: None. A. Pansa: None. L. Cardarelli: None. V. Bertini: None. E. Errichiello: None. M. Delledonne: None. O. Zuffardi: None.

P01.21A The influence of chromosomal microarray and NIPT on the diagnostic yield in 6811 high risk pregnancies without The influence of chromosomal microarray and NIPT on the diagnostic yield in 6811 high risk pregnancies without ultrasound anomalies

M. I. Srebniak1, M. F. C. M. Knapen2, M. Polak3, M. Joosten1, K. E. M. Diderich1, W. F. J. van IJcken4, R. Heydanus5, A. Dijkman6, T. Toolenaar7, F. A. T. de Vries1, J. Knijnenburg1, A. T. J. I. Go8, R. H. Galjaard1, D. Van Opstal1

1Erasmus MC, Clinical Genetics, Rotterdam, Netherlands, 2Erasmus MC, Department of Obstetrics and Gynecology, Rotterdam, Netherlands, 3Institute of Psychology, Erasmus University Rotterdam, Rotterdam, Netherlands, 4Erasmus MC, Rotterdam, Netherlands, 5Department of Obstetrics and Gynecology, Amphia Hospital, Breda, Netherlands, 6Department of Obstetrics and Gynecology, Reinier de Graaf Gasthuis, Delft, Netherlands, 7Department of Gynecology, Albert Schweitzer Hospital Dordrecht, Dordrecht, Netherlands, 8Department of Obstetrics and Gynecology, Erasmus MC, Rotterdam, Netherlands

Introduction: Prenatal diagnostics has been impacted by technological changes in the past decade, which have affected the diagnostic yield. The aim of this study was to evaluate the impact of SNP array and non-invasive prenatal testing (NIPT) on the diagnostic yield and the number of invasive tests in our center.

Material and Methods: The frequency of pathogenic fetal unbalanced chromosome aberrations was studied in 6811 high risk pregnancies without ultrasound anomalies referred for prenatal testing in 2009-2015 due to advanced maternal age, abnormal first trimester screening results (with nuchal translucency < 3.5mm) or recurrence risk for chromosome aberration. Chromosomal SNP microarray analysis replaced karyotyping in in 2012 and since 2014 a choice between NIPT and diagnostic testing with microarray was offered to women with an increased risk for common aneuploidy (as a part of a national TRIDENT study).

Results: The introduction of microarray led to an additional yield of submicroscopic pathogenic chromosome aberrations in 1.9% in fetuses without ultrasound anomalies. The introduction of NIPT led to a decrease of invasive tests, but also of the diagnostic yield.

Conclusions: Since 33% of pathogenic fetal chromosome aberrations were different from the common aneuploidies and triploidy, whole genome analysis should be offered after invasive sampling. Because NIPT (as a second screening) has resulted in a decreased diagnostic yield it should be accompanied by an appropriate pre-test counseling in high risk pregnancies.

M.I. Srebniak: None. M.F.C.M. Knapen: None. M. Polak: None. M. Joosten: None. K.E.M. Diderich: None. W.F.J. van IJcken: None. R. Heydanus: None. A. Dijkman: None. T. Toolenaar: None. F.A.T. de Vries: None. J. Knijnenburg: None. A.T.J.I. Go: None. R.H. Galjaard: None. D. Van Opstal: None.

P01.22B Chromosomal microarray analysis in fetuses with double renal collecting system

A. Singer1, I. Maya2, A. Frumkin3, S. Zeligson4, R. Berger5, S. Ben Shachar6, C. Vinkler7, L. Sagi-Dain8

1Community Genetics, Public Health Services, Ministry of Health, Jerusalem, Israel, 2Recanati Genetics Institute, Beilinson Hospital, Rabin Medical Center, Petach Tikva, Israel, 3. Department of Genetic and Metabolic Diseases, Hadassah, Hebrew University Medical Center, Jerusalem, Israel, 4Medical Genetics Institute, Shaare Zedek Medical Center, Jerusalem, Israel, 5Maccabi Health Services, Rehovot, Israel, 6. Genetics Institute, Sorasky Medical Center, Tel Aviv, Israel, 7Genetic Institute, "Wolfson", Holon, Israel, 8Genetics Institute, Carmel Medical Center, Haifa, Israel

Duplication of the renal collecting system is one of the most common variants of urinary tract anatomy with an estimated incidence of about 1% . This condition is characterized by two pelvicalyceal units draining a single kidney. It can be either complete or partial. The objective of our study was to examine the rate for chromosomal aberrations in isolated prenatal sonographic finding. Data from all chromosomal microarray analyses (CMA) reported to the Ministry of Health between January 2013 and December 2016 were retrospectively obtained from a computerized database. All pregnancies with sonographic diagnosis of isolated duplex renal collecting system and documentation of CMA result were included. Rate of abnormal CMA findings in these cases was compared to that of the general population risk, based on a systematic review encompassing 9272 cases with normal ultrasound, and a local data of 5541 pregnancies undergoing CMA due to maternal request. One pathogenic CMA finding was found amongst 98 pregnancies with double collecting system (1.02%), not significantly different from the risk for abnormal CMA results in the general population. In addition, two variants of unknown significance were demonstrated (2.04%). This is the first report describing the rate of chromosomal anomalies in pregnancies with isolated duplex renal collecting system. It is suggested that routine invasive prenatal testing with CMA analysis in such cases is no more useful than in the general population. Prospective larger studies are needed to guide the optimal management of pregnancies with isolated duplex renal collecting system.

A. Singer: None. I. Maya: None. A. Frumkin: None. S. Zeligson: None. R. Berger: None. S. Ben Shachar: None. C. Vinkler: None. L. Sagi-Dain: None.

P01.23C A component of sperm fibrous sheath has major effect on testis morphology and functionality

L. Sun1, L. Huang2, Z. Chen3, N. Li2

1Department of Reproductive Medicine,Guangzhou Women and Children's Medical Center, Guangzhou, China, 2Guangzhou Institute of Pediatrics,Guangzhou Women and Children's Medical Center, Guangzhou, China, 3Department of Reproductive Medicine,Guangzhou Women and Children's Medical Center, Guangzhou, China, Guangzhou, China

The fibrous sheath is a unique cytoskeletal structure located in the principle piece of the sperm flagellum with more than 13 protein components. AKAP4 is the most abundant protein in the fibrous sheath, which interacts with at least 3 other proteins. The molecular structure and functionality of the fibrous sheath are largely unknown. We collected a clinic sample of sperms featured by dysplasia of fibrous sheath (DFS), leading to a failure of natural conception. The sperm donor is an offspring of consanguineous family, and we identified an inherited homozygous truncating mutation in his genome by whole-exome sequencing. The affected protein is one of the components of fibrous sheath, and this mutation caused a shortened protein which lost part of the original functions. The mice model with this mutation introduced by CRISPR-Cas9 technique showed similar phenotype to the human, with sperms of reduced number and lower motility due to flagellum malfunction. Strikingly, we observed that, unlike AKAP4 knock-out mice model, the knock-out mice model we constructed for the novel gene exerted major effect on testis, manifested by significant size/weight reduction and azoospermia. Microscopic observation of testis slices and in-situ hybridization showed abnormal cross-section of the seminal vesicles and disorganized progression from spermatogonia to spermatid, when comparing the knock-out mice model with the control ones. Therefore, we concluded that this gene is of critical importance to normal spermatogenesis and testis development.

L. Sun: None. L. Huang: None. Z. Chen: None. N. Li: None.

P01.24D Use of prenatal exome sequencing in fetuses with ultrasound anomalies

M. Segura-Puimedon, B. Rodríguez-Santiago, A. Vallmajó, M. Codina-Solà, B. Campos, D. Datta, I. Banchs, H. Mattlin, Y. Sarria, O. Abad, J. Rodríguez, L. Pérez-Jurado, L. Armengol

Quantitative Genomic Medicine Laboratories, qGenomics, Esplugues de Llobregat, Spain

Introduction: Whole exome sequencing is a diagnostic tool in postnatal settings for individuals with a suspected genetic condition. Recently, its application in prenatal settings has increased and is sporadically used as a diagnostic tool. We present here our experience using this approach in a prenatal setting.

Material and Methods: Exome sequencing was performed in 42 fetal samples carrying different ultrasound anomalies. 30 samples were from evolutive pregnancies and 12 were from legal pregnancy interruptions. In 10 of the samples previous prenatal CGH-array was performed with negative result. Segregation studies were performed in cases with a candidate variant when possible.

Results: Common reasons for referral were skeletal anomalies, polymalformated fetuses, cerebral anomalies or monogenic disorder suspicion (Noonan syndrome). Pathogenic variants were identified in n = 8 (19%) of samples, being previously described or de novo in the index case. In n = 9 (21%) cases, variants of unknown significance were identified, and in two of them inheritance was consistent with expected pattern. In more than half of the cases (52%) we were not able to identify any candidate variant. Diagnostic yield was highest in fetuses with skeletal anomalies, where pathogenic variants were identified in (n = 6) 60% of cases, and in fetuses with clinical suspicion of Noonan syndrome, where a pathogenic variant was found in 75% of the samples. No pathogenic variants were found in polymalformated fetuses or fetuses with cerebral anomalies.

Conclusion: Exome sequencing is a valuable diagnostic tool in fetuses with ultrasound anomalies, especially when skeletal anomalies are present or Noonan syndrome is suspected.

M. Segura-Puimedon: A. Employment (full or part-time); Significant; Quantitative Genomic Medicine Laboratories, qGenomics, Barcelona, Spain. B. Rodríguez-Santiago: A. Employment (full or part-time); Significant; Quantitative Genomic Medicine Laboratories, qGenomics. A. Vallmajó: A. Employment (full or part-time); Significant; Quantitative Genomic Medicine Laboratories, qGenomics. M. Codina-Solà: A. Employment (full or part-time); Significant; Quantitative Genomic Medicine Laboratories, qGenomics. B. Campos: A. Employment (full or part-time); Significant; Quantitative Genomic Medicine Laboratories, qGenomics. D. Datta: A. Employment (full or part-time); Significant; Quantitative Genomic Medicine Laboratories, qGenomics. I. Banchs: A. Employment (full or part-time); Significant; Quantitative Genomic Medicine Laboratories, qGenomics. H. Mattlin: A. Employment (full or part-time); Significant; Quantitative Genomic Medicine Laboratories, qGenomics. Y. Sarria: A. Employment (full or part-time); Significant; Quantitative Genomic Medicine Laboratories, qGenomics. O. Abad: A. Employment (full or part-time); Significant; Quantitative Genomic Medicine Laboratories, qGenomics. J. Rodríguez: A. Employment (full or part-time); Significant; Quantitative Genomic Medicine Laboratories, qGenomics. L. Pérez-Jurado: E. Ownership Interest (stock, stock options, patent or other intellectual property); Significant; Quantitative Genomic Medicine Laboratories, qGenomics. F. Consultant/Advisory Board; Significant; Quantitative Genomic Medicine Laboratories, qGenomics. L. Armengol: E. Ownership Interest (stock, stock options, patent or other intellectual property); Significant; Quantitative Genomic Medicine Laboratories, qGenomics.

P01.26B Challenges and opportunities in variant interpretation in NGS-based expanded carrier screening. First results from an Argentinian cohort

S. Menazzi, M. Fabbro, D. Lorenzi, M. Bilinski, C. Fernandez, M. Galain, P. Nicotra, V. Chekherdemian, F. Nodar, S. Papier

NOVAGEN, Buenos Aires, Argentina

Introduction: In the past decade, NGS-based technologies have been disruptive in many areas of clinical genetics, mainly related to the diagnosis of known entities. Reproductive medicine has not been excluded from these advances, and expanded carrier screening (ECS) has become increasingly used, both for couples at risk and general population.Here we describe challenges and opportunities of an NGS-based ECS panel for recessive disorders.Material and Methods: After variant calling of 120 cases using an in-house developed pipeline for processing data of 483 genes sequenced on a Nextseq 550 platform, variants were classified according to ACMG guidelines and our clinical geneticists’ and molecular biologists’ criteria.Results: 39.2% of the patients were found to be carriers for at least one pathogenic/likely pathogenic variant for 48 different diseases. A pathogenic variant por G6PD (X-linked haemolytic anemia) was found in an apparently healthy man. 91 unique variants of uncertain significance (39 classified as “Conflict of interpretation” in ClinVar) were found in a homozygous/hemizygous state, which when present in genes related to recessive full-penetrance and early-onset disease were deemed likely benign.Conclusions:NGS-based ECS represents an unique opportunity to identify couples at risk of having children with recessive conditions but also to calculate variant frequencies from countries underrepresented in global consortiums, diagnose individuals with low-penetrance disorders and classify homozygous/hemizygous variants of uncertain significance as benign. ACMG guidelines are mainly focused on diagnosis of affected individuals, but variant classification for ECS must be based on data not related to the phenotype.

S. Menazzi: A. Employment (full or part-time); Significant; NOVAGEN. M. Fabbro: A. Employment (full or part-time); Significant; NOVAGEN. D. Lorenzi: A. Employment (full or part-time); Significant; NOVAGEN. M. Bilinski: A. Employment (full or part-time); Significant; NOVAGEN. C. Fernandez: A. Employment (full or part-time); Significant; NOVAGEN. M. Galain: A. Employment (full or part-time); Significant; NOVAGEN. P. Nicotra: A. Employment (full or part-time); Significant; NOVAGEN. V. Chekherdemian: A. Employment (full or part-time); Significant; NOVAGEN. F. Nodar: A. Employment (full or part-time); Significant; NOVAGEN. S. Papier: A. Employment (full or part-time); Significant; NOVAGEN.

P01.27C NGS expanded carrier screening in the Netherlands: initial implementation results

K. M. Abbott, T. Dijkhuizen, M. Veldhuis, J. Schuurmans, I. van Langen, R. J. Sinke

University Medical Center Groningen, Groningen, Netherlands

Introduction: Expanded carrier screening (ECS) has broadened in recent years from high risk population-targeted testing to general public screening. and the main challenge now is choosing the most applicable test design for the intended population. Here we describe the ECS test developed at our department of Genetics and our initial results.

Materials and Methods: Based on focus group discussions, we designed and implemented a couple-based ECS multi-gene test for 70 rare, early onset and serious recessive Mendelian conditions using NGS technologies. Concentrating on couple-based screening, emphasis was on the combined risk for having affected children. The a priori risk of being a carrier couple is approximately 1 in 150 and increases for those referred for medical reasons (e.g. consanguinity). Only results with high predictive value regarding affected offspring were reported in the combined result.

Results: A total of 169 couples were tested, 52 potential high-risk couples and 117 general public couples as part of an population-based implementation study. Five couples, referred for diagnostic reasons, shared carriership of one of the diseases tested. All remaining couples tested normal. Reporting times averaged at 38 days, and in some cases even within 2 weeks.

Conclusions: Our combined approach for ECS testing allows for a fast, simplified procedure to report a combined risk to couples, forestalling the burden of individual findings. Broader implementation (e.g. general public via their GP) seems warranted, and is supported by these first results. Future international discussions will guide further development of such important screening tests.

K.M. Abbott: None. T. Dijkhuizen: None. M. Veldhuis: None. J. Schuurmans: None. I. van Langen: None. R.J. Sinke: None.

P01.28D Molecular autopsy is an important tool in the diagnosis of lethal fetal disorders and structural anomalies

A. Yeung1,2,3, M. Regan1,4, M. Hunter1,3

1Monash Genetics, Monash Health, Melbourne, Australia, 2Victorian Clinical Genetics Services, Murdoch Children's Research Institute, Parkville, Australia, 3Department of Paediatrics, Monash University, Melbourne, Australia, 4Family Cancer and Genetics, Melbourne Health, Parkville, Australia

Introduction: Genomic sequencing is emerging as an important tool in the diagnosis of lethal fetal disorders and structural malformations. We sought to determine the clinical utility of genomic sequencing as an adjunct to standard antenatal imaging and fetal autopsy, as well as the impact of molecular diagnosis on clinical care.

Materials and Methods: We performed a retrospective review of perinatal cases referred to the Monash Genetics Unit between 2015 and 2017 in the setting of structural malformations or fetal-death-in-utero. 19 fetuses were identified in whom genomic testing had been performed following a normal microarray and findings on antenatal imaging and autopsy suggestive of an underlying monogenic disorder. Testing comprised either a targeted panel or whole exome sequencing in a clinically accredited laboratory.

Results: A diagnosis was established in 9/19 cases (47%) on the basis of likely pathogenic or pathogenic variants detected in genes with a concordant Mendelian phenotype: RIT1, RAF1, L1CAM, AGRN, MTM1, CHRNB1, CEP290, COL1A1, and PKHD1. Variants of unknown significance were detected in 2/19. A significant impact on clinical management was noted in the families who received a molecular diagnosis: 2/9 families were informed that a causative variant was de novo, restoring reproductive confidence; 3/9 patients were counselled regarding increased recurrence risk and proceeded to prenatal diagnosis in subsequent pregnancies, and 3/9 couples were referred to an IVF service for preimplantation genetic diagnosis.

Conclusion: Genomic sequencing enhances the diagnostic yield of standard fetal imaging and autopsy and improves patient care.

A. Yeung: None. M. Regan: None. M. Hunter: None.

P01.29A NGS studies in structurally abnormal fetuses with a normal chromosomal microarray analysis. Clinical experience

A. Borrell1, M. Pauta1, V. Borobio1, B. Marquès1, C. Badenas2, M. Milà2

1BCNatal.Hospital Clínic Barcelona, Barcelona, Catalonia, Spain, 2CDB.Hospital Clínic Barcelona, Barcelona, Catalonia, Spain

Objective: The elective genetic testing for structurally abnormal fetuses is chromosomal microarray analysis (CMA). We investigated the value of next generation sequencing (NGS) studies in fetuses with selected structural anomalies and normal CMA.

Methods: During a 30-month period, NGS studies were performed on fetal DNA extracted from amniocytes or chorionic villi in 25 structurally abnormal fetuses with a normal CMA. NGS studies included a single gene analysis (n = 6), gene panel (n = 14), or clinical exome sequencing (n = 5). Single gene analysis was performed in suspected syndromes caused by a single candidate gene (thanatophoric dysplasia, CHARGE, Mowat-Wilson...) and when a panel or exome was not available in our center (Noonan, lissencephaly). A gene panel was used when for a specific fetal syndrome or sign have multiple candidate genes (large echogenic kidneys, Noonan syndrome, craneosynostosis). Finally, clinical exome sequencing was performed in recurrent or multisystem anomalies with no specific syndrome suspicion.

Results: In 40% (10/25) of the cases, NGS provided definitive diagnoses. It was provided in 1/6 (17%) of single gene analyses (thanatophoric dysplasia), in 8/14 (57%) of gene panels (4 by CAKUT panel, 2 by Noonan, one by a craniosynostosis and one by skeletal dysplasia panels), and in 2/5 (40%) of clinical exomes (primary microcephaly and Bohring-Opitz syndrome).

Conclusion: NGS provides a definitive diagnosis in 40% of fetuses with selected structural anomalies and normal CMA results. The most frequent diagnoses were specific skeletal dysplasias, Noonan syndrome in persistent nuchal fold +/- hydrops, and autosomal recessive renal diseases in large echogenic kidneys.

A. Borrell: None. M. Pauta: None. V. Borobio: None. B. Marquès: None. C. Badenas: None. M. Milà: None.

P01.30B Fetal exome sequencing: yield and limitations in a single tertiary center

H. Daum, V. Meiner, O. Elpeleg, T. Harel

Hadassah, Jerusalem, Israel

Objective: To explore the indications and diagnostic outcomes of fetal exomes in a single referral center.

Methods: 77 unrelated fetal samples underwent exome sequencing between 2012-2017. Indications, turnaround time, diagnostic rates, and pregnancy outcomes were analyzed.

Results: The most common indication for fetal exome sequencing was multiple malformations (21/77, 27%), followed by isolated brain malformations (15/77, 19%). Twelve fetuses (15%) were referred for isolated increased nuchal translucency (IINT). Exome analysis was diagnostic for 16 fetuses (21%); when sub-classified to fetal malformations vs. IINT it became clear that exome analysis did not reveal any known or probable pathogenic variants in IINT whereas among the remaining fetuses, a molecular diagnosis was reached in 16/65 (25%). Proband-only cases received a diagnosis more often than trio exomes.

Conclusion: Exome sequencing has the potential to provide molecular diagnoses in cases where conventional prenatal cytogenetic testing is negative. A referral bias of consanguineous cases could account for the high diagnostic rate for proband-only sequencing. Syndrome-specific prognostic information enables parents to make informed decisions, whereas challenges include time limitations and variant interpretation in the setting of non-specific fetal findings. As we report only established disease-gene associations, further segregation and functional studies in a research setting are expected to significantly increase the diagnostic yield.

H. Daum: None. V. Meiner: None. O. Elpeleg: None. T. Harel: None.

P01.31C Low fetal fraction and digynic triploidy: products-of-conception testing supports Fetal-Fraction-Based Risk model for non-invasive prenatal testing

S. Krinshpun, W. DiNonno, T. McKanna, A. Ryan, K. Martin

Natera, Inc., San Carlos, CA, United States

Introduction: Pregnancies receiving a ‘no result’ on non-invasive prenatal testing (NIPT) due to low fetal fraction (‘no result’ LFF) are at increased risk for trisomies 18 and 13 (T18, T13) and digynic triploidy (DT). The Fetal-Fraction–Based Risk (FFBR) method has recently been validated to assess SNP-based NIPT ‘no result’ LFF pregnancies given fetal fraction, maternal weight, and gestational age. This method demonstrated high sensitivity for pregnancies affected by T18, T13, and DT (high risk, ≥1%). The objective of this study was to retrospectively apply the FFBR model to SNP-based NIPT ‘no result’ LFF cases that later had products-of-conception (POC) testing.

Materials and Methods: Over 30,000 consecutive POC samples submitted January 2014-December 2017 were reviewed. Pregnancies with both SNP-based NIPT ‘no result’ LFF and SNP-based POC results, which included parent of origin, were selected for application of the FFBR model.

Results: A total of 338 POC cases also had NIPT performed. Of these cases, 48 (14.2%) had ‘no result’ LFF (expected given its association with pregnancy loss). The prevalence of chromosome abnormalities in the ‘no result’ LFF cases was 66.7% (32/48); DT was the most common (43.8% [14/32]). All DT cases (100%) received a high FFBR score.

Conclusion: DT was a significant source of chromosome abnormalities among pregnancy losses that previously had a SNP-based NIPT ‘no result’ LFF. Retrospective application of FFBR demonstrated that these cases could have been identified as at-risk at time of SNP-based NIPT, allowing for a more informed genetic counseling and prenatal management.

S. Krinshpun: A. Employment (full or part-time); Significant; Natera, Inc. W. DiNonno: A. Employment (full or part-time); Significant; Natera, Inc. T. McKanna: A. Employment (full or part-time); Significant; Natera, Inc. A. Ryan: A. Employment (full or part-time); Significant; Natera, Inc. K. Martin: A. Employment (full or part-time); Significant; Natera, Inc.

P01.33A Age-dependent gonosomal mosaicism in fertile women

A. Tarlycheva1, Z. Markova1, T. Volkova2, N. Shilova1

1Federal Budgetary State Institution "Research Centre for Medical Genetics", Moscow, Russian Federation, 2Clinic "Moja sem'ja", Moscow, Russian Federation

Introduction: One of the most significant features of the human genome is high variability. Genomic variations occur during ontogenesis in various tissues and organs leading to the tissue-specific mosaicism. However, phenomenon and mechanisms of a low-level gonosomal mosaicism in women of reproductive age have not been properly described and clarified.

Materials and Methods: preparations from buccal smear (34) and peripheral venous blood (32) each woman had at least one healthy child. Three groups were formed that included woman of different ages: 20-29 years (1), 30-39 years (2) and 40-49 years (3). FISH-analysis with centromeric probes on chromosomes X and 18 in accordance with the standard protocol.

Results: The study found that in the blood of healthy women there is a physiological low-level mosaicism with a clear trend in increased proportion of abnormal cells associated with the increasing age to 1.83%, 2.23% and 5.88% for groups 1, 2 and 3, respectively (P= 0.0042). The buccal smear also exhibited physiological pattern of a low-level mosaicism, however, the level of mosaicism was statistically insignificant in different age groups and, on average, was 4.01% (P = 0.530). It is shown that mosaicism in buccal smear is represented by two cell lines: one is with disomy and another one includes monosomy on chromosome X.

Conclusion: The obtained data can be a reference for the evaluation of low-level mosaicism in fertile women.

A. Tarlycheva: None. Z. Markova: None. T. Volkova: None. N. Shilova: None.

P01.34B Exome sequencing reveals novel IGSF10 variation in patients with hypergonadotropic hypogonadism

R. Colombo1,2, A. Jolly3, Y. Bayram3, E. Karaca3, N. Di Simone4, G. Scambia4, T. Tos5, S. Jhangiani6, Z. C. Akdemir6, J. E. Posey3, J. R. Lupski3,6,7

1Institute of Clinical Biochemistry, Faculty of Medicine, Catholic University and Policlinico Agostino Gemelli, Rome, Italy, 2Center for the Study of Rare Hereditary Diseases, Niguarda Ca' Granda Metropolitan Hospital, Milan, Italy, 3Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, United States, 4Department of Obstetrics and Gynecology, Faculty of Medicine, Catholic University and Policlinico Agostino Gemelli, Rome, Italy, 5Department of Medical Genetics, Sami Ulus Children’s Hospital, Ankara, Turkey, 6Human Genome Sequencing Center, Baylor College of Medicine, Houston, TX, United States, 7Department of Pediatrics, Baylor College of Medicine, Houston, TX, United States

Introduction: Hypergonadotropic hypogonadism (HH) is characterized by hypogonadism due to an impaired response of the gonads to gonadotropins (Gn) and a secondary lack of sex steroid production and elevated Gn levels. HH can be caused by environmental factors and congenital disorders that affect ovarian development and function, as well as syndromic and non-syndromic single gene disorders. However, in most cases of gonadal dysfunction the molecular etiology remains an enigma.

Subjects and Methods: To identify novel molecular causes in HH we applied whole exome sequencing (WES) to 33 affected female individuals from 30 unrelated families including 22 with parental consanguinity.

Results: WES revealed likely pathogenic variants in known HH-associated genes in 7/30 families (23%) including AR, NOBOX, MCM8, PADI6, PSMC3IP, and TG. In seven unrelated families, we identified likely pathogenic variants in candidate disease genes. In three of these families, each with one apparently sporadic case, biallelic variation in IGSF10 was found. Two of the three families had reported parental consanguinity. IGSF10 encodes a 2623-amino-acid member of the immunoglobulin superfamily that is likely involved in controlling early migration of neurons expressing gonadotropin-releasing hormone. All three missense IGSF10 variants affect conserved amino acid residues, were predicted to be deleterious by several SNV scoring algorithms, and are present in genomic databases with a frequency <2x10-5.

Conclusions: A WES approach enabled the identification of novel IGSF10 mutations in females with HH, thus expanding the spectrum of genes involved in impaired ovarian response to Gn.

R. Colombo: None. A. Jolly: None. Y. Bayram: None. E. Karaca: None. N. Di Simone: None. G. Scambia: None. T. Tos: None. S. Jhangiani: None. Z.C. Akdemir: None. J.E. Posey: None. J.R. Lupski: None.

P01.35C A case of Down syndrome with isodicentric chromosome 21 misdiagnosed by noninvasive prenatal testing (NIPT)

J. Pietrzak1, C. Wleczyk1, K. Bernatowicz2, A. Kashyap2, E. Studniak1, W. Bonda1, M. Kossowski1, M. Ryłów1, M. Bryśkiewicz1, S. Zajączek2

1Cytogenetic Unit, Department of Laboratory Diagnostics, Pomeranian Medical University, Szczecin, Poland, 2Department of Genetics and Pathology, Pomeranian Medical University, Szczecin, Poland

Isodicentric chromosome 21 is an extremely rare chromosomal aberration. Only several cases have been reported worldwide. The phenotype of patients with 46,idic(21)(q22.3) karyotype is generally concordant with patients who have simple trisomy 21.

We report a girl born in 38-th week from first pregnancy; prenatal screening (USG and a double test) performed in 13-th week of gestation predicted a 6-fold increased risk of Down syndrome. Noninvasive prenatal testing using cffDNA from mother's blood did not show increased risk of trisomy 21 in fetus. Invasive prenatal testing was not performed. After birth, phenotypic features of trisomy 21 were observed in the child.

Cytogenetic testing performed from the periferal blood revealed an unbalanced karyotype 46,XX,idic(21)(q22.3). The result were confirmed using Methods: FISH, and MLPA. Microarray additionally revealed a terminal microdeletion sized 11.2 kbp on chromosome 21. Both parents were tested and confirmed negative for any chromosomal aberration from blood and fibroblasts and they have been informed about phenomena of germinal mosaicism in gonadal DNA.

In this case, NIFTY test could not identify the abnormality. Further studies are needed to assess sensitivity of NIPT in the cases of Down syndrome, not caused by simple trisomy 21.

We conclude that invasive prenatal diagnosis should be proposed in all pregnancies with increased trisomy risk, even if NIPT results are negative. High resolution microarray testing can be helpful in identification of microdeletions in patients with idic 21 and could delineate genotype -phenotype correlations.

J. Pietrzak: None. C. Wleczyk: None. K. Bernatowicz: None. A. Kashyap: None. E. Studniak: None. W. Bonda: None. M. Kossowski: None. M. Ryłów: None. M. Bryśkiewicz: None. S. Zajączek: None.

P01.36D De novo mutations in idiopathic male infertility

A. Hodić1, A. Maver1, B. Zorn2, D. Plaseska-Karanfilska3, M. Ristanović4, I. Novaković4, B. Peterlin1

1Clinical Institute of Medical Genetics, University Medical Centre Ljubljana, Ljubljana, Slovenia, 2Andrology Unit, Reproductive Unit, Department of Obstetrics and Gynecology, University Medical Centre Ljubljana, Ljubljana, Slovenia, 3Research Centre for Genetic Engineering and Biotechnology, Skopje, Macedonia, The Former Yugoslav Republic of, 4Institute of Human Genetics, Medical Faculty, University of Belgrade, Belgrade, Serbia

Introduction: Infertility affects about 5% of adult human males and despite efforts in understanding genetic basis of male infertility, a large number of cases still remain to be explained. Previous methods of detecting disease-related genes included large family studies. In disorders, such as infertility, natural selection prevents transmission of mutations, and therefore many genes whose mutations cause infertility are not yet known.

Materials and Methods: In order to investigate potential roles of de novo mutations in male infertility we performed trio whole exome re-sequencing in 13 infertile males and their parents. All infertile males were diagnosed with idiopathic azoospermia. For all subjects library preparation was performed with Nextera Coding Exome Capture Kit (Illumina, San Diego, CA), with subsequent sequencing on Illumina HiSeq 2500 platform.

Results: We identified de novo mutations in three infertile males. Among genes with de novo mutations, two (NEURL4, BRD2) were previously implicated in reproduction in animal models. Previous studies have shown that NEURL4 contributes to germ cell formation in Drosophila, while the BRD2 is essential for chromatin remodeling during spermatogenesis in mice. The third gene with de novo mutation (SEMA5A) has not yet been implicated in reproduction, but it shows expression in testis.

Conclusions: We identified potentially new genes and mechanisms involved in the pathogenesis of male infertility.

A. Hodić: None. A. Maver: None. B. Zorn: None. D. Plaseska-Karanfilska: None. M. Ristanović: None. I. Novaković: None. B. Peterlin: None.

P01.37A Interphase Chromosome Profling (ICP) as a rapid, sensitive and cost-effective diagnostic tool for Prenatal and Postnatal settings

S. K. Bhattacharya, V. Lal

Dr. Lal PathLabs Ltd., New Delhi, India

Karyotyping has an important role in the genetic work-up of POC specimens, since approximately one half of miscarriages are due to chromosomal imbalances. The three primary methods used to obtain karyotype are 1) Classical cytogenetics 2) Targeted FISH and 3) aCGH. Each of these methods has its advantages and disadvantages. Additionally, the TAT is significantly long. The targeted FISH, only covers 5-7 chromosomes and therefore provides incomplete information. It cannot detect any structural abnormalities. Prenatal diagnosis by karyotype determination is done mostly to provide assurance, since majority of the pregnancies would have a normal karyotype. Therefore, fast and accurate information is highly critical for management of the pregnancy. However, the same limitations mentioned for POC also apply to prenatal diagnosis. To overcome these challenges, we recently validated and adapted a novel cytogenetic technology ‘Interphase Chromosome Profiling (ICP) (InteGen LLC, USA) to assess the molecular karyotype of 200 miscarriage material and 80 amniotic fluid samples using interphase nuclei. For POC and AF samples, using ICP probes, all numerical, most balanced and unbalanced structural aberrations, and all Robertsonian translocations can be detected. Using ICP, we obtained results from all (100%) samples and the TAT was significantly reduced to less than 1 day. However, with a proper workflow, results can be delivered in less than 2 hours.

S.K. Bhattacharya: A. Employment (full or part-time); Significant; Dr. Lal PathLabs Ltd. V. Lal: A. Employment (full or part-time); Significant; Dr. Lal PathLabs Ltd..

P01.38B The impact of chromosomal microarray analysis on resolving intra-utrine fetal death cases in Israeli population

O. Lobel, S. Zeligson, M. Bar Meir, R. Michaelson- cohen, S. Koka, P. Schwed, A. Samueloff, O. Weiss, M. Ben Uziyahu, E. Levy-Lahad, R. Segel

Shaare Zedek Medical Center, Jerusalem, Israel

Background: 30-50% of clinically recognized pregnancies end in intra-uterine fetal death (IUFD), 20% within the second and the third trimester, 25-60% without determined cause. Genetic work-up may lower the uncertainty, acknowledge the recurrence risks and enable wiser management of future pregnancies. Microarray technology (CMA) plays a major role in identifying genetic etiology of prenatal and postnatal pathologies, raising detection rate compared with karyotyping. An advantage of CMA in IUFD workup, is usage of directly extracted DNA without a culture, therefore without viability issues. The CMA chip includes copy number and singe nucleotide polymorphism probes, enabling identification of small copy number changes, mosaicism and homozygous regions throughout the genome. We charachterized CMA findings in IUFD cases in the Israeli population to find: - the characteristics and frequency of the chromosomal aberrations in IUFD compared to livebirths. - Do maternal and fetal determinants have impact on chromosomal aberration prevalence in IUFD cases? - Can finding of higher percentage of homozygous regions in IUFD explain the etiology?

Methods: We performed CMA on placental tissue of IUFD, gathered information regarding mothers and fetuses charachteristics, and compared with information of women who underwent prenatal diagnosis during the same period.

Results: CMA finding was an independent predictor with higher prevalence in IUFD than in live-born pregnancies. The chance of finding a pathogenic aberration in in IUFD with congenital anomalies was higher than other women. Homozygosity analysis had no advantage over CNV analysis.

Conclusions: CMA resolves more IUFD cases and therefore should be implicated in such cases.

O. Lobel: None. S. Zeligson: None. M. Bar Meir: None. R. Michaelson- cohen: None. S. Koka: None. P. Schwed: None. A. Samueloff: None. O. Weiss: None. M. Ben Uziyahu: None. E. Levy-Lahad: None. R. Segel: None.

P01.39C Extended genetic analyses in infertile men with non-obstructive azoospermia

F. Tüttelmann1, C. Krallmann2, Y. Stratis1, M. Hoffmann1, L. Hankamp1, S. Burkhardt1, C. Dreier1, C. Ruckert1, J. Gromoll2, P. F. Wieacker1, S. Kliesch2, A. Röpke1

1Institute of Human Genetics, University of Münster, Münster, Germany, 2Centre of Reproductive Medicine and Andrology (CeRA), University Hospital Münster, Münster, Germany

Male infertility is a clinically and genetically highly heterogeneous disease, mostly caused by spermatogenetic failure. The most severe form is non-obstructive azoospermia (NOA). In the majority of NOA cases, a genetic origin is suspected but current genetic testing, comprising cytogenetic analysis and AZF deletion screening, only discovers the cause in about 17%.

Recently, we expanded our analyses of NOA patients that attended the Centre of Reproductive Medicine and Andrology (CeRA). First, the routine chromosomal and AZF analyses were performed. In a second step, sequence analysis of three genes was carried out.

Chromosomal analyses were performed in 399 patients of whom 60 (15.0%) were identified with numerical (47,XXY; 47,XYY) or structural aberrations (46,XX; aberrant Y chromosomes; translocations; inversions). AZF deletions were found in 1.8% (6 of 335).

The coding sequence of TEX11, NR5A1 and DMRT1 was analysed in 123 patients. Potentially pathogenic variants were identified in 6 patients (4.9%). One mutation in TEX11 (c.450C>T) and one in NR5A1 (c.712G>A) were already published, whereas novel mutations were detected in NR5A1 (1x c.1079C>T) and in DMRT1 (2x c.308A>G, 1x c.436C>G).

In conclusion, the basic genetic analyses in men with NOA by conventional cytogenetic analysis and AZF screening revealed the expected number of aberrations. Through sequencing of three genes, which have been confirmed as responsible for spermatogenetic failure, an additional 5% of men carrying possibly pathogenic variants were identified.

This work was carried out within the frame of the DFG Clinical Research Unit ‘Male Germ Cells: from Genes to Function‘ (CRU 326).

F. Tüttelmann: None. C. Krallmann: None. Y. Stratis: None. M. Hoffmann: None. L. Hankamp: None. S. Burkhardt: None. C. Dreier: None. C. Ruckert: None. J. Gromoll: None. P.F. Wieacker: None. S. Kliesch: None. A. Röpke: None.

P01.40D Profile of copy number variants in Estonian men with impaired spermatogenesis

A. Punab1, L. Kasak1, M. Punab2, E. Laasik1, A. Valdner1, M. Laan1

1Institute of Biomedicine and Translational Medicine, University of Tartu, Tartu, Estonia, 2Andrology Centre, Tartu University Hospital, Tartu, Estonia

Introduction: Infertility affects 5-7% of men. The current pipeline at the andrology clinic is able to assign a definite cause for 40% of patients, including genetic errors in 8% of cases, whereas 60% of them remain idiopathic (Punab M et al. 2017 Hum Reprod). Considering the complexity of spermatogenesis, it is likely that a substantial proportion of this uncharacterised aetiology may be explained by unknown genetic factors. Currently, the role of genomic copy number variants (CNVs) in male infertility is not well defined. We aimed to characterise genome-wide profile of CNVs among Estonian men with idiopathic infertility.

Material and Methods: All patients (n = 211) and controls (n = 100) were recruited and clinically characterized at the Andrology Centre, Tartu University Hospital. Cases comprised of idiopathic non-obstructive azoospermia or oligozoospermia patients. Controls represented partners of pregnant women. CNV calling utilized genome-wide SNP data (HumanOmniExpress-24 BeadChip) and was performed using alternative CNV prediction algorithms in parallel. CNVs were validated with TaqMan (specific loci) or aCGH (microdeletions/duplications).

Results: Infertility patients and fertile men did not differ in their overall CNV load. However, an enrichment of asymptomatic carriers of known microdeletions and microduplications was observed among patients. Additionally, a novel recurrent CNV overlapping an uncharacterized testis-specific gene, was detected solely in seven infertility cases. Replication analysis for its association with male infertility is ongoing in a larger Estonian cohort.

Conclusions: Diagnostic yield for the patients with impaired spermatogenesis may be increased via introducing profiling of genome-wide genomic rearrangements into clinical routine.

Grants: IUT34-12, Happy Pregnancy project.

A. Punab: None. L. Kasak: None. M. Punab: None. E. Laasik: None. A. Valdner: None. M. Laan: None.

P01.41A Two novel CEP290 pathological variants prenatally identified by targeted next-generation sequencing using a custom Meckel-Gruber gene panel

M. Alameda Garcia1, E. Del Nuevo Martinez1, S. Garcia Gomez1, M. Labrador Ranz1, S. Izquierdo Alvarez2, A. Rodríguez Valle2, M. Miramar Gallart2, A. Sesto Yague1, J. Puente-Prieto1

1LabGenetics, San Sebastian de los Reyes, Spain, 2Hospital Universitario Miguel Servet, Zaragoza, Spain

Meckel-Gruber syndrome (MKS) is a lethal autosomal recessive disorder characterized by a classic ultrasound triad of occipital encephalocele, polycystic kidneys and postaxial polydactyly. anomalies of the central nervous system, dysplasias and malformations. The mortality is 100%.. In this case report, a prenatal sample from a fetus with MKS clinical features was screened for 21 genes using a targeted next-generation sequencing panel using a Ion PGM System (Thermo Fisher Scientific).

Two novel pathological variants, both resulting in stop codons, p.Ser1198* (c.3593C>A) and p.Ser1648* (c.4943C>G), were found in the CEP290 gene which codes for a centrosomal protein of 290 kDa involved in early and late steps of cilia formation. Sanger sequencing confirmed the carrier status of the parents.

Our results show that our Meckel-Gruber targeted next-generation sequencing 21-gene panel is an effective tool for the identification of pathological variants involved in this syndrome and confirms the possibility of obtaining a faster and accurate prenatal genetic diagnosis.

M. Alameda Garcia: Other; Significant; LabGenetics. E. Del Nuevo Martinez: Other; Significant; LabGenetics. S. Garcia Gomez: Other; Significant; LabGenetics. M. Labrador Ranz: Other; Significant; LabGenetics. S. Izquierdo Alvarez: None. A. Rodríguez Valle: None. M. Miramar Gallart: None. A. Sesto Yague: Other; Significant; LabGenetics. J. Puente-Prieto: Other; Significant; LabGenetics.

P01.43C Comparison of microRNA profiles in infants born with and without assisted reproduction techniques

B. Gozum, A. Toylu, B. Nur, M. Sakinci, M. Özekinci, O. A. Clark, E. Mihci

Akdeniz University, Antalya, Turkey

The incidence of congenital anomalies in ART babies is higher than that of babies born with spontaneous pregnancy. The cause of this situation is unknown. Three mechanisms are known to cause congenital anomalies in ART pregnancies: point mutation, chromosomal disorders, epigenetic abnormalities. An important factor in epigenetics is microRNA. Studies have shown microRNAs are associated with fertility and development.The aim of this study to demonstrate whether infertility-related miRNAs are different in children born with spontaneous pregnancy compared to those born with ART. The other aim of the study to show whether miRNAs are associated with anomalies and dysmorphic findings in patients. A total of 38 term newborns included the study. In Akdeniz University Hospital, a baby born with 21 ART and 17 spontaneous pregnancies within one year was included in the study. Plasma samples were taken from newborns. All newborn’s physical examinations were performed and enrolled. The total microRNA isolated from plasma samples was reverse transcribed into the cDNA. Quantitative real time -PCR was performed with specific primers for miR-16 reference gene and miR-17, 21, 23, 92, 141, 145, 191, 483 target genes. REST software was used for the normalization of relative expression values. The plasma levels of the target miRNA molecules were showed comparable difference between control and ART groups. All three target miRNA molecule were displayed significantly higher levels of expression in ART babies than controls. In our preliminary results signed that infertility-causing miRNAs in parents might be cause of congenital anomalies in newborns. Project code:TTU-2016-1709

B. Gozum: None. A. Toylu: None. B. Nur: None. M. Sakinci: None. M. Özekinci: None. O.A. Clark: None. E. Mihci: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Significant; Akdeniz University Scientific Research Projects Foundation.

P01.44D Feasibility and concerns of preimplantation genetic diagnosis for mitochondrial DNA disorders

J. Steffann1, S. Monnot1, N. Gigarel1, A. Rotig2, M. Rio1, N. Frydman3, L. Hesters3, A. Munnich1, J. Bonnefont1

1Paris Descartes University and Necker Hospital, Paris, France, 2INSERM UMR1163 Imagine Institute, Paris, France, 3Paris-Sud and Paris-Saclay University, Antoine-Béclère Hospital, Clamart, France

Preimplantation genetic diagnosis (PGD) is an alternative procedure to prenatal diagnosis for couples at-risk to have children affected with a severe genetic disease, such as a mitochondrial DNA (mtDNA) disorder. PGD relies on the genetic analysis of one or a few cells sampled from in-vitro fertilized embryos, between day 3 and day 5 of development. In the case of mtDNA disorders, quantification of the mutant load on these cells is performed in order to assess the risk for the embryo to develop a severe mitochondrial disease, either in utero or in childhood. Our 15-year experience supports the reliability of such procedure. Overall 15 heteroplasmic patients were included in our PGD program. A total of 26 cycles were started, 25 oocytes retrievals and 16 embryo transfers were performed, resulting in 3 pregnancies and birth of 3 children. The mutant load assessed on a single blastomere sampled from 94 embryos was very close to the mutant load of the remaining cell/embryo, for all mutations tested: m.8344A>G, m.3243A>G, m.8993T>G, m.8993T>C, m.9185T>C, m.10197G>A. Most of the transferred embryos (17/25) were heteroplasmic, and 2/3 neonates carried the maternal mtDNA mutation, questioning the long-term prognosis of these patients. PGD remains a cumbersome procedure with a low success rate, which cannot be applied to homoplasmic or critically homoplasmic patients. There is therefore a strong need to develop alternative procedures such as nuclear transfer.

J. Steffann: None. S. Monnot: None. N. Gigarel: None. A. Rotig: None. M. Rio: None. N. Frydman: None. L. Hesters: None. A. Munnich: None. J. Bonnefont: None.

P01.45A Detecting confined placental and fetal mosaicism using cell-free DNA sequencing on maternal plasma

I. Lund1,2,3, E. Vestergaard1,2,3, N. Becher1,2,3, D. Lildballe1,2, P. Schelde4, L. Hatt4, R. Singh4, O. Petersen5,2, N. Uldbjerg5, I. Vogel1,2,3

1Aarhus University Hospital, Department of Clinical Genetics, Aarhus N., Denmark, 2Center for Fetal Diagnostics, Aarhus University, Aarhus University Hospital, Aarhus N., Denmark, 3Department of Biomedicine, Aarhus University, Aarhus N., Denmark, 4Arcedi Biotech Aps, Vejle, Denmark, 5Aarhus University Hospital, Department of Gynecology and Obstetrics, Aarhus N., Denmark

Introduction: Mosaicism is characterized by a normal and an abnormal cell-line. Test results on cell-free DNA (cfDNA) in maternal plasma can be compromised as the fraction of abnormal cells may be too low for detection. We wanted to explore the detection rate of both confined placental and fetal mosaicism using cfDNA sequencing on maternal plasma.

Methods and Material: We retrieved data on invasive samples from mosaic pregnancies obtained from 2014 to 2017. On maternal plasma, we retrospectively performed cfDNA testing by genome-wide massive parallel sequencing and VeriSeq-NIPT analysis software.

Results: CfDNA detected placental mosaicism in 59% (n = 16). The false negative rate of placental mosaicism using cfDNA testing was 41% (n = 11). The mean level of mosaicism in the invasive samples was 72.0% in the detected cases and 20% in the false negative cases (p < 0.05). Fetal mosaicism, confirmed by amniocentesis, was detected by cfDNA sequencing in 63% (5/8 cases). A mosaic trisomy 21 case which was confirmed both by CVS (84% T21 cells) and AC was missed by cfDNA.

Conclusion: CfDNA sequencing is capable of detecting placental mosaicism in 59% of the cases. It seems that the level of mosaicism in the invasive samples predicts whether or not cfDNA testing is able to detect the abnormal cell-line; however a high-level mosaicism of trisomy 21 was missed. In the future, we need to learn more about placental mosaicism in general and in particular the comparability between the detection rates of the new non-invasive methods.

I. Lund: None. E. Vestergaard: None. N. Becher: None. D. Lildballe: None. P. Schelde: A. Employment (full or part-time); Significant; Arcedi Biotech Aps. L. Hatt: A. Employment (full or part-time); Significant; Arcedi Biotech Aps. R. Singh: A. Employment (full or part-time); Significant; Arcedi Biotech Aps. O. Petersen: None. N. Uldbjerg: None. I. Vogel: None.

P01.46B A homozygous donor splice-site mutation in the meiotic gene MSH4 causes primary ovarian insufficiency

C. Carlosama1, M. Elzaiat2, L. C. Patiño1, H. E. Mateus1, R. A. Veitia2, P. Laissue1

1Center For Research in Genetics and Genomics-CIGGUR, GENIUROS Research Group, School of Medicine and Health Sciences, Universidad del Rosario, Bogota, Colombia, 2Institut Jacques Monod, Université Paris Diderot, Paris, France

Premature ovarian insufficiency (POI) is a pathology affecting women under 40 years of age characterized by an early cessation of menses and high FSH levels. Despite recent progresses in molecular diagnosis, the etiology of POI remains idiopathic in most cases. Whole-exome sequencing of members of a Colombian family affected by POI allowed us to identify a novel homozygous donor splice-site mutation in the meiotic gene MSH4 (MutS Homolog 4). The variant followed a strict mendelian segregation within the family and was absent in a cohort of 135 women over 50 years of age without history of infertility, from the same geographical region as the affected family. Exon trapping experiments showed that the splice-site mutation induced skipping of exon 17. At the protein level, the mutation p.Ile743_Lys785del is predicted to lead to the ablation of the highly conserved Walker B motif of the ATP-binding domain, thus inactivating MSH4. Our study describes the first MSH4 mutation associated with POI and increases the number of meiotic/DNA mismatch repair genes formally implicated as being responsible for this condition.

This work was supported by the Universidad del Rosario grant: [CS/ABN062/GENIUROS 017] and by the Fondation pour la Recherche Médicale grant: [DEQ20150331757]. C.C. and M.E. on the one hand, and R.A.V. and P.L. on the other hand contributed equally to this work.

C. Carlosama: None. M. Elzaiat: None. L.C. Patiño: None. H.E. Mateus: None. R.A. Veitia: None. P. Laissue: None.

P01.47C Clinical application of paired-end MPSS for cfDNA screening of common aneuploidies

V. Cirigliano, E. Ordoñez, L. Rueda, S. Nicolas, M. Grau, I. Castilla, C. Puertollano, M. Lechuga, M. Cañadas

Synlab, Esplugues de Llobregat, Spain

Paired-end MPSS allows digital counting of plasma cfDNA while also measuring fragments length. cfDNA size differences can be used to determine fetal fraction (FF) and to improve sensitivity by additionally applying counting statistics on short (fetal) fragments.

NeoBona is the first test using such approach, we evaluated its performance by screening a large cohort of consecutive average risk gestations.

Prospective study of 19151 pregnancies (575 twins) screened for common trisomies, including XY aneuploidies in 57% of cases.

NeoBona test was used to determine the likelihood of aneuploidy (Tscore) based on FF, counting statistics and cfDNA size distribution where cut-offs were applied to classify normal and aneuploid cases.

Test results were provided in 99.2% gestations, 288 T21, 63 T18 and 27 T13, in 23 cases detected with FF between 0.8 and 3%. Invasive procedures were performed in 99% risk pregnancies, 5 false positives were observed for T21, 2 T18 and 3 for T13 (FPR 0.03%, 0.01% and 0.02%); 1 T21 was missed (DR 99.7%). XY aneuploidies were reported in 39 cases, follow-up available for 14 with 4 FP results (FPR 0.13%). Vanishing twins of discrepant sex were suspected in 5 cases and 4 maternal X aneuploidies were identified.

Paired-end MPSS and the bioinformatics approach of NeoBona allowed detecting aneuploidies even at fetal fractions below 1% while reducing FPR. Removing the need of a lower FF limit allowed cfDNA analysis to be successful on a high proportion of clinical cases extending the benefits of cfDNA screening to a larger population of pregnancies.

V. Cirigliano: None. E. Ordoñez: None. L. Rueda: None. S. Nicolas: None. M. Grau: None. I. Castilla: None. C. Puertollano: None. M. Lechuga: None. M. Cañadas: None.

P01.48D Map of maternal copy number variation highlighted by NIPT analysis

A. Marichal, B. Grisart, J. Billard, S. Brohée, D. Feret, C. Hougardy, S. Mary, S. Rombout, P. Hilbert, C. Meunier, N. Simonis, K. Dahan

Institut de Pathologie et de Génétique, Gosselies, Belgium

Since July 2017 reimbursement of NIPT is entirely covered during pregnancy for all pregnant women in Belgium. In our institute we processed more than 12000 samples over a 6 month period. Our in house NIPT workflow allows the identification of trisomies involving chromosome 13, 18 and 21 but also trisomies affecting other autosomes as well as sex chromosomes aneuploidies. Intrachromosomal rearrangements are also investigated using a 1 Mb sliding window approach. This allowed us to identify fetal rearrangements in a number of cases but also maternal rearrangements with an unpreceded power. Indeed given the prevalence of maternal cfDNA in purified cfDNA prepared from the blood of pregnant mothers, some rather small rearrangements can be pin-pointed quite reliably. Interestingly, some unusually large maternal rearrangements (>1 Mb) have also been observed. Maternal CNVs can usually be distinguished from fetal ones by the level of significance of the intrachromosomal Z-score. Some of these CNVs were futher confirmed by CGH arrays using maternal constitutional DNA. The majority of these CNVs are duplications (68 duplications and 43 deletions) some of which overlap syndromic genes. Some correspond to large known CNVs. However, some large CNVs ranging from 1 to 5 Mb seem to be rare familial deletions or duplications probably not associated with any pathogenic phenotype. Indirect screening of the whole maternal population using NIPT offers a unique opportunity to identify large probably benign CNVs. A map of these rare familial CNVs characterized by CGH will presented.

A. Marichal: None. B. Grisart: None. J. Billard: None. S. Brohée: None. D. Feret: None. C. Hougardy: None. S. Mary: None. S. Rombout: None. P. Hilbert: None. C. Meunier: None. N. Simonis: None. K. Dahan: None.

P01.49A External assessment of the quality of cell free fetal DNA non-invasive prenatal testing for aneuploidies

Z. C. Deans1, F. Khawaja1, R. Hastings2, K. Rack2, S. Patton3, W. Gutowska-Ding3, L. Jenkins4, S. Allen5, L. S. Chitty6, E. Sistermans7

1UK NEQAS for Molecular Genetics, Edinburgh, United Kingdom, 2CEQAS, Oxford, United Kingdom, 3EMQN, Manchester, United Kingdom, 4Great Ormond Street NHS Foundation Trust, London, United Kingdom, 5Birmingham Women’s and Childrens NHS Foundation Trust, Birmingham, United Kingdom, 6UCL Great Ormond Street Institute of Child Health, London, United Kingdom, 7VUmc medical Center Amsterdam, Amsterdam, Netherlands

Introduction: To deliver a high standard of laboratory testing, external quality assessment (EQA) is required to provide important information for clinicians, laboratories and patients, demonstrating that accurate testing is being performed and reported. Providing an EQA for cell free fetal DNA (cffDNA) testing is challenging as sample acquisition (availability and scalability) is a limiting factor. The delivery and results of a large international pilot EQA for laboratory NIPT for aneuploidy using maternal plasma samples is described.

Materials and Methods:Eighty-six maternal plasma samples from pregnancies with known outcomes (low/high-risk for common aneuploidy) were obtained from the RAPID sample bank. Three EQA providers (CEQAS, EMQN, and UKNEQAS for Molecular Genetics) delivered the pilot assessing NIPT and reporting The submitted reports were assessed and feedback provided for genotyping, interpretation and clerical accuracy.

Results:Ninety-five laboratories from 30 countries participated. The use of maternal plasma allowed any testing methods to be applied. Two critical errors were reported; one false positive and an incorrect high-risk trisomy 18 result. Reports lacked details of methods, limitations, and many formats made it difficult to identify key clinical recommendations.

Conclusions: Growing international demand for participation demonstrates the clinical need for an independent evaluation of NIPT practice. This pilot EQA has demonstrated that the use of real maternal samples distributed at ambient temperature has enabled global participation with very low sample failure rate (2%). The genotyping accuracy was good but review of the large number of reports submitted highlighted the need for further standardisation and guidance on NIPT reporting.

Z.C. Deans: None. F. Khawaja: None. R. Hastings: None. K. Rack: None. S. Patton: None. W. Gutowska-Ding: None. L. Jenkins: None. S. Allen: None. L.S. Chitty: None. E. Sistermans: None.

P01.50B Validation of novel bioinformatic algorithm SCAR for fetal sex determination in twin pregnancies

M. Hýblová1, J. Budiš2, F. Ďuri3, M. Kucharík2, G. Minarik1, T. Szemes2

1Medirex, Bratislava, Slovakia, 2Geneton, Bratislava, Slovakia, 3CVTI, Bratislava, Slovakia

Introduction: Currently used approaches for determination of fetal sex in noninvasive prenatal testing (NIPT) have shown limitations in correct prediction of fetal sex in cases of twin pregnancies. According to recent information only SNP based tests of NIPT category are able to determine fetal sex for each of the twin.

Aim: To test the feasibility and to validate the new bioinformatic algorithm called SCAR to predict sex of both fetuses in twin pregnancies with utilisation of whole genome coverage genomic scan of circulating DNA from pregnant plasma.

Materials and Methods: Low coverage whole genome sequencing analysis was performed on MiSeq and NextSeq platforms on circulating DNA of 76 pregnant women with twins according to previously published protocol. For each of the fetuses sex determination algorithm SCAR predicted the most probable combination of twin sexes: girl-girl; girl-boy or boy-boy according to fetal fraction counted from fragment lengths and reads mapped on Y chromosome. All predictions were verified after delivery.

Results: Among 76 twin pregnancies, 70 were identified correctly and 6 cases were found as uninformative. All of 6 samples fell to the group with fetal fraction lower than 10%.

Conclusion: Novel algorithm SCAR predicted correctly 92% cases however fetal fraction under 10% critically affected reliable sex determination in boy-girl and boy-boy pregnancies.

M. Hýblová: None. J. Budiš: None. F. Ďuri: None. M. Kucharík: None. G. Minarik: None. T. Szemes: None.

P01.52D Chromosomal microarray coupled to genome-wide non-invasive prenatal testing (NIPT) to minimize the number of unnecessary invasive procedures

B. Oneda, P. Sirleto, R. Baldinger, M. Taralczak, P. Joset, M. Zweier, D. Niedrist, S. Azzarello-Burri, K. Steindl, A. Rauch

Institute of Medical Genetics, University of Zurich, Schwerzenbach, Switzerland, Zurich, Switzerland

Prospective clinical results on genome-wide NIPT are still few and there is little follow up and little data available on test accuracy. We received 1921 samples including 108 twin pregnancies for NIPT. 93.6% of the cases were analyzed genome-wide. Additionally, in order to assess test limitations, we performed NIPT retrospectively in 90 cases with a variety of segmental aberrations. In the prospective cohort, 176 samples showed chromosomal abnormalities, 144 of small size. In the latter, we performed chromosomal microarray analysis on maternal DNA obtained from the NIPT tube and suggested invasive testing only for aberrations not of maternal origin. We found maternal CNVs in 7.8% of the total cases which were surprisingly large in several instances. We detected and confirmed pathologic chromosomal abnormalities in 32 samples, six of which would not have been detected if NIPT had been restricted to common trisomies. The positive predictive value for the common trisomies was 100% and a retrospective questionnaire for quality control showed no evidence for a false negative result. The positive predictive value for non-maternal segmental anomalies was 50%. Thus the number of ‘’unnecessary’’ invasive procedures provoked by genome wide NIPT was as low as 0.3%. We correctly detected all chromosomal aberrations bigger than 6.3 Mb in size in the retrospective cohort. Altogether, we demonstrate that genome wide NIPT does not lead to a significant loss of specificity, if in cases with segmental abnormalities, maternal chromosomal microarray testing is performed prior to invasive testing.

B. Oneda: None. P. Sirleto: None. R. Baldinger: None. M. Taralczak: None. P. Joset: None. M. Zweier: None. D. Niedrist: None. S. Azzarello-Burri: None. K. Steindl: None. A. Rauch: None.

P01.53A Non-invasive prenatal testing of microdeletion syndromes

K. Tsangaras, P. Mina, M. Ioannides, C. Loizides, A. Achilleos, E. Kypri, G. Koumbaris, P. C. Patsalis

NIPD Genetics, Nicosia, Cyprus

Introduction: The discovery of cffDNA in maternal plasma has greatly facilitated the development of NIPT of fetal aneuploidies. However, sub-chromosomal copy number change detection still remains a challenge. Towards this goal, we employed a proprietary hybrid capture-based technology and novel bioinformatics pipeline for the detection of microdeletion syndromes. By leveraging the inherent high enrichment uniformity and high read depth of this in-solution hybridization NIPT method we achieved accurate non-invasive detection of fetal microdeletion syndromes. The assay combines multiple depth of coverage-based and fragment size-based ploidy detection engines to detect 1p36, DiGeorge, Wolf-Hirschhorn, and Smith-Magenis microdeletion syndromes with high sensitivity and specificity.

Materials and Methods: cfDNA was extracted from 752 unaffected first trimester pregnancy plasma samples and 29 affected prenatal and synthetic samples. Enrichment probes were designed to span the syndromes’ critical regions avoiding low copy repeats and repetitive elements. All samples were enriched using hybrid capture technology as previously described. Enriched sequencing libraries were analyzed using a proprietary statistical analysis pipeline developed to test for deletions in each of the syndromes.

Results: The assay was able to correctly classify all abnormal and normal samples resulting in 100% specificity and specificity.

Conclusions: Using a proprietary target capture enrichment technology and novel multi-engine copy number detection pipeline we accurately detected all normal and abnormal samples. This novel microdeletion NIPT method overcomes the limitations of other methodologies and increases the number of diseases that can be reliably detected by NIPT, thus offering more choices to couples towards an informed management of their pregnancy.

K. Tsangaras: A. Employment (full or part-time); Significant; NIPD Genetics. P. Mina: A. Employment (full or part-time); Significant; NIPD Genetics. M. Ioannides: A. Employment (full or part-time); Significant; NIPD Genetics. C. Loizides: A. Employment (full or part-time); Significant; NIPD Genetics. A. Achilleos: A. Employment (full or part-time); Significant; NIPD Genetics. E. Kypri: A. Employment (full or part-time); Significant; NIPD Genetics. G. Koumbaris: A. Employment (full or part-time); Significant; NIPD Genetics. P.C. Patsalis: A. Employment (full or part-time); Significant; NIPD Genetics.

P01.54B Development of a novel noninvasive prenatal test (NIPT) of fetal aneuploidies, microdeletions and 50 single gene diseases

M. Nicolaou, C. Loizides, M. Ioannides, K. Tsangaras, P. Mina, A. Achilleos, E. Kypri, G. Koumbaris, P. C. Patsalis

NIPD Genetics, Nicosia, Cyprus

Introduction: We hereby present a novel NIPT of major aneuploidies, microdeletions and 50 monogenic diseases with moderate and severe phenotypes, including Hematological, Kidney, Opthalmological, Neurological, Inherited Metabolic Diseases, such as Thalassaemia, Cystic Fibrosis, Phenylketonuria and Tay-Sachs.

Methods: cfDNA was obtained from 300 pregnancies referred for NIPT at 10th-15th week of gestation for identification of 651 causative mutations in 50 disease associated genes. A study including another 1000 pregnancies using cfDNA and paternal DNA is ongoing for NIPT of major aneuploidies, microdeletions and 50 monogenic diseases. An enriched sequencing library was prepared using custom TArget Capture Sequences (TACS) as previously described. TACS were designed based on genomic locations of known causative mutations for monogenetic diseases under investigation. Enriched products were sequenced using NGS and the data was processed using a custom bioinformatics pipeline.

Results: For the initial 300 samples, a high number of causative mutations was identified and a selection of those was confirmed using Sanger sequencing. For the ongoing 1000 samples, causative mutations were identified and the fetal risk for aneuploidies, microdeletions and monogenic disorders was determined.

Conclusions: This is the first time that NIPT is made available for a high number of single gene diseases together with aneuploidies and microdeletions, opening a new chapter in prenatal screening. The cumulative risk for the fetus is estimated to be as high as 1/125. This novel NIPT is expandable to hundreds of single gene diseases and can be taken potentially by all pregnant women as early as the 10th week of gestation

M. Nicolaou: A. Employment (full or part-time); Significant; NIPD Genetics. C. Loizides: A. Employment (full or part-time); Significant; NIPD Genetics. M. Ioannides: A. Employment (full or part-time); Significant; NIPD Genetics. K. Tsangaras: A. Employment (full or part-time); Significant; NIPD Genetics. P. Mina: A. Employment (full or part-time); Significant; NIPD Genetics. A. Achilleos: A. Employment (full or part-time); Significant; NIPD Genetics. E. Kypri: A. Employment (full or part-time); Significant; NIPD Genetics. G. Koumbaris: A. Employment (full or part-time); Significant; NIPD Genetics. P.C. Patsalis: A. Employment (full or part-time); Significant; NIPD Genetics.

P01.55C Non-invasive prenatal testing (NIPT): how to handle secondary findings of maternal chromosomal abnormalities

M. Baetens, T. Sante, S. Vergult, M. De Smet, S. Janssens, O. Vanakker, B. Callewaert, B. Poppe, A. Dheedene, B. Menten

Center for Medical Genetics Ghent, Ghent, Belgium

NIPT has become a widely implemented screening test for the detection of fetal trisomy 13, 18 and 21. Over 8000 NIPT analyses have been performed thus far at the Center for Medical Genetics Ghent, using shallow whole genome sequencing (sWGS) protocol. Around 0,6% samples showed an increased risk for trisomy 13, 18 or 21. Also in 0,6% analyses, we reported another chromosomal abnormality, including other fetal aneuploidies but also several clinically relevant maternal CNVs. The detection and disclosure of these (secondary) maternal aberrations poses ethical dilemmas. Aberrations such as the unfortunate detection of a (predisposition to) malignancy or other incidental findings are not always straightforward to disclose.

We recently identified a possible malignancy in a 25-year old women. The chromosome profile resembles aberrations previously seen in patients with colon carcinoma (loss of 8p, gain of 8q and 20). During subsequent colonoscopy, a possible precursor adenomatous polyps was removed. Follow-up is ongoing. Also, some well-known (maternal) CNVs have been identified, such as Hereditary Neuropathy with liability to Pressure Palsies (HNPP) deletions (OMIM162500). Furthermore, several deletions and duplications with unknown clinical significance have been detected that affected the chromosomal Z-scores of the NIPT analysis. In five cases thus far, the presence of an extra X-chromosome in the mother was present, these were communicated.

With the plummeting costs of NGS on the one hand and the advent of better bio-informatic tools for analysis on the other hand, guidelines are clearly needed to guide us in this new genetic, healthcare landscape of secondary findings.

M. Baetens: None. T. Sante: None. S. Vergult: None. M. De Smet: None. S. Janssens: None. O. Vanakker: None. B. Callewaert: None. B. Poppe: None. A. Dheedene: None. B. Menten: None.

P01.56D Clinical experience with noninvasive prenatal testing (NIPT) for rare autosomal trisomies

F. M. Liao1, D. Huynh1, S. Kim2, V. Corey2, K. Curnow2, W. Seltzer1, S. Beruti2, S. Bhatt2

1Illumina, Inc, Redwood City, CA, United States, 2Illumina, Inc, San Diego, CA, United States

Objective: Using a whole-genome sequencing NIPT approach, our laboratory began offering screening for rare autosomal trisomies (RATs) in 2017. This study presents our initial clinical experience.

Method: Maternal blood samples from over 10,000 singleton pregnancies were analyzed in the CLIA-certified Illumina Laboratory (Redwood City, CA) by the Verifi™ Plus Prenatal Test. Sequencing data was computationally processed with chromosome-specific quantitative scores determined using sequence coverage and fetal fraction. Classification thresholds for each chromosome were derived to maximize specificity while accounting for differences in prevalence for each RAT.

Results: 43 cases (0.4%) were reported as RAT screen positive. The most common RAT identified was trisomy 22, followed by trisomies 7 and 9. The average maternal age (35.0 years) and gestational age (12.4 weeks) of the screen positive cohort were similar to the whole study cohort. However, some high-risk indications, abnormal ultrasound (1.8x) and history suggestive of increased risk for aneuploidy (5.5x), were more frequently listed in the screen positive cohort than in the whole study cohort. Clinical outcome was available in 8 cases (18.6%): 2 confirmed positives (1 full trisomy 9; 1 segmental 9p duplication), 2 false positives, 3 miscarriages, and 1 elective termination without confirmatory testing; >99% of pregnancies are ongoing.

Conclusions: Our 0.4% screen-positive frequency is consistent with previous studies, though some differences were noted in the relative RAT prevalence.1,2 Results obtained through NIPT early in pregnancy can be valuable for clinical management. Ongoing outcome collection will provide more insight into the biological aspects of RATs.

F.M. Liao: A. Employment (full or part-time); Significant; Illumina, Inc. D. Huynh: None. S. Kim: A. Employment (full or part-time); Significant; Illumina, Inc. V. Corey: A. Employment (full or part-time); Significant; Illumina, Inc. K. Curnow: A. Employment (full or part-time); Significant; Illumina, Inc. W. Seltzer: F. Consultant/Advisory Board; Significant; Illumina, Inc. S. Beruti: A. Employment (full or part-time); Significant; Illumina, Inc. S. Bhatt: F. Consultant/Advisory Board; Significant; Illumina, Inc.

P01.57A Outcome of high risk for digynic triploidy results on SNP-based non-invasive prenatal testing

T. McKanna1, J. Chaperon1, A. Ryan1, S. Leonard1, K. Martin1, H. Hedriana2

1Natera, Inc., San Carlos, CA, United States, 2University of California, Davis, CA, United States

Introduction: Single nucleotide polymorphism (SNP)-based non-invasive prenatal testing (NIPT) is uniquely able to identify the extra haplotype and parental origin in triploid pregnancies. The objective of this study was to establish a positive predictive value (PPV) for pregnancies suspected to be at high risk for digynic (maternal) triploidy (DT) via SNP-based NIPT. For apparent false positive cases, possible maternally-derived causes were investigated.

Materials and Methods: Retrospective outcome data were collected for SNP-based NIPTs performed between January 1, 2015 and December 31, 2017 and coded as high risk for DT. IRB-approved outcomes comprised: number of fetuses, ultrasound findings, results of cytogenetic testing including parental origin of triploidy, and maternal medical findings.

Results: A total of 39 cases with suspected DT were identified and outcome data were obtained for 30 (77%) cases (see Table). The PPV for confirmed or suspected triploidy was 23.3% (7/30). Maternal neoplasm was found in 26.7% (8/30) cases.

Conclusions: This post hoc analysis of SNP-based NIPT data revealed a PPV of 23.3% for pregnancies determined to be at high risk for DT. An additional and unexpected finding was the similar number of maternal neoplasm cases. While a small cohort, these results suggest that maternal neoplasm should be included in the differential diagnosis of high risk DT results on SNP-based NIPT.

Table. Outcomes from suspected DT pregnancies determined via SNP-based NIPT

Outcomes, n (%)

Cases (N=30)

Normal fetal and maternal outcome

12 (40.0)

Maternal neoplasma

8 (26.7)

Triploidy suspected by ultrasound

4 (13.3)

Confirmed triploidy

3 (10.0)

Complete molar pregnancy

1 (3.3)

Early fetal demise

1 (3.3)

Ongoing early gestation

1 (3.3)

  1. a 4 lymphoma, 1 colon cancer, 1 Stage IV cholangiocarcinoma diagnosed a year after delivery, 1 ovarian teratoma, and 1 unspecified.

T. McKanna: A. Employment (full or part-time); Significant; Natera, Inc. J. Chaperon: A. Employment (full or part-time); Significant; Natera, Inc. A. Ryan: A. Employment (full or part-time); Significant; Natera, Inc. S. Leonard: None. K. Martin: None. H. Hedriana: F. Consultant/Advisory Board; Modest; Natera, Inc.

P01.58B Widespread use of Non Invasive Prenatal Testing(NIPT) : experience of a Belgian genetic Center

B. Grisart, J. Billard, S. Brohée, A. Marichal, D. Feret, C. Hougardy, S. Mary, S. Rombout, P. Hilbert, C. Meunier, N. Simonis, K. Dahan

Centre de Génétique Humaine, Institut de Pathologie et de Génétique, Charleroi (Gosselies), Belgium

Non Invasive Prenatal Testing was developed in our institute in 2014 using an in house whole genome approach. For more than 3 years, this test was open for all pregnant women at their own expense. During this period we tested 7041 maternal blood with a positive screening rate for chromosome 13, 18 and 21 of 0,10%, 0,20% and 1,3% respectively. Since July 2017 NIPT has been reimbursed in Belgium for all pregnant women. This resulted in a sharp increase in activity as in 6 months more than 12000 samples were processed. The rates of trisomy 13, 18 and 21 over this period were 0.05%, 0.06% and 0.4% respectively. Beside these common trisomies other trisomies involving chromosomes 4, 6, 7, 8, 14, 15, 16, 20 and 22 were identified, most of them (>94%) were not confirmed on invasive samples. In case of trisomy for chromosome 6, 7, 11, 14, 15 and 20, uniparental disomy was evaluated in the normal fetus. Intrachromosome analysis using a 1 Mb sliding window approach, allowed to identify some micro rearrangements affecting the fetus. These ones ranged from a few megabases to several tenth of megabases and were confirmed by CGH on amniocytes. Sex chromosome aneuploidies could be technically identified but a Belgian prenatal consortium (www.BeSHG.be) decided not to report these sex chromosomes aneuploidies. Indeed generalization of NIPT would screen almost the whole population for these sex aneuploidies as well as for susceptibility loci. This raised ethical questions which have to be addressed.

B. Grisart: None. J. Billard: None. S. Brohée: None. A. Marichal: None. D. Feret: None. C. Hougardy: None. S. Mary: None. S. Rombout: None. P. Hilbert: None. C. Meunier: None. N. Simonis: None. K. Dahan: None.

P01.59C Making NIPT available to all pregnant women

Å. Janfalk Carlsson

Vanadis Diagnostics – a PerkinElmer company, Sollentuna, Sweden

Non-Invasive Prenatal Testing (NIPT) is increasing in interest for detection of aneuploidies due to these tests giving a more reliable result than obtained from traditional first trimester screening. NIPT should not only be available to high-risk pregnancies but for all women. With Vanadis NIPT, the aim was to fulfill this criterium, making NIPT available for all women by creating a fully automated method with simple preparation need and minimal hands-on time and thereby reducing both complexity and cost. Vanadis NIPT is a sequencing and PCR free, probe-based technology used to label targets on chromosome 13, 18, 21 and Y, thereby allowing for trisomy screening (13, 18 and 21) as well as sex determination. The assay consists of four enzymatic steps resulting in Rolling Circle Amplification Products (RCPs) for each of these four chromosomes. The RCPs are labeled with four different dyes, one for each chromosome, and deposited onto a nano-pore filter from where the labeled objects are counted by imaging. We will present performance characteristics for the Vanadis NIPT assay, including data for real clinical samples, to show the analytical precision and clinical feasibility to correctly identify trisomy 13, 18 and 21 as well as the sex of the fetus.

Å. Janfalk Carlsson: None.

P01.60D Non-invasive prenatal diagnosis (NIPD) of single gene disorders by relative haplotype dosage (RHDO): review of 18 months of clinical service

E. C. Young1, B. Bowns1, A. Gerrish1, M. Parks2, S. Court1, S. Clokie1, C. Mashayamombe-Wolfgarten1, J. Hewitt1, D. Williams1, T. Cole1, M. Griffiths1, S. Allen1

1West Midlands Regional Genetics Service, Birmingham, United Kingdom, 2Nonacus Ltd., Birmingham, United Kingdom

Introduction: We have developed and implemented a relative haplotype dosage (RHDO)- based method for NIPD of multiple single gene disorders (SGD), including spinal muscular atrophy (SMA), Duchenne and Becker muscular dystrophies (DMD/BMD), cystic fibrosis (CF) and congenital adrenal hyperplasia (CAH). Diagnostic services for SMA and DMD/BMD were launched in September 2016, followed by the launch of a CF service in December 2017.

Materials and Methods: The test involves targeted enrichment of thousands of SNPs across multiple genomic regions and massively parallel sequencing (Illumina MiSeq) of cfDNA followed by RHDO analysis. Maternal, paternal and proband genomic DNA samples are tested alongside cfDNA for haplotype phasing and to measure fetal fraction. Our method can test 2-3 pregnancies on a single MiSeq run, thus centralising testing and decreasing costs. The development of an automated analysis pipeline has increased capacity further. There is no requirement to confirm positive results.

Results: To date, we have performed NIPD for 48 referrals (UK and international) and reported 16 normal, 18 unaffected carrier and 10 affected pregnancies. For 4 cases, a complete result could not be issued due to persistent low fetal fraction, a recombination event or lack of informative SNPs. Of the 48 diagnostic tests, we have so far received postnatal confirmation of 15 results, with no discrepancies.

Conclusions: NIPD by RHDO is a robust assay, which is feasible to provide in a clinical setting for both X-linked and autosomal recessive disorders. The assay could be extended to increase the availability of NIPD for many monogenic disorders.

E.C. Young: None. B. Bowns: None. A. Gerrish: None. M. Parks: None. S. Court: None. S. Clokie: None. C. Mashayamombe-Wolfgarten: None. J. Hewitt: None. D. Williams: None. T. Cole: None. M. Griffiths: None. S. Allen: None.

P01.62B Predicting fetoplacental chromosomal mosaicism during non-invasive prenatal testing

N. Brison1, M. Neofytou1, L. Dehaspe1, B. Bayindir1, K. Van Den Bogaert1, L. Dardour2, H. Peeters1, H. Van Esch1, G. Van Buggenhout1, A. Vogels1, J. Breckpot1, T. de Ravel1, E. Legius1, K. Devriendt1, J. R. Vermeesch1

1Centre for Human Genetics - UZ Leuven, Leuven, Belgium, 2Department of Human Genetics, Faculty of Medicine “Ibn Al Jazzar”, Sousse, Tunisia

Objective:Non-invasive prenatal detection of trisomies 21, 18 and 13 can be achieved with high accuracy through sequencing of cell-free DNA (cfDNA) found in maternal blood. Using a genome-wide approach, fetal aneuploidies other than the common trisomies can also be detected. Fetoplacental mosaicism is the main cause for false positive/negative NIPT results. We further improved the analytical power of genome-wide cfDNA screening by enabling the detection of fetoplacental mosaicism.

Method: Aneuploidy detection was combined with fetal fraction estimation to enable the detection of placental chromosomal mosaicism. This pipeline was applied to whole genome sequencing data derived from ~20.000 maternal plasma samples. Following an abnormal NIPT, test results were validated by conventional invasive prenatal or postnatal genetic testing.

Results: The new analysis pipeline identified 134, 24 and 7 non-mosaic trisomies 21, 18 and 13 respectively. All for whom follow-up information was available were confirmed upon invasive testing. The incidence of other, rare autosomal trisomies (RATs) was ~0.3%, with trisomy 7 and 16 being the most prevalent. Three of these RATs, predicted as full trisomies in the placenta, were found to be mosaic in the fetus; 25 other RATs were predicted to be mosaic, 8 of which have been confirmed in placental tissue. The new pipeline also correctly predicted twin pregnancies with discordant fetal sex.

Conclusions: This improved analysis pipeline permits the detection of autosomal aneuploidies and pinpoints pregnancies at risk of fetoplacental mosaicism. This knowledge can influence estimation of the risk for miscarriage, aid in genetic counselling and improve prenatal management.

N. Brison: None. M. Neofytou: None. L. Dehaspe: None. B. Bayindir: None. K. Van Den Bogaert: None. L. Dardour: None. H. Peeters: None. H. Van Esch: None. G. Van Buggenhout: None. A. Vogels: None. J. Breckpot: None. T. de Ravel: None. E. Legius: None. K. Devriendt: None. J.R. Vermeesch: E. Ownership Interest (stock, stock options, patent or other intellectual property); Modest; Cartagenia. Other; Modest; Collaboration with Cartagenia.

P01.63C Pregnancy outcome for fetuses with increased nuchal translucency but normal karyotype and CMA: impact on the counselling and need for further genetic investigations

R. Ficarella1, M. Mucciolo2, C. Votino1, F. R. Lepri2, M. F. Antonucci1, A. L. Buonadonna1, V. Lanari2, P. Volpe1, M. Gentile1, A. Novelli2

1ASL Bari, Bari, Italy, 2Bambino Gesù Paediatric Hospital, Roma, Italy

Objectives: To investigate the outcome for fetuses with nuchal translucency (NT) ≥3.5 mm but normal karyotype/CMA.

Methods: All patients were referred to our institution for NT≥3.5 mm from 2012 to 2016. We followed prenatally all patients until delivery and pregnancy outcome was recorded. Targeted Resequencing was performed using a panel including 15 RASopathies genes.

Results: We identified 74 fetuses. An adverse perinatal outcome was observed in 27% of cases. US follow-up showed 10 cases with cardiac malformations (major in 6/10); diaphragmatic hernia (1); Dandy Walker Malformation plus skeletal dysplasia (1); pyelectasis (1), and IUGR (1 monochorionic twins). 54% of these cases (40/74) were analyzed using our customized panel of RASopathies genes. Four variants were identified, the aminoacid substitutions Val1432Phe and Arg2452Cys, in the NF1 gene, and a Thr7Arg and Thr159Pro, in the LZTR1 gene. Four additional intronic variants were also identified, but no one altered the splice site according to the prediction tools. Thirty additional fetuses with NT ≥ 3.5 mm had been previously analyzed, leading to the identification of two variants, the Gln506Pro in the PTPN11 gene, and the Glu63Lys in the KRAS gene.

Conclusion: Even with normal karyotype/CMA, a NT>99th centile is associated with an adverse pregnancy outcome, as in one third of cases a congenital malformation and/or a miscarriage was observed. More interestingly our study demonstrate a RASopathy gene variant in 4/10 (10%) fetuses, in the absence of ultrasound markers that could address the diagnostic suspicion.

R. Ficarella: None. M. Mucciolo: None. C. Votino: None. F.R. Lepri: None. M.F. Antonucci: None. A.L. Buonadonna: None. V. Lanari: None. P. Volpe: None. M. Gentile: None. A. Novelli: None.

P01.64D Novel pathogenic splice variant in PALB2 gene causing anemia Fanconi identified by transcriptomic analysis

I. Viakhireva1, E. Musatova1,2, E. Pomerantseva3, Y. Shcherbatyuk4, S. Korobkov4, S. Zhikriveckaya2, F. Konovalov5, M. Skoblov1,6

1Research Centre for Medical Genetics, Moscow, Russian Federation, 2Center of Genetics and Reproductive Medicine "Genetico", LLC, Moscow, Russian Federation, 3Center of Genetics and Reproductive Medicine, Moscow, Russian Federation, 4Hospital Lapino, MD Medical Group, Moscow, Russian Federation, 5Genomed, Ltd, Moscow, Russian Federation, 6Moscow Institute of Physics and Technology, Dolgoprudny, Russian Federation

Fanconi anemia is rare congenital disease caused by mutations in genes responsible for DNA repair and expressing in chromosomal instability. Although the main symptom is anemia, clinical pattern differs in parents with mutations in genes according to different complementation group. The most severe clinical pattern is described in cases with PALB2 gene mutations. These children develop severe anemia and early onset of different oncological diseases such as medulloblastoma, Wilms tumor, different leukemias. We report clinical case of a child with Fanconi anemia died because of medulloblastoma at the age 4 years 10 months. The disease was caused by frameshifting mutation in PALB2 gene c. 172_175del inherited from mother and novel intronic deletion NC_000016.9:g. 23625423delAAAAATA inherited from father. Frameshift mutation was identified by exome sequencing of the affected child. The deletion was identified in father’s blood by transcriptomic analysis. Functional analysis of mutation in minigene system confirmed its pathogenicity. As the family was interested in having a healthy child, understanding of molecular causes of disease in this family allowed to perform preimplantation genetic testing for monogenic disease (PGT-M). One IVF cycle was performed and 10 embryos were biopsied for PGT-M. According to the PGT-M results, it was determined that 3 embryos had both variants in heterozygose stage, 1 embryo inherited only c. 172_175del variant, 4 embryos inherited only NC_000016.9:g. 23625423delAAAAATA variant and 2 embryos did not inherit either of two variants. Preimplantation testing for aneuploidies was performed for these two embryos and they defined as euploid and were recommended for transfer.

I. Viakhireva: None. E. Musatova: None. E. Pomerantseva: None. Y. Shcherbatyuk: None. S. Korobkov: None. S. Zhikriveckaya: None. F. Konovalov: None. M. Skoblov: None.

P01.66B Preimplantation genetic diagnosis for chromosomal rearrangements using shallow whole genome sequencing at the blastocyst stage

A. Dheedene1, I. De Croo2, E. Van den Abbeel2, P. De Sutter2, K. Tilleman2, B. Menten1

1Center for Medical Genetics Ghent, Ghent University Hospital, Ghent, Belgium, 2Department for Reproductive Medicine, Ghent University Hospital, Ghent, Belgium

Preimplantation genetic diagnosis (PGD) for chromosomal rearrangements is used to avoid the transfer of embryos with genomic aberrations to the uterus and hence to improve implantation rate and avoid miscarriage or the birth of children with congenital anomalies. Currently, genomic microarrays are predominantly used for the detection of unbalanced structural abnormalities and aneuploidies in embryos from parents at risk. There are however several limitations to the use of microarrays such as constraints on resolution and throughput. With the advent of massive parallel sequencing (MPS), we investigated the use of shallow whole genome sequencing for PGD (CNVseq) on trophectoderm biopsies in our clinical diagnostic workflow. PGD was performed on embryos of translocation carriers in combination with vitrification and frozen embryo transfer in non-stimulated cycles. Data were collected from January 2016 onwards.

In total 45 PGD cycles have been performed for reciprocal (n = 39) and Robertsonian (n = 5) translocation and inversion (n = 1) carriers (total number of embryos = 185). Almost 60% of the analysed embryos showed chromosomal aberrations, which is in line with our earlier results with microarrays (Christodoulou et al., Fertilty&Sterility, 2017). The CNVseq protocol shows especially for small chromosomal segments better results than microarrays, and a resolution of ~5 Mb is achieved. Furthermore, many samples can be processed in batch leading to higher throughput. Besides abnormalities due to the parental rearrangement, also other chromosome abnormalities were detected in our cohort.

We describe the successful implementation of CNVseq on blastocysts in patients with a chromosomal rearrangement to identify euploid embryos for transfer.

A. Dheedene: None. I. De Croo: None. E. Van den Abbeel: None. P. De Sutter: None. K. Tilleman: None. B. Menten: None.

P01.67C Whole-exome sequencing identifies novel causative variants and expands the phenotypic spectrum of PLK4-related primary microcephaly

P. Boonsawat1, R. Asadollahi1, D. Niedrist1, P. Joset1, J. Wisser2, H. Budka3, P. K. Bode4, H. Sticht5, K. Steindl1, A. Rauch1

1Institute of Medical Genetics, Schlieren-Zurich, Switzerland, 2Department of Obstetrics, Zurich, Switzerland, 3Institute of Neuropathology, Zurich, Switzerland, 4Institute of Pathology and Molecular Pathology, Zurich, Switzerland, 5Institute of Biochemistry, Erlangen, Germany

Loss-of-function variants in PLK4, encoding a key regulator of centriole duplication, cause autosomal recessive microcephaly and chorioretinopathy 2 (MCCRP2). Currently, only 13 cases from six families have been reported harboring four recessive variants. Following whole-exome sequencing analysis in 61 microcephalic cases, we identified novel causative PLK4 variants in two aborted sib fetuses and an additional unrelated child. In the two fetuses, we found a nonsense variant and a serine substitution in compound heterozygous (CH) state, which the latter likely creates an additional phosphorylation site in the phosphodegron element of PLK4, leading to reduced protein level via accelerated autodestruction. Autopsy examination of the fetuses revealed white matter neuronal heterotopia and cerebellar vermis hypoplasia in one, and absence of corpus callosum in the other, apart from facial dysmorphism and microcephaly. Additional physical anomalies included 2-lobed right lung and accessory spleen, which have not been previously reported in MCCRP2. Furthermore, we identified (likely) pathogenic CH variants in the unrelated child, presenting with primary microcephaly, facial dysmorphism and moderate speech delay. Brain MRI showed simplified cortical gyri, dysplastic corpus callosum, and novel finding of large cerebellum-brain stem relative to the supratentorial region. Considering our cases and those previously reported, we consistently observed simplified gyri, abnormal corpus callosum and neuronal heterotopia, suggesting the importance of PLK4 in the regulation of neuronal migration. Moreover, we report a novel MRI finding as well as additional organ anomalies in MCCRP2, and describe the first deleterious missense variant located in the phosphodegron element outside the main PLK4 domains.

P. Boonsawat: None. R. Asadollahi: None. D. Niedrist: None. P. Joset: None. J. Wisser: None. H. Budka: None. P.K. Bode: None. H. Sticht: None. K. Steindl: None. A. Rauch: None.

P01.68D Evidence for key roles of transcription factors and microRNAs in orchestrating placental gene expression patterns in common pregnancy complications

S. Sõber1, M. Reiman1, K. Rull1,2,3, M. Laan1

1Institute of Biomedicine and Translational Medicine, University of Tartu, Tartu, Estonia, 2Department of Obstetrics and Gynaecology, University of Tartu, Tartu, Estonia, 3Women’s Clinic of Tartu University Hospital, Tartu, Estonia

Introduction: Approximately 5% of pregnancies suffer from pre-eclampsia (PE) and 3% of couples are affected by recurrent pregnancy loss (RPL).

Aims: To identify mechanisms affecting gene expression in common pregnancy complications PE and RPL.

Methods: We sequenced transcriptomes and miRNomes from term placental samples from normal (n = 8) and PE (n = 8) pregnancies and 1st trimester samples from 2 RPL cases and electively terminated pregnancies (ETP; n = 8). Differential expression was tested using DESeq and DESeq2. g:Profiler was used for enrichment analysis.

Results: In placentas of RPL cases, we detected 195 transcripts with altered expression in RPL compared to ETP (1). Over 60% of genes with altered expression in RPL possess binding sites for E2F transcription factors. E2F regulates the cell cycle and coordinates the mammalian endocycle and placental development.

Expression of 215 genes was altered in PE (2). Promoters of down-regulated genes (n = 173) exhibited strong enrichment for binding sites for transcription factors AP2, SP1 and LRF. Promoters of 77 genes (44.5%) contain potential response elements for all three transcription factors.

Correlation analysis between microRNA and gene expression identified an extensive network of coordinated expression involving multiple transcripts and microRNAs.

Conclusions: The E2F family of transcription factors represents a potential central coordinator of the shut-down of nuclear and cellular functions leading to fetal demise. Inadequate AP2, SP1 and LRF activity along with altered microRNA levels may drive gene expression changes in pre-eclampsia.

(1)Sõber et al. SciRep (2016): 38439.

(2)Sõber et al. SciRep (2015): 13336.

Grants: IUT34-12 (Estonian Research Agency), Happy Pregnancy (SA Archimedes).

S. Sõber: None. M. Reiman: None. K. Rull: None. M. Laan: None.

P01.69A Preimplantation genetic testing for cystic fibrosis and aneuploidy in clinical practice

J. Diblík, I. Soldátova, M. Sekowská

GENNET s.r.o., Praha, Czech Republic

Introduction: Cystic fibrosis (CF) due to mutations in the CFTR gene is the most frequent reason for preimplantation genetic testing of monogenic disorders (PGT-M). Many women requiring PGD for CF are at advanced age that is a limiting factor of IVF success.

Materials and methods: We have performed PGT for CF in 81 IVF cycles for 51 couples between the years 2007 and 2017. The total number of examined embryos is 438. We have examined 317 samples of blastomeres from cleavage stage embryos and 121 samples of trophectoderm from blastocysts. The PGT-M for was performed by haplotyping using whole genome amplification (WGA) and multiplex fluorescence PCR analysis of short tandem repeat polymorphisms (STR markers) linked to the CFTR gene. We have recently added the aneuploidy detection (PGT-A) by NGS using Ion Torrent Proton as a second step in the evaluation of trophectoderm samples.

Results: We have amplified the DNA from 295 out of 317 single blastomeres (93%) and 104 out of 121 trophectoderm samples (86%). We have found 246 embryos not affected by CF (62%) or other abnormalities of chromosome 7 (monosomy, trisomy) using the haplotype analysis. We have further analysed the amplified DNA from 72 trophectoderm samples and detected aneuploidy in 31 out of them (43 %).

Conclusions: We have successfully used trophectoderm biopsy and whole genome amplification to combine the preimplantation genetic testing of a monogenic disorder (PGT-M) with aneuploidy detection (PGT-A) in clinical practice.

J. Diblík: None. I. Soldátova: None. M. Sekowská: None.

P01.70B Implementation of target capture enrichment on single and few cells for the robust detection of embryo abnormalities

M. Ioannides1, A. Achilleos1, K. Tsangaras1, C. Loizides1, P. Mina1, E. Kypri1, C. Sismani2, G. Koumbaris1, P. C. Patsalis1

1NIPD Genetics, Nicosia, Cyprus, 2The Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus

Introduction: High throughput non-invasive prenatal testing (NIPT) technologies have demonstrated safe, accurate and reliable results for the detection of fetal abnormalities, relying on the detection and analysis of cell free fetal DNA (cffDNA) in maternal plasma. However, such analysis is often limited by the low abundance of DNA, as in the case of fertilized embryos. Therefore, the development of novel, sensitive approaches which can provide reliable results from single/few cells is necessary.

Materials and Methods: Amplified DNA isolated from seven and 17 embryos was obtained from 3-day and 5-day biopsy cases. TArget Capture Sequences (TACS) were designed at a median resolution of 1Mb spanning all chromosomes and were used to perform in-solution hybridization capture enrichment as previously described1. Novel bioinformatics algorithms were also developed to determine the ploidy status of the samples.

Results: All samples were correctly classified and all abnormalities were detected including numerical and structural rearrangements. Results obtained were in agreement with array CGH.

Conclusions: Targeted sequencing is the preferred method for applications requiring high read depth. This assay in combination with a novel bioinformatics pipeline can be used for the genome-wide screening of fertilized embryos (PGS/PGD). It can also be used in cases where limited number of cells from affected tissues/individuals are available.

M. Ioannides: A. Employment (full or part-time); Significant; NIPD Genetics. A. Achilleos: A. Employment (full or part-time); Significant; NIPD Genetics. K. Tsangaras: A. Employment (full or part-time); Significant; NIPD Genetics. C. Loizides: A. Employment (full or part-time); Significant; NIPD Genetics. P. Mina: A. Employment (full or part-time); Significant; NIPD Genetics. E. Kypri: A. Employment (full or part-time); Significant; NIPD Genetics. C. Sismani: None. G. Koumbaris: A. Employment (full or part-time); Significant; NIPD Genetics. P.C. Patsalis: A. Employment (full or part-time); Significant; NIPD Genetics.

P01.71C Various approaches to preimplantation genetic testing - experience from seven monogenic disorders

R. Staneva1,2, S. Hadjidekova1,2, S. Andonova3, A. Savov3, S. Bitchev3, S. Yaneva2, M. Pancheva4, M. Serafimova2, T. Chaushev2, K. Nikolova2, D. Toncheva1, G. Stamenov2

1Department of Medical Genetics, Medical University of Sofia, Sofia, Bulgaria, 2Women's Health Hospital "Nadezhda", Sofia, Bulgaria, 3National Genetic Laboratory, UHOG “Maichin dom”, Sofia, Bulgaria, 4Women's Health Hospital, Sofia, Bulgaria

Background: In the last decade preimplantation genetic testing (PGT) for severe genetic diseases emerged as a viable alternative to prenatal testing and termination of pregnancy for couples where one or both partners is a carrier or suffering from a debilitating genetic condition.

Materials & Methods: 7 couples opted for IVF-PGT after extensive genetic counseling. Indications were beta-thalassemia, epidermolysis bullosa, myotonic dystrophy type 1, Huntingtion disease, Fragile-X syndrome, hemophilia A and Duchenne muscular dystrophy. Trophectoderm biopsy of 32 5-day embryos was performed. DNA amplification was achieved by Repli-g (Qiagen). For all trinucleotide repeat disorders allele size was determined by fragment length analysis (TNR Diagnostics). RT-PCR was applied for epidermolysis bullosa and beta-thalassemia (Microsynth), Sanger sequencing for haemophilia A and sex determination by aCGH for DMD. All protocols were tested in advance on donated unviable embryos.

Results: DNA from 31 embryos was available for testing after amplification (96,9%). DNA analysis was successful for all 31 embryos (100%). There were 21 unaffected embryos and 3 females (for the DMD case). Four of the couples (57,1%) achieved pregnancy after the first transfer, 2 couples (28,6%) after the second and only one (14,3%) did not get pregnant after three transfers, giving a 85,7% success rate of the IVF-PGT procedure. Except one pregnancy that has not yet reached time for prenatal confirmation, all PGT results were confirmed by DNA testing of CVS samples, 6 healthy babies were delivered.

Conclusion:IVF-PGT is a valid option for couples at risk to have a child with severe genetic condition.

R. Staneva: None. S. Hadjidekova: None. S. Andonova: None. A. Savov: None. S. Bitchev: None. S. Yaneva: None. M. Pancheva: None. M. Serafimova: None. T. Chaushev: None. K. Nikolova: None. D. Toncheva: None. G. Stamenov: None.

P01.72D Preimplantation genetic testing for polycystic kidney disease is an option for affected families

V. Berckmoes1, P. Verdyck1, P. De Becker1, A. De Vos2, G. Verheyen2, P. Van der Niepen3, W. Verpoest2, I. Liebaers1, M. Bonduelle1, M. De Rycke1

1Centre for Medical Genetics, Brussel, Belgium, 2Centre for Reproductive Medicine, Brussel, Belgium, 3Nephrology & Hypertension Department, Brussel, Belgium

Introduction: In this study, preimplantation genetic testing (PGT) data for polycystic kidney disease (PKD) from 2005 until 2016 are reported. As males affected with autosomal dominant PKD (ADPKD) may present with reproductive system abnormalities and infertility, the clinical outcome was compared between couples with the female partner affected with ADPKD and couples with the male partner affected with ADPKD.

Materials and Methods: Sixteen single-cell clinical tests for PKD based on multiplex PCR of STR markers were applied for 91 PGT cycles for 43 couples.

Results: A diagnosis was obtained for 93.3% of the analysed embryos of which 36.8% were genetically transferable. Transfer of 74 embryos in 53 fresh cycles and transfer of 34 cryopreserved embryos in 33 frozen-warmed embryo transfer cycles resulted in a live birth delivery rate of 38.4% per transfer with 31 singleton live births, 2 twin live births and 1 ongoing pregnancy. The observed cumulative delivery rate was 57.8% per couple after five treatment cycles. The clinical pregnancy rate and live birth delivery rate was significantly lower for couples with the male partner affected with ADPKD compared with couples with the female partner affected with ADPKD. However, female age was the only variable significantly associated with live birth delivery rate.

Conclusions: This study shows that PGT for PKD performed in our centre offers good reproductive outcomes from both fresh and frozen embryo transfers. Males affected with ADPKD who suffer from infertility should be advised to seek treatment on time to improve their chances of conceiving a child.

V. Berckmoes: None. P. Verdyck: None. P. De Becker: None. A. De Vos: None. G. Verheyen: None. P. Van der Niepen: None. W. Verpoest: None. I. Liebaers: None. M. Bonduelle: None. M. De Rycke: None.

P01.73A DNA copy number variations in a cohort of 216 Italian women with premature ovarian failure

K. Teearu1, O. ilina1, O. Tšuiko1,2, A. Marozzi3, P. Finelli3,4, I. Bestetti3,4, D. Toniolo5, A. Salumets2,6, A. Kurg1

1Institute of Molecular and Cell Biology, University of Tartu, Tartu, Estonia, 2Competence Center on Health Technologies, Tartu, Estonia, 3Department of Medical Biotechnology and Translational Medicine Università degli Studi di Milano, Milan, Italy, 4Medical Cytogenetics and Molecular Genetics Lab, IRCCS Istituto Auxologico Italiano, Milan, Italy, 5Genetics of Common Disorders, Division of Genetics and Cell Biology, San Raffaele Scientific Institute, Milan, Italy, 6Chair of Obstetrics and Gynaecology, University of Tartu, Tartu, Estonia

Premature ovarian failure (POF) is considered as a multifactorial and heterogeneous condition affecting approximately 1% women of reproductive age. Despite the extensive research, the considerably complex pathogenesis of POF is still not well understood. POF can develop as result of a broad spectrum of pathogenic mechanisms including genetic, autoimmune and iatrogenic causes that leads to follicular dysfunction or depletion. In recent years, many research studies are trying to find out the genetic component of the disease by using different high-resolution methods. We have performed a case-control genetic association study, using high-resolution SNP microarrays to investigate DNA copy number variations (CNVs) of 216 Italian women presenting POF phenotype and 240 women from the Italian general population as a control. All patient and control samples were collected at the Division of Genetics and Cell Biology, San Raffaele Scientific Institute and University of Milan, Italy and genotyped by Illumina PsychArray BeadChips at the Estonian Genome Center University of Tartu Genotyping core in Tartu, Estonia. Microarray data, analyzed using different algorithms, revealed both, genomic regions containing genes previously associated with POF (e.g. 26.8Mb 1q41-q44 duplication affecting FMN2 gene) and novel potentially clinically significant CNVs (e.g. 11p15.2 microdeletion). In addition to autosomal CNVs, we identified several POF critical regions on the X chromosome. The currently known POF genes only account for a small proportion of patients, while the majority remain without a genetic diagnosis. Using whole-genome DNA microarrays, the present study provides novel insight into the implications of CNVs in genetic aetiology of POF.

K. Teearu: None. O. ilina: None. O. Tšuiko: None. A. Marozzi: None. P. Finelli: None. I. Bestetti: None. D. Toniolo: None. A. Salumets: None. A. Kurg: None.

P01.74B The diagnostic yield of exome sequencing in the prenatal setting: A clinical laboratory experience

A. Telegrafi, C. Yates, K. G. Monaghan, E. Ryan, B. Friedman, H. Sroka, R. Willaert, R. Chikarmane, J. Juusola

GeneDx, Gaithersburg, MD, United States

Introduction: Fetal ultrasound abnormalities pose a unique diagnostic challenge. In order to help increase the diagnostic yield of underlying genetic etiologies exome sequencing (ES) is gaining use in the prenatal setting.

Methods: We retrospectively analyzed ES results on 233 deceased fetuses and 33 ongoing pregnancies with ultrasound anomalies.

Results: Of the 233 deceased fetuses, 76% of testing was performed as proband-parent trios. Fifty-six percent were male and 44% female. Most cases had multiple congenital anomalies (MCA) (78%). The most common ultrasound findings were central nervous system (CNS) anomalies (51%), hydrops (33%), skeletal abnormalities (30%), cardiovascular defects (28%), neuromuscular findings (26%), and genitourinary anomalies (24%). A definitive molecular diagnosis was identified in 28% of cases, a possible diagnosis in 36%, only a candidate gene was reported in 10%, and 26% of cases had no reportable variants. We also analyzed 33 prenatal specimens from ongoing pregnancies. All cases were trios and had non-diagnostic standard genetic testing prior to ES. Seventy-three percent were male and 27% female. Most cases had MCA (79%). Common ultrasound findings included cardiovascular anomalies (33%), genitourinary abnormalities (33%), CNS defects (30%), and hydrops (30%). For all ongoing pregnancies, a definitive molecular diagnosis was identified in 30.3% of cases, a possible diagnosis in 33.3%, only a candidate gene was reported in 6.1%, and 30.3% had no reportable variants.

Conclusion: Although interpretive and ethical issues remain a concern in the prenatal setting, ES may identify genetic variants responsible for fetal anomalies and impact prognosis, medical management, and recurrence risks.

A. Telegrafi: A. Employment (full or part-time); Significant; GeneDx. C. Yates: A. Employment (full or part-time); Significant; GeneDx. K.G. Monaghan: A. Employment (full or part-time); Significant; GeneDx. E. Ryan: A. Employment (full or part-time); Significant; GeneDx. B. Friedman: A. Employment (full or part-time); Significant; GeneDx. H. Sroka: A. Employment (full or part-time); Significant; GeneDx. R. Willaert: A. Employment (full or part-time); Significant; GeneDx. R. Chikarmane: A. Employment (full or part-time); Significant; GeneDx. J. Juusola: A. Employment (full or part-time); Significant; GeneDx.

P01.76D Prenatal array CGH: comparison of 400kb and 3Mb resolution

K. Mann, J. Ahn, S. Bint, C. Brown, C. Mackie Ogilvie

Guy’s and St Thomas’ NHS Foundation Trust, London, United Kingdom

There is currently no clear consensus on the cost-benefit of detailed genome-wide copy-number analysis for pregnancies with ultrasound anomalies. Decisions regarding test resolution should consider evidence concerning diagnostic yield, cost (technical and analytical), reporting times and the number of uncertain and incidental findings. From 2012 to 2017 we investigated pregnancies with ultrasound anomalies using a prenatal array testing strategy designed to minimise uncertain results and incidental findings whilst identifying clinically significant abnormalities; a 3Mb backbone resolution was supplemented with high resolution analysis of 23 regions known to be associated with severe, fully penetrant syndromes. Review of 480 of these cases (after anonymization) at an average of 120kb resolution found no severe, fully penetrant imbalance had been missed. European guidelines now recommend a genome-wide resolution of at least 400kb (the evidence base for this guideline is unclear). To comply with this, in 2017 we introduced a 400kb analysis strategy, with high resolution analysis of 25 syndrome regions; only findings associated with the ultrasound anomalies and actionable or severe, early-onset incidental findings were reported. Of 201 prenatal samples tested, 22 were reported as abnormal, including one incidental finding, 178 as normal and one failed. Only one case (0.5% of samples), a de novo 1.6 Mb 15q25 deletion (OMIM 614294) would not have been identified using our previous analysis strategy. Fourteen unique imbalances (7% of samples) were not reported following detailed variant classification; in addition, four susceptibility loci (class 4 variants) were not disclosed. A cost-benefit comparison of these strategies will be discussed.

K. Mann: None. J. Ahn: None. S. Bint: None. C. Brown: None. C. Mackie Ogilvie: None.

P01.77A Case report: Gonosomal placental mosaicism leads to a false-positive NIPT result

T. Harasim1, A. Wagner1, U. Heinrich1, E. Krimmel1, M. Delius2, I. Rost1, H. Klein1

1Center for Human Genetics and Laboratory Diagnostics, Dr. Klein, Dr. Rost and Colleagues, Martinsried, Germany, 2Department of Gynecology and Obstetrics of the hospital of the Ludwig-Maximilians-University, Munich, Germany

Introduction: A 37 years old pregnant woman with no fetal ultrasound abnormalities received a NIPT result at early gestational age indicating a monosomy X. For confirmatory purpose, amniocentesis including aCGH was performed: the fetal karyotype was reported to be normal, male (46,XY) instead of the expected 45,X. Perinatally, another NIPT with high sequencing depth was requested (Prenatalis®, MVZ Martinsried) in concert with postpartal FISH of placental villi to shed light into these discordant results.

Method: Prenatalis® was performed with ~ 21 million reads used for detection of aneuploidy 13, 18, 21, X and Y. Postpartal FISH analysis was done on nuclei of placental villi by using the centromere specific DXZ1-, DYZ3- and D18Z- probes (Cytocell, Cambridge, UK).

Results: Prenatalis® confirmed the initial monosomy X result at a fetal fraction of 39% (GA:36+6). FISH-analysis revealed placental mosaicism with a dominant X0-cell line (86% of all nuclei) in combination with a XY cell line (10%). 4% of nuclei showed two X-chromosomes, probably a contamination of maternal cells. The patient delivered a phenotypically normal, male baby, who was not karyotyped further.

Conclusion: The results presented above describe confined placental mosaicism of a dominant monosomy X cell line in concert with a low level XY-cell line. It can be deduced from the NIPT results, that the placenta released cfDNA exclusively from 45,X loci, since y-chromosomal cfDNA could not be detected during NIPT. Since placental cfDNA only serves as a proxy for the fetus, confirmation of positive NIPT results are highly recommended.

T. Harasim: None. A. Wagner: None. U. Heinrich: None. E. Krimmel: None. M. Delius: None. I. Rost: None. H. Klein: None.

P01.78B Rapid whole exome sequencing; implementation in the prenatal setting

I. Feenstra1, Y. Arens2, S. de Munnik1, A. C. Deden1, A. C. J. Gijsbers1, V. van der Schoot2, W. van Zelst-Stams1, E. Sikkel1, D. Smeets1, K. Neveling1, M. Nelen1, H. Yntema1

1Radboud university medical centre, Nijmegen, Netherlands, 2Maastricht University Medical Centre, Maastricht, Netherlands

The introduction of whole exome sequencing (WES) in genome diagnostics has dramatically changed the current practice in clinical genetics. It is expected that WES and ultimately whole genome sequencing will replace current routine clinical practice (array based technologies), not only after birth but also during pregnancy. The implementation of WES in a prenatal setting for genetic analysis of fetuses with multiple congenital abnormalities has the potential to increase the diagnostic yield and thereby improving prognostic information for professionals and expectant parents. However, there are a number of factors that need to be taken into account before WES could be part of the prenatal diagnostic workup. These include technical and practical aspects like long turn-around-times (TATs), costs, difficulties in interpreting variants and limited possibilities to determine the fetal phenotype. Intensive collaboration within a multi-disciplinary team consisting of a molecular laboratory specialist, a clinical geneticist and a fetal medicine specialist is required. Furthermore, the possibility of detecting variants of unknown significance (VOUS) or incidental findings (IF) may lead to ethical dilemmas and demands careful pre- and post-test counselling.

Until now, rapid WES with short TATs has been performed on a case-by-case basis in our centre in a small number of pregnancies in the second or third trimester. We will provide an overview of the workflow, the challenges and the difficulties encountered. To warrant an accurate, more widespread implementation of prenatal WES, there is a strong need for clear criteria, data-sharing and an (inter)national guideline.

I. Feenstra: None. Y. Arens: None. S. de Munnik: None. A.C. Deden: None. A.C.J. Gijsbers: None. V. van der Schoot: None. W. van Zelst-Stams: None. E. Sikkel: None. D. Smeets: None. K. Neveling: None. M. Nelen: None. H. Yntema: None.

P01.79C Multilevel regression modeling improves STR classification in QF-PCR analysis for common fetal chromosomal aneuploidies

P. Noveski, M. Terzic, M. Vujovic, M. Kuzmanovska, E. Sukarova Stefanovska, D. Plaseska-Karanfilska

Research Center for Genetic Engineering and Biotechnology “Georgi D. Efremov”, Macedonian Academy of Sciences and Arts, Skopje, Macedonia, The Former Yugoslav Republic of

The quantitative fluorescent polymerase chain reaction (QF-PCR) has proven to be a reliable method for detection of common fetal chromosomal aneuploidies, with advantages over conventional karyotyping such as cost-effectiveness, reliability and requirement of only small amount of material. However, there are some technical shortcomings, involving the necessity to perform two or more multiplex PCR reactions simultaneously for a given sample or the uncertainty of aneuploidy determination when the STR (short tandem repeats) height ratio is unusual due to large size difference between alleles. Here, we present an in-house one-tube multiplex QF-PCR method including 20 PCR markers (14 STR markers and 6 fixed size) for rapid prenatal diagnosis of chromosome 13, 18, 21, X and Y aneuploidies. In order to improve the aneuploidy classification of a given diallelic STR marker, we used a total of 7630 diallelic genotypes (88 trisomic and 7542 normal) from 871 samples to employ multilevel logistic regression analysis using "height ratio" and "allele size difference" as fixed effects and "marker" as random effect. We employed two regression models, one for the 2:1 height ratio (n = 47) and second for the 1:2 height ratio (n = 41) of the trisomic diallelic markers. Both models achieved 100% specificity for marker aneuploidy classification on training data as compared to 98.3% (2:1 ratio) and 97.9% (1:2 ratio) specificity when using only height ratio for classification. In conclusion, adjusting for the allele size difference and marker type improves the STR classification, eliminates sample re-testing and reinforces the robustness of the QF-PCR method for prenatal testing.

P. Noveski: None. M. Terzic: None. M. Vujovic: None. M. Kuzmanovska: None. E. Sukarova Stefanovska: None. D. Plaseska-Karanfilska: None.

P01.80D High-resolution array-CGH analysis and Targeted Whole Exome Sequencing on patients affected by Primary Ovarian Insufficiency (POI) identified new genes involved in oocyte grow and differentiation

I. Bestetti1,2, C. Barbieri3, A. Sironi1,2, C. Castronovo1, C. Caslini2, R. Rossetti4, A. Pistocchi2, A. Rajkovic5,6,7, C. Sala3, D. Toniolo3, L. Persani4,8, A. Marozzi2, P. Finelli1,2

1Lab. of Medical Cytogenetics and Molecular Genetics, IRCCS Istituto Auxologico Italiano, Cusano Milanino, Milan, Italy, 2Dep. of Medical Biotechnology and Translational Medicine, University of Milan, Milan, Italy, 3Division of Genetics and Cell Biology, San Raffaele Research Institute and Vita Salute University, Milan, Italy, 4Lab. of Endocrine and Metabolic Research and Division of Endocrine and Metabolic Diseases, IRCCS Istituto Auxologico Italiano, Cusano Milanino, Milan, Italy, 5Dep. of Obstetrics, Gynecology, and Reproductive Sciences, Magee-Womens Research Institute, University of Pittsburgh, Pittsburgh, PA, United States, 6Dep. of Pathology, University of Pittsburgh, Pittsburgh, PA, United States, 7Dep. of Human Genetics, University of Pittsburgh, Pittsburgh, PA, United States, 8Dep. of Clinical Sciences and Community Health, University of Milan, Milan, Italy

POI is a heterogeneous group of disorders that affect women fertility whose genetic origin has been clarified in less than 30% of cases. To unveil new causative genes essential for ovarian function we searched for rare high-penetrance Copy Number Variants (CNVs) in a cohort of 67 46,XX patients affected by the most severe phenotype (primary amenorrhea). High-resolution array-CGH analysis detected 72 rare CNVs according to the Database of Genomic Variants in 49 patients. CNVs gene content analysis and disease prioritization selected 37 CNVs involving 2 POI-associated genes and 42 putative candidate genes (e.g. TP63, VLDLR). The research of this CNVs in an ad-hoc cohort of 134 control women supported their actual rarity. Despite the presence of ovary genes also in the ad-hoc cohort, chi-q and Wilcoxon tests showed in patients a significant enrichment of ovary-related CNVs/genes (P=0.0132/P=0.0126) supporting array-CGH as a valuable tool in identifying novel POI molecular defects. Array-CGH genes identified together with their predicted interactors, and other known ovary/POI-related genes (n = 226) were then screened in a targeted-WES analysis of 102 secondary amenorrhea patients. After filtering variants (MAF<0.005; LoF SNVs inclusion) a total of 375 possibly pathogenic SNVs were found in 31 array-genes, 12 interactor-genes, and 83 ovary/POI-related genes. Burden test analysis versus 1000G_EUR control women confirmed a statistical significance for 1 interactor-TP63 gene (P=1.65E-07) and 2 ovary/POI-related genes (FSHR, P=1.66E-05;FOXO3, P=7.31E-05). This combined approach allowed to increase the knowledge about POI pathogenesis and will probably provide the basis for a more accurate genetic diagnosis of POI patients.

I. Bestetti: None. C. Barbieri: None. A. Sironi: None. C. Castronovo: None. C. Caslini: None. R. Rossetti: None. A. Pistocchi: None. A. Rajkovic: None. C. Sala: None. D. Toniolo: None. L. Persani: None. A. Marozzi: None. P. Finelli: None.

P01.81A Genetic analysis of products of conception of couples with recurrent miscarriage using QF-PCR and array-CGH testing strategy

L. Lovrečić1, N. Pereza2, H. Jaklič1, S. Ostojić2, B. Peterlin1

1Clinical Institute of Medical Genetics, University Medical Center Ljubljana, Department of Gynaecology and Obstetrics, Ljubljana, Slovenia, 2Faculty of medicine, University of Rijeka, Department of biology and medical genetics, Rijeka, Croatia

Introduction: Although previous studies have shown that embryonic chromosome aberrations are the most common cause of recurrent miscarriage (RM), the comprehensive genetic evaluation using the combination of quantitative fluorescence-polymerase chain reaction (QF-PCR) and array-comparative genomic hybridisation (aCGH) was not used systematically for their detection in the clinical setting. We aimed to investigate the frequency and type of chromosome aberrations in POCs of couples with at least one previous miscarriage using the QF-PCR and a-CGH strategy.

Materials and Methods: This retrospective study was conducted on 73 first-trimester POCs (September 2014-February 2017). The POCs were collected from 73 women with at least one previous miscarriage and analysed for chromosomal anomalies using QF-PCR and aCGH as part of the routine clinical evaluation.

Results: Chromosome aberrations were detected in 52/73 POCs (71.2%), of which 41 (56.2%) were identified by QF-PCR and an additional 11 (15.1%) by aCGH. Numerical aberrations constituted the majority (92.3%) of abnormalities, with trisomies as the most common subtype (72.9%). Causative structural aberrations were found in three samples (5.8%) and variant of unknown significance in one sample. The frequency of chromosome aberrations was not dependent on the number of previous miscarriages, whereas it significantly increased with advanced maternal age.

Conclusions: The results of our comprehensive genetic analyses of POCs of RM couples confirm the QF-PCR and aCGH combination as an effective diagnostic strategy. Considering the high frequency of chromosome aberrations, a routine genetic analysis of POCs should be considered, which could improve the clinical approach to couples with miscarriage.

L. Lovrečić: None. N. Pereza: None. H. Jaklič: None. S. Ostojić: None. B. Peterlin: None.

P01.82B Telomere shortening as the main indicator of non-viable fetus elimination

I. Tkach1, N. Huleyuk1, D. Zastavna1,2, M. Tyrka2

1Institute of Hereditary Pathology, NAMS of Ukraine, Lviv, Ukraine, 2Department of Biotechnology and Bioinformatics, Faculty of Chemistry, Rzeszow University of Technology, Rzeszow, Poland

Background: Telomeres are transcriptionally inactive genomic areas, which, if shortened, are associated with pathological processes, unsuccessful fertilization, aging, and death. Telomere dysfunction has also been linked to chromosomal rearrangements and genomic instability. The role of telomeres in postnatal life has been extensively studied and discussed both in physiological as well as in pathological processes. However, the role of telomere length in prenatal development is still poorly understood, and mainly concerns the preimplantation stage. The aim of this study was to estimate relative telomere length in spontaneously eliminated human embryos between 5th and 12th week of gestation.

Results: Relative telomere length was measured from total genomic DNA using a real-time polymerase chain reaction approach. In this study, we examined relative telomere length in 80 spontaneously eliminated embryos and in 25 embryos eliminated due to induced abortions. Relative telomere length in spontaneous abortions was significantly lower (P = 0.000001) compared to the induced abortions. Spontaneous abortions with aneuploid anomalies (monosomy X, trisomy 21, trisomy 16 and triploidy) were characterized by shorter telomeres, compared to spontaneous abortions, subgroup with euploid (46,XN) karyotype.

Conclusion: Spontaneously lost pregnancies are characterized by shortened telomeres, especially in embryos with aneuploidies. We hypothesize that the shortening of telomeres is involved in the processes leading to spontaneous abortions.

I. Tkach: None. N. Huleyuk: None. D. Zastavna: None. M. Tyrka: None.

P01.83C Targeted cfDNA Analysis Using DANSR assays for Determination of Fetal RHD Status

S. Saini1, R. Foley1, C. Kingsley1, E. Wang1, M. Schmid1, P. Bogard1, A. Ramos2

1Ariosa Diagnostics Inc., Roche Sequencing Solutions Inc., San Jose, CA, United States, 2Roche Sequencing Solutions, San Jose, CA, United States

Objectives: To develop a targeted cell-free (cfDNA) test, the Harmony® prenatal test, enhancement that allows determination of fetal RhD status in RhD-negative pregnant women.

Method: 12 simulated pregnancy plasma samples with known RHD genotype were prepared by titrating non-pregnant, RHD-positive cfDNA (fetal source) into non-pregnant, female RHD-negative cfDNA (maternal source) to simulate fetal fractions of 5%, 10% and 15%. A 0% sample served as a negative control. Digital Analysis of Selected Regions (DANSR) assays targeting exons 2, 3, 4, 5 and 7 of the RHD gene were added to existing DANSR assays and the generated DANSR products were hybridized onto a custom DNA microarray for analysis of fetal fraction and determination of fetal RHD status using the fetal fraction optimized algorithm FORTE.

Results: In all 12 simulated pregnancy samples, RHD sequences were detected. The RHD signal in each case correlated with fetal fraction and was therefore consistent with an RHD-positive fetal source on the background of an RHD-negative maternal source. As expected, no RHD sequences were detected for samples with 0% fetal fraction.

Conclusion: Targeted cfDNA testing using DANSR assays has the potential to determine fetal RHD status and be used as a noninvasive screening method to identify pregnancies at increased risk for RhD immunization.

S. Saini: A. Employment (full or part-time); Significant; Ariosa Diagnostics Inc., Roche Sequencing Solutions Inc. R. Foley: A. Employment (full or part-time); Significant; Ariosa Diagnostics Inc., Roche Sequencing Solutions Inc. C. Kingsley: A. Employment (full or part-time); Significant; Ariosa Diagnostics Inc., Roche Sequencing Solutions Inc. E. Wang: A. Employment (full or part-time); Significant; Ariosa Diagnostics Inc., Roche Sequencing Solutions Inc. M. Schmid: A. Employment (full or part-time); Significant; Ariosa Diagnostics Inc., Roche Sequencing Solutions Inc. P. Bogard: A. Employment (full or part-time); Significant; Ariosa Diagnostics Inc., Roche Sequencing Solutions Inc. A. Ramos: None.

P01.84D Prenatal diagnosis of mosaic ring chromosome 16 - a rare event with uncertain prognosis

F. Brito1, M. Silva1, C. Alves1, C. Ferreira1, S. Serafim1, L. Simão1, B. Marques1, S. Pedro1, A. Tarelho1, J. Furtado1, P. Lopes1, N. Silva1, M. Viegas1, A. Fernandes2, F. Teixeira2, S. Gomes3, H. Correia1

1Instituto Nacional de Saúde Dr. Ricardo Jorge, Unidade de Citogenética, Lisboa, Portugal, 2Hospital Central do Funchal, Serviço de Pediatria, Funchal, Portugal, 3Hospital Central do Funchal, Serviço de Obstetrícia, Funchal, Portugal

Ring chromosomes are rare cytogenetic findings (prenatal frequency ~ 0.0075%) often associated with an abnormal phenotype, depending of the chromosomal origin, genetic content and the presence of a mosaic. Supernumerary ring chromosome 16 [r(16)] is rarely observed and mosaicism makes the genotype/phenotype correlation difficult.

We report a de novo mosaic r(16) detected after prenatal diagnosis in a woman referred for advanced maternal age. Multiplex ligation-dependent probe amplification (MLPA) for aneuploidy testing of chromosomes 13, 18, 21 and X was normal. Karyotype was 47,XX,+r[10]/46,XX[15]. Chromosomal microarray analysis (CMA) on DNA obtained from long-term cultured amniocytes did not detect any alterations. MLPA with a pericentromeric probe kit on an uncultured sample showed a chromosome 16 gain, encompassing 16p11.2 and 16q11.2 regions, including TGFB1I1, AHSP, VPS35 and ORC6 genes, leading to partial characterization of the r(16). Although no phenotype has been correlated with overexpression of these genes, the 16p11.2 region is associated with neurodevelopmental disorders. Nevertheless individuals with microduplication of 16p11.2 and normal development have been described.

The lack of a precise definition of genetic content of the r(16) and its mosaic form leads to uncertain prognosis of clinical outcome.

After genetic counseling the couple opted to continue the pregnancy. At birth no major malformations were observed and a lower level of mosaic r(16) was observed in peripheral blood.

The mosaicism, as well as limitations of CMA in those cases, prevent a refined characterization of these genomic imbalances and pose a challenge in genetic counseling.

F. Brito: None. M. Silva: None. C. Alves: None. C. Ferreira: None. S. Serafim: None. L. Simão: None. B. Marques: None. S. Pedro: None. A. Tarelho: None. J. Furtado: None. P. Lopes: None. N. Silva: None. M. Viegas: None. A. Fernandes: None. F. Teixeira: None. S. Gomes: None. H. Correia: None.

P01.85A A novel partial deletion of the NR5A1 gene in a female patient with 46, XY disorder of sex development

O. Nagy1, J. Kárteszi2, M. Hartwig2, M. Tihanyi2, É. Erhardt3, A. Patócs4, A. Ujfalusi1

1Division of Clinical Genetics, Department of Laboratory Medicine, Faculty of Medicine, University of Debrecen, Debrecen, Hungary, 2Hospital of Zala County, Zalaegerszeg, Hungary, 3Department of Pediatrics, University of Pécs, Pécs, Hungary, 4Department of Laboratory Medicine, Endocrin Genetics Laboratory, Semmelweis University, Budapest, Hungary

NR5A1 (Steroidogenic factor 1, SF-1) is a transcriptional regulator of genes required for normal adrenal and gonadal development and function. Mutations in NR5A1 have been identified in patients with various forms of disorders of sex development (DSD), including gonadal dysgenesis with or without adrenal insufficiency. To date microdeletion or partial deletion involving the NR5A1 gene have been reported in only a few of cases with DSD.We present a patient with female external genitalia, clitoromegaly, bilateral ingiunal hernia containing testicles, mixed internal genitalia (uterus, Fallopian tube, epididymis), minor facial dysmorphism, normal adrenal function, low testosterone, high FSH levels. Family history is negative for disorders of sex development or premature ovarian failure. Chromosome analysis revealed a 46,XY karyotype. Array CGH did not detect pathogenic copy number variations. Next generation sequencing of the most common DSD genes did not identify pathogenic variants. Genomic DNA was screened for small deletion/duplication of genes commonly affected in DSD using the SALSA Intersex MLPA kit (MRC-Holland) according to manufacturer’s protocol. We identified a novel partial deletion encompassing the exons 5 and 6 of the NR5A1 gene leading to haploinsufficiency of the gene, that is alone sufficient to cause the patient’s abnormal sexual development.This report expands upon the range of mutations associated with NR5A1 gene, further confirms the role of NR5A1 deletions in 46,XY DSD and emphasises the utility of MLPA as a genomic screening tool in the workup of DSDs of unclear etiology.This study was supported by Ministry of National Economy, Hungary GINOP-2.3.2-15-2016-00039

O. Nagy: None. J. Kárteszi: None. M. Hartwig: None. M. Tihanyi: None. É. Erhardt: None. A. Patócs: None. A. Ujfalusi: None.

P01.87C A novel next generation sequencing assay for the detection of SMA carrier status and the number of copies of SMN2

R. Hrdlickova, J. Nehyba, C. Clear, D. Fox, A. Kothandaraman

Bioo Scientific, Austin, TX, United States

Spinal muscular dystrophy (SMA) is an inherited autosomal recessive neuromuscular disease caused by a defective SMN1 gene. SMN1 codes for a protein involved in RNA processing. The disease affects 1 out of 6000 newborns, while about 1 out of 50 individuals are carriers.

SMN1 has a paralog SMN2, which differs in coding sequence by just 1 base. SMN2 produces a functional protein though less efficiently than SMN1. These genes are located 500 kb apart on chromosome 5, which permits frequent recombination events that result in deletions, duplications or chimeras. Evolution has selected for multiple copies of SMN2 since having several copies can partially compensate for a non-functional SMN1 gene. Because of the severity of SMA, carrier and newborn screening is important. To be utilized in carrier screening, an assay has to detect one copy of SMN1 with 100% sensitivity, which necessitates distinguishing every SMN1 copy from SMN2.

An NGS assay was developed to detect one copy of SMN1 and the number of SMN2 copies in SMA carriers in an easy and scalable protocol suitable for automatization. The assay is performed in one tube, using a ligation-free method. Testing of clinical research samples with known numbers of SMN1 and SMN2 has demonstrated the precision of the assay to detect the number of SMN copies. The protocol can also be easily expanded to include all SMN1 mutations and/or be combined with assays to detect variants in other known carrier-screening genes for future clinical research.

Research use only. Not for diagnostic use.

R. Hrdlickova: None. J. Nehyba: None. C. Clear: None. D. Fox: None. A. Kothandaraman: None.

P01.88D A new candidate biomarker in Spermatogonial Stem Cell maturation : Dynamin 2


1Ankara University, School of Medicine, Department of Medical Biology, Ankara, Turkey, 2Ankara University, School of Medicine, Department of Urology, Ankara, Turkey

Dynamin 2 (DNM2) belongs to the GTPase superfamily. Beside having important roles in the regulation of membrane fission and fusion events dynamins induce differentiation of germ cells in spermatogenesis.

In this study, our aim was to investigate dynamin 2 gene expression and the relationship between this biomarker with impaired spermatogenesis in non-obstructive azoospermia (NOA) and obstructive azoospermia (OA) as control group. NOA group consisted of 20 hypospermatogenesis (HS), 20 maturation arrest (MA), 20 Sertoli Cell Only syndrome (SCO) patients. The biomarker was analyzed by RT-PCR array.

When compared with the control group, DNM2 gene expression in HP, MA and SCO groups showed 1.60 ± 0.30 (p > 0.05), 0.28 ± 0.05 (p < 0.001) and 0.86 ± 0.18 (p > 0.05) fold changes, respectively.

The dynamin family of proteins has important regulatory roles in membrane remodelling and endocytosis. By this way they also affects the cell viability. Thus, the decrease in DNM2 gene expression in MA group suggests that clinically DNM2 expression deficiency may cause maturation arrest. DNM2 is thought to be an important marker for the understanding the etiology of male infertility and in the development of treatment protocols for azoospermic patients with MA.

Y. Yukselten: None. O.S. Aydos: None. A. Sunguroglu: None. K. Aydos: None.

P01.89A Expression profiles of Spermatogonial Stem Cells’ marker genes in azoospermic patients with different pathologies


1Ankara University, School of Medicine, Department of Medical Biology, Ankara, Turkey, 2Ankara University, School of Medicine, Department of Urology, Ankara, Turkey

Spermatogonial stem cells (SSCs) have major roles on spermatogenesis and male fertility. Identification of SSCs and determination of their effects on impaired spermatogenesis are very important for elucidation of the etiology of male infertility. In this study, our aim was to investigate CD90, CD29, CD49f and POU5F1 genes expressions and the relationship between these biomarkers with impaired spermatogenesis in non-obstructive azoospermia (NOA) and obstructive azoospermia (OA) as control group. NOA group consisted of 20 hypospermatogenesis (HS), 20 maturation arrest (MA), 20 Sertoli Cell Only syndrome (SCO) patients. The biomarkers were analyzed by RT-PCR array. All groups were compared with the control group. CD29 and CD49f gene expressions showed 0.08 ± 0.01 and 0.65 ± 0.15 fold decreases (p < 0.05), in the HS group, while CD90 and POU5F1 genes showed 1.20 ± 0.21 and 0.83 ± 0.24 fold changes, respectively (p > 0.05). In the MA group, CD90, CD49f and POU5F1 gene expressions showed 0.40 ± 0.05, 0.22 ± 0.03 and 0.16 ± 0.02 fold decrease (p < 0.05) respectively, while CD29 gene showed 0.85 ± 0.10 fold change (p > 0.05). In the SCO group, CD90, CD29 and POU5F1 gene expressions showed 4.73 ± 1.03, 2.10 ± 0.47 and 3.56 ± 0.68 fold increases, (p <0.05) respectively, while CD49f gene expression showed 1.67 ± 0.37 fold change (p > 0.05). As a conclusion, it was found that SSCs biomarkers exhibit different profiles in NOA patients. We believe that the curative protocols which will be developed against the effects of these markers in spermatogenic defects will be very valuable in terms of human reproductive health.

S.O. Aydos: None. Y. Yukselten: None. A. Sunguroglu: None. K. Aydos: None.

P01.90B Robust preimplantation genetic diagnosis of spinal muscular atrophy combining triplex SMN1 detection with multi-microsatellite haplotyping

S. S. Chong1,2,3, M. Zhao1, M. Lian2, F. S. H. Cheah2

1Department of Paediatrics, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore, 2Khoo Teck Puat - National University Children's Medical Institute, National University Health System, Singapore, Singapore, 3Department of Laboratory Medicine, National University Hospital, Singapore, Singapore

Background: Preimplantation genetic diagnosis (PGD) of spinal muscular atrophy (SMA) is vulnerable to misdiagnosis arising from allele drop-out or exogenous DNA contamination. We describe a PGD strategy that detects SMA with high diagnostic confidence and accuracy.

Methods: A triplex PCR-minisequencing assay was developed for reliable detection of SMN1 and SMN2. A single-tube assay was developed to simultaneously amplify 13 highly polymorphic microsatellite markers located within 0.5 Mb of the 1.7 Mb duplicated SMN region on chromosome 15q13.2. Single cells were subjected to whole genome amplification, and separate aliquots were used for triplex SMN1/2 detection and for tridecaplex microsatellite-based haplotyping. The strategy was validated on 24 single cells isolated from cell lines of an SMA case-parent trio.

Results: Triplex SMN1/2PCR-minisequencing reliably detected single-copy SMN1 even in the presence of five SMN2 copies. Only SMN2 was detected in all SMA-affected samples. Observed heterozygosities of the 13 flanking microsatellite markers ranged from 0.56 to 0.8, and 98.4% of genotyped individuals were heterozygous for 2 or more markers on either side of SMN1. Triplex SMN1/2PCR-minisequencing results were completely correlated with observed marker diplotypes in all tested samples.

Conclusion: Triplex detection of SMN1 and SMN2, combined with linked multi-marker diplotyping, improves diagnostic confidence for SMA PGD. The highly polymorphic tridecaplex microsatellite panel can potentially be informative in most if not all at-risk couples.

S.S. Chong: None. M. Zhao: None. M. Lian: None. F.S.H. Cheah: None.

P01.91C Numerical and structural genomic aberrations in spontaneous abortions, detected by array CGH analysis

K. Belemezova1,2, M. Rizov1, M. Hristova-Savova1, E. Nikolova1, T. Timeva3,4, T. Milachich3, M. Yunakova3, P. Andreeva3,5, P. Chaveeva3, A. Shterev3, V. Djonov6, I. Dimova1,7

1Genetic Laboratory, SAGBAL „Dr Shterev“, Sofia, Bulgaria, 2Clinical Immunology Laboratory, St Ivan Rilski Hospital, Medical University Sofia, Sofia, Bulgaria, 3SAGBAL „Dr Shterev“, Sofia, Bulgaria, 4"Angel Kanchev" University of Ruse, Ruse, Bulgaria, 5South-West University "Neofit Rilski", Blagoevgrad, Bulgaria, 6Institute of Anatomy, University of Bern, Bern, Switzerland, 7Laboratory of Genomic diagnostics, Medical University Sofia, Sofia, Bulgaria

Background: Chromosomal abnormality in the product of conception is the reason for 50-70% of abortions. Chromosomal microarray analysis of fetal tissue has been proposed as a technique to evaluate the cause of isolated and recurrent early pregnancy loss (miscarriages) and later pregnancy loss (intrauterine fetal demise).

Materials and Methods: In this study, we applied array CGH method for analysis of chromosomal imbalances at a high resolution in 24 samples of spontaneous abortions.


Genomic aberration


Trisomy 22


Trisomy 16


Monosomy Х


Trisomy 18


Trisomy 19


Trisomy 20


Trisomy 21


Trisomy 15


Double trisomy 18 and 19


Duplication 7р


Tetrasomy 9р






Conclusion: In 15 of 24 spontaneous abortions (64%), genomic anomalies were discovered by array CGH analysis. Two thirds of them could be detected by the rapid DNA analysis (offered in our country as a rapid QF-PCR analysis for aneuploidies 13, 15, 16, 18, 21, 22, X and Y). Numerical anomalies were detected in 73% of aberrant cases, and in 27% - structural aberrations/chromothripsis were revealed. For couples with recurrent pregnancy loss and evidence of a structural genetic abnormality in one of the parents, preimplantation genetic diagnosis with transfer of unaffected embryos or the use of donor gametes might be considered for therapy.

Acknowledgment: SNSF grant No IZ73Z0_152454.

K. Belemezova: None. M. Rizov: None. M. Hristova-Savova: None. E. Nikolova: None. T. Timeva: None. T. Milachich: None. M. Yunakova: None. P. Andreeva: None. P. Chaveeva: None. A. Shterev: None. V. Djonov: None. I. Dimova: None.

P01.92D Phenotype variability of NR5A1 (SF1) gene mutations in DSD patients

V. B. Chernykh1, N. Y. Raygorodskaya2, N. V. Bolotova2, J. V. Paltseva3

1Research Centre for Medical Genetics, Moscow, Russian Federation, 2Saratov State Medical University, Saratov, Russian Federation, 3Clinical Hospital of Medical University named SR Mirotvortsev, Saratov, Russian Federation

Background: NR5A1(Steroidogenic Factor 1, SF1) gene mutations result in various forms of disorders of sex development (DSD), adrenal insufficiency, primary hypogonadism, male and female infertility.

The Aim: To evaluate the clinical variability of ambiguous phenotypes and the gender assignment in DSD patients with SF1 mutations.

Materials and Methods: Clinical examination, hormonal tests, ultrasound, laparoscopy, standard cytogenetic examination, and DNA analyses, including Sanger and next-generation sequencing.

Results: Case 1. A 46,XY female patient, aged 18 months with clitorophallus. A small testis was detected in the right labioscrotal fold. Hormonal tests showed LH - 0.25 IU/l, FSH - 12.9 IU/l, and testosterone after hCG stimulation - 0.3 nmol/l. Pelvic ultrasonography and laparoscopy showed the uterus, fallopian tubes and dysgenetic abdominal gonad on the left side. DNA analysis detected the heterozygous nonsense mutation c.256delA of the NR5A1 gene. Case 2. A 46,XY boy, aged 5 months, with undermasculinized Prader III genitalia, perineal hypospadias and small testes in bifid scrotum. Uterus was not found. Hormonal tests showed testosterone - 5.6 nmol/l, LH - 2.2 IU/l, FSH - 5.0 IU/l, and AMH - 43.8 ng/ml. DNA analysis found heterozygous p.R313C mutation of the NR5A1 gene.

Conclusions: The 46,XY patients with heterozygous NR5A1 mutations presented various phenotypes of gonadal development. The patient with nonsense mutation presented female phenotype with gonadal dysgenesis and Müllerian derivates. The male patient with NR5A1 missense mutation developed genitalia ambiguous with moderated undermasculinization and no Müllerian structures, that is characteristic for partial 46,XY gonadal (testicular) dysgenesis.

V.B. Chernykh: None. N.Y. Raygorodskaya: None. N.V. Bolotova: None. J.V. Paltseva: None.

P01.93A Higher levels of circulating mRNA for Tenascin X (TNXB) gene in maternal plasma at the second trimester of pregnancy in isolated congenital ventricular septal defects

C. Lapucci1, D. Morano2, S. Berto1, L. Walczer Baldinazzo1, D. Prandstraller3, F. Cro'1, A. Farina4

1Genetic department, Synlab Italy, Castenedolo, Brescia, Italy, 2Department of Morphology, Surgery and Experimental Medicine, Section of Obstetrics and Gynecology, University of Ferrara, Azienda Ospedaliero-Universitaria S.Anna, Cona, Ferrara, Italy, 3Pediatric Cardiology and Adult Congenital Unit, Sant'Orsola Malpighi Hospital, Bologna, Italy, 4Division of Obstetrics and Prenatal Medicine, DIMEC Sant'Orsola Malpighi Hospital, University of Bologna, Bologna, Italy

Introduction: Maternal plasma is a source of circulating placental nucleic acids; therefore it is a powerful tool for prenatal screening for congenital heart diseases (CHD). Previous studies showed that the identification of circulating mRNAs of MAPK1, IQGAP1 and Visfatin in maternal plasma had different gene expressions between CHD foetuses and control foetuses. This method allowed us to further investigate the expression of Tenascin X gene (TNXB) published as involved in CHD. Levels of circulating mRNA for the TNXB were investigated in pregnancies with ventricular septal defects during 2nd trimester of pregnancy.

Materials and Methods: The circulating mRNA assay was isolated from blood samples collected in several Institutes in Italy from March 2016 to July 2017. TNXB gene expression was investigated by RT-PCR in 10 women carrying foetus with ventricular septal defects and 31 controls at 19-24 weeks of gestation.

Results:RT-PCR showed that TNXB gene expression was higher in foetuses with ventricular septal defects with a 2-ΔΔCT value of 4.38  ±  3.01 vs 1.00  ±  0.80 in controls. CSH2 was used as housekeeping gene which expression value was normalised based on several gestational ages.

Conclusions: The data confirmed a link between circulating mRNA and CHD; ventricular septal defects could be associated with abnormal level of TNXB mRNA during the 2nd trimester of pregnancy. In future, the variation of TNXB gene expression will be used to identify the foetuses with CHD, however this molecular marker will be assessed throughout prospective studies in a wider population.

C. Lapucci: None. D. Morano: None. S. Berto: None. L. Walczer Baldinazzo: None. D. Prandstraller: None. F. Cro': None. A. Farina: None.

P01.94B Genetics of sex hormone-binding globulin and testosterone levels in fertile and infertile men of reproductive age

M. Grigorova1, M. Punab2, O. Poolamets2, M. Adler1,2, V. Vihljajev2, M. Laan1

1Institute of Biomedicine and Translational Medicine, University of Tartu, Tartu, Estonia, 2Andrology Unit, Tartu University Hospital, Tartu, Estonia

Introduction: Testosterone (T) is a central androgenic hormone. Sex hormone-binding globulin (SHBG) modulates T bioactivity. The current understanding of genetic variation contributing to male reproductive hormone levels is moderate. We aimed to confirm or reject genetic associations of top-loci (SHBG, GCKR, SLCO1B1, JMJD1C) from GWA-studies for SHBG and T, and uncover additional genetic effects on male fertility-related parameters (J Endocr Soc. 2017 1(6): 560-576).

Material and Methods: Study groups: young men (n = 540; 19.3 ± 1.8 years), severe idiopathic male infertility patients (n = 641; 31.6 ± 6.0 years), male partners of pregnant women (n = 324; 31.9 ± 6.6 years). Recruitment: Andrology Unit, Tartu University Hospital, Estonia. Analysis: genetic associations with reproductive parameters (linear regression, meta-analysis).

Results. Robust associations with SHBG for SHBG rs1799941 (meta-analysis: P=3.7x10-14; beta = 4.67(0.62) nmol/L), SHBG rs727428 (P=7.3x10-11; -3.74(0.57), SHBG Pro185Leu (rs6258; P=1.2x10-4, -12.2(3.17) and GCKR Pro446Leu (rs1260326; P=1.5x10-4; -2.2(0.59). Total T correlates with genetically modulated SHBG levels (r=0.48-0.74, P < 0.0001), guaranteeing stable availability of free T. Among infertile men, SHBG Pro185Leu shows downstream effect on LH (P=5.1x10-5; -1.66(0.57) IU/L) and FSH (P=3.4x10-3; -2.48(1.23) IU/L). No associations for SHBG Asp327Asn (rs6259), SLCO1B1 Val174Ala (rs4149056), JMJD1C rs7910927.

Conclusions. We replicated previously reported associations and detected additional effects for four of seven analysed GWAS hits. These variants are promising candidates for the future studies of hypotestosteronemia in aging men, as well as promising pharmacogenetic targets for hormone replacement therapy in males with fertility problems.

Funding: European Union through the European Regional Development Fund (project Happy Pregnancy, 3.2.0701.12-0047) and Estonian Research Council (IUT34-12, ETF9030, PUT181).

M. Grigorova: None. M. Punab: None. O. Poolamets: None. M. Adler: None. V. Vihljajev: None. M. Laan: None.

P01.95C Evaluation of an in-house-developed HRM based approach for Non-invasive prenatal diagnosis of beta thalassaemia

M. Karapanou, M. Vlahou, A. Floratos, A. Balassopoulou, E. Boutou

Laiko Hospital, Athens, Greece

Introduction: Thalassaemias are the most common monogenic disorders worldwide. In the majority of cases prenatal diagnosis of embryos at risk is performed through an invasive procedure that has 1-2% probability of miscarriage. We have previously developed a non-invasive prenatal testing (NIPT) approach, based on analyzing free fetal DNA. Here, we present the results of 32 cases where the parental beta globin gene mutations are inherited by the fetus. Our method is based on High Resolution Melting (HRM) analysis and covers all possible combinations of fetal inheritance.

Materials and Methods: ffDNA is isolated from 2ml maternal plasma and screened for the presence of β globin gene parental mutations based on an in-house developed HRM approach. Previously described SNPs in the vicinity of β globin gene, for which parents presented distinct haplotypes, were also determined and used for identification of the fetal fraction. Sex determination was also helpful in case of male embryos.

Results: Our hitherto results (32 cases) cover all possible combinations like male or female embryos that inherited either the paternal or the maternal mutation and cases where the embryo has inherited both parental mutations. Our results succeeded in obtaining the same diagnosis with the preceded analysis of corresponding chorionic villus sampling, showing up to now 100% diagnostic capacity.

Conclusions: The described approach, after further evaluation, may allow its application at the diagnostic level as a primary or secondary method for NIPT for beta thalassaemia and if accordingly adopted it may be used for other monogenic diseases as well.

M. Karapanou: None. M. Vlahou: None. A. Floratos: None. A. Balassopoulou: None. E. Boutou: None.

P01.96D Performance of the VeriSeq NIPT Solution - a novel PCR-free, paired-end sequencing-based noninvasive prenatal screening test

S. Duenwald1, D. Vavrek2, K. Meier2, C. Deciu2, M. Garcia-Ransom3, M. Halks-Miller4

1Illumina, Inc., San Francisco, CA, United States, 2Illumina, Inc., San Diego, CA, United States, 3N/A, San Francisco, CA, United States, 4Grail, San Francisco, CA, United States

Objectives: To develop and evaluate performance of a novel and highly automated, paired-end sequencing-based noninvasive prenatal test (NIPT) for fetal chromosomal aneuploidies on chromosomes 21, 18, 13, X, and Y.

Methods: We developed a PCR-free, paired-end sequencing-based NIPT, the VeriSeq™ NIPT Solution, that estimates fetal fraction (FF) and screens for fetal chromosomal aneuploidies in maternal plasma samples. Performance of VeriSeq NIPT Solution was determined by analyzing 3057 frozen maternal plasma samples that had been tested previously using a single-end sequencing-based NIPT (Verifi™ Prenatal Test, Illumina, Inc.); samples were blinded prior to reanalysis. Clinical outcomes (cytogenetic analysis or newborn physical examination) were available for all cases used to determine assay sensitivity and specificity.

Results: At a sequencing depth of 8M reads on a NextSeq™ 500 or 550, the limit of detection was below 3% FF for each chromosome-of-interest. Test performance was determined using 3107 samples, of which 21 (0.7%) were not reported because of QC failure on the only plasma aliquot available. Sensitivity was 98.9% (90/91), 90.0% (18/20), and 100% (8/8) for trisomy 21, trisomy 18, and trisomy 13, respectively. Specificities were ≥99.9% for all three trisomies; there were 8 false-positive results (one trisomy 21, three trisomy 18, and four trisomy 13). Concordance for sex chromosome aneuploidies ranged from 80.0-100%.

Conclusions: VeriSeq NIPT Solution showed good test performance for the detection of fetal chromosomal aneuploidies, even at low fetal fractions. This paired-end sequencing-based NIPT assay detects fetal chromosomal aneuploidies with high sensitivities and specificities and a low assay failure rate.

S. Duenwald: A. Employment (full or part-time); Significant; Illumina, Inc. E. Ownership Interest (stock, stock options, patent or other intellectual property); Modest; Illumina, Inc. D. Vavrek: A. Employment (full or part-time); Significant; Illumina, Inc. E. Ownership Interest (stock, stock options, patent or other intellectual property); Modest; Illumina, Inc. K. Meier: A. Employment (full or part-time); Significant; Illumina, Inc. E. Ownership Interest (stock, stock options, patent or other intellectual property); Modest; Illumina, Inc. C. Deciu: A. Employment (full or part-time); Significant; Illumina, Inc. E. Ownership Interest (stock, stock options, patent or other intellectual property); Modest; Illumina, Inc. M. Garcia-Ransom: E. Ownership Interest (stock, stock options, patent or other intellectual property); Modest; Illumina, Inc. M. Halks-Miller: E. Ownership Interest (stock, stock options, patent or other intellectual property); Modest; Illumina, Inc.

P01.97A The compare results of different whole genome amplification methods for purposes of cell-based noninvasive prenatal diagnosis

E. Musatova, A. Tveleneva, Z. Markova, N. Shilova


Introduction: Trophoblast cells in blood of pregnant women are a potential target for non-invasive prenatal testing, but the number of trophoblasts is very low. Obtaining the quantity and quality of DNA sufficient for subsequent analysis is key point in the possibility of studying the genetic material of single cells. To compare three different methods of whole genome amplification in model experiment was performed.

Materials and Methods: There were used 10 samples of artificially created mixtures. The artificial mixtures were prepared by mixing samples of peripheral venous blood of adult with cells’ samples of chorionic villus with known karyotype. There was used filtration through polycarbonate filters to isolate trophoblasts from artificial mixtures. Isolated cells were fixed with paraformaldehyde during sample preparation. Detection of trophoblasts on the filters was carried out by immunocytochemical staining with monoclonal antibodies to cytokeratin 7. Isolation of single cytokeratin7-positive cells was performed by laser microdissection. WGA performed by three different Methods: linker adapter PCR (LA-PCR), degenerate oligonucleotide primed PCR (DOP-PCR) and multiple displacement amplification (MDA). Analysis of molecular karyotype of WGA products was performed by comparative genomic hybridization. Lengths and concentrations of WGA products and results of comparative genomic hybridization results were compared.

Results:LA-PCR showed the highest sensitivity, specificity and uniformity of amplification in comparison with other methods.

Conclusion: The results of model experiment show that not all the WGA methods used are applicable for the analysis of single trophoblast cells, isolated by filtration and fixed with paraformaldehyde in the process of sample preparation.

E. Musatova: None. A. Tveleneva: None. Z. Markova: None. N. Shilova: None.

P01.98B Rare mosaicism with ring X chromosome in female patient with Turner syndrome and normal fertility: a case report

S. D. Teofilov, T. P. Ostojic, M. R. Bulatovic, J. D. Jovanovic, O. V. Miljanovic

Clinical Center of Montenegro, Podgorica, Montenegro

Introduction: Turner syndrome (TS) is a chromosomal condition that affects development in females, caused by the loss of an X-chromosome or X-structural abnormalities in the X-chromosome.This condition occurs in about 1 in 2,500 newborn girls. The most frequent constitutional karyotype of TS patients is 45,X. Small percentage individuals with TS also have another cell line with 46 chromosomes due to presence of a ring chromosome X instead of normal X chromosome

Materials and Methods: We present a case of a 37-year-old TS women who had normal fertility and three documented pregnancies. Cytogenetic analysis were based on the analysis of 200 metaphases. According to standard procedures, metaphase chromosomes were obtained from phytohaemagglutinin stimulated lymphocyte cultures from peripheral blood. The chromosomes were analyzed by Giemsa banding and karyotype according to the Use of the International System for Human Cytogenetic Nomenclature.

Results: Female patient with a phenotype of TS contacted a genetic counselor after two lost pregnancy. The result of cytogenetic analysis: 45,X[188]/46,X,r(X)(p22.1q28)[12]. After a few months, the patient came pregnant and prenatal diagnosis was conducted. The third pregnancy was successfully completed by the birth of a healthy boy.

Conclusions:TS women typically experience gonadal dysfunction that results in amenorrhea and sterility. Most likely, an ovum with the ring X chromosome can be fertile and can produce a viable zygote.TS associated with an X ring chromosome, r (X), is rare and this view is our country first case report on normal pregnancy in TS with 45,X/46,X,r(X) mosaicism.

S.D. Teofilov: None. T.P. Ostojic: None. M.R. Bulatovic: None. J.D. Jovanovic: None. O.V. Miljanovic: None.

P02 Sensory disorders (eye, ear, pain)

P02.02A Coding and non-coding structural variants of ABCA4 contribute to the missing heritability in Stargardt disease, a prevalent inherited retinal disease

M. Bauwens1, S. Naessens1, C. Van Cauwenbergh1,2, T. Van Laethem1, S. De Jaegere1, I. Balikova2, Y. Sznajer3, J. De Zaeytijd2, B. P. Leroy2,4, E. De Baere1

1Center for Medical Genetics Ghent, Ghent University, Ghent, Belgium, 2Department of Ophthalmology, Ghent University and Ghent University Hospital, Ghent, Belgium, 3Centre de Génétique Humaine, Cliniques universitaires St. Luc, Université catholique de Louvain, Brussels, Belgium, 4Division of Ophthalmology and Center for Cellular & Molecular Therapeutics, The Children's Hospital of Philadelphia, Philadelphia, PA, United States

Stargardt disease (STGD1) is a prevalent autosomal recessive inherited retinal disease (IRD), hallmarked by a large proportion of mono-allelic cases with one coding ABCA4 mutation, representing an interesting cohort to elucidate missing heritability. We aimed to find the second pathogenic allele in eleven mono-allelic STGD1 patients without pathogenic coding or non-coding sequence variant after ABCA4 locus resequencing. Targeted copy number variant (CNV) analysis using a customized platform (arrEYE) interrogating coding and non-coding regions of IRD genes, revealed four novel ABCA4 CNVs in unrelated STGD1 patients. A 10 kb deletion spanning ABCA4 exons 10-11 was identified in one patient. An out-of-frame deletion of exon 40-50 (19.112 bp) was identified in another patient and eliminates the 3’UTR of the gene, most likely rendering the resulting transcript unstable and prone to nonsense-mediated decay. An in-frame tandem duplication (exons 2-6) of 26 kb was identified in another patient. A non-coding intronic tandem duplication of 7 kb was identified in intron 1, occurring in trans with a mild mutation. We hypothesize a regulatory or splicing effect as a consequence of the duplication. All CNVs were delineated and investigated via bioinformatics analyses, pointing to a replicative-based mechanism for three out of the four CNVs and to non-homologous end joining for one CNV. These findings add to the ABCA4 mutational spectrum, characterized by a paucity of CNVs, with eight deletions reported so far. Finally, we highlight the importance of investigating non-coding regions of ABCA4 in mono-allelic STGD1 patients.

M. Bauwens: None. S. Naessens: None. C. Van Cauwenbergh: None. T. Van Laethem: None. S. De Jaegere: None. I. Balikova: None. Y. Sznajer: None. J. De Zaeytijd: None. B.P. Leroy: None. E. De Baere: None.

P02.03B Two novel homozygous splicing mutations in ARL2BP cause autosomal recessive Retinitis Pigmentosa

A. Fiorentino1, J. Yu2, G. Arno1,3, N. Pontikos1, S. Halford2, S. Broadgate2, M. Michaelides3,1, K. J. Carss4,5, F. L. Raymond5,6, M. E. Cheetham1, A. R. Webster1,3, S. M. Downes7, A. J. Hardcastle1, NIHR-BioResource Rare Diseases Consortium, UK Inherited Retinal Dystrophy Consortium

1UCL Institute of Ophthalmology, London, United Kingdom, 2University of Oxford, Oxford, United Kingdom, 3Moorfields Eye Hospital, London, United Kingdom, 4Department of Haematology, University of Cambridge, Cambridge, United Kingdom, 5Cambridge University Hospitals NHS Foundation Trust, Cambridge, United Kingdom, 6Department of Medical Genetics, University of Cambridge, Cambridge, United Kingdom, 7Oxford Eye Hospital, John Radcliffe Hospital, Oxford, United Kingdom

Introduction: Retinitis pigmentosa is the most common inherited retinal dystrophy, affecting approximately 1 in 4,000 individuals. Mutations in ARL2BP, encoding ADP-ribosylation factor-like 2 binding protein, have recently been implicated as a cause of autosomal recessive retinitis pigmentosa (arRP) with 3 homozygous variants identified to date (c.101-1G>C, c.134T>G [p.Met45Arg], c.207+1G>T) in cases of Arab-Muslim, European and Moroccan origin.

Materials and Methods:Whole-genome sequencing (WGS) or whole-exome sequencing (WES) was performed in 1051 unrelated individuals recruited to the UK Inherited Retinal Disease Consortium and NIHR-BioResource Rare Diseases research studies. Sanger sequencing was used to validate the next generation sequencing data, and RT-PCR analysis was performed on RNA extracted from blood from affected individuals to test for altered splicing of ARL2BP.

Results: Homozygous variants in ARL2BP (NM_012106.3) were identified in two unrelated individuals with RP. The variants, c.207+1G>A and c.390+5G>A, at conserved splice donor sites for intron 3 and intron 5 respectively, were predicted to alter the pre-mRNA splicing of ARL2BP. RT-PCR spanning the affected exon/intron boundaries showed both variants caused abnormal splicing of ARL2BP in samples from affected individuals compared to controls.

Conclusions: Our study identified 2 homozygous variants in ARL2BP as a rare cause of arRP. Further studies are required to define the underlying disease mechanism causing retinal degeneration as a result of mutations in ARL2BP.

A. Fiorentino: None. J. Yu: None. G. Arno: None. N. Pontikos: None. S. Halford: None. S. Broadgate: None. M. Michaelides: None. K.J. Carss: None. F.L. Raymond: None. M.E. Cheetham: None. A.R. Webster: None. S.M. Downes: None. A.J. Hardcastle: None.

P02.04C ATXN3 in retina: from ataxia to candidate gene for retinal dystrophies

V. Toulis1, M. Costa2, G. Marfany1,3,4

1Departament de Genètica, Microbiologia i Estadística, Facultat de Biologia, Universitat de Barcelona, Barcelona, Spain, 2Department of Neurology, Medical School, University of Michigan, Ann Arbor, MI, United States, 3Institut de Biomedicina de la Universitat de Barcelona (IBUB), Barcelona, Spain, 4Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Instituto de Salud Carlos III, Barcelona, Spain

Introduction: An expanded CAG repeat on the ATXN3 gene, encoding the deubiquitinating enzyme ATXN3, causes Spinocerebellar Ataxia Type 3 (SCA3), a late onset autosomal dominant neurodegenerative disorder. The physiological role of the wild-type ATXN3 protein, however, is not completely understood. Since deubiquitination seems to play a crucial role in photoreceptor development and differentiation, we aimed to define the function of ATXN3 in retinal formation combining animal and cell models.

Methods: Functional analysis in Atxn3 murine models was performed and the retinal phenotype was studied by immunofluorescence detection, transmission electron microscopy and photoreceptor isolation for a more detailed description. A cell model of ATXN3 knockdown was generated using RNAi in human ARPE-19 cells, which was analyzed by western-blot and immunofluorescence detection.

Results: The Atxn3 knockout mouse retinas showed a significant elongation of the photoreceptors, mislocalization of cone-specific phototransduction proteins, with concomitant elongation of the connecting cilium -the gate through which the proteins are transported to the photoreceptor outer segment-, and diminished phagocytosis of the photoreceptor outer segments by the retinal pigmented epithelium (RPE). These results were also confirmed in vitro in ARPE-19 cells.

Conclusions: Our work supports the key role of ATXN3 in retinal development and maintenance, particularly in the ciliogenesis and protein trafficking in photoreceptors, thereby highlighting ATXN3 as a good candidate gene for inherited retinal dystrophies.

Grants: V.T. is in receipt of a FPI fellowship (BES-2014-068639, MINECO). Work is supported by SAF2013-49069-C2-1-R (Ministerio de Economía y Competitividad, Spain), La Marató TV3 (project 201417.30), SGR2014-0932 (Generalitat de Catalunya).

V. Toulis: None. M. Costa: None. G. Marfany: None.

P02.05D First audiological characteristics of autosomal dominant hearing loss caused by a novel TBC1D24 gene alteration

D. Oziębło1,2, G. Tacikowska3, K. Kochanek4, H. Skarżyński5, M. Ołdak1

1Department of Genetics, Institute of Physiology and Pathology of Hearing, Warsaw, Poland, 2Postgraduate School of Molecular Medicine, Medical University of Warsaw, Warsaw, Poland, 3Department of Otoneurology, Institute of Physiology and Pathology of Hearing, Warsaw, Poland, 4Department of Experimental Audiology, Institute of Physiology and Pathology of Hearing, Warsaw, Poland, 5Oto-Rhino-Laryngology Surgery Clinic, Institute of Physiology and Pathology of Hearing, Warsaw, Poland

Background: To date different genetic variants in TBC1D24 gene were causally involved in the development of neurological syndromes and profound prelingual hearing loss (HL) inherited in a recessive manner (DFNB86). In 2014 the first and so far only TBC1D24 pathogenic variant has been linked with postlingual autosomal dominant HL (DFNA65).

Material and methods:Five-generation Polish family participated in the study. Clinical exome sequencing on the proband’s DNA and family segregation analysis of the identified variants were performed. Audiological assessment included pure tone audiometry (PTA), impedance audiometry, transient evoked otoacoustic emissions (TEOAE) and auditory brainstem responses (ABRs). Vestibular system function was evaluated using ocular and cervical vestibular evoked myogenic potentials (oVEMP, cVEMP). Temporal bone computed tomography was also performed.

Results: Genetic testing revealed a novel probably pathogenic c.553G>A (p.Asp185Asn) TBC1D24 variant, which fully segregated with HL in the studied family. Clinically, progressive HL involving mainly high frequencies was observed. No TEOAE were recorded in the study subjects and no or increased threshold of the stapedial muscle reflex was found. Function of the vestibulocochlear nerve measured by ABR was normal. No vestibular dysfunction and anatomical abnormalities of cochleovestibular system were detected.

Conclusions: Our results represent the first independent confirmation of TBC1D24 involvement in the development of autosomal dominant HL and the first thorough clinical characteristics of TBC1D24-induced autosomal dominant HL. The identified TBC1D24 variant affects the cochlear component of the auditory system and results in a high frequency HL usually observed in the third decade of life.

Supported by: 2016/22/E/NZ5/00470

D. Oziębło: None. G. Tacikowska: None. K. Kochanek: None. H. Skarżyński: None. M. Ołdak: None.

P02.06A Integrative identification of miRNA regulatory hubs as candidate molecular discriminators of chronic otitis media pathologies

I. Jovanovic1, T. Djuric1, M. Zivkovic1, S. Jesic2,3, A. Stankovic1

1Laboratory for Radiobiology and Molecular Genetics, Vinca Institute of Nuclear Sciences, University of Belgrade, Belgrade, Serbia, 2Medical Faculty, University of Belgrade, Belgrade, Serbia, 3Clinic for Otorhinolaryngology and Maxillofacial Surgery, Clinical Centre of Serbia, Belgrade, Serbia

Introduction: Chronic otitis media is followed by irreversible tissue damage and destruction of the middle ear structures. Cholesteatoma is an expanding, destructive epithelial lesion within the middle ear, commonly associated with chronic otitis media. Molecular mechanisms of chronic otitis media with cholesteatoma (COMch) and without cholesteatoma (COM) are challenging subject in research. The aim of this study was to perform transcriptome profiling of tissue from COMch and COM, and subsequent network analysis, to identify candidate miRNA regulatory hubs that can potentially serve as molecular discriminators of these two pathologies.

Materials and Methods: Transcriptome data were obtained from COMch (n = 2 patients) and COM tissue (n = 4 patients), by employing Illumina iScan microarray technology. Differentially expressed genes (DEGs) were identified using R/Bioconductor limma package. Functional analysis of DEGs and determination of network miRNAs by reverse mapping of target DEGs was done using miRNet software. Nonspecific miRNAs were excluded.

Results: Transcriptome analysis identified 169 DEGs. Significant biological processes involving DEGs covered epithelial cell and keratinocyte differentiation and extracellular structure organization. miRNet determined hsa-miR-8485, hsa-miR-24-3p, hsa-miR-34a-5p, hsa-miR-9-5p, hsa-miR-375, hsa-miR-145-5p, hsa-miR-21-5p as top hub miRNas with centrality degree ≥8.

Conclusions: The identified hub miRNAs should be the guide mark for future studies on mechanisms of miRNA activity in chronic otitis media pathologies. Also, hub miRNAs should be evaluated for their utilization in prevention and therapy, based on the growing potential of miRNAs to become clinically applicable biomarkers and therapeutic targets.

Acknowledgements: This work was funded by Serbian Ministry of Education, Science and Technological development Grant OI175085.

I. Jovanovic: None. T. Djuric: None. M. Zivkovic: None. S. Jesic: None. A. Stankovic: None.

P02.07B A study of the genetics of cholesteatoma through systematic review and whole exome sequencing

B. A. Jennings1, C. Philpott1, M. F. Bhutta2, D. Swan3, G. Willis4, J. Woods5, P. Prinsley5

1UEA, Norwich, United Kingdom, 2Brighton and Sussex University NHS Trust, Brighton, United Kingdom, 3NCIMB, Aberdeen, United Kingdom, 4Norfolk and Norwich University Hospital, Norwich, United Kingdom, 5James Paget University Hospital, Great Yarmouth, United Kingdom

Introduction A cholesteatoma is a mass of keratinising epithelium in the middle ear. It is a rare disorder, associated with significant morbidity. Its OMIM entry (#604183) cites minimal evidence for Mendelian inheritance, but we have observed 31 multiply affected families in Norfolk; including individuals with bilateral disease, suggesting a genetic component for its aetiology.

Methods We conducted a systematic literature review (SR) to identify any published studies about the genetics of cholesteatoma and established a national biobank for subsequent whole exome sequencing (WES) studies of familial disease. We have also completed a pilot sequencing study to identify candidate variants that segregate with the disease phenotype (using NimbleGen exome capture; and the Illumina HiSeq4000 platform).

Results In our SR, we identified 8 case-series with multiply-affected families and associations with congenital malformation syndromes. DNA and clinical data have been collected from 42 participants (from 9 multiply affected Norfolk families) to date. In 2018, participants will also be recruited from 10 additional UK centres. Our pilot WES study of 16 participants from 4 families identified 95,437 variants. Variant filtering, using pedigree analysis, has identified 430 candidate genes for further filtering using the Ensembl Variant Effect Predictor.

Conclusion We have completed our SR (see PROSPERO register CRD42015023579) and established the first biobank to explore the genetics-of-cholesteatoma. A WES strategy and bioinformatics pipeline have been developed in the pilot study; and preliminary filtering has identified candidate variants that could have an impact on TGF β signalling and inflammatory processes.

B.A. Jennings: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Modest; The Bernice Bibby Research Trust. C. Philpott: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Modest; Rosetrees Trust (A1136), The Bernice Bibby Research Trust. M.F. Bhutta: None. D. Swan: None. G. Willis: None. J. Woods: None. P. Prinsley: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Modest; Royal College of Surgeons, The Bernice Bibby Research Trust.

P02.08C The relationship between defects at the middle ear bone chain and NLRP3 and OPG gene polymorphisms at chronic otitis media patients

S. Keskin1, P. Ata2, B. Erkal2, T. Dogan2, A. Eren3, A. Tatlipinar4

1Fatih Sultan Mehmet Research and Training Hospital, Istanbul, Turkey, 2Marmara University School of Medicine, Istanbul, Turkey, 3Koc University, Istanbul, Turkey, 4Okan University School of Medicine, Istanbul, Turkey

Introduction: The purpose of this research is to investigate the relation between osteoprotogerin, NLRP3 inflammasome gene polymorphisms and ossicular chain defects.

Materials and Methods: There were 96 participants, composed of 30 patients with type 1 tympanoplasty due to chronic otitis media between 01/06//2013 - 01/06/2016, had intact ossicular chain; 20 patients who had ossiculoplasty because of ossicular chain erosion, and 46 healthy controls. DNA was isolated from peripheral blood using Puregene Qiagen kit. OPG and NLRP3 were analyzed at real-time PCR reaction with melting curve analysis. Also inner ear swabs were used for RNA isolation and OPG and NLRP3 expression were analyzed with Real-time quantitativePCR.

Results: There were 37 males (38.5%) and 59 females (61.5%) with an age range of 15 - 70 years, The average age was 35.05 ± 14.43 years. There was no statistically significant difference between three groups according to OPG c.226A>C (p.Thr76Pro) and NLRP3 c.592G>A (p. Val198Met) polymorphisms. But when analyzed between the controls and patient group as a whole there was a significant difference of OPG c.226A>C (p.Thr76Pro) polymorphism. The expression level of OPG was higher at the tympanoplasty group compared to ossiculoplasty group

Conclusion: There was no statistically significant difference of OPG and NLRP3 gene polymorphisms among three groups. But TNFRSF11B (OPG) c.226A>C (p.Thr76Pro) polymorphism was higher at tympanoplasty and ossiculoplasty patients as a whole compared to controls. The level of OPG at inner ear swabs taken from tuba Eustachi opening at nasopharynx was increased at tympanoplasty group.

S. Keskin: None. P. Ata: None. B. Erkal: None. T. Dogan: None. A. Eren: None. A. Tatlipinar: None.

P02.09D CIB2 gene in the pathogenesis of hearing loss - results of pooled DNA high throughput sequencing

A. Pollak1, J. Gzik1, A. Jacoszek2,3, U. Lechowicz1, P. Stawiński1, R. Ploski2, H. Skarżynski1, M. Oldak1

1Institute of Physiology and Pathology of Hearing, Warsaw, Poland., Warsaw, Poland, 2Department of Medical Genetics, Warsaw Medical University, Warsaw, Poland, Warsaw, Poland, 3Postgraduate School of Molecular Medicine, Warsaw Medical University, Warsaw, Poland, Warsaw, Poland

Hearing loss (HI) is a highly heterogenic and frequent disability that affects human senses. Calcium- and integrin-binding protein 2 (CIB2) is one of the genes involved in HI pathogenesis. The CIB2 protein is responsible for maintaining Ca2+ homeostasis in cells and interacting with integrins. The aim of presented study was to establish the frequency of CIB2-related hearing impairment based on pooled DNA sequencing. DNA samples derived from 900 patients with HI were pooled and 180 pools were obtained (5 DNA samples/pool). The whole CIB2 coding region was amplified in the DNA pools and sequenced on MiSeq apparatus (Illumina). Ten novel, potentially pathogenic variants were identified within the pools. DNA samples derived from pools with detected variants were separately resequenced via direct sequencing to confirm the presence of the variants as well as to assign the variants to a particular sample. Five of the primarily detected variants were confirmed in the heterozygous form, which was in line with the NGS results, the following five variants are currently being verified. Thus, our results confirmed that pool-seq is a cost and time effective approach that can be used for screening of causative HI variants in a large group of patients. This study was supported by National Science Centre grant 2011/03/D/NZ5/05592.

A. Pollak: None. J. Gzik: None. A. Jacoszek: None. U. Lechowicz: None. P. Stawiński: None. R. Ploski: None. H. Skarżynski: None. M. Oldak: None.

P02.10A PAX6 sequence variants affecting splicing cause сongenital aniridia

A. Filatova1, T. Vasilyeva1, M. Skoblov1,2, A. Marakhonov1,2, A. Voskresenskaya3, R. Zinchenko1,4

1Research Centre of Medical Genetics, Moscow, Russian Federation, 2Moscow Institute of Physics and Technology (State University), Dolgoprudny, Russian Federation, 3Cheboksary branch of S. Fyodorov Eye Microsurgery Federal State Institution, Cheboksary, Russian Federation, 4Pirogov Russian National Research Medical University, Moscow, Russian Federation

Introduction: Aniridia is a rare autosomal dominant panocular disorder caused by mutations in the PAX6 gene or chromosome 11p13 rearrangements.

Materials and Methods: DNA samples for analysis were obtained from patients with congenital aniridia (110 patients from 84 unrelated families). The search for mutations in the PAX6 gene was carried out by Sanger sequencing, MLPA and analysis of heterozygosity loss (LOH) in proband. To determine the effects of identified SNVs on PAX6 pre-mRNA splicing we used in vitro minigene assay.

Results: Molecular analysis of a large cohort of aniridia patients from Russia conducted earlier revealed a significant proportion of PAX6 mutations affecting splicing (14 from 81 mutations). We focused on 8 SNVs affected slicing: 6 deep-intronic and 2 exonic. These variants were classified as variant of unknown significance (VUS), benign or likely pathogenic according to ACMG recommendations. Human Splicing Finder and IntSplice on-line tools analysis predict them to disrupt PAX6 pre-mRNA splicing. To validate this hypothesis we used a minigene system and showed that all investigated sequence variants except one affect splicing. These variants result in open reading frame shifting, premature termination codon formation following by RNA degradation by nonsense-mediated decay. Thus, investigated SNVs produce a null allele and haploinsufficiency of the PAX6-function. So putative mutations were reclassified as loss of function.

Conclusions: Using functional in vitro analysis we confirmed the pathogenicity of 7 PAX6 mutations affecting splicing. Our results emphasized the necessity of such analysis and advanced search for PAX6 mutations. This work is supported by RFBR grant 17-04-00475.

A. Filatova: None. T. Vasilyeva: None. M. Skoblov: None. A. Marakhonov: None. A. Voskresenskaya: None. R. Zinchenko: None.

P02.11B Analysis through exome sequencing in patients with non familial primary congenital cataract

O. M. Messina-Baas1, C. Leon-Oviedo2, J. M. Valdes-Miranda3, M. R. Rivera-Vega2, R. Vega-Gama2, N. Xilotl-DeJesus2, V. Martínez-Montoya4, M. G. Tovar-Ayala2, L. M. Gonzalez-Huerta2, S. A. Cuevas-Covarrubias5

1Oftalmologia, Hospital General de Mexico, Mexico DF, Mexico, 2Genetica, Hospital General de Mexico, Mexico DF, Mexico, 3Gentica, Hospital General de Mexico, Mexico DF, Mexico, 4Genética, Hospital General de Mexico, Mexico DF, Mexico, 5Genetica, Hospital General de Mexico, Facultad de Medicina, Universidad Nacional Autonoma de Mexico, Mexico DF, Mexico

Cataracts are one important cause of blindness around the world. Primary congenital cataract has several patterns of inheritance and can be syndromic and non-syndromic. More than 100 loci have been associated with primary congenital cataract; autosomal dominant is the principal form of inheritance. Cataract is due to opacities of the lens resulting in defects of the refractive index. This produces alterations in the lens structure resulting in light scattering with a significant concentration of high-molecular-weight protein aggregates. The aim of the present study was to analyze, through exome sequencing, a sample of Mexican patients with non-familial primary congenital cataract. Genomic DNA was extracted through conventional methods and analyzed through exome sequencing; Sanger direct sequencing was performed to confirm the exome results. Molecular analysis identified defects in the HSF4, BFSP1, GCNT2, GJA8, BFSP1, NHS and MAF genes. These alterations were not found in healthy members of the families and 100 normal controls. This study allowed to describe the genes associated with primary congenital cataract in a sample of Mexican patients and enriched the spectrum of these molecular defects associated with hereditary cataract.

O.M. Messina-Baas: None. C. Leon-Oviedo: None. J.M. Valdes-Miranda: None. M.R. Rivera-Vega: None. R. Vega-Gama: None. N. Xilotl-DeJesus: None. V. Martínez-Montoya: None. M.G. Tovar-Ayala: None. L.M. Gonzalez-Huerta: None. S.A. Cuevas-Covarrubias: None.

P02.12C Evaluation of genetic variants associated with congenital stationary night blindness 2 (CSNB2)

A. Mihalich1, G. Cammarata2, E. Ponti1, G. Tremolada2, S. Bianchi Marzoli2, A. Di Blasio1

1Istituto Auxologico Italiano, Lab. of Medical Genetics, Milano, Italy, 2Istituto Auxologico Italiano, Dept. of Neurophthalmology and Electrophysiology, Milano, Italy

Introduction: CSNB2 is a non-progressive retinal disorder with clinical features that include reduced visual acuity, nystagmus and variable myopia or hypermetropia. Characteristic abnormalities are detected upon electroretinography, autofluorescence and infrared imaging. The results of these tests indicate that vision impairment in CSNB2 patients may derive from a decreased synaptic transmission between photoreceptor and second-order neurons. The Cav1.4 channel is involved in this process and CACNA1F gene encodes the pore-forming subunit α1. Since CACNA1F is located on the X chromosome, Cav1.4 channelopathies are typically affecting male patients.

Materials and Methods: We performed NGS of 32 known retinal disease genes on DNA of 3 male patients with CSNB2 and different symptoms of the disease. In two patients CACNA1F mRNA was also studied in peripheral lymphocytes.

Results: We have identified 3 unreported CACNA1F variants : in exon 4 c.425dupC, in exon 43 c.G5123C and in exon 48 c. 5800delG. The first variant causes insertion of a stop codon that could determine mRNA degradation by Nonsense Mediated Decay (NMD). However, mRNA evaluation revealed that the transcript was present. The second variant is located in a splicing site and skipping of exon 43 was demonstrated by mRNA sequencing. The third variant determinates a frameshift. In this patient mRNA was not available.

Conclusions: These results confirm that, in CSNB2 patients, variants in the same gene may be associated with different phenotypic characteristics. Moreover, they highlight the importance of studying gene expression and protein function to assess the biological significance of the variants detected.

A. Mihalich: None. G. Cammarata: None. E. Ponti: None. G. Tremolada: None. S. Bianchi Marzoli: None. A. Di Blasio: None.

P02.13D Large-scale molecular analysis of Hereditary Hearing Loss genes in Argentinean deaf patients: lookingfora needle in a haystack

P. I. Buonfiglio1, C. D. Bruque2, V. Lotersztein3, E. Goldschmidt4, A. B. Elgoyhen1, V. K. Dalamón1

1Instituto de Investigaciones en Ingeniería Genética y Biología Molecular “Dr. Héctor Torres” - INGEBI/CONICET, CABA, Argentina, 2Centro Nacional de Genética Médica A.N.L.I.S. Dr. Carlos G. Malbrán, CABA, Argentina, 3Servicio de Genética del Hospital Militar Central Cirujano Mayor Dr. Cosme Argerich, CABA, Argentina, 4Servicio de Genética del Hospital General de Agudos Dr. Juan A. Fernández, CABA, Argentina

Hereditary Hearing Loss (HHL) is a common trait affecting 1 in 2000 new born children. The presence of over 100 different genes involved in HHL, lead us to go on board with Whole Exome Sequencing (WES) in order to search for the causative mutations. The main objective of this project was to diagnose Argentinean deaf families and discover novel mutations or new genes involved in pathology. We designed a flowchart to exclude all the spurious variations obtained and target for few candidates. To approach this, we filtered results from WES, and candidate variations were segregated throughout family members. Variations positively selected, were analyzed using bioinformatic predictors and tracked in public databases. Additionally, conservation studies, structure and functional domain analysis in proteins, and in-vivo studies were performed. Using this strategy we analysed 15 WES results. We identified 16 causative mutations in 12 families with syndromic and non-syndromic hearing loss (11 missense, 4 frameshift and 1 splicing site mutations). Six were novel and functional studies of some of the identified mutations, using Zebra fish models, are under way. In the remaining 3 families, variables of uncertain significance were detected (Vous). To our knowledge this is the first study using WES to diagnose deaf patients in Argentina. We show in the present study that our flowchart is advantageous and noteworthy for large-scale molecular analysis in deaf patients. These findings clearly highlight the importance of genetic studies followed by in-sílico and in-vivo validation to better understand the genetic basis of Hereditary Hearing loss.

P.I. Buonfiglio: None. C.D. Bruque: None. V. Lotersztein: None. E. Goldschmidt: None. A.B. Elgoyhen: None. V.K. Dalamón: None.

P02.14A Personalized stem cell therapy to correct corneal defects due to a unique homozygous-heterozygous mosaicism of Ectrodactyly-Ectodermal dysplasia-Clefting syndrome

P. Nespeca1, V. Barbaro2, A. Nasti1, P. Raffa1, A. Migliorati1, S. Ferrari2, E. Palumbo3, M. Bertolin2, C. Breda2, F. Miceli4, A. Russo3, L. Caenazzo1, D. Ponzin2, G. Palù1, C. Parolin1, E. Di Iorio1

1Departments of Molecular Medicine, Padova, Italy, 2Fondazione Banca degli Occhi, Venezia, Italy, 3Departments of Biology, Padova, Italy, 4Departments of Neuroscience, Napoli, Italy

Introduction:Ectrodactyly-Ectodermal dysplasia-Clefting syndrome is a rare autosomal dominant disease caused by mutations in the p63 gene. To date, approximately 40 different p63 mutations have been identified, all heterozygous. No definitive treatments are available to counteract and resolve the progressive corneal degeneration due to a premature aging of limbal epithelial stem cells. Here, we describe a unique case of a young EEC patient, who was, surprisingly, homozygous for a novel and de novo R311K missense mutation in the p63 gene.

Material and Methods: Genetic analysis was performed in p63 gene to identify the disease-causing mutation. FISH, qRT-PCR and SNPs assay were executed to discover an additional alteration in p63. From oral biopsy, the heterozygous mutant cells were expanded performing clonal analysis.

Results: The analysis of the degree of somatic mosaicism in leukocytes and oral mucosal epithelial stem cells (OMESCs) showed that the 80% were homozygous mutant cells and 20% were heterozygous. Cytogenetic and molecular analyses excluded genomic alterations, thus suggesting a de novo mutation followed by an allelic gene conversion of the wild-type allele by de novo mutant allele as a possible mechanism to explain the homozygous condition. The R311K-p63 OMESCs were able to generate well-organized and stratified epithelia in vitro, resembling the features of healthy tissues.

Conclusion: This study supports the rationale for the development of cultured autologous oral mucosal epithelial stem cell sheets obtained by selected heterozygous R311K-p63 stem cells, as an effective and personalized therapy for this unique case of EEC syndrome, thus bypassing gene therapy approaches.

P. Nespeca: None. V. Barbaro: None. A. Nasti: None. P. Raffa: None. A. Migliorati: None. S. Ferrari: None. E. Palumbo: None. M. Bertolin: None. C. Breda: None. F. Miceli: None. A. Russo: None. L. Caenazzo: None. D. Ponzin: None. G. Palù: None. C. Parolin: None. E. Di Iorio: None.

P02.15B Mutations in F-Box and WD40 Domain Protein 11 (FBXW11) are associated with developmental eye and digit anomalies

F. Ceroni1, R. M. Young2, R. J. Holt1, B. Crespo3, N. Chassaing4,5, D. A. Bax1, C. Zazo Seco6, P. Calvas4,5, D. Gerrelli3, S. W. Wilson2, N. K. Ragge1,7

1Faculty of Health and Life Sciences, Oxford Brookes University, Oxford, United Kingdom, 2Department of Cell and Developmental Biology, University College London, London, United Kingdom, 3Great Ormond Street Institute of Child Health, University College London, London, United Kingdom, 4Service de Génétique Médicale, Hôpital Purpan, CHU Toulouse, Toulouse, France, 5UMR 1056 Inserm - Université de Toulouse, Toulouse, France, 6UDEAR, Université de Toulouse, UMRS 1056 INSERM-Université Paul Sabatier, Toulouse, France, 7West Midlands Regional Clinical Genetics Service and Birmingham Health Partners, Birmingham Women’s and Children’s NHS Foundation Trust, Birmingham, United Kingdom

Defects in eye morphogenesis can lead to a spectrum of structural ocular disorders, including anophthalmia, microphthalmia and coloboma (AMC). These conditions, affecting approximately 6-13 per 100,000 individuals, are clinically and genetically heterogeneous. The number of genes known to be involved in AMC is rapidly growing, yet only approximately 25% of patients receive a genetic diagnosis. Following whole-exome sequencing of 53 individuals with developmental eye anomalies, we identified one patient with bilateral microphthalmia and polydactyly carrying a de novo nonsynonymous variant (NM_012300.2:c.1087C>T, p.[Arg363Trp]) in FBXW11. This gene codes for an F-box protein involved in the regulation of cytoplasmic levels of β-catenin and therefore plays an important role in the Wnt signalling, a fundamental growth-control pathway also implicated in eye and limb development. Screening of FBXW11 in a further 252 patients with developmental eye anomalies identified three previously unreported 3’ untranslated region (UTR) variants in three unrelated individuals. In situ hybridisation experiments showed FBXW11 expression in developing human eye and brain, including the retinal neuroepithelium, telencephalon and the cerebellum. We also demonstrated that zebrafish models with reduced expression of the FBXW11 orthologues fbxw11a and fbxw11b display smaller, misshapen and underdeveloped eyes, and abnormal pectoral fin and jaw development.

Our results support the role of FBXW11 in eye and limb development, likely via modulation of the Wnt pathway, and therefore this gene represents an important candidate for human developmental disorders.

F. Ceroni: None. R.M. Young: None. R.J. Holt: None. B. Crespo: None. N. Chassaing: None. D.A. Bax: None. C. Zazo Seco: None. P. Calvas: None. D. Gerrelli: None. S.W. Wilson: None. N.K. Ragge: None.

P02.16C Molecular diagnosis in paediatric retinal dystrophies

M. Borregán

Hospital Sant Joan de Déu, Esplugues de Llobregat, Spain

Introduction: Hereditary retinal dystrophies (HRD) are a group of conditions with a great clinical and genetic heterogeneity; more than 300 responsible genes have been identified. The most prevalent HRD is retinitis pigmentosa (RP) (1: 4000).

Materials and Methods: Prospective study that includes 98 patients were evaluated in a tertiary hospital from 2010 to 2017. The clinical diagnosis was made through clinical exploration: visual acuity, biomicroscopy, fundus exploration and electrophysiological tests. According to the patient's collaboration, computerized campimetry, wide-field retinography, optical coherence tomography and microperimetry were performed. For the genetic diagnosis, a customized panel of 155 genes (IRD150) was designed by next generation sequencing and applied to children with clinical diagnosis of HRD.

Results: Identification of the causative variant was achieved in 70% of cases. In our cohort, mutations in ABCA4 gene were the most frequent, followed by the BEST1 gene (Best's disease) and RPGR (X-linked RP). 33% of molecular findings have not been previously described. Variant segregation within families was performed in order to increase evidence of pathogenicity. Adequate genetic counselling was offered in all cases. Regarding therapeutic options, 5 families with a diagnosis of ACL and mutations in RPE65 that could be candidates for gene therapy have been identified. In 97% of cases molecular diagnosis was concordant with clinical diagnostic orientation.

Conclusion: The identification of the molecular cause in HRD is essential for diagnosis and genetic counselling to the patient and the family. In some cases, a visual prognosis and potential treatment could be provided.

M. Borregán: None.

P02.17D The improved DNA diagnostics and genetic causes of early onset hereditary hearing loss in the Czech Republic

D. Safka Brozkova, S. Poisson Markova, P. Seeman

2nd Medical School and University Hospital Motol, Prague, Czech Republic;, Prague 5, Czech Republic

Hereditary hearing loss (HL) is the most common sensory deficit. About 60% of cases have genetic origin. The largest group of HL patients shows autosomal recessive inheritance. Up to 40% of HL in the Czech Republic is caused by biallelic mutations in the GJB2 gene. We have enlarged the diagnostics for the second most frequent type of HL - DFNB16. We use a simple and fast quantitative comparative fluorescent PCR and MLPA for detection of deletions or CNVs of the STRC gene. For patients who are not homozygous for the STRC deletion we use masivelly parallel sequencing (MPS) of panel of all genes already associated with autosomal recessive and X -linked types of NSHL. HaloPlex or SureSelect technology was used for library preparation.Overall 90 patients were tested by 3 consecutively updated gene panel versions (62, 67 and 70 genes were included). The causal variants on both alleles were found in 35% of patients (32 of 90). The most frequent causes of HL detected in the Czech population were biallelic probably pathogenic variants in following genes STRC, MYO15A, CDH23 and OTOG. Detection rate for each panel testing was different, namely 34%, 40% and 12.5%. In the first two panels most patients with at least one affected sibling were included. On the contrary only patients with sporadic HL were included in the last panel. Possible explanation for lower detection rate is that patients with acquired or autosomal dominant inherited HL were included in the last panel. Supported- by MH CR AZV 16-31173A

D. Safka Brozkova: None. S. Poisson Markova: None. P. Seeman: None.

P02.18A HARS2 sequence variants identified in young individuals with severe sensorineural hearing impairment

H. Karstensen1, N. Rendtorff1, L. Hindhede1, A. Stein2, R. Hartmann-Petersen2, K. Lindorff-Larsen2, A. Højland3,4, M. Petersen3,4, L. Tranebjærg1,5

1Kennedy Centre, Clinical Genetics Clinic, Copenhagen University hospital, Glostrup, Denmark, 2The Linderstrøm-Lang Centre for Protein Science, Department of Biology, University of Copenhagen, Copenhagen, Denmark, 3Department of Clinical Genetics, Aalborg University hospital, Aalborg, Denmark, 4Department of Clinical Medicine, Aalborg University hospital, Aalborg, Denmark, 5Department of Clinical Medicine, Panum Institute, University of Copenhagen, Copenhagen, Denmark

The genetics of hearing impairment (HI) is complex and highly heterogeneous, and variation in the same gene may be associated with different types of both syndromic and non-syndromic HI. Here we focus on HI, in association with the HARS2 gene, where biallelic missense variants previously have been associated with Perrault syndrome in two studies. The published HARS2 variants were all identified in patients, ascertained clinically because of the combination of HI and gonadal dysgenesis, the clinical hallmarks of Perrault syndrome. We have investigated the presence of HARS2 sequence variants in 79 cases (42 females; mean age 23  ±  21.5 years) with HI. Using Next Generation Sequencing we have identified three potential pathogenic missense variants. In a family with three affected siblings (two girls age 12 and 15, and one boy age 19), we identified compound heterozygosity for the variants: p.Lys58Glu and p.Arg150Cys, and in a 6 year-old girl we identified compound heterozygosity for the variants: p.Arg150Cys and p.Arg327Gln. Supporting evidence for the functional defects of the variants were obtained using in silico modeling of HARS2 variants identified in this and previous studies, by predicting both the change in protein stability (ΔΔG) and of potential loss of function using a co-variation-based analysis of multiple HARS2 sequence alignments. The three female patients with HARS2 variants, compatible with autosomal recessive inheritance, are young for determining signs of ovarian dysgenesis, but will be followed carefully. The present study raises the possibility for non-syndromic HI being the only phenotypic effect of biallelic HARS2 variation.

H. Karstensen: None. N. Rendtorff: None. L. Hindhede: None. A. Stein: None. R. Hartmann-Petersen: None. K. Lindorff-Larsen: None. A. Højland: None. M. Petersen: None. L. Tranebjærg: None.

P02.19B Genetic testing of hearing loss related genes in Slovene patients with next generation sequencing

K. Trebušak Podkrajšek1,2, T. Obreza1, J. Kovač1, S. Bertok1, T. Battelino1,2, S. Battelino3,2

1University Medical Centre Ljubljana, University Children´s Hospital, Ljubljana, Slovenia, 2University of Ljubljana, Faculty of Medicine, Ljubljana, Slovenia, 3University Medical Centre Ljubljana, Department of Otorhinolaryngology and Cervicofacial Surgery, Ljubljana, Slovenia

Introduction: Hereditary hearing loss (HL) is characterized by a vast phenotypic and genetic heterogeneity. Up to date it was associated to 119 genes; 73 with non-syndromic and 46 with syndromic HL (Hereditary Hearing Loss Homepage, http://hereditaryhearingloss.org, February 2018). In Slovenians, 26.6% of congenitally deaf patients1 and 11% of progressive HL patients2 had biallelic GJB2 mutations, where other genetic causes were limitedly exploited so far.

Material and Methods: 58 HL patients (12 congenital, 46 progressive HL) with negative GJB2 and GJB6 testing were subjected to targeted NGS with TruSightOne Sequencing Panel on the MiSeq platform followed by interpretation of variants in selected 111 HL associated genes and subsequent Sanger sequencing confirmation. Novel variants were evaluated with in silico prediction tools (Mutation Taster, CADD).

Results: Causative variants were detected in 33 patients in 16 genes (COL2A1, DIAPH3, ILDR, MYH14, MYO15A, MYO6, MYO7A, PDZD7, POU4F3, SLC17A8, TBC1D24, TCOF1, TECTA, USH2A, WFS1, TMPRSS3), most frequently in USH2A (19%), TMPRSS3 (16%) and SLC17A8 (9,7%). Six variants were not reported so far.

Conclusions: Causative variants explaining clinical presentation were detected in 28(48,3%) of GJB2/GJB6 negative patients, a diagnostic yield comparable to other reports. In remaining patients, molecular diagnosis could not be achieved, where five patients were carrying one likely causative variant in genes associated with autosomal recessive HL. Identification of genetic cause delivers an important information for the genetic counseling and to some extend enables HL clinical prediction and verbal communication improvement with cochlear implants.

1Battellino Int Adv Otol 2011;7:372-378

2Battelino J Laryngol Otol 2012;126:763-9

K. Trebušak Podkrajšek: None. T. Obreza: None. J. Kovač: None. S. Bertok: None. T. Battelino: None. S. Battelino: None.

P02.20C Evidence against TMPRSS3/GJB2 digenic inheritance of hearing loss - practical lesson learned in the era of high throughput sequencing

M. Oldak1, U. Lechowicz1, A. Pollak1, D. Oziębło1,2, H. Skarżyński3

1Department of Genetics, Institute of Physiology and Pathology of Hearing, Warsaw, Poland, 2Postgraduate School of Molecular Medicine, Medical University of Warsaw, Warsaw, Poland, 3Oto-Rhino-Laryngology Surgery Clinic, Institute of Physiology and Pathology of Hearing, Warsaw, Poland

Background: Hearing loss (HL) is the most common disability of human senses and genetic factors significantly contribute to its development. For some HL genes digenic inheritance has been documented. Recently it has been proposed for TMPRSS3 and GJB2 recessive pathogenic variants. As the data were not convincing, we aimed to verify the hypothesis.

Material and methods: From our genetic database of HL patients with at least one TMPRSS3 pathogenic variants (n = 42) we have selected individuals with additional pathogenic variants in the GJB2 gene (n = 3) and recruited for the study all of the available family members. Segregation analysis of the respective TMPRSS3 and GJB2 pathogenic variants within the families was performed on genomic DNA by Sanger sequencing.

Results: The strategy has allowed to identify four individuals who were double heterozygous for pathogenic TMPRSS3 and GJB2 variants. Two individuals from two different families had GJB2 c.35delG and TMPRSS3 c.208delC and in two other individuals from one family GJB2 c.35delG together with TMPRSS3 c.1343T>C variants were found. None of these subjects has ever reported hearing problems and their hearing status was normal.

Conclusions: Access to a large genetic database of HL patients allowed us to identify as many as four individuals with concomitant pathogenic variants in TMPRSS3 and GJB2 genes and without HL. Our data provide evidence against TMPRSS3/GJB2 digenic inheritance of HL. As high throughput sequencing is increasingly used for genetic testing, particular caution should be taken to not overinterpret the findings.

Supported by: 2011/03/D/NZ5/05592

M. Oldak: None. U. Lechowicz: None. A. Pollak: None. D. Oziębło: None. H. Skarżyński: None.

P02.21D Molecular Diagnostics of Hereditary Hearing Loss using Next-generation sequencing (NGS) in Estonian patients

R. Teek1, H. Roomere1, S. Pajusalu1,2, K. Õunap1,2

1Department of Clinical Genetics, United Laboratories, Tartu University Hospital, Tartu, Estonia, 2Department of Clinical Genetics, Institute of Clinical Medicine, University of Tartu, Tartu, Estonia

Introduction: Hearing loss (HL) is the most common sensory disorder worldwide. Despite this, DNA diagnostics of HL is complicated due to the heterogeneity of the disease. The most common cause for autosomal-recessive non-syndromic HL in the Caucasian population is mutations in the GJB2 gene. We know from our previous study that the most frequent mutations found among Estonian children with early onset HL were c.35delG and p.M34T (36%) in GJB2 gene. Still, other genes causing HL are rarely investigated in Estonian patients with HL. Study group consist of 60 probands with HL as main complaint or one of the the clinical features and who referred to geneticist during 2015-2017. In patients with isolated HL mutations in the GJB2 gene were excluded by GJB2 gene sequencing. Among 60 cases in 46 patients HL was the main complaint and in rest of 14 cases HL was one of the symptoms in addition to other clinical symptoms.

Methods: the NGS subpanel of genes associated with HL was performed (107 genes, Illumina TruSightOne panel) in 46 cases. For the other 14 cases with non-isolated HL only selected gene analysis or different subpanels were done.

Results: we found that the mutated genes were most commonly associated with Usher, Waardenburg and Alport syndrome. There were some mutations in genes related to non-syndromic hearing loss: MYO15A, TECTA, MARVELD2, MYO6 and STRC gene.

Conclusion: The using of NGS gene subpanel analysis for HL is valuable and increased the detection of genetic etiology of HL for 33% (20 patients).

R. Teek: None. H. Roomere: None. S. Pajusalu: None. K. Õunap: None.

P02.22A Targeted Re-Sequencing (TRS) and high density SNP array for the molecular characterisation of Hereditary Hearing Loss (HHL)

S. Lenarduzzi1, A. Morgan2, S. Cappellani1, V. Pecile1, M. Morgutti1, E. Orzan1, U. Ambrosetti3, M. La Bianca1, F. Faletra1, E. Grosso4, F. Sirchia1, A. Sensi5, C. Graziano6, M. Seri6, P. Gasparini1,2, G. Girotto1,2

1IRCSS Burlo Garofolo, Trieste, Italy, 2University of Trieste, Trieste, Italy, 3Univ. Milano U.O.C. Audiologia/Fondazione IRCCS Cà Granda Ospedale Maggiore Policlinico, Milano, Italy, 4Città della Salute e della Scienza University Hospital, Medical Genetics Unit, 10126, Torino, Italy, 5Department of Clinical Pathology, Medical Genetics Unit, Pievesestina, 47522, Cesena, Italy, 6Unit of Medical Genetics, S. Orsola-Malpighi Hospital, Bologna, Italy

Introduction: HHL is characterized by a high genetic heterogeneity hampering accurate molecular diagnosis that is essential for a proper genetic counselling and therapeutic options.

Methods: 166 Italian HHL cases (46 familial and 120 sporadic) were screened with a TRS panel of 96 deafness genes, using Ion Torrent PGM™. Annotated variants were filtered according to the pattern of inheritance, frequency, and pathogenicity. In negative cases, HD-SNPs arrays were used to identify CNVs using Genome Studio software for allele detection and genotype calling followed by PennCNV analysis.

Results: Approximately 40% of cases (66/166) were positive for GJB2 mutations, In the remaining 100 cases, TRS and SNPs array led to the characterization of approximately 30% of cases (30/100). In particular, we identified: 1) the first case of UPD involving LOXHD1, with the presence of both a small isodisomy segment spanning LOXHD1 and a heterodisomy on the remaining parts of Chr18, suggesting that the UPD is the result of a non-disjunction in meiosis I, followed by trisomy rescue, 2) two large deletions in OTOA and STRC, 3) four patients (13%) with mutations in TECTA which is thus the second major player in the Italian population (in both autosomal recessive and dominant families), followed by TMPRSS3 (3/100,10%), MYO6, MYO7A, MYO15A, PDZD7, and ACTG1 (2/100,7% each).

Conclusions: Thanks to this approach approximately 58% (96/166) of cases were characterized confirming the large mutation’s spectrum of HHL genes, as well as the efficacy of TRS and HD-SNPs arrays in helping an accurate molecular diagnosis.

S. Lenarduzzi: None. A. Morgan: None. S. Cappellani: None. V. Pecile: None. M. Morgutti: None. E. Orzan: None. U. Ambrosetti: None. M. La Bianca: None. F. Faletra: None. E. Grosso: None. F. Sirchia: None. A. Sensi: None. C. Graziano: None. M. Seri: None. P. Gasparini: None. G. Girotto: None.

P02.23B Genetic analysis of Pakistani families with hereditary retinal dystrophies

Z. Ravesh1,2, B. Wissinger2, M. Ansar1

1Department of Biochemistry, Lab of Genomics, Quaid-i-Azam University, Islamabad, Pakistan, 2Institute for Ophthalmic Research, Tübingen, Germany

Introduction: Hereditary retinal dystrophies (RD) are a group of heterogeneous disorders caused by mutations in over 200 genes. RDs can be subdivided into different groups based on the primary degeneration of rod or cone photoreceptor cells. This study was conducted to investigate the underlying RD genes and mutation in consanguineous families from Pakistan.

Material and Methods: Families were recruited after informed consent. Peripheral blood was collected and genomic DNA was extracted according to standard procedures. Homozygosity mapping was performed using Affymetrix Gene Chip Human Mapping 250 K-NspI arrays. The data were analyzed using Homozygosity Mapper software. Primers for amplifying all exons and intron boundaries were designed with Primer3plus software followed by PCR and Sanger sequencing. Minigene splicing assay and DNA walking were performed on respective samples.

Results: Homozygosity mapping identified a novel locus in family A, one novel gene (C8ORF37) in family B and a large novel genomic deletion of the (LCA5) gene in family C.

Conclusion: Our study indicates the heterogeneous nature of retinal dystrophies in Pakistan. Although earlier studies explored number of RD families, but underlying genes are still unknown for significant proportion of Pakistani families. Theoretically the traditional screening methods were successful, however genome wide scan were further implemented to improve the detection of underlying variations in Retinal Dystrophies. Therefore the amalgamation of traditional and modern molecular techniques is required for accurate identification of mutations. It is anticipated that these findings will contribute to future genetic testing in Pakistani families to minimize the risk of recessive disorders.

Z. Ravesh: None. B. Wissinger: None. M. Ansar: None.

P02.24C Variants in the FLRT3 and SLC35E2B identified using whole-exome sequencing in seven high myopia families from Central Europe

J. Swierkowska1, J. A. Karolak1,2, T. Gambin3,4, M. Rydzanicz5, A. Frajdenberg6, M. Mrugacz7, M. Podfigurna-Musielak8, P. Stankiewicz9, J. R. Lupski10,11,12, M. Gajecka1,2

1Institute of Human Genetics, Polish Academy of Sciences, Poznan, Poland, 2Poznan University of Medical Sciences, Poznan, Poland, 3Institute of Computer Science, Warsaw University of Technology, Warsaw, Poland, 4Baylor College of Medicine, Department of Molecular & Human Genetics, Houston, TX, United States, 5Medical University of Warsaw, Warsaw, Poland, 6Linköping University, Linkoping, Sweden, 7Medical University of Bialystok, Bialystok, Poland, 8Medical Centre Vigor Med, Leszno, Poland, 9Baylor College of Medicine, Houston, TX, United States, 10Baylor College of Medicine, Department of Molecular & Human Genetics and Department of Pediatrics, Houston, TX, United States, 11Human Genome Sequencing Center, Baylor College of Medicine, Houston, TX, United States, 12Texas Children's Hospital, Houston, TX, United States

Objectives: High myopia is an eye disorder with a refractive error greater than -6 diopters (D) with both environmental and genetic factors involved. Although a number of high myopia loci have been identified, the pathogenic genes in the general population have not been determined. The aim of the study was to identify sequence variants causative for high myopia in families from Central Europe.

Methods: Seventeen individuals from seven unrelated Central European families with hereditary high myopia were assessed using whole-exome sequencing analyses. Selected variants were further evaluated using Sanger sequencing and the segregation analyses in other family members.

Results: Out of a total of 72 variants identified, two novel missense variants c.1642G>C in FLRT3 and c.938C>T in SLC35E2B segregate with high myopia in the HM-78 family.

Conclusions: FLRT3 and/or SLC35E2B could represent disease candidate genes and may be potentially responsible for high myopia in the HM-78 family. Our data suggest that there are different genetic backgrounds of high myopia in each multiplex family, indicating the complex genetic causes of high myopia. This study was supported by National Science Centre in Poland, (Doctoral Scholarship Etiuda 2013/08/T/NZ5/00754), and Ministry of Science and Higher Education of Poland (27/GM/2017) and United States National Institutes of Health, National Human Genome Research Institute/National Heart Lung and Blood Institute (UM1HG0067542).

J. Swierkowska: None. J.A. Karolak: None. T. Gambin: None. M. Rydzanicz: None. A. Frajdenberg: None. M. Mrugacz: None. M. Podfigurna-Musielak: None. P. Stankiewicz: None. J.R. Lupski: None. M. Gajecka: None.

P02.25D Exome-based RetNet panel analysis in a Belgian cohort with inherited retinal disease (IRD) expands the molecular and phenotypic spectrum of recently identified IRD genes

S. Van de Sompele1, K. Van Schil1, C. Van Cauwenbergh1,2, T. Rosseel1, S. De Jaegere1, T. Van Laethem1, I. Balikova2, B. P. Leroy1,2,3, F. Coppieters1, E. De Baere1

1Center for Medical Genetics, Ghent University and Ghent University Hospital, Ghent, Belgium, 2Department of Ophthalmology, Ghent University and Ghent University Hospital, Ghent, Belgium, 3Division of Ophthalmology, The Children’s Hospital of Philadelphia, Philadelphia, PA, United States

Inherited retinal diseases (IRD) are a major cause of early-onset blindness, caused by mutations in >250 disease genes.

We investigated the currently known IRD genes (RetNet panel, https://sph.uth.edu/retnet) in 253 IRD probands using whole-exome sequencing (WES).

We identified (likely) causative variants in 55% of IRD probands, in frequently as well as rarely mutated RetNet genes. Specifically, two unrelated probands with simplex retinitis pigmentosa (RP) were homozygous for a novel frameshift variant in RAX2, in which so far only mono-allelic mutations have been described in dominant cone-rod dystrophy and macular dystrophy, suggesting a novel disease association to a known IRD gene. Secondly, ARL3 was recently proposed as a novel disease gene for autosomal dominant RP (ADRP). Interestingly, we identified the same ARL3 variant in an ADRP family, as well as a novel missense variant in a simplex RP patient, corroborating its involvement in ADRP. Lastly, in a proband diagnosed with non-syndromic IRD, bi-allelic truncating variants in CEP164 were identified. Previous observations suggest a strong genotype-phenotype correlation of CEP164-disease, with truncating mutations causing early-onset phenotypes of dysplasia and malformation in different organs, and hypomorphic alleles causing late-onset degenerative phenotypes including IRD. Our observations therefore challenge the hypothesis that null mutations underlie the most severe end of the phenotypic spectrum.

Overall, RetNet-WES analysis revealed an underlying genetic defect in 55% of a Belgian IRD cohort, expanding their disease associations (RAX2), supporting their role in IRD (ARL3), and providing new insights into phenotypic consequences of bi-allelic loss-of-function alleles (CEP164).

Funding: BOF15/GOA/011; 01D04716; AUGE/13/023; FWO.

S. Van de Sompele: None. K. Van Schil: None. C. Van Cauwenbergh: None. T. Rosseel: None. S. De Jaegere: None. T. Van Laethem: None. I. Balikova: None. B.P. Leroy: None. F. Coppieters: None. E. De Baere: None.

P02.26A Diagnostics of inherited retinal disorders: Slovenian experience using clinical exome sequencing

M. Volk1, A. Maver1, A. Fakin2, H. Jaklic1, M. Hawlina2, B. Peterlin1

1Clinical Institute of Medical Genetics, UMC Ljubljana, Ljubljana, Slovenia, 2Eye Hospital, UMC Ljubljana, Ljubljana, Slovenia

Introduction: Inherited retinal disorders are diagnostically challenging group of clinically and genetically heterogeneous diseases that may affect the entire retina or may be restricted to the macula. In addition, they may also be an ophthalmic manifestation of a systemic condition. There are approximately 261 genes associated with inherited retinal degeneration (https://sph.uth.edu/retnet/). We present our experiences using clinical exome sequencing for diagnostics of inherited retinal disorders.

Materials and Methods: 40 Slovenian patients with referral of suspected inherited retinal disorder were submitted to our institution. We performed targeted analysis of retinopathy-associated genes in the exome sequencing data. Mitochondrial sequence was also analysed based on the off-target exome reads. Filtered variants were analysed according to population frequency, characterization in ClinVar database, putative impact of the variant and predicted pathogenicity.

Results: Causative pathogenic variants were detected in 25 patients (63%). Of these, we confirmed the referral diagnosis in 23 cases, and reclassified initial diagnosis in two cases, one to mitochondriopathy and one to 16q11.2 microdeletion syndrome. In addition, we found variants of unknown clinical significance (VUS) in 6 cases and 9 cases with negative results. Altogether, we identified 19 known pathogenic and 12 novel pathogenic or likely pathogenic variants, 11 VUS and a microdeletion.

Conclusion: Our results demonstrate high diagnostic yield using clinical exome sequencing in diagnostics of inherited retinal disorders. Clinical exome sequencing should be a first-tier investigation not only because of genetic heterogeneity but also because of the potential to identify novel findings.

M. Volk: None. A. Maver: None. A. Fakin: None. H. Jaklic: None. M. Hawlina: None. B. Peterlin: None.

P02.27B Whole-gene panel sequencing in patients with inherited retinal dystrophies and mono-allelic variants: contribution of deep-intronic mutations and CNVs

M. Martín-Sánchez1, M. González-del Pozo1,2, N. Bravo-Gil1,2, C. Méndez-Vidal1, E. Rodríguez-de la Rúa3,4, S. Borrego1,2, G. Antiñolo1,2

1Department of Maternofetal Medicine, Genetics and Reproduction, Institute of Biomedicine of Seville, University Hospital Virgen del Rocio/CSIC/University of Seville, Seville, Spain, 2Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Seville, Spain, 3Department of Ophthalmology, University Hospital Virgen Macarena, Seville, Spain, 4Retics Patologia Ocular. OFTARED. Instituto de Salud Carlos III (ISCIII), Madrid, Spain

Introduction: Inherited Retinal Dystrophies (IRDs) are a group of rare disorders characterized by photoreceptors degeneration. Phenotypic and genetic heterogeneity hamper the diagnosis of IRDs. Although NGS technologies have shown to be helpful in this endeavor, some cases remain unsolved even after analyzing the whole exome. Remarkably, a number of patients carry only one mutated allele in a recessive gene, suggesting that screening non-coding regions could improve the diagnosis.

Materials and Methods: We performed a targeted sequencing study of 29 patients, 25 harboring mono-allelic mutations. The design comprised the entire genomic sequence of three genes (USH2A, ABCA4 and CEP290), the coding exons and flanking intronic bases of 76 IRDs related genes and two disease-associated intronic regions.

Results:Thirty-two mutations (8 novel) were identified in 18 probands (diagnostic rate: 62.07%). Among the variants, two were CNVs in USH2A, comprising one deletion (exons 22-55) and one duplication (exons 46-47) in two different patients. No deep-intronic mutations were detected.

Conclusions: Sequencing entire genes represents an intermediate strategy between exon-targeted and whole-genome sequencing, while reducing costs, time and effort. In our cohort, deep-intronic mutations may not play a significant role in the etiopathogenesis of IRDs in contrast to CNVs, which account for about 10% of the total mutations. Our approach to discover the second mutant allele in patients with mono-allelic mutations, together with our reliable CNVs detection algorithm, marks a step forward to increase diagnosis rate of IRDs.

Funding:PI15-01648 (ISCIII and European Union ERDF/ESF, “Investing in your future”), ER17P1AC702/2018 (CIBERER), CTS-1664 (Government of Andalusia).

M. Martín-Sánchez: None. M. González-del Pozo: None. N. Bravo-Gil: None. C. Méndez-Vidal: None. E. Rodríguez-de la Rúa: None. S. Borrego: None. G. Antiñolo: None.

P02.28C Clinical exome sequencing as a genomic approach for the diagnosis of unsolved cases of inherited retinal dystrophies

N. Bravo-Gil1,2, M. Martín-Sánchez1, M. González-del Pozo1,2, C. Méndez-Vidal1,2, S. Borrego1,2, G. Antiñolo1,2

1Department of Maternofetal Medicine, Genetics and Reproduction, Institute of Biomedicine of Seville, University Hospital Virgen del Rocio/CSIC/University of Seville, Seville, Spain, 2Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Seville, Spain

Introduction: Genomic approaches based on the analysis of previously disease-associated genes have shown a high diagnostic efficiency in Inherited Retinal Dystrophies (IRD). However, around 40-50% of cases remain unsolved after their application.

Materials and Methods: Targeted enrichment of around 5000 clinically relevant genes (SureSelect Focused Exome, Agilent) and Illumina HiSeq 3000 sequencing, were used for the molecular diagnosis of 12 unsolved IRD cases previously analyzed by a retinal gene panel.

Results: Application of our data analysis pipeline allowed the identification of causal mutations in 4 out of the 12 cases. Three of these cases carried variants in IRD-associated genes not initially included in the panel (FAM161A, MFRP and RP1L1) and 1 harbored a mutation in the orf15 of RPGR that, although it was included in the panel, represents a highly repetitive region technically difficult to capture. Furthermore, in 2 other families, we found candidate mutations in genes not previously associated with IRD for which segregation and/or additional studies are currently pending.

Conclusions: These results allowed us to improve the efficiency of our retinal panel and suggest that most of unsolved cases could carry understudied mutations in IRD genes (deep intronic or large rearrangements) or variants in genes not yet associated with any phenotype. In this regard, it will be increasingly difficult to identify new genotype-phenotype correlations of already known genes. Therefore, in our experience, genome sequencing would be the most appropriate approach for these cases.

Funding:PI15-01648 (ISCIII and European Union ERDF/ESF, “Investing in your future”), CIBERER, CTS-1664 (Andalusian Government).

N. Bravo-Gil: None. M. Martín-Sánchez: None. M. González-del Pozo: None. C. Méndez-Vidal: None. S. Borrego: None. G. Antiñolo: None.

P02.29D Iterative Sequencing and Variant Screening (ISVS) as a novel pathogenic mutations search strategy

U. Lechowicz1, T. Gambin2,3, A. Pollak1, A. Podgorska1, P. Stawinski1, A. Franke4, B. Petersen4, M. Firczuk5, M. Oldak1, H. Skarzynski6, R. Ploski7

1Department of Genetics, Institute of Physiology and Pathology of Hearing, Warsaw, Poland, 2Institute of Computer Science, Warsaw University of Technology,, Warsaw, Poland, 3Department of Medical Genetics, Institute of Mother and Child at Warsaw, Warsaw, Poland, 4Institute of Clinical Molecular Biology, Kiel University, Kiel, Germany, 5Department of Immunology, Center of Biostructure Research, Medical University of Warsaw, Warsaw, Poland, 6Oto-Rhino-Laryngology Surgery Clinic, Institute of Physiology and Pathology of Hearing, Warsaw, Poland, 7Department of Medical Genetics, Centre of Biostructure, Medical University of Warsaw, Warsaw, Poland

Autosomal recessive diseases (ARD) are typically caused by a limited number of mutations whose identification is challenged by their low prevalence. Our purpose was to develop a novel approach allowing an efficient search for mutations causing ARD and evaluation of their pathogenicity without a control group. We developed Iterative Sequencing and Variant Screening (ISVS) approach based on iterative cycles of gene sequencing and mutation screening, and ISVS Simulator software (http://zsibio.ii.pw.edu.pl/shiny/isvs/) for assessment of detected variants’ significance. As shown by simulations, ISVS efficiently identifies and correctly classifies pathogenic mutations except for cases where the gene of interest has extremely high number of low frequency nonpathogenic variants. By applying ISVS, we found 4 known and 9 novel (p.C73Y, p.S124L, p.C194Mfs*17, c.782 + 2 T > A, c.953-5 A > G, p.L325Q, p.D334Mfs*24, p.R436G, p.M448T) TMPRSS3 variants among deaf patients. For 3 known and 5 novel variants the disease association was supported by ISVS Simulator odds >90:1. Pathogenicity of 6 novel mutations has been supported by in-silico predictions of variants’ deleteriousness. By directly comparing variant prevalence in patients and controls, disease association was demonstrated only for two variants and it was relatively weak (P < 0.05). Summarizing, ISVS strategy and ISVS Simulator are useful for detection of genetic variants causing AR diseases.

Research was supported by the grants 2011/03/D/NZ5/05592 and 2014/13/B/NZ2/01248.

U. Lechowicz: None. T. Gambin: None. A. Pollak: None. A. Podgorska: None. P. Stawinski: None. A. Franke: None. B. Petersen: None. M. Firczuk: None. M. Oldak: None. H. Skarzynski: None. R. Ploski: None.

P02.30A Multiple differentially methylated regions specific to keratoconus overlap known keratoconus linkage loci

M. Gajecka1,2, J. A. Karolak1,2, M. Rydzanicz3, P. Gasperowicz3, R. Ploski3, J. P. Szaflik4, M. Kabza1

1Poznan University of Medical Sciences, Poznan, Poland, 2Institute of Human Genetics, Polish Academy of Sciences, Poznan, Poland, 3Medical University of Warsaw, Warsaw, Poland, 4Medical University of Warsaw, Warsawa, Poland

Keratoconus (KTCN) is a complex degenerative eye disorder in which development both genetic and environmental or behavioral components are involved. In order to verify if DNA methylation may also play a role in KTCN etiology, reduced representation bisulfite sequencing (RRBS) of human corneas obtained from five KTCN and five non-KTCN individuals was performed. Multiple differentially methylated regions (DMRs) specific to KTCN were detected and many of them overlap previously identified KTCN linkage loci (3p14.3, 15q24.1, 20p13, 5q35.2, 13q32.3) and chromosome arms (2q, 4q, 5p, 9p, 14q, and 17q). For many of these regions candidate genes and variants were never identified and our results may allow for significant narrowing of the genomic regions of interest and reduce the list of putative KTCN genes. We also reanalyzed the previously described RNA-Seq dataset of 25 KTCN and 25 non-KTCN human corneas and found that 12 genes downregulated in KTCN (IQGAP2, SYNJ2, CYP1B1, MYO1G, WNT5A, PARVB, MGLL, CDC25B, PSG3, FHL2, CAMK1D, and THEMIS) and six upregulated genes (WNT3, RB1, AC098617.1, RPS6KA2, PELI2, and PLXNA4) overlapped or were located in the near vicinity of the identified DMRs. Particularly interesting were the DNA methylation changes in two genes encoding Wnt ligands (Wnt3 and Wnt5A), as they provide a potential explanation for the Wnt signaling pathway deregulation observed in KTCN. Supported by National Science Centre in Poland, 2012/05/E/NZ5/02127.

M. Gajecka: None. J.A. Karolak: None. M. Rydzanicz: None. P. Gasperowicz: None. R. Ploski: None. J.P. Szaflik: None. M. Kabza: None.

P02.31B Mutations in TUBB4B cause a distinctive sensorineural disease

S. Méchaussier1, R. Luscan2, A. Paul2, G. Tian3, X. Gérard1, S. Defoort-Dhellemmes4, N. Loudon5, I. Audo6, S. Bonin7, J. LeGargasson8, J. Dumont9, N. Goudin10, M. Garfa-Traore10, M. Bras11, A. Pouliet12, B. Bessières13, N. Boddaert14, S. Lyonnet2, N. J. Cowan3, J. Rozet1, S. Marlin2, I. Perrault1

1Laboratory of Genetics in Ophthalmology (LGO), INSERMU1163 INSTITUT IMAGINE, Paris Descartes University, Paris, France, 2Laboratory of Embryology and genetics of human malformation, INSERMU1163 INSTITUT IMAGINE, Paris Descartes University, Paris, France, 3Department of Biochemistry & Molecular Pharmacology, NYU Langone Medical Center., New York NY 10016, NY, United States, 4Service d'Exploration de la Vision et Neuro-ophtalmologie. Pôle d'Imagerie et Explorations Fonctionnelles, CHRU de Lille, Hôpital Roger Salengro, Lille, France, 5Pediatric ENT Department, Hôpital Necker-Enfants Malades, APHP and Paris Descartes University, Paris, France, 6Centre Hospitalier National d'Ophtalmologie des Quinze-Vingts, Paris, France, 7Ophthalmology Department, Hôpital Lariboisière, APHP and Paris Diderot University, Paris, France, 8Visual Exploration Department, Hôpital Lariboisière, APHP, Paris, Diderot University, Paris, France, 9Cell Division and Reproduction. Institut Jacques Monod, CNRS, University Paris Diderot, Paris, France, 10Cell Imaging Core Facility of the Structure Fédérative de Recherche Necker INSERM US24/CNRS UMS3633 Imagine and Paris Descartes University, Paris, France, 11Bioinformatics Platform, Imagine and Paris Descartes University, Paris, France, 12Genomics Platform, Imagine and Paris Descartes University, Paris, France, 13Unité d'Embryo-foetopathologie, Hôpital Necker-Enfants Malades, APHP and Paris Descartes University, Paris, France, 14Department of Pediatric Radiology, Hôpital Necker-Enfants Malades, APHP, Paris, Descartes University, Paris, France

Leber congenital amaurosis (LCA) is a neurodegenerative disease of photoreceptor cells that causes blindness within the first year of life. It occasionally occurs in syndromic metabolic diseases and plurisystemic ciliopathies. Using exome sequencing in a multiplex family and three sporadic cases with an atypical association of LCA with early-onset hearing loss, we identified two heterozygous mutations affecting Arg391 in the β-tubulin 4B isotype-encoding gene (TUBB4B). Inspection of the atomic structure of the microtubule (MT) protofilament reveals that the β-tubulin Arg391 residue contributes to a binding pocket that interacts with α-tubulin contained in the longitudinally adjacent αβ-heterodimer, consistent with a role in maintaining MT stability. Functional analysis in cultured cells overexpressing FLAG-tagged wild-type or mutant TUBB4B and in patient skin-derived fibroblasts showed that the mutant TUBB4Bs were able to fold, form αβ-heterodimers and co-assemble into the endogenous MT lattice. However, the dynamics of growing MTs were consistently altered, showing that the mutations have a significant dampening impact on normal MT growth. Our findings provide a link between sensorineural disease and anomalies in MT behavior, and describe a syndromic LCA unrelated to ciliary dysfunction.

Acknowledgements: We are grateful to the families for their participation in the study. This work was supported by “S’entendre”, by grants from the Retina France to IP, UNADEV- AVIESAN ITMO MNP to JMR, FRM (DEQ20160334869) to JD, and by a grant (GM097376) from the NIH to NJC.

S. Méchaussier: None. R. Luscan: None. A. Paul: None. G. Tian: None. X. Gérard: None. S. Defoort-Dhellemmes: None. N. Loudon: None. I. Audo: None. S. Bonin: None. J. LeGargasson: None. J. Dumont: None. N. Goudin: None. M. Garfa-Traore: None. M. Bras: None. A. Pouliet: None. B. Bessières: None. N. Boddaert: None. S. Lyonnet: None. N.J. Cowan: None. J. Rozet: None. S. Marlin: None. I. Perrault: None.

P02.32C Progressive shrinking of the eye and visual impairment caused by biallelic variants in the MARK3 gene

E. Ranza1, H. Chung2,3, M. Ansar4, Y. M. Waryah5, P. Makrythanasis4,6, E. Falconnet4, M. Guipponi1, A. K. Narsani7, F. A. Santoni4,8, A. M. Waryah5, H. Bellen2,3,9, S. E. Antonarakis4

1Service of Genetic Medicine, University Hospitals of Geneva, Geneva, Switzerland, 2Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, United States, 3Jan and Dan Duncan Neurological Research Institute, Texas Children's Hospital, Houston, TX, United States, 4Department of Genetic Medicine and Development, University of Geneva, Geneva, Switzerland, 5Molecular Biology and Genetics Department, Medical Research Center, Liaquat University of Medical and Health Sciences, Jamshoro, Pakistan, 6Biomedical Research Foundation of the Academy of Athens, Athens, Greece, 7Institute of Ophthalmology, Liaquat University of Medical and Health Sciences, Jamshoro, Pakistan, 8Department of Endocrinology Diabetes and Metabolism, University Hospital of Lausanne, Lausanne, Switzerland, 9Howard Hughes Medical Institute, Houston, TX, United States

Developmental eye birth defects often severely reduce vision. We studied a cohort of more than 150 Pakistani consanguineous families with eye birth defects with at least two affected individuals. Families were analyzed by a combination of exome sequencing and homozygosity mapping. In one family (F105) three affected individuals were reported with progressive eye phthisis and visual impairment, and we identified a non-synonymous homozygous variant (NM_001128918.2:c.1708C>G:p.Arg570Gly) in MARK3. Given that MARK3 is highly conserved in flies (I: 55%; S: 67%) the fly homologue, par1, was knocked down in the eye during development. This resulted to a significant reduction in eye size and reduced amplitude of the electroretinograms, suggesting an impairment in visual transduction. Overexpression of the wild type Par1 protein in the developing eye caused a mild defect in eye morphology and a mild reduction in ERG amplitude. Expression of the Par1 Arg792Gly mutation, the corresponding mutation observed in the patients, induced severely reduced eye size that are rough, and cause a near complete loss of the amplitude of the ERG. Our data in flies and human indicate that the MARK3 variants correspond to a loss of function variant in human and flies, although the precise nature of the allele remains to be determined. GeneMatcher search did not detect additional cases. MARK3 is also known to interact with Mitf, which causes the COMMAD syndrome (MIM 617306) that includes severe microphthalmia. We conclude that MARK3 is a novel candidate for visual impairment with affected ocular anatomy.

E. Ranza: None. H. Chung: None. M. Ansar: None. Y.M. Waryah: None. P. Makrythanasis: None. E. Falconnet: None. M. Guipponi: None. A.K. Narsani: None. F.A. Santoni: None. A.M. Waryah: None. H. Bellen: None. S.E. Antonarakis: None.

P02.33D Spanish rare variants with unknown significance in candidate hearing loss genes for Meniere disease in Spain

A. Gallego-Martinez1, T. Requena1, P. Román-Naranjo1, D. Bobbili2, P. May2, J. Dopazo3, J. Lopez-Escamez1,2,4

1Centro Pfizer-Universidad de Granada-Junta de Andalucía de Genómica e Investigación Oncológica (GENYO), Granada, Spain, 2Luxembourg Centre for Systems Biomedicine (LCSB), Université du Luxembourg, Esch-sur-Alzette, Luxembourg, 3Clinical Bioinformatics Research Area, Fundación Progreso y Salud, Hospital Virgen del Rocío, Sevilla, Spain, 4Department of Otolaryngology, Instituto de Investigación Biosanitaria ibs.GRANADA, Hospital Universitario Virgen de las Nieves, Granada, Spain

Introduction: The genetic architecture in Meniere’s disease (MD), an inner ear disorder defined by episodic vertigo, sensorineural hearing loss (SNHL) and tinnitus, is not known. A panel of 69 hearing loss genes have been sequenced targeting rare variants in MD.

Materials and Methods: Nine hundred thirty DNA samples (890 cases and 40 controls) were pooled (each pool = 10 samples) and libraries were generated by HaloPlex PCR target enrichment system. Paired-end sequencing was performed in a Nextseq500 instrument. BWA and GATK were used for alignment and quality control. Variant calling was made through VarScan2. The estimated minor allelic frequencies (MAF) were compared with public references values in multiple populations, including Spanish variant server database.

Results: An enrichment of certain rare variants in cases was observed in genes such as MARVELD2. Some intronic variants with unknown significance showed a higher MAF compared with available data from Spanish population, including SLC12A2, TRIOBP, KCNQ1 and KCNE3 genes. Prioritizing pathogenicity prediction tools suggest that some of them should be consider as MD candidate variants. So, a novel synonymous variant in MARVELD2 gene in 3 unrelated individuals was found and validated by Sanger sequencing.

Conclusions: Spanish population has a specific enrichment of rare variants in some hearing loss genes. The involvement of MARVELD2 variant in MD has to be investigated. The functional role of the rest of the variants in SNHL and MD remains to be established.

Acknowledgments: Funded by 2016-Target sequencing from the Meniere Society, UK and Luxembourg National Research Fund (INTER/Mobility/17/11772209).

A. Gallego-Martinez: None. T. Requena: None. P. Román-Naranjo: None. D. Bobbili: None. P. May: None. J. Dopazo: None. J. Lopez-Escamez: None.

P02.34A Novel and ultrarare allelic variants in DIABLO and SLC7A8 genes in familial Meniere’s disease

P. Roman-Naranjo1, A. Gallego-Martinez1, M. C. Moleon-Gonzalez2, D. R. Bobbili3, T. Requena-Navarro1, P. May3, J. A. Lopez-Escamez1,2,3

1Otology & Neurotology Group CTS495, Department of Genomic Medicine, Centre for Genomics and Oncological Research - Pfizer/University of Granada/Andalusian Regional Government (GENYO), Granada, Spain, 2Department of Otolaryngology, Instituto de Investigación Biosanitaria ibs.GRANADA, Hospital Universitario Virgen de las Nieves, Granada, Spain, 3Luxembourg Centre for Systems Biomedicine (LCSB), University of Luxembourg, Esch-sur-Alzette, Luxembourg

Introduction: Meniere’s disease (MD), an inner ear disorder characterized by vertigo, sensorineural hearing loss and tinnitus, involves 7.5 cases in 100.000 people. MD shows familial aggregation and we have found rare variants in FAM136A, DTNA, PRKCB and SEMA3D genes in single families, showing genetic heterogeneity. We present a new family with autosomal dominant MD segregating novel variants for this condition.

Materials and Methods: A Spanish family including 3 affected women with MD, suggestive of an autosomal-dominant pattern of inheritance, was diagnosed. DNA was isolated from blood samples to perform whole-exome sequencing in the 3 cases. Single nucleotide variants (SNVs) and insertions/deletions (indels) were identified and annotated by GATK and Scalpel after quality controls. These variants were filtered by exome data from 1579 Spanish controls and the cut-off for minor allele frequency (MAF) was 0.001. Variants were prioritized according to pathogenicity by multiple bioinformatics tools.

Results: A total of 2822 rare SNPs and 779 rare indels segregated the phenotype. We identified 7 candidate variants with a MAF lower than 0.001. A novel non-synonymous SNV in DIABLO gene (c.C353G;p.T118R) and a non-synonymous SNV in SLC7A8 gene (rs146946494) were the two best rated variants.

Conclusions: Familial MD shows genetic heterogeneity. This study is the basis for a forthcoming functional study to evaluate how these variants are involved in MD.

Acknowledgments: Funded by 2016-Target sequencing from the Meniere Society, UK and Luxembourg National Research Fund (INTER/Mobility/17/11772209).

P. Roman-Naranjo: None. A. Gallego-Martinez: None. M.C. Moleon-Gonzalez: None. D.R. Bobbili: None. T. Requena-Navarro: None. P. May: None. J.A. Lopez-Escamez: None.

P02.35B The role of the gut microbiome in osteoarthritis and joint pain

C. G. Boer, D. Radjabzeh, C. Medina-Gomez, D. Schiphof, P. Arp, F. Rivadeneira, A. G. Uitterlinden, J. P. Hays, R. Kraaij, J. B. J. van Meurs

Erasmus MC, Rotterdam, Netherlands

Introduction: Osteoarthritis a degenerative joint disease is predominantly thought to be due to mechanical and genetic factors. However, also chronic inflammation plays a causal role in Osteoarthritis and Osteoarthritis related joint pain. A recently proposed hypothesis suggests the involvement of the gastrointestinal (gut) microbiome in (obesity-related) knee Osteoarthritis pain, through a low grade systemic inflammation mediated by bacterial endotoxins from the gut microbiome.

Materials and Methods: Gut microbial composition was determined by 16S ribosomal RNA-sequencing (n = 1,427, Rotterdam Study population). Association analysis were done in MaAs. We used the relative abundancy of gut microbiome taxonomies, adjusted for age, sex, technical covariates, and BMI. Knee joint pain measures are based on the standardised pain questionnaires(WOMAC) pain score.

Results: We find four highly significant associations (FDR<0.05) with knee joint pain on different taxonomic levels (Class-Order-Family-Genus) leading to the bacterial genus of Streptococcus (FDR= 1.96E-05,). Additional adjustment for BMI did not affect the identified association Also, the relative abundancy of Streptococcus in the gut is significantly associated with amount of effusion (assessed by MRI), a measure for knee joint inflammation (FDR=1.1E-2, n = 373).

Conclusions: We identified a significant positive association between Streptococcus abundance and knee joint pain and inflammation in the knee. This association seems independent of BMI. Streptococcus species are known to potentially cause osteomyelitis and rheumatic fever, an inflammatory joint disease affecting the heart and articular joints, indicating that Streptococcus itself or released components (such as membrane vescicles), can directly target the joint.

C.G. Boer: None. D. Radjabzeh: None. C. Medina-Gomez: None. D. Schiphof: None. P. Arp: None. F. Rivadeneira: None. A.G. Uitterlinden: None. J.P. Hays: None. R. Kraaij: None. J.B.J. van Meurs: None.

P02.36C Mutation screening and tissue expression patterns implicate SRY-box 14 (SOX14) in human eye and brain developmental anomalies

R. J. Holt1, D. Gold Diaz2, N. Chassaing3,4, L. E. Valdivia2,5, A. W. Wyatt6, J. Plaisancié2, D. Bourgeois4, C. Vincent-Delorme7, R. Osborne8, D. A. Bax1, C. Santos2, S. Broadgate9,1, L. Cooper-Charles10, L. E. Allen10, D. McMullan10, S. W. Wilson2, D. Gerrelli2, P. Calvas4,3, N. K. Ragge1,11

1Oxford Brookes University, Oxford, United Kingdom, 2University College London, London, United Kingdom, 3Université de Toulouse, Toulouse, France, 4Hôpital Purpan, Toulouse, France, 5Universidad Mayor, Santiago, Chile, 6University of British Columbia, Vancouver, BC, Canada, 7Hôpital Jeanne de Flandre, Lille, France, 8Wellcome Trust Sanger Institute, Cambridge, United Kingdom, 9University of Oxford, Oxford, United Kingdom, 10Birmingham Women’s Hospital, Birmingham, United Kingdom, 11West Midlands Regional Clinical Genetics Service and Birmingham Health Partners Birmingham Women’s and Children’s Hospital NHS Foundation Trust, Birmingham, United Kingdom

Anophthalmia, microphthalmia and coloboma (AMC) are developmental eye anomalies which occur in approximately 3 in 10,000 births. They are a genetically heterogeneous group of conditions, with over 300 genes having been identified as underlying them. However, only approximately 25% of patients receive a genetic diagnosis, depending on phenotype. The most frequent genetic cause of severe AMC are alterations in SOX2, a member of the SOXB family of transcription factors which have important functions in early central nervous system development. Both SOX2 and SOX14 bind the same transcription factor binding site, acting as enhancers and repressors, respectively. Therefore, SOX14 may mediate SOX2 targeted gene transcription and so be a candidate for AMC. We screened SOX14 in 306 individuals with developmental eye anomalies and identified four families carrying variants: a de novo heterozygous c.242G>T (p.Arg81Leu), a maternally inherited frameshift c.722delA, a de novo deletion of 7.78Mb including SOX14, and a paternally inherited SOX14 duplication. However, the link between the identified variations and the ocular phenotype still remain to be demonstrated. Furthermore, in situ hybridisation experiments using human embryonic tissue demonstrated that SOX14 is expressed in the eye and regions of the brain, including the hindbrain and diencephalon. Although, we developed a zebrafish model carrying CRISPR-induced mutations of sox14, these fish showed no alterations in eye development or gross anatomical abnormalities. We consider SOX14 to be a likely important candidate in mammalian nervous system development and should be considered a candidate for AMC disorders.

R.J. Holt: None. D. Gold Diaz: None. N. Chassaing: None. L.E. Valdivia: None. A.W. Wyatt: None. J. Plaisancié: None. D. Bourgeois: None. C. Vincent-Delorme: None. R. Osborne: None. D.A. Bax: None. C. Santos: None. S. Broadgate: None. L. Cooper-Charles: None. L.E. Allen: None. D. McMullan: None. S.W. Wilson: None. D. Gerrelli: None. P. Calvas: None. N.K. Ragge: None.

P02.37 Dmyopia and late-onset progressive cone dystrophy associate to LVAVA/MVAVA exon 3 interchange haplotypes of opsin genes on chromosome

O. Orosz1, I. Balogh1, G. Losonczy2

1Divison of Clinical Genetics, Department of Laboratory Medicine, Debrecen, Hungary, 2Department of Ophthalmology, Zuyderland-Eyescan, Sittard-Geleen, Netherlands

Introduction: Rare interchange haplotypes in exon 3 of the OPN1LW and OPN1MW opsin genes cause X-linked myopia, color vision defect, and cone dysfunction. The severity of the disease varies on a broad scale from nonsyndromic high myopia to blue cone monochromatism. Here, we describe a new genotype-phenotype correlation attributed to rare exon 3 interchange haplotypes simultaneously present in the long- and middle-wavelength sensitive opsin genes (L- and M-opsin genes).

Materials and Methods: A multigenerational family with X-linked high myopia and cone dystrophy was investigated by clinical exome sequencing.

Results: Affected male patients had infantile onset myopia with normal visual acuity and color vision until their forties. Visual acuity decreased thereafter, along with the development of severe protan and deutan color vision defects. A mild decrease in electroretinography response of cone photoreceptors was detected in childhood, which further deteriorated in middle-aged patients. Rods were also affected, however, to a lesser extent than cones. Clinical exome sequencing revealed the LVAVA and MVAVA toxic haplotypes in the OPN1LW and OPN1MW opsin genes, respectively.

Conclusions: Here, we show that LVAVA haplotype of the OPN1LW gene and MVAVA haplotype of the OPN1MW gene cause apparently nonsyndromic high myopia in young patients but lead to progressive cone dystrophy with deuteranopia and protanopia in middle-aged patients corresponding to a previously unknown disease course. To the best of our knowledge, this is the first report on the joint effect of these toxic haplotypes in the two opsin genes on chromosome X. Supported by Ministry of National Economy, Hungary GINOP-2.3.2-15-2016-00039.

O. Orosz: None. I. Balogh: None. G. Losonczy: None.

P02.38A A recurrent intergenic variant upstream of PRDM13 causes autosomal dominant progressive bifocal chorioretinal atrophy in two unrelated pedigrees

G. Arno1,2, R. S. Silva1,2, N. Pontikos1,2, V. Cipriani1,2, S. Defoort-Dhellemmes3, A. Kalhoro1,2, K. J. Carss4,5, F. L. Raymond4,6, V. van Heyningen1, A. T. Moore1,2,7, B. Puech3, A. R. Webster1,2

1UCL Institute of Ophthalmology, London, United Kingdom, 2Moorfields Eye Hospital, London, United Kingdom, 3Exploration de la Vision et Neuro-Ophtalmologie, Centre Hospitalier Universitaire, Lille, France, 4NIHR Bioresource – Rare Diseases, Cambridge University Hospitals NHS Foundation Trust, Cambridge Biomedical Campus, Cambridge, United Kingdom, 5Department of Haematology, University of Cambridge, NHS Blood and Transplant Centre, Cambridge, United Kingdom, 6Department of Medical Genetics, Cambridge Institute for Medical Research, University of Cambridge, Cambridge, United Kingdom, 7Ophthalmology Department, UCSF School of Medicine, San Francisco, CA, United States

Background: Progressive bifocal chorioretinal atrophy (PBCRA) is characterised by macular (and subsequent nasal) chorioretinal atrophic lesions evident during infancy. Prior genetic linkage pinpointed the disease locus to chromosome 6q14-16.2 overlapping the North Carolina Macular Dystrophy (NCMD) locus (MCDR1), a non-progressive developmental macular dystrophy in which mutations upstream of PRDM13 have been implicated.

Methods: Whole genome sequencing was performed to interrogate structural and single nucleotide variants in 5 PBCRA affected individuals from 2 unrelated families, including the 6q14-16.2 linked family (family 1) to gain insight into the cause of PBCRA.

Results: Seven novel variants were identified on the disease haplotype (chr6:98117898-103695199) in 3 individuals from family 1. Eleven novel variants were shared between the 2 affected individuals of family 2 at the same locus. A single variant (chr6:100046804T>C), 7.8kb upstream of the PRDM13 gene, was identified in both families, haplotype analysis confirmed that the variant arose independently.

Discussion: We report the likely pathogenic variant in two unrelated PBCRA families and expand the non-coding variant spectrum upstream of PRDM13; the PBCRA variant lies 5.7kb closer to PRDM13 than the 3 variants previously implicated in NCMD. Duplications encompassing PRDM13 have also been implicated in NCMD.

Taken together this suggests altered spatio-temporal expression of PRDM13 is a candidate disease mechanism in the phenotypically distinct NCMD and PBCRA. Since both disorders affect the macula at birth, exploring the functional distinctions between these variants will be key to understanding the disease mechanisms and the importance of PRDM13 in the context of normal retinal development.

G. Arno: None. R.S. Silva: None. N. Pontikos: None. V. Cipriani: None. S. Defoort-Dhellemmes: None. A. Kalhoro: None. K.J. Carss: None. F.L. Raymond: None. V. van Heyningen: None. A.T. Moore: None. B. Puech: None. A.R. Webster: None.

P02.39B Recurrence of the SLC22A4 p.C113Y deafness-causing mutation in North Africans

C. Chiereghin1, M. Robusto2, L. Mauri3, P. Primignani3, P. Castorina4, U. Ambrosetti4, S. Duga1,2, R. Asselta1,2, G. Soldà1,2

1Humanitas Clinical and Research Center, Rozzano, Italy, 2Humanitas University, Department of Biomedical Sciences, Pieve Emanuele, Italy, 3S.S. Genetica Medica, ASST Grande Ospedale Metropolitano Niguarda, Milano, Italy, 4Dipartimento di Scienze Cliniche e di Comunità, Università degli Studi di Milano and Fondazione IRCCS Cà Granda Ospedale Maggiore Policlinico, UO Audiologia, Milano, Italy

Introduction: Nonsyndromic sensorineural hearing loss (NSHL) is one of the most common congenital disorders in humans and is characterized by a high genetic heterogeneity. Recently, an homozygous missense variant (NM_003059.2:c.338G>A:p.C113Y) in the SLC22A4 gene (DNFB60 locus on chromosome 5), which encodes the organic cation transporter OCTN1, has been described in two Tunisian families affected by autosomal recessive NSHL.

Materials and Methods:Genome-wide linkage analysis with OmniExpressExome-8 v1.4 BeadChip array (Illumina) was performed on a large consanguineous NSHL family of Moroccan origin to highlight the genomic loci most likely to be involved in the disease. Whole-exome sequencing (WES) was then carried out on two affected siblings.

Results:Genome-wide linkage analysis on the pedigree pointed to a unique strong linkage signal peak (LOD > 3.5) in an interval of about 3Mb on chromosome 5q23.3-q31.1, encompassing the SLC22A4 gene, delimited by markers rs11241999 and rs2237060. Moreover, WES analysis identified the presence, at the homozygous state, of the previously described p.C113Y mutation in both affected siblings. Finally, Sanger sequencing confirmed the presence of the p.C113Y variant in all 6 affected relatives, and one unaffected sibling. The entire SLC22A4 gene was screened in additional 7 NSHL patients coming from North African countries, but no likely pathogenic variants were found.

Conclusion: This represents the first independent replication of the involvement of SLC22A4 in autosomal recessive NSHL, highlighting the importance of this gene, and of the p.C113Y variant, at least in the North African population. This study was supported by Fondazione Cariplo (grant#2013-0825).

C. Chiereghin: None. M. Robusto: None. L. Mauri: None. P. Primignani: None. P. Castorina: None. U. Ambrosetti: None. S. Duga: None. R. Asselta: None. G. Soldà: None.

P02.41D Autosomal dominant nystagmus in a large family associated to a novel mutation in the PAX6 gene

R. Vega-Gama1, L. M. Gonzalez-Huerta2, M. R. Rivera-Vega2, J. M. Valdes-Miranda2, N. Xilotl-DeJesus2, V. Martínez-Montoya2, M. Tovar-Ayala2, S. A. Cuevas-Covarrubias3

1Hospital General de Mexico, Mexico DF, Mexico, 2Genetica, Hospital General de Mexico, Mexico DF, Mexico, 3Genetica, Hospital General de Mexico, Facultad de Medicina, Universidad Nacional Autonoma de Mexico, Mexico DF, Mexico

Introduction: Congenital nystagmus is the most common eye movement disorder, with bilateral and involuntary oscillations of the eye, visual alteration and erroneous postures of the head. Nystagmus has been related to alterations in different genes with several patterns of inheritance. Apparently, X-linked inheritance pattern is the most common. Nystagmus can be a syndromic or non-syndromic entity. The PAX6 gene has been associated with different eye diseases such as optic nerve/eye colobomas, aniridia, anterior dysgenesis, cataract with corneal dystrophy, foveal hypoplasia, keratitis and optic nerve hypoplasia. The PAX6 gene is also involved in congenital nystagmus with photophobia, posterior embryoxoton and foveal hypoplasia.

Objective: In this study, we described a large Mexican family of four generations with idiopathic congenital nystagmus and no other alterations of the structures of the eye and a novel PAX6 gene mutation.

Material and Methods: Genomic DNA was extracted from peripheral blood of 15 members of a Mexican family and 100 normal controls. They were analyzed through exome sequencing.

Results: It was detected in PAX6 gene the mutation c.382C>T. This mutation was not found in healthy members of the family, normal controls and world databases.

Conclusion: The result include a mutation not previously reported in the literature. This mutation involves the PAX6 gene associated to nystagmus congenital with an autosomal dominant pattern and no other clinical manifestations. This is of great relevance for the genetic diagnosis of idiopathic congenital nystagmus associated to PAX6 gene.

R. Vega-Gama: None. L.M. Gonzalez-Huerta: None. M.R. Rivera-Vega: None. J.M. Valdes-Miranda: None. N. Xilotl-DeJesus: None. V. Martínez-Montoya: None. M. Tovar-Ayala: None. S.A. Cuevas-Covarrubias: None.

P02.42A Functional study of a PAX6non-stop mutation causing autosomal dominant Retinitis Pigmentosa

W. Lin1, C. Liu1, L. Hu1, C. Chung1, J. Chien2, M. Lin1, S. Huang1, Y. Ching1

1Tzu Chi University, Hualien, Taiwan, 2National Tsinghua University, Hsinchu, Taiwan

Introduction: Retinitis Pigmentosa (RP) is a group of genetically heterogeneous conditions of retinal dystrophies. PAX6 is a highly conserved transcription factor expressing in the eyes and controls the development of eyes. Null mouse mutation of Pax6 exhibit Small eye (Sey) phenotype, similar to the heterozygous mutations of Cad, a gene involved in de novo pyrimidine biosynthesis and tightly regulated during the cell cycle. In our previous study demonstrated that PAX6 regulated transcription of Cad. Functional studies of PAX6 mutations were largely unknown, our study is to investigate whether the PAX6non-stop mutation affects the gene expression of CAD as a way of functional analysis.

Materials and Methods: Structural modeling was used to predict the influence of the non-stop mutation. In vitro luciferase promoter assay and in vivo zebrafish knockdown and rescue experiments were performed to test the function of PAX6 mutation.

Results: The non-stop mutation we identified from a human RP family were predicted to have additional 36 a.a. forming an α-helical structure. We hypothesized that the mutation would lead to improper binding of PAX6 to the CAD promoter and lose transactivation ability. The luciferase promoter assay, PAX6 non-stop mutation abolished CAD promoter activity. The in vivo zebrafish morpholino knockdown with overexpressing the non-stop mutation PAX6 developed retinal abnormalities.

Conclusions: Our studies suggest that the non-stop mutation in PAX6 may reduce the expression of CAD leading to an insufficient development of the retina.

W. Lin: None. C. Liu: None. L. Hu: None. C. Chung: None. J. Chien: None. M. Lin: None. S. Huang: None. Y. Ching: None.

P02.43B Perrault-like phenotypes may account for some of the genetic and phenotypic heterogeneity within Perrault syndrome

L. A. M. Demain1,2, D. Antunes3, A. Heiberg4, J. O’Sullivan1,2, S. S. Bhaskhar1,2, R. T. O’Keefe5, W. G. Newman1,2

1Division of Evolution and Genomic Sciences, Faculty of Biology, Medicine and Health, School of Biological Sciences, University of Manchester, Manchester, United Kingdom, 2Manchester Centre for Genomic Medicine, Manchester University NHS Foundation Trust, Manchester, United Kingdom, 3Medical Genetics Department, Hospital de Dona Estefânia, Centro Hospitalar Lisboa Central, Lisbon, Portugal, 4Department of Medical Genetics, Oslo University Hospital, Oslo, Norway, 5Division of Cellular & Molecular Function, Faculty of Biology, Medicine and Health, University of Manchester, Manchester, United Kingdom

Perrault syndrome is a rare recessive condition characterised by sensorineural hearing loss (SNHL) and primary ovarian insufficiency (POI). Additional phenotypes, most commonly neurological, may also present. Perrault syndrome is clinically and genetically heterogeneous. Six causative genes have been identified to date, five of which function in mitochondrial homeostasis. In a number of cases no causative variants have been identified in known Perrault syndrome genes. Perrault syndrome may be difficult to clinically distinguish from overlapping phenotypes such as mitochondrial DNA depletion syndrome 7, which causes SNHL, POI and severe neurological dysfunction. We identified two cases of apparent Perrault syndrome due to variants in genes associated with other conditions. We identified a homozygous known pathogenic variant RMND1 c.713A>G, p.(Asn238Ser) in a proband with SNHL, POI and renal acidosis. RMND1 is essential for mitochondrial translation and recessive variants in RMND1 have been associated with renal defects, neurological phenotypes and SNHL. In a proband with SNHL, POI and mild intellectual disability we identified compound heterozygous rare variants in XPNPEP3; c.263A>G, p.Gln88Arg and c.1261C>G, p.His421Asp as the likely cause of the phenotype. XPNENP3 is involved in mitochondrial protein processing. Homozygous loss of function variants in XPNENP3 have been linked to nephronophthisis with some individuals also affected by SNHL. POI has not been reported secondary to variants in RMND1 or XPNENP3 and reflects the later onset and sex-limited nature of this phenotype. Overlapping phenotypes such as those reported here may account for some of the genotypic and phenotypic variation seen in Perrault syndrome.

L.A.M. Demain: None. D. Antunes: None. A. Heiberg: None. J. O’Sullivan: None. S.S. Bhaskhar: None. R.T. O’Keefe: None. W.G. Newman: None.

P02.44C 12q21.33 deletion in a patient with posterior amorphous corneal dystrophy

J. Lenk1, J. Porrmann2, A. Kahlert2, M. Smitka3, I. Eger4, E. Schröck2, K. Hackmann2, R. Herber1, F. Raiskup1, A. Tzschach2

1Department of Ophthalmology, Universitätsklinikum Carl Gustav Carus, Dresden, Germany, 2Technische Universität Dresden, Institute of Clinical Genetics, Dresden, Germany, 3Children´s hospital, Universitätsklinikum Carl Gustav Carus, Dresden, Germany, 4Department of Neuropediatrics, Städtisches Klinikum Görlitz, Görlitz, Germany

Posterior amorphous corneal dystrophy (PACD) (OMIM 612868) is a rare autosomal dominant disorder characterized by partial or complete posterior lamellar corneal opacification, decreased corneal thickness and flattening of the corneal curvature. Onset of the disease is in the first years of life. PACD is associated with heterozygous deletions in chromosome band 12q21.33-q22 harbouring the genes DCN (Decorin, OMIM 125255), KERA (Keratocan, OMIM 603288), LUM (Lumican, OMIM 600616) and EPYC (Epiphycan, OMIM 601657) which encode small leucine-rich proteoglycans. Only four families with deletions of this region have been published to date. We report on a 7-year-old male patient with PACD in whom an interstitial deletion in 12q21.33 was detected by array CGH. Subsequent FISH analyses in the parents revealed a balanced insertional translocation of this 12q21.33 segment into the long arm of one chromosome 13 in the mother. This family corroborates the association of 12q21.33 deletions with PACD and constitutes the first example of the involvement of a balanced chromosome aberration that predisposes to this rare disorder.

J. Lenk: None. J. Porrmann: None. A. Kahlert: None. M. Smitka: None. I. Eger: None. E. Schröck: None. K. Hackmann: None. R. Herber: None. F. Raiskup: None. A. Tzschach: None.

P02.45D First independent confirmation of the PTPRQ gene involvement in autosomal dominant hearing loss

D. Oziębło1,2, A. Adamiok1, A. Sarosiak1,2, H. Skarżyński3, M. Ołdak1

1Department of Genetics, Institute of Physiology and Pathology of Hearing, Warsaw, Poland, 2Postgraduate School of Molecular Medicine, Medical University of Warsaw, Warsaw, Poland, 3Oto-Rhino-Laryngology Surgery Clinic, Institute of Physiology and Pathology of Hearing, Warsaw, Poland

Background: Hearing loss (HL) is the most common birth defect affecting about 1-6/1000 births and the most common disability of human senses. Genetic factors play an important role in the development of HL. The PTPRQ gene has been previously reported in the context of autosomal recessive HL and in 2017 for the first time in the development of autosomal dominant HL.

Material and methods: A five-generation Polish family with progressive, high frequency autosomal dominant HL was recruited for the study. Genomic DNA was isolated from peripheral blood samples or buccal swabs of available family members. Clinical exome sequencing was conducted in the proband’s DNA sample. Family segregation analysis of the identified variants was performed using Sanger sequencing.

Results: Molecular genetic testing showed the presence of probably pathogenic c.6881G>A (p.Trp2294*) variant in the PTPRQ gene, which fully segregated with HL observed in the family. The identified variant is located in the last coding exon of the PTPRQ gene and introduces a premature stop codon. The c.6881G>A transition has not been reported in population databases. To date, the PTPRQ variant has been described in one family worldwide and is the only PTPRQ genetic variant causally involved in autosomal dominant HL.

Conclusions: Identification of the c.6881G>A variant provides an independent confirmation of the PTPRQ involvement in autosomal dominant HL, which is progressive, affects high frequencies and is usually diagnosed in the first decade of life.

Supported by: 2016/22/E/NZ5/00470

D. Oziębło: None. A. Adamiok: None. A. Sarosiak: None. H. Skarżyński: None. M. Ołdak: None.

P02.46A Using UK Biobank for common conditions of poorly characterised genetic aetiology: the example of retinal detachment

T. S. Boutin1, D. G. Charteris2, A. Chandra3, D. Mitry4, V. Vitart1

1MRC HGU, MRC Institute of Genetics and Molecular Medicine, Edinburgh, United Kingdom, 2Moorfields Eye Hospital, London, United Kingdom, 3Southend University Hospital, Westcliff-on-Sea, United Kingdom, 4Department of Ophthalmology, Royal Free NHS Foundation Trust, London, United Kingdom

Introduction: Rhegmatogenous retinal detachment (RRD) is a common cause of emergency ophthalmic intervention. There is some evidence for a genetic contribution to idiopathic RRD but studies have been limited. Here we evaluated the use of the UK Biobank Resource to increase insight into RRD genetic aetiology.

Material and Methods: This research has been conducted using the UK Biobank (UKBB) resources and two sets of clinically ascertained RRD cases (close to 1000 genotyped cases). A discovery genome-wide association study was carried out for retinal detachment using the largest dataset, UKBB, using BOLT-lmm which allows rapid analysis of large (N> 5000) datasets. Associated conditions, high myopia and cataract, were analysed in UKBB, to evaluate the amount of common genetic underpinning. RRD associations were replicated using the datasets of clinically ascertained cases and controls.

Results: Discovery GWAS was performed using N=3977 self reported or hospital record linked retinal detachment cases and revealed three genome-wide significant signals as well as a low but significant heritability, 24% on the liability scale. High myopia showed substantially higher heritability, with, potentially mechanistically interesting, some but not all the top signals also influencing retinal detachment. Two of the three genome-wide significant retinal detachment associations seem specific to this phenotype with no association with high myopia, cataract, nor any phenotypes from publicly available PheWAS databases. One of those hits, in the FAT3 gene, was replicated in the clinically ascertained datasets.

Conclusions. Biobank resources especially linkage to health records are a very promising complement to studies of well ascertained cases.

T.S. Boutin: None. D.G. Charteris: None. A. Chandra: None. D. Mitry: None. V. Vitart: None.

P02.47B Whole exome sequencing of a cohort of Polish patients with retinal disorders - NeuStemGen project

R. Szymańczak1, P. Łyszkiewicz1, A. Wąsowska1, E. Matczyńska1, W. Krysa1, K. Kamińska1, E. Ewa Suchecka1, J. Kosakowski1, M. Jurkowska1, A. Pałucha1, M. Jędrzejowska1, S. Teper2, E. Wylęgała2, E. Pius-Sadowska3, A. Machalińska3, A. M. Boguszewska-Chachulska1

1Genomed SA, Warsaw, Poland, 2Chair and Ophthalmology Department, II School of Medicine with the Division of Dentistry in Zabrze, Medical University of Silesia, Katowice, Poland, 3Pomeranian Medical University, Szczecin, Poland

The goal of the NeuStemGen project is to identify novel biomarkers, which may serve as diagnostic and prevention targets in degenerative disorders. Identification of recurrent pathogenic variants will allow to develop new therapies, such as genome editing and gene therapy, for patients with progressive, untreatable retinal dystrophies and degenerations.

The specific approach adopted, based on WES, served to identify not only pathogenic variants in genes associated retinal disorders but also in novel genes.

WES was performed for the cohort of 94 patients with clinical symptoms of retinal dystrophies while the POLGENOM genomic database of long-lived Poles was used as the control group. An in-house bioinformatic pipeline was applied to analyse SNVs and InDels. Further analysis involved Gemini, enabling an analysis of the full set of samples in search for rare pathogenic variants in the whole exome. CNVs were identified using XHMM, these results were validated using aCGH arrays (180K), showing conformity for larger structural variants.

Pathogenic variants were identified predominantly in genes already known to cause retinal degenerations (such as ABCA4, USH2A, EYS, RHO) although variants in novel genes, previously reported as single cases, were also uncovered. A gain of chr 8 was identified as a possible cause of retinal dystrophy in one patient.

The results of this analysis will support further development of the targeted retinal panel covering most pathogenic variants occurring in the Polish population and allowing for a fast, low-cost genetic analysis, preceding selection of a personalised therapy.

Supported by NCBiR, Project No.STRATEGMED1/234261/2/NCBR/2014.

R. Szymańczak: None. P. Łyszkiewicz: None. A. Wąsowska: None. E. Matczyńska: None. W. Krysa: None. K. Kamińska: None. E. Ewa Suchecka: None. J. Kosakowski: None. M. Jurkowska: None. A. Pałucha: None. M. Jędrzejowska: None. S. Teper: None. E. Wylęgała: None. E. Pius-Sadowska: None. A. Machalińska: None. A.M. Boguszewska-Chachulska: None.

P02.48C Mutation in the intracellular chloride channel CLCC1 associated with autosomal recessive retinitis pigmentosa

I. D'Atri1, L. Li2, X. Jiao2, F. Ono3, R. Nelson4, C. Chan5, N. Nakaya6, Z. Ma2, Y. Ma2, X. Cai7, L. Zhang7, S. Lin1, A. Hameed8, B. A. Chioza1, H. Hardy1, G. Arno9, S. Hull9, M. Khan10, J. Fasham1, G. V. Harlalka1, M. Michaelides9, A. T. Moore9, Z. Akdemir11, S. Jhangiani12, J. R. Lupski11, F. P. M. Cremers12, R. Qamar10, A. Salman13, J. K. Chilton1, J. Self13, F. Kabir14, M. Naeem14, M. Ali14, J. Akram15, P. A. Sieving16, S. Riazuddin14, S. Riazuddin14, J. Hejtmancik2, E. L. Baple1, A. H. Crosby1

1RILD Wellcome Wolfson Centre, Royal Devon & Exeter NHS Foundation Trust, University of Exeter, Exeter, United Kingdom, 2Ophthalmic Genetics and Visual Function Branch, National Eye Institute, NIH, Bethesda, MD, United States, 3Section on Model Synaptic Systems, Laboratory of Molecular Physiology, National Institute on Alcohol Abuse and Alcoholism, NIH, Bethesda, MD, United States, 4Unit on Neural Circuits, National Institute of Neurological Disorders and Stroke, NIH, Rockville, MD, United States, 5Laboratory of Immunology, National Eye Institute, NIH, Bethesda, MD, United States, 6Section of Molecular Mechanisms of Glaucoma, Laboratory of Molecular and Developmental Biology National Eye Institute, NIH, Bethesda, MD, United States, 7School of Life Sciences, University of Science and Technology of China, Hafei, China, 8Institute of Biomedical and Genetic Engineering (IBGE), Islamabad, Pakistan, 9University College London, Institute of Ophthalmology, London, United Kingdom, 10Faculty of Science, COMSATS Institute of Information Technology, Islamabad, Pakistan, 11Department of Molecular and Human Genetics, Baylor College of Medicine, Huston, TX, United States, 12Department of Human Genetics, Donders Institute for Brain, Cognition and Behaviour, Radboud University Medical Center, Nijmegen, Netherlands, 13Faculty of Medicine, University of Southampton, Southampton, United Kingdom, 14National Centre of Excellence in Molecular Biology, University of the Punjab, Lahore, Pakistan, 15Allama Iqbal Medical College, University of Health Sciences, Lahore, Pakistan, 16National Eye Institute, NIH, Bethesda, MD, United States

Retinitis pigmentosa (RP) is an inherited eye disease characterised by photoreceptor death and retinal degeneration, resulting in vision loss. This condition affects ~1:4000 individuals worldwide and is highly clinically and genetically heterogeneous, presenting with variable symptoms and inheritance patterns. We identified a homozygous missense alteration (c.75C>A, p.D25E) in the CLCC1 gene, which encodes a presumptive intracellular chloride channel highly expressed in the retina, associated with autosomal recessive RP in eight consanguineous families from Pakistani and the UK. The p.D25E alteration decreased CLCC1 channel function accompanied by accumulation of mutant protein in granules within the ER lumen. In keeping with these findings, Clcc1+/- KO mice displayed depressed electroretinogram and photoreceptor number. Together these findings define a single founder gene mutation as a cause of RP in families of Pakistani descent, and strongly suggest that CLCC1 function is crucial for maintaining retinal integrity and function. This work was supported by National Eye Institute Grant R01EY021237-01 (SAR), National Human Genome Research Institute (NHGRI)/National Heart Lung and Blood Institute (NHLBI) to the Baylor Hopkins Center for Mendelian Genomics (UM1 HG006542, JRL), Medical Research Council UK (G1002279), the Newlife Foundation for Disabled Children (SG/15-16/12), Fight For Sight (Ref 2027), Wellcome Trust 209083/Z/17/Z.

I. D'Atri: None. L. Li: None. X. Jiao: None. F. Ono: None. R. Nelson: None. C. Chan: None. N. Nakaya: None. Z. Ma: None. Y. Ma: None. X. Cai: None. L. Zhang: None. S. Lin: None. A. Hameed: None. B.A. Chioza: None. H. Hardy: None. G. Arno: None. S. Hull: None. M. Khan: None. J. Fasham: None. G.V. Harlalka: None. M. Michaelides: None. A.T. Moore: None. Z. Akdemir: None. S. Jhangiani: None. J.R. Lupski: None. F.P.M. Cremers: None. R. Qamar: None. A. Salman: None. J.K. Chilton: None. J. Self: None. F. Kabir: None. M. Naeem: None. M. Ali: None. J. Akram: None. P.A. Sieving: None. S. Riazuddin: None. S. Riazuddin: None. J. Hejtmancik: None. E.L. Baple: None. A.H. Crosby: None.

P02.49D Genetic screening for Italians patients affected with Retinitis Pigmentosa

V. Errichiello1, S. Carboni2, G. Pagliaroli2, V. Caputo1, C. Strafella1,3, G. Campoli2, C. Peconi2, F. Sangiuolo1, G. Novelli1, R. Cascella1,4, E. Giardina1,2

1Department of Biomedicine and Prevention, “Tor Vergata” University, Roma, Italy, 2Laboratory of Genomic Medicine UILDM, IRCCS Santa Lucia Foundation, Roma, Italy, 3Emotest Laboratory, Pozzuoli, Italy, 4Department of Chemical Pharmaceutical and Biomolecular Technologies, Catholic University “Our Lady of Good Counsel” Laprakë, Rruga Dritan Hoxha, Tirana, Albania

Introduction: Retinitis Pigmentosa (RP, OMIM #268000) is a degenerative disorder affecting peripheral retina which is caused by a progressive loss of photoreceptors. The genetics of RP is highly heterogeneous, with several associated genes mainly implicated in the phototransduction cascade. RP shows autosomal dominant, autosomal recessive, X-linked and mitochondrial inheritance patterns. One of the most investigated gene associated with autosomal RP is RHO (3q21-q24), which encodes for Rodopsin and is essential for vision in low-light conditions. In this context, the genetics of RP was studied considering the screening of RHO as a first-level analysis and a panel of 24 putative genes as second-level step.

Matherial and Methods: 100 Italian RP patients were analyzed by this two steps-analysis, through direct sequencing and NGS on IonTorrent S5 (Thermo Fisher). Concerning NGS analysis, a 20X coverage was fixed as minimum depth of coverage.

Results: the first-step analysis identified a variant in the fourth exon of RHO gene, namely c.G760T. The variant was found in 1 patient, while the remaining 99 patients were negative for this level of analysis. These patients were therefore subjected to the second-step analysis, which reported a number of variants. At the moment, the resulting variants are under investigation and validation.

Conclusion: the present study highlights a major burden of genes other than RHO associated with the disease in the Italian population. Given the genetic heterogeneity of RP, it would be highly helpful and faster to perform a large-scale screening of genes compared to the slower and labor-intensive traditional approaches.

V. Errichiello: None. S. Carboni: None. G. Pagliaroli: None. V. Caputo: None. C. Strafella: None. G. Campoli: None. C. Peconi: None. F. Sangiuolo: None. G. Novelli: None. R. Cascella: None. E. Giardina: None.

P02.50A Haplotypes constructed by Whole exome sequencing to map and identify a novel disease-causing RP2 gene variant from a recessive X linked Retinities Pigmentosa family

Y. Ching1, W. Fan2, W. Lin1, W. Tsai1, L. Hu1, S. Huang1, R. Chung3

1Molecular Biology and Human Genetics, Hualien, Taiwan, 2Whole-Genome Research Core Laboratory of Human Diseases, Chang Gung Memorial Hospital, Keelung, Taiwan, 3National Health Research Institutes, Zhunan, Taiwan

Introduction: Retinitis pigmentosa (RP) is a genetically heterogeneous with more than 70 RP loci currently known. A three-generation RP family exhibiting X linked recessive pattern were studied.

Materials and Methods: Linkage analysis was performed using 18 microsatellite markers. Whole exome sequencing was used for mutational analysis.

Results: Polymorphic microsatellite markers were used to map the disease interval to a 48 Mb region on the X chromosome between the markers DXS1068 and DXS1196. Whole exome sequencing (WES) was performed on a selected sib-pair within the family. Total 1973 SNPs were found to be located within the critical interval. Among these SNPs, 139 were shared between the obligated carrier and the affected male offspring but not the phenotypically normal male offspring. We also utilized the WES identified polymorphic SNPs mapped within the critical disease interval to construct detailed haplotype for the sib-pair. Additional crossing-over evens were revealed on and the disease interval were mapped into two intervals: chrx: 38,911,177 -68890047; and chr X: 69,155,432-69,261,818). The causative mutation (RP2 c.102G>A; Lys34Lys, a RP2 splicing donor site mutation) was then identified.

Conclusion: We have combined the positional information obtained from the linkage and haplotype analysis to identify the genetic intervals harboring the disease-causing gene, and also utilized DNA polymorphisms identified from WES as additional genetic markers to further mapped the crossing-over evens as a creative strategy for positional cloning.

MOST: 104-2311-B-320 -001 -

Y. Ching: None. W. Fan: None. W. Lin: None. W. Tsai: None. L. Hu: None. S. Huang: None. R. Chung: None.

P02.51B Improved genetic diagnostics of RPGRORF15-associated retinal dystrophy

J. Känsäkoski1, S. Tuupanen1, J. Sistonen1, P. Siivonen1, K. Kämpjärvi1, M. Mehine1, M. Valori1, P. Salmenperä1, E. Sankila2, E. Salminen1, S. Myllykangas1, T. Alastalo1, J. W. Koskenvuo1

1Blueprint Genetics, Helsinki, Finland, 2Helsinki University Eye Hospital, Helsinki, Finland

Retinitis pigmentosa (RP) is the most common form of inherited retinal degeneration affecting around 1:3,000 individuals worldwide. Classical RP is characterized by progressive rod-cone dysfunction. Patients initially present with night blindness and tunnel vision, followed by decreased visual acuity and macular affectation. RP is clinically and genetically heterogeneous, and it can be inherited in an autosomal dominant, autosomal recessive, and X-linked manner. The majority of the X-linked RP, associated with a severe phenotype, is caused by mutations in the RPGR gene. All known mutations causing RPGR-related retinal dystrophies are found to affect the RPGRORF15 isoform, which contains a unique C-terminal 567-aa exon called ORF15. ORF15 is a mutational hotspot for RPGR-associated RP, accounting for two-thirds of all disease-causing mutations. The exon ORF15, however, includes a highly repetitive, purine-rich sequence, which generally performs poorly in next-generation sequencing (NGS)-based assays. To address the clinical importance of the RPGR ORF15 and the lack of high quality NGS-based diagnostics, we have developed a novel test to detect variants in the ORF15 region. This test combines NGS analysis with Illumina NovaSeq 6000 platform and Sanger sequencing, specifically optimised for this region. We have validated the test using samples with known RPGR ORF15 variants, and additionally tested patients with a clinical suspicion of X-linked RP, who have remained negative in previous genetic testing. We show that the test has high clinical sensitivity and specificity for detecting RPGR ORF15 variants, and that it improves the genetic diagnostics of inherited retinal dystrophies.

J. Känsäkoski: None. S. Tuupanen: A. Employment (full or part-time); Significant; Blueprint Genetics. J. Sistonen: A. Employment (full or part-time); Significant; Blueprint Genetics. P. Siivonen: A. Employment (full or part-time); Significant; Blueprint Genetics. K. Kämpjärvi: A. Employment (full or part-time); Significant; Blueprint Genetics. M. Mehine: A. Employment (full or part-time); Significant; Blueprint Genetics. M. Valori: A. Employment (full or part-time); Significant; Blueprint Genetics. P. Salmenperä: A. Employment (full or part-time); Significant; Blueprint Genetics. E. Sankila: F. Consultant/Advisory Board; Modest; Blueprint Genetics. E. Salminen: A. Employment (full or part-time); Significant; Blueprint Genetics. S. Myllykangas: E. Ownership Interest (stock, stock options, patent or other intellectual property); Significant; Blueprint Genetics. T. Alastalo: E. Ownership Interest (stock, stock options, patent or other intellectual property); Significant; Blueprint Genetics. J.W. Koskenvuo: E. Ownership Interest (stock, stock options, patent or other intellectual property); Significant; Blueprint Genetics.

P02.52C SVAF retrotransposon insertion in BBS1 gene, leading to Bardet- Biedl Syndrome

E. Tavares1, A. Vig1,2, S. Li1, G. Billingsley1, W. Sung3, A. Vincent1,2,4, B. Thiruvahindrapuram3, E. Héon1,2,4

1Genetics and Genome Biology, Toronto, ON, Canada, 2Institute of Medical Science, University of Toronto, Toronto, ON, Canada, 3The Centre for Applied Genomics, Toronto, ON, Canada, 4Ophthalmology and Vision Sciences. The Hospital for Sick Children, Toronto, ON, Canada

Introduction: Active transposable elements (TE) account for over 0.02% of the human genome. One de novo insertion is believed to happen with each 10-100 live births. TEs can cause disease by inserting themselves into coding or regulatory portions of genes, facilitating duplications and deletions, among other mechanisms. Due to their repetitive nature, traditional sequencing methods often fail to detect TEs. Here we report a Bardet-Biedl syndrome (BBS) patient who is compound heterozygous for the most frequent disease-causing mutation in BBS1 and an inserted SVAF retrotransposon.

Methods and Results:Whole-genome sequencing (WGS) was performed for a female BBS patient with an identified maternally inherited mutation M390R. No other mutation was identified in BBS1 using dense microarray analysis, qPCR assays, and whole exome sequencing. An exonic TE insertion was detected in exon 13 of BBS1 in by visual inspection of the WGS read files. Following Sanger sequencing, we determined the insertion to be a paternally inherited SVAF element of ~2 kb. We have not identified the same SVAF insertion upon screening twenty-four BBS negative patients, 2 with disease-causing mutations in BBS1. However, screening for other TE insertions are yet to be completed.

Conclusions:Disease-causing TE insertions can be missed by traditional genetic testing methods. However, they can be detected by using WGS data with specific methods, not systematically implemented. Large -scale implementation of such algorithms will allow the discovery of more disease-causing TE insertions and better assessment of the frequency of this category of mutational events.

E. Tavares: None. A. Vig: None. S. Li: None. G. Billingsley: None. W. Sung: None. A. Vincent: None. B. Thiruvahindrapuram: None. E. Héon: None.

P02.53D In vitro functional analysis of a novel RP2 alleles

Y. KAO1, W. Fan2, R. Chung3, W. Lin1, W. Tsai1, L. Hu1, S. Huang1, Y. Ching1

1Tzu Chi University, Hualien, Taiwan, 2Chang Gung Memorial Hospital, Keelung, Taiwan, 3National Health Research Institutes, Zhunan, Taiwan

Introduction: Retinitis pigmentosa (RP), a hereditary heterogeneous disease with a prevalence of 1/4000, characterized by the degeneration of photoreceptors leading to progressive loss of vision in patients. We have identified a three-generation X-linked RP family.

Materials and Methods: Eighteen X chromosome microsatellite markers were used to locate disease locus, and one of the Sibling-pairs within the family were used to performed Whole-exome sequencing.

Results: The disease locus was mapped between markers DXS1068 and DXS1196. Whole-exome sequencing identified 139 possible SNPs located within the critical diseases-causing intervals. Considering that co-segregation of mutation and genes expression patterns, the RP2_c.102G> A point mutation might be the responsible mutation of this family's disease. It is a novel allele of RP2 gene with allele frequency <1%, and no known functional impact. At protein level the mutation appears to be no functional impact (Lys34> Lys), however, c.102G> A located at the last nucleotide of the splicing donor site of exon1, suggesting that this mutation might cause intron retention at the transcriptional level. A MiniGene assay was designed to validate the functional consequences of this mutation.

Conclusion: We have identified a novel splicing-site mutation of RP2 gene from an X-Link RP family. In vitro functional analysis using MiniGene assay has been performed to verify the functional impact of the mutation.

Y. Kao: None. W. Fan: None. R. Chung: None. W. Lin: None. W. Tsai: None. L. Hu: None. S. Huang: None. Y. Ching: None.

P02.54A An integrated molecular approach to characterize the genetic bases of hearing loss in an Italian cohort

F. Cesca1, E. Bettella1, R. Polli1, E. Leonardi1, M. C. Aspromonte1, S. Bigoni2, R. Santarelli3, A. Murgia1

1Laboratory of Molecular Genetics of Neurodevelopment, Department of Women’s and Children’s Health, University of Padova, Padova, Italy, 2Medical Genetics Unit, Ferrara University Hospital, Ferrara, Italy, 3Audiology and Phoniatric Service, Treviso Regional Hospital, Treviso, Italy

Non-syndromic hearing loss is characterized by a vast genetic heterogeneity with more than 160 loci described in humans and 100 genes so far identified. With the aim of targeting genes strongly associated, in Caucasians, with NSHL or with SHL which onset is usually characterized by isolated deafness (i.e. Pendred and Usher syndrome), we developed an NGS targeted panel of 59 genes, to obtain an advanced efficient diagnostic tool. The Ion Torrent PGMTM platform combined with a customized bioinformatics pipeline was used for the analysis of 87 DNA samples collected from clinically highly selected Italian subjects negative for GJB2 mutations/GJB6 deletions. An etiological diagnosis was established in 41 of these subjects, with an overall diagnostic yield of 47%. An early molecular diagnosis of Usher syndrome was achieved in 3 unrelated children carrying mutations in ADGRV1, CDH23 and USH2A genes. NGS allowed the identification of a homozygous as well as a heterozygous deletion in the STRC gene; the latter deletion was found in-trans with a known STRC pathogenic variant. The deletions were confirmed by q-PCR with primers that excluded the highly homologous STRC pseudogene. The novel likely causative variants identified were located in the following genes: ACTG1, ADGRV1, CDH23, CEACAM16, COCH, COL11A2, EYA4, GJB3, KCNQ4, MYO7A, PCDH15, PTPRQ, SLC17A8, TMPRSS3. Our targeted panel coupled with a solid bioinformatics pipeline has proved a sensitive molecular tool with a high diagnostic yield. We demonstrate the importance and efficacy of integrating the powerful NGS technology with a comprehensive data processing and a careful clinical evaluation.

F. Cesca: None. E. Bettella: None. R. Polli: None. E. Leonardi: None. M.C. Aspromonte: None. S. Bigoni: None. R. Santarelli: None. A. Murgia: None.

P02.55B Stereocilin gene mutations associated with vertigo: Expansion of the DFNB16 phenotype

C. A. Frykholm1, J. Klar1,2, T. Tomanovic3, A. Ameur2, N. Dahl1,2

1Department of Immunology, Genetics and Pathology, Uppsala, Sweden, 2Science for Life Laboratory, Biomedical Centre, Uppsala University, Uppsala, Sweden, 3Department of Hearing and Balance Disorders, Karolinska University Hospital, Stockholm, Sweden

Introduction: Vestibular disorders comprise a group of diseases with transient or permanent loss of vestibular function characterized by vertigo and imbalance. Isolated vestibulopathy is rare and more often associated with migraine, Ménière disease, ataxia or sensorineural hearing loss.

Materials and Methods: We examined two siblings and their first cousin with childhood onset of episodic vertigo and sensorineural hearing loss. Hearing loss and vestibular dysfunction was investigated by audiometry, SVH, cVEMP and oVEMP. DNA was analyzed using exome sequencing and SNP-array.

Results and Conclusions: Clinical investigations confirmed pathological vestibular responses in two siblings and hearing loss in all three affected individuals. The cousin had a history compatible with a vestibular disorder. DNA analysis revealed that the siblings were homozygous for a STRC stop variant (c.4027C>T) and their cousin was compound heterozygous for the stop variant and a 90kb deletion spanning the STRC gene. These results are consistent with DFNB16. STRC, encoding stereocilin, is expressed in the cochlea and in the vestibular organ where it ensheats the kinocilium suggesting a role for the protein in sensing balance and spatial orientation. Our findings support such a function for stereocilin in the vestibular organ and expand the phenotype associated with DFNB16.

Grants: Supported by the Swedish Research Council (2015-02424).

C.A. Frykholm: None. J. Klar: None. T. Tomanovic: None. A. Ameur: None. N. Dahl: None.

P02.56C Genetic, epigenetic and environmental contributors to Age-related Macular Degeneration susceptibility

C. Strafella1,2, V. Errichiello1, V. Caputo1, F. Sangiuolo1, F. Ricci3, G. Novelli1, A. Cusumano3, R. Cascella4,5, E. Giardina1,4

1Department of Biomedicine and Prevention, “Tor Vergata” University, Rome, Italy, 2Emotest Laboratory, Pozzuoli, Italy, 3UOSD Retinal Pathology PTV Foundation “Policlinico Tor Vergata”, Rome, Italy, 4Laboratory of Genomic Medicine UILDM, IRCCS Santa Lucia Foundation, Rome, Italy, 5Department of Chemical Pharmaceutical and Biomolecular Technologies, Catholic University “Our Lady of Good Counsel”, Tirana, Albania

Introduction: Susceptibility to Age-related Macular Degeneration (AMD) strictly depends on genetic, epigenetic and environmental factors. Previous results highlighted prominent differences concerning genetic contributors to AMD in Italian population compared to worldwide groups. Among genetic variables, SNPs of CFH, ARMS2, IL-8, TIMP3, SLC16A8, RAD51B, VEGFA and COL8A1 were significantly associated with the risk of AMD in our cohort. Given these data, this study aimed to evaluate the contribution of genetic, epigenetic (SNPs of miRNA-146a, miRNA-31, miRNA-23a, miRNA-27, miRNA-20a and miRNA-150 genes) and environment factors (age, sex, smoking, diet) to exudative AMD.

Materials and Methods: 976 exudative AMD patients and 1000 controls were subjected to an epigenotyping analysis through Real-Time PCR and direct sequencing. Biostatistical analysis was performed to calculate association and estimate the contribution of genetics, epigenetics and environment to AMD susceptibility. Gene-environment interactions were evaluated by bioinformatic tools.

Results: The SNPs rs11671784 (miRNA-27, G/A) and rs2910164 (miRNA-146a, C/G), advanced age, smoking and dietary habits were significantly associated with AMD risk (p<0.05). Genetic/epigenetic variants appeared to contribute to AMD susceptibility for 23% while non-genetic variants accounted for 10% of disease. Concerning gene-environment interactions, we found that AMD-associated genes may be involved in the alteration of Bruch's membrane and angiogenesis, contributing to the exacerbation of aging and environmental damages.

Conclusions: Our study provides an overview of genetic/epigenetic and non-genetic factors characterizing AMD susceptibility in Italian population. These data may be applied to develop a “population-specific precision medicine” approach able to prevent AMD or improve patients’ quality of life.

C. Strafella: None. V. Errichiello: None. V. Caputo: None. F. Sangiuolo: None. F. Ricci: None. G. Novelli: None. A. Cusumano: None. R. Cascella: None. E. Giardina: None.

P02.58A Discovery of new Hereditary Hearing Loss (HHL) genes by Whole Exome Sequencing (WES) and in vitro/in vivo functional studies: five years of experience

G. Girotto1,2, A. Morgan1, M. Brumat1, M. Di Stazio1, S. Cappellani2, E. Campana1, U. Ambrosetti1, M. La Bianca2, E. Orzan2, P. Gasparini1,2

1University of Trieste, Trieste, Italy, 2IRCCS-Burlo Garofolo, Trieste, Italy

Introduction: HHL is genetically heterogeneous and at least 40% of cases are not characterized by mutations in known genes. Thus, we applied WES followed by “in vitro” and “in vivo” functional studies for the discovery of new genes.

Methods: 14 Italian HHL families, negative for mutations in 96 deafness-genes, were analyzed by WES (Ion Proton™). Variants were filtered according to: a) pattern of inheritance, b) frequency, c) pathogenicity. Functional studies on new candidates were carried out by in vitro/in vivo experiments.

Results: WES allowed the discovery of five new HHL-genes: PSIP1 (https://doi.org/10.1038/srep18568), TBL1Y, SPATC1L, PLS1 and ATP2B2. As regards PSIP1, a nonsense variant in a 3-generation family was identified; RNAseq and immunolabeling confirmed gene expression in mouse inner ear. For TBL1Y, a missense variant was detected in a large Y-linked family; functional experiments demonstrated TBL1Y expression in human cochlea and an early degradation of the mutated protein. For SPATC1L, a nonsense variant in a 3-generation family was identified; protein modeling revealed a reduced structural stability (loss of part of the C-terminus) confirmed by western blot (presence of a shorter protein isoform). Finally, for PLS1 and ATP2B2 (known as a CDH23 modifier) Zebrafish KI models of the identified variants (a missense and a nonsense variant, respectively, in two dominant HHL families) are at the final stages of validation. WES data of the remaining 9 families are now under investigation.

Conclusions: Our approach, based on WES followed by functional studies, already proved to be effective for the discovery of new HHL-genes.

G. Girotto: None. A. Morgan: None. M. Brumat: None. M. Di Stazio: None. S. Cappellani: None. E. Campana: None. U. Ambrosetti: None. M. La Bianca: None. E. Orzan: None. P. Gasparini: None.

P02.59B Dominant WFS1 mutations in a new congenital phenotype

A. Chaussenot1,2, C. Rouzier1,2, C. Vincent-Delorme3, M. Bonnet-Dupeyron4, G. Auge1, V. Paquis-Flucklinger1,2

1Department of Medical Genetics, National Centre for Mitochondrial Diseases, Archet 2 Hospital, Nice, France, 2Nice Sophia-Antipolis University, CNRS UMR 7284, INSERM U1081, Institute for Research on Cancer and Aging, Nice, France, 3Jeanne de Flandre hospital, Lille, France, 4Laboratoire de Biologie Médicale, Centre hospitalier de Valence, Valence, France

Introduction: Dominant phenotypes related to WFS1 mutations were known to be less severe than the Wolfram syndrome (WS) recessive phenotype, characterized by diabetes mellitus, optic atrophy, diabetes insipidus and deafness. Dominant phenotypes included isolated low-frequency sensorineural hearing loss, optic atrophy and hearing impairment, isolated adult-onset diabetes and isolated congenital nuclear cataracts. More recently, De Franco et al. (Diabetes 2017) described a severe congenital Wolfram-like syndrome (CWLS), characterized by congenital progressive hearing loss, neonatal diabetes mellitus and cataract, due to de novo dominant mutations in WFS1.

Materials and Methods: We reported 3 unrelated cases with this new dominant phenotype, among our French cohort of 116 patients carrying at least 1 WFS1mutation.

Results: The dominant known p.Glu809Lys mutation was found de novo for 2 unrelated girls, respectively 12 and 3 years-old, who presented CWLS during the first year of life, associated with psychomotor retardation, failure to thrive, amblyopia, dysmorphic features and cerebellar hypoplasia. We also described a 20 years-old patient, who had developed with diabetes and deafness before 1 year of age and bilateral cataract diagnosed at 18 months, associated with glaucoma, amblyopia, cerebellar ataxia, short stature, hypothyroidism and hypogonadism. The clinical presentation was very suggestive of CWLS. We found a heterozygous p.His860Asp WFS1 mutation, that was previously described at compound heterozygous state in a case of WS, but with congenital deafness and occuring De Novo, asking the question of its pathogenicity.

Conclusions: We highlight the expanding clinical spectrum of WFS1-related disorders with 3 case reports of severe congenital dominant phenotype.

A. Chaussenot: None. C. Rouzier: None. C. Vincent-Delorme: None. M. Bonnet-Dupeyron: None. G. Auge: None. V. Paquis-Flucklinger: None.

P02.60C Multidisciplinary team work to get differential diagnosis of a reverse phenotype in retinal disease

L. Candita1, B. Boschi1, E. Contini1, I. Passerini1, A. Sodi2, A. L. Nutini1, O. Colavecchio1, T. Morgani1, E. Ronconi1, E. Pelo1

1SOD Diagnostica genetica, AOU Careggi, Firenze, Italy, 2Dipartimento di Chirurgia e Medicina Traslazionale, Clinica Oculistica, AOU Careggi, Firenze, Italy

We report a case of a 23 year old woman referred to our center with a diagnosis of Leber congenital amaurosis (LCA). An accurate clinical re-evaluation detected dystrophy of retinal pigment epithelium, Franceschetti sign, exotropia and nistagmus. Clinical signs and symptoms were therefore not suggestive for LCA. A genetic counseling was then offered to this patient. We performed NGS analysis of 137 retinal dystrophy associated genes. Mutational and CNV analysis were performed. Pathogenic variants c.1666del in heterozygosis in CEP290 gene and c.484G>A in heterozygosis in CRB1 gene and probably pathogenic variant c.1054C>T in heterozygosis in CACNA2D4 gene were detected. Variant c.1666del was reported in association with LCA (Hui Wang et al., 2015) in compound heterozygosis with a pathogenic variant in CEP290 gene; variant c.484G>A in CRB1 gene was reported in one family with pigmented paravenous chorioretinal atrophy (PPCRA) (McKay et al., 2005) dominantly inherited. The cosegregation analysis showed that proband’s father carried all the three familial variants. No defined clinical symptoms of impairment visual loss were reported. Clinical and molecular findings are not always clear: therefore we recommend a multidisciplinary patient management in rare pathology. NGS data should lead to clinical revaluation, early diagnosis in mild symptomatic relatives and sometimes lead to achieve reverse phenotype. In conclusion, we requested clinical revaluation of the proband and her parents by our physicians, to gain also a clinical PPCRA diagnosis, which should justify a CRB1 dominant disease with variable expression or incomplete penetrance.

L. Candita: None. B. Boschi: None. E. Contini: None. I. Passerini: None. A. Sodi: None. A.L. Nutini: None. O. Colavecchio: None. T. Morgani: None. E. Ronconi: None. E. Pelo: None.

P03 Internal organs & endocrinology (lung, kidney, liver, gastrointestinal)

P03.01D High diagnostic yield in well-defined cohort of ADPKD patients by conventional sequencing and NGS

M. Losekoot1, A. Tholens1, M. Phylipsen1, E. Meijer2, F. W. Visser2,3, J. P. H. Drenth4, J. W. de Fijter5, J. F. Wetzels6, R. Zietse7, R. T. Gansevoort2, D. J. M. Peters8

1Dept. of Clinical Genetics, Leiden University Medical Center, Leiden, Netherlands, 2Dept. Nephrology, University Medical Center Groningen, Groningen, Netherlands, 3Dept. Internal Medicine, Ziekenhuisgroep Twente, Almelo, Netherlands, 4Dept. Gastroenterology and Hepatology, Radboud University Medical Center, Nijmegen, Netherlands, 5Dept. Nephrology, Leiden University Medical Center, Leiden, Netherlands, 6Dept. Nephrology, Radboud University Medical Center, Nijmegen, Netherlands, 7Dept. Nephrology, Erasmus Medical Center Rotterdam, Rotterdam, Netherlands, 8Dept. of Human Genetics, Leiden University Medical Center, Leiden, Netherlands

Autosomal dominant polycystic kidney disease is an inherited disease characterized by progressive cyst formation in both kidneys and renal function loss, which ultimately leads to end-stage renal failure. A well-defined clinical cohort of 339 ADPKD patients that gave informed consent and were screened for participation in a clinical trial was analysed for the presence of PKD1 and PKD2 mutations. The molecular analysis is relevant for the trial since disease progression is partly determined by mutation type. Mutation analysis was performed by Sanger sequencing and MLPA which resulted in a mutation detection ratio of 94%. Mutation negative patients were analysed for mutations in GANAB, HNF1B or PKHD1 but no mutation was detected. Four mutation negative patients were sequenced with a NGS approach (Illumina HiSeq4000 platform, targets captured using custom-designed gene panel specific Agilent SureSelectXTClearseq enrichment kit). Data analysis was performed using an in house developed pipeline (stringent post-sequencing annotation pipeline based on BWA, GATK and VEP and various filtering steps in LOVD+). In 3 out of 4 patients a pathogenic mutation was detected in PKD1 or PKD2. Mutations were missed by Sanger sequencing because of allelic drop-out, or a gap in the overlapping Sanger sequencing fragments. The remaining 16 patients are currently being analyzed with the same NGS approach.In conclusion, mutation analysis in a well-defined ADPKD cohort has an extremely high mutation detection rate (94%). The NGS approach is a very useful addition to standard mutation analysis techniques especially in very polymorphic regions of the genome like the PKD1 genomic region.

M. Losekoot: None. A. Tholens: None. M. Phylipsen: None. E. Meijer: None. F.W. Visser: None. J.P.H. Drenth: None. J.W. de Fijter: None. J.F. Wetzels: None. R. Zietse: None. R.T. Gansevoort: None. D.J.M. Peters: None.

P03.02 ANGS allele drop out in a family with NPHP4 related nephronophthisis

A. V. Kirov1, L. Grozdanova2, T. Todorov3,1, A. Todorova1,3

1IMDL Genome Centre Bulgaria, Sofia, Bulgaria, 2Department of Medical Genetics, Medical University Hospital “St. George”, Plovdiv, Bulgaria, 3GMDL Genica, Sofia, Bulgaria

Here we report a family with one child died soon after birth due to bilateral renal agenesis and one terminated early pregnancy due to the same reason. The family was referred for genetic counseling and molecular-genetic testing to our lab. DNA from the affected children was not available so we perform whole exome sequencing of both parents. We analyzed the data searching for heterozygous genetic variant in the known nephronophthisis and polycystic kidney disease associated genes. However, only one heterozygous variant in exon 23 of NPHP4 gene was detected in the mother: NM_015102.4: c.3292G>A (p.Ala1098Thr). Some authors reported few patients with only one NPHP4 mutation so we could not exclude the possibility of incomplete penetrance of the genetic variant. We confirm the genetic finding with standard Sanger sequencing of both parents. Surprisingly, the father was also a heterozygous carrier of the same variant. We double check the finding and the NGS coverage (above 100x in both patients) and again it was missing from the NGS data of the father. It is well known that both cytosine methylation and DNA structures known as G-quadruplexes (G4s) in some region contributed to allelic dropout (ADO) in PCR based reactions and NGS library preparation. However the genetic region in proximity to our genetic variant is not GC-rich and as far as we know this region is also not methylated. Obviously ADO in NGS era is something that we still need to keep in mind and continue to investigate in our routine laboratory work.

A.V. Kirov: None. L. Grozdanova: None. T. Todorov: None. A. Todorova: None.

P03.03B COL4A5 G624D: abundance of the Alport syndrome mutation in Russia along with Greek, Hungarian and Slovenian populations suggests it is a frequent mutation in Eastern Europe with mild phenotype

L. I. Shagam1, V. S. Sukhorukov1,2, T. A. Kuznetsova1, M. E. Aksenova1, V. V. Dlin1

1Veltishev Pediatric Clinical Research Institute of Pirogov Russian National Research Medical University, Moscow, Russian Federation, 2Sechenov University, Moscow, Russian Federation

Introduction: Alport syndrome (AS) is a familial hematuria caused by mutations in COL4A3, COL4A4 and/or COL4A5 genes which lead to defects in glomerular filtration barrier. So far, some founder mutations have been identified with many of them being region- or ethnicity-specific. COL4A5 c.1871G>A, p.(Gly624Asp) pathogenic variant is known to be prevalent in AS patients from Slovenia (6 out of 17), Hungary (3 out of 10) and Greece and lead to late age at onset of end stage renal disease. In contrast to these populations, the mutation is considered to be rare in the US, Northern and Western Europe or Japan. Here we show that the mutation was detected in 7 AS patients out of 49 with genetically confirmed diagnosis from Russia.

Materials and Methods: The population sample contained 76 apparently unrelated pediatric patients (1 to 17 years old) from diverse range of regions in the European part of Russian Federation with confirmed or suspected diagnosis of AS according to current guidelines. NGS sequencing was performed using Ion PGM (AmpliSeq panel).

Results: We confirmed the diagnosis in 49 patients including 43 with X-linked AS (harboring COL4A5 gene mutations) and 1 with digenic COL4A5 and COL4A3 inheritance. Seven of them were bearing the COL4A5 c.1871G>A, p.(Gly624Asp) mutation which corresponds to 14% frequency in the sample. We demonstrate that the mutation is characterized by late age at onset of hematuria (>48 months) and absence of proteinuria in childhood. The research was supported by RFBR grant 18-34-00708 to L.I.S. and Minzdrav government grant №115022070016.

L.I. Shagam: None. V.S. Sukhorukov: None. T.A. Kuznetsova: None. M.E. Aksenova: None. V.V. Dlin: None.

P03.04C Identification of the genetic background of Polish patients with suspected Alport Syndrome using next-generation sequencing

P. Halat-Wolska1, E. Ciara1, J. Antoniewicz2, K. Gadomska-Prokop2, L. Obrycki2, M. Rydzanicz3, J. Kosińska3, D. Siestrzykowska1, B. Chałupczyńska1, P. Stawiński3,4, D. Jurkiewicz1, D. Piekutowska-Abramczuk1, M. Pelc1, P. Kowalski1, D. Wicher1, A. Cieślikowska1, P. Iwanowski1, M. Gierla2, A. Łuba2, A. Niemirska2, D. Runowski2, W. Jarmużek2, J. Lesiak2, M. Gorzkowska-Paczwa2, A. Borowski2, J. Latoszyńska2, M. Podymniak-Grzeszykowska2, R. Grenda2, K. Chrzanowska1, R. Płoski3, M. Krajewska-Walasek1, M. Litwin2

1Department of Medical Genetics, The Children's Memorial Health Institute, Warsaw, Poland, 2Department of Nephrology, The Children's Memorial Health Institute, Warsaw, Poland, 3Department of Medical Genetics, Warsaw Medical University, Warsaw, Poland, 4Department of Genetics, Institute of Physiology and Pathology of Hearing, Warsaw, Poland

Introduction: Alport syndrome (AS) is a clinically and genetically heterogeneous nephropathy associated with sensorineural hearing loss and ocular anomalies, with thin basement membrane nephropathy being at the mildest end of the disorder spectrum. In most AS cases pathogenic variants can be found in COL4A5 (XL~80%) whereas COL4A3 and COL4A4 are associated with autosomal recessive (AR~15%) and dominant (AD~5%) forms.

Materials and Methods: In our study we examined a group of 36 unrelated Polish patients with suspected AS. To identify the molecular basis of the disease, we conducted next-generation sequencing (NGS) using Illumina TruSight One Sequencing Panel, which allowed simultaneous analysis of all genes encoding the subunits of type IV collagen.

Results: A genetic etiology was established in 32 patients. Overall, 31 pathogenic or likely pathogenic variants in COL4A3, COL4A4 (both AR~12% and AD~12%) and COL4A5 (XL~75%) were identified, including 10 known mutations and 21 novel variants expanding the list of COL4A3-5 alterations. Changes were randomly distributed across all coding regions of COL4A3-5, with no indices of a genetic “hot spot”, however we revealed variants repeated four times (COL4A5: c.1871G>A) or twice (COL4A3: c.2083G>A; COL4A5: c.2414G>T, c.3399delA) in our study group.

Conclusions: Diagnosis of patients with suspected AS was confirmed at the molecular level in ~89% cases. Our findings correspond to the data reported worldwide. NGS is efficient, reduces screening time and cost, and provides an expanding diagnostic tool to investigate the genetic backgrounds of AS, which will improve personalized diagnostics, genetic and prognostic counseling for the patients and their relatives.

P. Halat-Wolska: None. E. Ciara: None. J. Antoniewicz: None. K. Gadomska-Prokop: None. L. Obrycki: None. M. Rydzanicz: None. J. Kosińska: None. D. Siestrzykowska: None. B. Chałupczyńska: None. P. Stawiński: None. D. Jurkiewicz: None. D. Piekutowska-Abramczuk: None. M. Pelc: None. P. Kowalski: None. D. Wicher: None. A. Cieślikowska: None. P. Iwanowski: None. M. Gierla: None. A. Łuba: None. A. Niemirska: None. D. Runowski: None. W. Jarmużek: None. J. Lesiak: None. M. Gorzkowska-Paczwa: None. A. Borowski: None. J. Latoszyńska: None. M. Podymniak-Grzeszykowska: None. R. Grenda: None. K. Chrzanowska: None. R. Płoski: None. M. Krajewska-Walasek: None. M. Litwin: None.

P03.05D Genome-wide association study of sepsis-induced acute respiratory distress syndrome: discovery stage

B. Guillen-Guio1, J. M. Lorenzo-Salazar2, A. Corrales1,3, E. Espinosa4, A. Muriel5, L. Lorente6, M. M. Martín7, C. Rodríguez-Gallego8, J. Solé-Violán9, A. Ambrós10, D. Carriedo11, J. Blanco5, J. M. Añón12, J. M. Añón12, J. Villar3,13, C. Flores1,2,3, the Genetics of Sepsis (GEN-SEP) Network

1Research Unit, Hospital Universitario N.S. de Candelaria, Universidad de La Laguna, Santa Cruz de Tenerife, Spain, 2Genomics Division, Instituto Tecnológico y de Energías Renovables (ITER), Santa Cruz de Tenerife, Spain, 3CIBER de Enfermedades Respiratorias, Instituto de Salud Carlos III, Madrid, Spain, 4Department of Anesthesiology, Hospital Universitario N.S. de Candelaria, Santa Cruz de Tenerife, Spain, 5Intensive Care Unit, Hospital Universitario Rio Hortega, Valladolid, Spain, 6Intensive Care Unit, Hospital Universitario de Canarias, La Laguna, Santa Cruz de Tenerife, Spain, 7Intensive Care Unit, Hospital Universitario N.S. de Candelaria, Santa Cruz de Tenerife, Spain, 8Department of Immunology, Hospital Universitario Dr Negrín, Las Palmas de Gran Canaria, Spain, 9Intensive Care Unit, Hospital Universitario Dr Negrín, Las Palmas de Gran Canaria, Spain, 10Intensive Care Unit, Hospital General de Ciudad Real, Ciudad Real, Spain, 11Intensive Care Unit, Complejo Hospitalario Universitario de León, León, Spain, 12Intensive Care Unit, Hospital Universitario La Paz, Madrid, Spain, 13Research Unit, Hospital Universitario Dr. Negrin, Las Palmas de Gran Canaria, Spain

Introduction: The acute respiratory distress syndrome (ARDS) is a complex syndrome of severe acute hypoxemic respiratory failure. Here, we describe the results of the discovery stage for the first genome-wide association study (GWAS) of sepsis-induced ARDS.

Materials and Methods: We performed a GWAS on 672 sepsis patients admitted into intensive care units. After quality control steps and variant imputation in the Haplotype Reference Consortium data, 7.8 million variants with a minor allele frequency ≥1% were analyzed. Logistic regressions were carried out based on the Wald test, considering sex, age and the APACHE II score as covariates. GCTA-COJO was used to identify the independent loci. Gene-set enrichment analysis was assessed with EnrichR.

Results:A suggestive association (p < 5.0e-5) with ARDS was found for 53 independent loci (lowest p = 2.6e-7). Top hits were significantly enriched in genes linked to VEGF ligand-receptor interactions (p = 3.5e-4), and located near genes previously associated with other respiratory traits including lung function, chronic obstructive pulmonary disease, asthma, and idiopathic pulmonary fibrosis.

Conclusions: We have identified putative novel genetic variants associated with sepsis-induced ARDS. Replication analyses are currently underway. These results will advance our understanding of ARDS pathogenesis and will likely allow identifying new therapeutic targets.

Funding: ISCIII (PI11/00623, PI16/00049) and co-financed by the European Regional Development Funds, “A way of making Europe” from the European Union; Agreement OA17/008 with ITER to strengthen scientific and technological education, training, research, development and innovation in Genomics, Personalized Medicine and Biotechnology; Fellowship from the ACIISI (TESIS2015010057) co-funded by European Social Fund to BGG.

B. Guillen-Guio: None. J.M. Lorenzo-Salazar: None. A. Corrales: None. E. Espinosa: None. A. Muriel: None. L. Lorente: None. M.M. Martín: None. C. Rodríguez-Gallego: None. J. Solé-Violán: None. A. Ambrós: None. D. Carriedo: None. J. Blanco: None. J.M. Añón: None. J.M. Añón: None. J. Villar: None. C. Flores: None.

P03.06A Autophagy inhibition ameliorates renal cystic disease in OFD type I syndrome

M. Morleo1,2, U. Formisano1, S. Brillante1, D. Iaconis1, S. Maione1, E. Damiano1, R. Tammaro1, C. Settembre1, B. Franco1,2

1Telethon Institute of Genetics and Medicine-TIGEM, Pozzuoli, Italy, 2Federico II University, Naples, Italy

Introduction:Oral-facial-digital type I syndrome is ascribed to cilia dysfunction and characterized by abnormalities of face, oral cavity and digits and by renal cystic disease (CK). The causative gene codifies for OFD1, a centrosomal/basal body protein, necessary for primary cilia formation. Interestingly, recent data established a link between CK, primary cilia, cilioproteins and autophagy, a self-degradative process.

Results: Mass spectrometry analysis identified autophagy-related proteins among putative OFD1 interactors. In addition, we demonstrated that OFD1-depleted renal cells show increased autophagic flux and that OFD1 exerts a direct functional role on autophagosome biogenesis in an mTOR and cilia-independent manner. To test the physiological relevance of these findings we moved to in vivo studies and demonstrated enhanced autophagic flux both at precystic and cystic stages in two Ofd1 mutants (Ofd1-IND and Ofd1;creKsp). Moreover, we achieved renal specific inactivation in kidneys of both Ofd1 and Atg7, a key player of autophagy. Histological analysis showed a significant reduction in the number and size of cysts in creKsp;Ofd1y/fl;Atg7fl/fl mutants compared to creKsp;Ofd1y/fl;Atg7+/+ mice, as revealed by quantification of the cystic index, suggesting that increased autophagy may be strictly associated to renal cystogenesis in OFD type I syndrome.

Conclusions: Altogether our data suggest that autophagy alterations may be a common pathogenic mechanism in CK. Dissection of the molecular mechanisms underlying cyst formation in OFD type I will allow elucidating the role of autophagy in CK and could disclose new therapeutic avenues for renal cystic disease. Supported by the Polycystic Kidney Disease Foundation and the Telethon Foundation

M. Morleo: None. U. Formisano: None. S. Brillante: None. D. Iaconis: None. S. Maione: None. E. Damiano: None. R. Tammaro: None. C. Settembre: None. B. Franco: None.

P03.07B Genotype/phenotype correlations in ADPKD

M. Audrezet1, E. Cornec-Legall1, Y. Le Meur1, C. FEREC1,2

1University of Brest, BREST, France, 2INSERM UMR1078, Brest, France

Autosomal dominant polycystic kidney disease (ADPKD) is one of the most common gene kidney disorder with a worldwide prevalence of 1/400 to 1/1000. ADPKD is a genetically heterogeneous disease with two main genes PKD1 and PKD2 responsible of the disorder .Recently a new gene,GANAB has been reported to be involved in polycystic diseases(Porath et al., AJHG 2016) . We have completely analyzed the coding sequence of a large cohort of more than 4500 ADPKD patients (The genkyst cohort from the western part of France (2500 patients) and patients from different centers of France,(2000 patients).The analysis were performed first by Sanger and more recently by New Generation Sequencing (NGS). We have found an overall detection rate of 93% in our cohort,75% being mutated in PKD1 and 18% in PKD2 .We have performed a genotype/phenotype correlation (Audrezet et al., Hum Mut,2012 ;Cornec-Le Gall et al., JASN,2013) and showed that the type of mutation in PKD1, truncating mutations was 55.6 years versus non truncating mutations were associated with a 12 years delay in ESRD. We have only found 6 families with a mutation in the GANAB gene .To investigate the molecular basis of prenatal form of ADPKD we screened 42 patients with early ADPKD and we showed that additional PKD variation inherited from the unaffected parent were identified in 37.2% of patients ..These results suggest that hypomorphic mutations could explain at least a part of the clinical variability observed in ADPKD (Audrezet et al., JASN,2015)

M. Audrezet: None. E. Cornec-Legall: None. Y. Le Meur: None. C. Ferec: None.

P03.08C A pathogenic PKD2 nonsense variant is highly prevalent in the Northern Portuguese population

L. Rocha1,2, S. Fernandes1, J. P. Oliveira1,2

1Genetics, Department of Pathology, Faculty of Medicine & Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal, 2Medical Genetics – São João Hospital Centre, Porto, Portugal

Introduction: Autosomal dominant polycystic kidney disease (ADPKD) is a multi-organic hereditary disorder, responsible for 7-10% incident cases of end-stage renal failure (ESRF). In most populations, pathogenic variants in PKD1 (OMIM#601313) and in PKD2 (OMIM#173910) account respectively for 85% and 15% of ADPKD cases. Although both forms of ADPKD have similar pathogenesis, the onset of clinical manifestations and progression to ESRF occurs at young ages in patients with PKD1 mutations, who reach ESRF on average 10-15 years earlier than those with PKD2 mutations. The allelic heterogeneity is high for both genes, making NGS an appropriate first-tier approach to genetic diagnosis.

Methods: DNA was extracted from 40 unrelated ADPKD patients. PKD1 and PKD2 genes were analyzed by NGS sequencing (Ion Torrent PGM). Heterozygosity for clinically relevant variants was confirmed by Sanger sequencing.

Results: Ten of the 40 patients (25%) were heterozygous for a single-nucleotide c.181C>T transition in PKD2 exon 1 - predicting the nonsense variant p.(Gln61Ter) - which co-segregated with a relatively mild form of ADPKD in the affected families, most of which are from a circumscribed region in the river Douro valley.

Discussion: The PKD2 p.(Gln61Ter) variant is reported in the Mayo Clinic ADPKD Mutation Database and is not recognized as polymorphic human variation. Its high prevalence in a limited region of the north of Portugal suggests a “founder” effect. Accordingly, we have changed our genotyping approach to mildly affected ADPKD families originating from the critical geographic region, and screen first for that PKD2 variant.

L. Rocha: None. S. Fernandes: None. J.P. Oliveira: None.

P03.09D Clinical and molecular aspects of the patients with Berardinelli-Seip congenital lipodystrophy types 1 and 4

N. Gunes1, T. Erkan1, T. Kutlu1, H. Onay2, T. Atik2, B. Tüysüz1

1İstanbul University, Cerrahpasa Medical Faculty, İstanbul, Turkey, 2Ege University, Faculty of Medicine, İzmir, Turkey

Introduction:Berardinelli-Seip congenital generalized lipodystrophy (BSCL) is a rare disorder due to homozygous mutations of AGPAT2 (type-1), BSCL2 (type-2), CAV1 (type-3) and CAVIN1 (type-4) genes and characterized by reduced adipose tissue, muscular hypertrophy, hepatomegaly, insulin resistance and hypertriglyceridemia. Here, we report the longitudinal observation and comparison of the patients with BSCL type-1 and 4.

Materials and Methods: Six patients clinically diagnosed with BSCL from four families were enrolled. Exons and exon-intron boundaries of AGPAT2 and CAVIN1 were studied by Sanger sequencing method.

Results: Homozygous two known mutations (c.685G>T; c.514G>A) and a novel mutation (c.316+1G>T) were detected in AGPAT2 in five patients from three families. They were admitted between 6 months and 11 years of age. The patients had reduced subcutaneous fat (5/5), muscular hypertrophy (5/5), enlarged hands and feet (3/5), acanthosis nigricans (2/5), hepatomegaly (3/5), hypertriglyceridemia (5/5), hyperinsulinemia (3/5) and low serum leptin level (1/5). During their follow-up period 4 to 7 years, hypertrophic cardiomyopathy and hyperinsulinemia have developed in only one patient. Last patient, a 2-year-old boy with pyloric stenosis operation history, had reduced subcutaneous fat, muscular hypertrophy, hypertrophic cardiomyopathy, and elevated CK value. Homozygous mutation in CAVIN1 (c.259C>T) was detected. Hypertriglyceridemia and hyperinsulinemia were observed at 5 years of age.

Conclusions: As distinct from other BSCL types, pyloric stenosis and high CK values in an infant should suggest BSCL type-4. Interestingly, a previously reported patient with the same mutation of our BSCL type-4 patient had similar features including achalasia/pyloric stenosis, developmental hip dysplasia and high CK levels.

N. Gunes: None. T. Erkan: None. T. Kutlu: None. H. Onay: None. T. Atik: None. B. Tüysüz: None.

P03.10A A chronic obstructive pulmonary disease and interaction between single nucleotide polymorphisms of FAM13A gene and smoking

S. Kim1, K. Y. Lee2, R. E. Kim1, C. Shin1,3

1Institute for Human Genomic Study, Seoul, Korea, Republic of, 2Department of Radiology, Korea University Ansan Hospital, Ansan, Korea, Republic of, 3Division of Pulmonary, Sleep and Critical Care Medicine, Department of Internal Medicine, Korea University Ansan Hospital, Ansan, Korea, Republic of

Background and aims: Although smoking is a primary risk factor for chronic obstructive pulmonary disease (COPD), genetic polymorphisms within the FAM13A gene have been consistently reported to association with pulmonary function and/or COPD in genome-wide association studies. We aimed to investigate the effect of FAM13A gene variants that interact with ever smoking on COPD and emphysema risk.

Methods: Using a community-based cohort, we analyzed the association between genetic variants of FAM13A gene and COPD (GOLD stage >1) / emphysema (e.g., total lung volume, emphysema volume and emphysema ratio on computed tomography) risks using multivariate logistic and linear regression models (total n = 3,400). Furthermore, similar analyses were conducted after stratification by smoking status

Results: Five common variants (rs1458551, rs2609264, rs2609261, rs2609260 and rs7671167) annotated to the FAM13A gene were shown to have an additive effect on COPD and emphysema risk, whereas rs3756050 was only associated with a significantly higher risk for COPD (risky homozygote odds ratio (OR) = 1.49 (95% CI 1.13-1.96)). Finally, we identified significant interaction between the rs3756050 and ever smoking (P for interaction = 0.01).

Conclusions: We confirmed the previously reported association of FAM13A with COPD as well as emphysema risk. The genetic variant of FAM13A gene also interacted with ever smoking to affect the risk of higher COPD risk. This study was supported by the Korea Centers for Disease Control and Prevention grant (2011-E71004-00, 2012-E71005-00, 2013-E71005-00, 2014-E71003-00), the National Research Foundation of Korea grant funded by the Korea government (NRF-2016R1A2B4012155 and NRF-2017R1A6A3A11034663), and the Korea University Grant.

S. Kim: None. K.Y. Lee: None. R.E. Kim: None. C. Shin: None.

P03.11B Novel susceptibility genes candidates of chronic pancreatitis identified by whole exome sequencing

A. M. Rygiel1, A. Kujko1, G. Oracz2, T. Gambin1, J. Kosińska3, K. Wejnarska4, K. Wertheim-Tysarowska1, E. Kołodziejczyk2, R. Płoski3, J. Bal1

1Institute of Mother and Child, Warsaw, Poland, 2The Children’s Memorial Health Institute, Warsaw, Poland, 3Medical University of Warsaw, Warsaw, Poland, 4The Children’s Memorial Health Institute, Warsaw, Poland

The early onset chronic pancreatitis (CP) is often associated with mutations in a subset of CP genes. Despite of a progress in a discovery of new CP genes, the genetic cause of the disease, in the majority of the patients, remains unknown.

Aim: To identify novel susceptibility genes in early onset CP patients using whole exome sequencing (WES).

Patients and Methods: Six patients (mean age at diagnosis 10 years) with idiopathic or hereditary/familial CP with undetermined cause of the disease and their relatives were included for WES. Before, Sanger sequencing was performed to exclude the genetic causes of CP. WES data (HiSeq 2500, Illumina) were compared between index patient and affected or/and unaffected relatives. The variants were selected taking into account: in silico prediction (SIFT, MutTaster), minor allele frequency (MAF <0.01), gene function and expression in the pancreas, and the variant co-segregation with CP.

Results:. We detected 3 variants in known CP genes: two recurrent CFTR variants (p.L997F and p.R75Q) and one novel p.L100V*21 variant (introducing a STOP codon) in CTRC. We identified potentially pathogenic variants in 5 novel genes: PNLIP (p.Q323L), GCK (p.V101M), TRPV6 (p.V239Sfs*53 and p.L576R), SCNN1G (p.I224V) and SERPINA12 (p.G327D). The TRPV6 and SERPINA12 variants showed autosomal dominant inheritance. In 5/6 of the index patients, the trans-heterozygous variants in two different genes were observed.

Conclusions: Using WES approach, we identified novel variants and susceptibility genes candidates in CP. Their clinical significance needs to be elucidated by the functional and the case-control studies.

Financed: National Science Center Poland:2015/19/B/NZ5/02224

A.M. Rygiel: None. A. Kujko: None. G. Oracz: None. T. Gambin: None. J. Kosińska: None. K. Wejnarska: None. K. Wertheim-Tysarowska: None. E. Kołodziejczyk: None. R. Płoski: None. J. Bal: None.

P03.12C The association between CEL-HYB1 allele and idiopathic/familial chronic pancreatitis in Polish pediatric patients

A. Kujko1, G. Oracz2, K. Fjeld3, K. Wejnarska2, K. Wertheim-Tysarowska1, E. Kołodziejczyk2, J. Bal1, D. Koziel4, A. Kowalik5, S. Gluszek4, A. Molven6, A. M. Rygiel1

1Institute of Mother and Child, Warsaw, Poland, 2The Children’s Memorial Health Institute, Warsaw, Poland, 3Haukeland University Hospital,, Bergen, Norway, 4Jan Kochanowski University of Kielce, Kielce, Poland, 5Oncology Center of the Holy Cross, Kielce, Poland, 6University of Bergen, Bergen, Norway

Introduction: The replacement of a part of the carboxyl ester lipase gene (CEL) and its pseudogene (CELP) can create CEL-HYB1 allele which has been recently shown to elevate susceptibility to chronic pancreatitis (CP) in adults patients of European but not Asian ancestry, suggesting that it is an European CP risk factor. However, the replication studies are lacking.

Aim: To evaluate the risk associated with the CEL-HYB1 allele in the pediatric Polish CP patients as compared to control group.

Patients and Method: We enrolled a single-center cohort of 147 unrelated CP children (mean age at diagnosis 12 years) including 64 idiopathic (ICP) or familial idiopathic (FCP) patients and 83 CP patients with various ethological risk factors (anatomical/physiological dysfunctions and/or genetics risk factors). The control group consisted of 331 ethnically matched individuals (mean age 45).The CEL-HYB1 allele was detected using PCR, confirmed by Sanger sequencing.

Results: The CEL-HYB1 allele is significantly overrepresented in ICP/FCP patients compared to controls (9.4% vs 2.1%, P=0.0098) with OR of 4.8 (95%Cl 1.5-13.3) but not in a CP group with known ethological factors (1.2% vs 2.1%, P>0.05, OR=0.57, 95% CI (0.05-3.4). We identified two CP families with CEL-HYB1 allele and confirmed the co-segregation of the allele with the disease.

Conclusions: Our replication study show that the CEL-HYB1 allele is a significant CP risk factor in idiopathic and familial CP pediatric patients. The screening for CEL-HYB1 allele should be considered in CP diagnostics in European ancestry patients.

Financed by National Science Center, Poland:2015/19/B/NZ5/02224

A. Kujko: None. G. Oracz: None. K. Fjeld: None. K. Wejnarska: None. K. Wertheim-Tysarowska: None. E. Kołodziejczyk: None. J. Bal: None. D. Koziel: None. A. Kowalik: None. S. Gluszek: None. A. Molven: None. A.M. Rygiel: None.

P03.13D Congenital adrenal hyperplasia, due to 21-hydroxylase deficiency (21-OHD), isolated or associated with Ehlers Danlos syndrome (EDS): technical and counseling problems related to molecular diagnosis

S. Menabò1, A. Balsamo1, A. Cassio1, L. Mazzanti1, M. Seri1, L. Baldazzi2

1Department of Medical and Surgical Sciences, University of Bologna, Bologna, Italy, 2Department of Women, Children and Urological Diseases, Medical Genetics Unit, "S.Orsola-Malpighi" University-Hospital, Bologna, Italy

The 21-OHD is caused by mutations in the CYP21A2 gene, that maps within a repeated module (RCCX) in a region (6p21) with a high recombination frequency. Some RCCX arrangements can complicate and significantly modify the interpretation of the genetic tests. Our goal was to obtain the correct diagnosis of complex cases by integrating: complete gene sequencing, segregation analysis of variants/SNPs, CNVs detection by MLPA. The analysis of > 1200 cases by this approach showed that: a) in the 6% of alleles with multiple mutations they are distributed on different genes (duplications); b) in the 33% of cases with the Q318X mutation there is a concomitant duplicated normal gene, resulting in a non-pathological allele; c) in 37% of cases with a whole gene deletion, this includes part of the contiguous TNXB gene, resulting in 21OHD potentially associated with EDS signs. In very rare cases (<1%) with particularly complex arrangements, interpretative doubts may persist. The complexity of 21OHD genetic analysis therefore requires the integration of multiple techniques and an excellent knowledge of the peculiarities of the locus. An incomplete investigation can lead to erroneous interpretations of the result with serious consequences in clinical management and genetic counseling. An accurate clinical evaluation of patients with 21OHD should always include the search for EDS signs. The approach described here has proved to be fundamental for a correct interpretation of complex arrangements and, especially in cases of preconceptional and prenatal tests, to improve the genetic counseling, as well as the clinical classification/management of the patients.

S. Menabò: None. A. Balsamo: None. A. Cassio: None. L. Mazzanti: None. M. Seri: None. L. Baldazzi: None.

P03.14A CYP21A2 mutations in congenital adrenal hyperplasia due to 21 hydroxylase deficiency in Turkish population

O. Cilingir1, E. Simsek2, B. Durak Aras1, E. Erzurumluoglu1, C. Binay3, M. A. Temena1, S. Kocagil1, S. Artan1

1Eskisehir Osmangazi University, Faculty of Medicine, Department of Medical Genetics, Eskisehir, Turkey, 2Eskisehir Osmangazi University, Faculty of Medicine, Department of Pediatric Endocrinology, Eskisehir, Turkey, 3Tekirdag Corlu State Hospital, Tekirdag, Turkey

Congenital adrenal hyperplasia (CAH) caused by 21-hydroxylase deficiency is a common autosomal recessive disorder due to mutations in the CYP21A2 gene. Defects in this gene lead to adrenal insufficiency, ambiguous genitalia, salt-wasting in classic form of CAH, and hyperandrogenism during childhood or early adulthood in milder form of CAH, known as nonclassic CAH (NCAH). Mutations are mostly caused by a rearrangement during intergenic recombination between CYP21A2 and its non-functional pseudogene CYP21AP with very high percentage of sequence homology. This study aimed at analyzing the frequency of 11 prevalent mutations in 183 patients. Mutations, including P30L, 8bp deletion, exon 6 cluster, L307 frameshift, R356W, R483P, I2 splice, I712N, V281L, Q318 and, P453S were analyzed by reverse-hybridization strip-based assay (CAH Strip Assay). While 62,3% of 183 patients (114/183) have wild type alleles, the revealed CAH-related variants are given in the table. Interestingly, homozygous Del8bpE3 and I2 splice mutations was found in two siblings. Segregation analysis showed that the mother was heterozygous for these compound mutations. After confirmation of paternity, we are now focussing on the possibility of uniparental disomy (UPD). Further studies for this case were planned to clarify the genetic mechanisms underlying this situation.

Table. Percentage of mutation frequencies










I2 Splice

Del 8bp E3


Del 8bp E3

I2 Splice


Del 8bp E3


I2 Splice




E6 Cluster



I2 Splice











I2 Splice








I2 Splice




Del 8bp E3

I2 Splice






I2 Splice

Del 8bp E3














I2 Splice

Del 8bp E3














O. Cilingir: None. E. Simsek: None. B. Durak Aras: None. E. Erzurumluoglu: None. C. Binay: None. M.A. Temena: None. S. Kocagil: None. S. Artan: None.

P03.16C Molecular characterization of congenital hypothyroidism with “gland in situ” in Macedonia

N. Zdraveska1, N. Schoenmakers2, V. Anastasovska1, M. Kocova1

1University Children's Hospital Skopje, Skopje, Macedonia, The Former Yugoslav Republic of, 2University of Cambridge, Metabolic Research Laboratories, Cambridge, United Kingdom

Introduction: Congenital hypothyroidism (CH), defined as lack of thyroid hormones at birth, is the most common neonatal endocrine disorder affecting 1 in 2000-4000 newborns worldwide. While up to 20% of CH cases are hereditary, the majority of cases are sporadic with unknown etiology. Mutations in at least 15 different genes have been associated with CH. The genetics of CH has not been studied in Macedonia previously.

Material and Methods: A multigenic sequencing of CH candidate genes including TG, TPO, DUOX2, DUOXA2, SLC5A5, SLC26A4, IYD and TSHR was performed in a cohort of 22 CH patients with “gland in situ” (GIS), both familial and sporadic cases, associated with permanent, transient or subclinical phenotype.

Results: Mutations were identified in 27% of patients in the TPO, TSHR and DUOX2 genes. In two siblings with severe persistent CH, monoallelic c.1187_1188insGCCG mutation in TPO gene was detected. A child with prenatally diagnosed goiter was compound heterozygous for two mutations in TPO gene (c.31_50dup/ c.1313G>A). Two novel mutations were detected in TSHR gene in children with subclinical hypothyroidism c.1516G>A and c.692+1_692+4delGTGA. A previously known mutation c.4637A>G in the DUOX2 gene was detected in heterozygous state in one child with transient hypothyroidism.

Conclusion: Genetic variants were not frequently found in Macedonian CH patients, thus the etiology of CH with GIS remains elusive. Factors other than known dyshormonogenesis-associated genes or the TSHR have to be considered, as well as future studies with whole exome sequencing for elucidating the cause of hypothyroidism.

N. Zdraveska: None. N. Schoenmakers: None. V. Anastasovska: None. M. Kocova: None.

P03.17D Development of a biochip array for the rapid detection of 32 common cystic fibrosis mutations

M. J. Latten1, D. A. Bailie2, C. A. Graham1, M. A. Crockard1, J. V. Lamont1, S. P. FitzGerald1

1Randox Laboratories Ltd, Crumlin, United Kingdom, 2Northern Ireland Regional Genetics Centre, Belfast Health and Social Care Trust, City Hospital, Belfast, United Kingdom

Introduction: Cystic Fibrosis (CF) is an autosomal recessive genetic condition which affects vital organs, mostly the lungs, by clogging them with thick, sticky mucus. Persistent infections can lead to chronic lung problems. The disorder is caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator gene (CFTR) and to date, over 2,000 activating mutations have been reported. In most populations, however, a panel of 30-35 mutations will detect over 90% of disease causing variants.

Materials and Methods: An assay was designed for detection of 32 common CF mutations and the intron 8 polypyrimidine tract variant 5,7,9 T, using genomic DNA. Following target-specific multiplex-PCR, amplicons were hybridised to a 7 x 7 array of Discrete Test Regions (DTR) on a biochip (Randox Laboratories Ltd, UK). Using the Evidence Investigator analyser. Biochips were imaged and analysed automatically. Assay run time was <3 hours. Pre-characterised samples (n = 50) containing known CF mutations were assessed and results compared against the predicate genotyping.

Results: The array was verified using 50 pre-characterised samples containing known CF mutations, as well as the intron 8 polypyrimidine tract, in order to confirm specificity of the biochip for detection of mutations in the CFTR gene. Each of the 32 targets was assessed at least once. 100% concordance was achieved.

Conclusions: This rapid screening technique enables the simultaneous analysis of 32 common mutations associated with cystic fibrosis. This will aid in the confirmation of suspected cases of CF and also in the identification of those who are carriers of a mutation.

M.J. Latten: A. Employment (full or part-time); Significant; Randox Laboratories Ltd. D.A. Bailie: C. Other Research Support (supplies, equipment, receipt of drugs or other in-kind support); Significant; Randox Laboratories Ltd. C.A. Graham: A. Employment (full or part-time); Significant; Randox Laboratories Ltd. M.A. Crockard: A. Employment (full or part-time); Significant; Randox Laboratories Ltd. J.V. Lamont: A. Employment (full or part-time); Significant; Randox Laboratories Ltd. S.P. FitzGerald: A. Employment (full or part-time); Significant; Randox Laboratories Ltd.

P03.18A In vivo validation of phage therapy against Pseudomonas aeruginosa infections using zebrafish as a new model for cystic fibrosis

M. Cafora1, F. Forti2, G. Deflorian3, L. Ferrari3, D. Ghisotti2, F. Briani2, A. Pistocchi1

1Dip. Biotecnologie Mediche e Medicina Traslazionale, Università degli Studi di Milano, Milan, Italy, 2Dip. Bioscienze, Università degli Studi di Milano, Milan, Italy, 3Istituto Fondazione FIRC di Oncologia Molecolare IFOM, Milan, Italy

To investigate the pathophysiology of cystic fibrosis (CF) several animal models have been developed including mouse, pig, and ferret; however, none of them perfectly recapitulates all human patient symptoms. On the contrary, zebrafish (Danio rerio) recently emerged as a powerful genetic model system to better understand CF onset and to develop new pharmacological treatments. Indeed, zebrafish embryos present only innate immune system and the zebrafish cftr gene is highly conserved with the human orthologue. cftr-loss-of-function zebrafish embryos mimic CF human defects in response to infection of P. aeruginosa, presenting a dampened respiratory burst response, a reduced neutrophil migration and defects in endocrine organs function.

In our previous work, we demonstrated that P. aeruginosa infection in mice and Galleria mellonella larvae could be cured by administration of phages, the natural enemies of bacteria. Phage therapy, used for decades in Eastern Europe, is gathering interest as a therapeutic alternative or a complementary treatment to antibiotics. The goal of this project is to in vivo validate the efficacy of phage therapy against P. aeruginosa infections using the CF-zebrafish animal model. Both wild-type and cftr-loss-of-function zebrafish embryos, were infected with P. aeruginosa by microinjection, followed by phage administration. The therapeutic effects of phages was evaluated, following embryo mortality, bacterial burden, neutrophil migration and immune response. In addition, we plan to combine the phage treatment with antibiotics to verify if combination of the two treatments has a positive outcome against P. aeruginosa infections.

Funding Grant: Italian Cystic Fibrosis Research Foundation FFC#22.2017

M. Cafora: None. F. Forti: None. G. Deflorian: None. L. Ferrari: None. D. Ghisotti: None. F. Briani: None. A. Pistocchi: None.

P03.19B Diagnostic contribution of in house designed next generation sequencing panel gene test for Disorders of Sexual Development from Turkey

A. Aghayev1, G. Toksoy1, S. Poyrazoglu2, B. Karaman1, S. Avcı1, Z. Yavas Abalı2, U. Altunoglu1, F. Bas2, F. Darendeliler2, S. Basaran1, Z. Uyguner1

1Dept. Med. Genet, Istanbul Med. Faculty, Ist. Uni., Istanbul, Turkey, 2Dept. Ped. Endocrinology, Istanbul Med. Faculty, Ist. Uni., Istanbul, Turkey

Introduction: Disorders of sexual development (DSD) are defined as congenital conditions covering a wide spectrum of clinical expressions from mild hypospadias to abnormal gonadal abnormalities causing complete sex reversal. Early diagnosis is valuable as it will impact the individual’s mental well-being and overall life quality, including the advisability for genetic counseling.

Materials and Methods: We evaluated the results of 44 DSD cases for gross deletion/duplication with MLPA and for sequence alteration via in-house-designed next generation sequencing (NGS) targeted gene panel, using an Ion Torrent platform, covering all exon and exon-intron boundaries of 31 DSD associated genes. Cases with chromosomal abnormalities except one case with 47,XXY were excluded from the study group. Screening of targeted regions that were missed by AmpliSeq primer design is covered by Sanger sequencing. Segregation analysis was performed for very rare and novel missense alterations.

Results: Seven previously described and 15 possible DSD associated rare variants were identified in 10 different genes within a total of 19 cases, lead our diagnostic rate to 43%. The structural alteration in one novel coding region was simulated in three dimensional protein modeling program.

Conclusions: This study revealed new data for known and novel associated variants and acknowledged mutation frequencies in cases with DSD from Turkey. Our results underscored the critical role of early diagnosis which is valuable for proper management of gonadal tumors, expedient treatments and definitive genetic counseling for families.

A. Aghayev: None. G. Toksoy: None. S. Poyrazoglu: None. B. Karaman: None. S. Avcı: None. Z. Yavas Abalı: None. U. Altunoglu: None. F. Bas: None. F. Darendeliler: None. S. Basaran: None. Z. Uyguner: None.

P03.20C Association between a promoter SNP in MUC5B and idiopathic pulmonary fibrosis in the Newfoundland population

M. O. Woods, A. Pirzada, G. Zhai, B. Fernandez

Memorial University of Newfoundland, St. John's, NL, Canada

Background: Idiopathic pulmonary fibrosis (IPF) is a late-onset, complex genetic disease characterized by inflammation and scarring of the lung parenchyma. To date, heterozygous causal variations in TERT, TERC, SFTPC, SFTPA2, that account for 2-20% of IPF, have been documented. More recently, a promoter variant (rs35705950) upstream of MUC5B has been shown to be associated with IPF.

Methods: All probands in this study were previously screened for variants in the four abovementioned PF genes. A TaqMan SNP Genotyping assay and a 7900HT Real-time PCR analyzer were used to genotype rs35705950 in our cohort. A case-control analysis was carried out using 110 affected individuals and 277 healthy controls from the Newfoundland population.

Results: There was a significant association between rs35705950 genotypes and IPF. The odds ratios for all individuals affected with IPF who were heterozygous and homozygous for the variant allele of this promoter polymorphism respectively were 5.4 (95% CI, 3.3 to 9.6, P < .001) and 12.2 (95% CI, 3.3 to 44.7, P < .001). Furthermore, two families displayed segregation of the variant allele with the phenotype. Using SISA, we showed that, in one of these families, the likelihood that the minor allele segregates with PF by chance is 1.56%.

Conclusions: The minor T allele of rs35705950 is associated with FPF and likely contributes to the significant proportion of FPF families without a known genetic predisposition.

Supported by the Regional Partnership Program of the CIHR; the NL Lung Association; and the generous donations from the family of Craig L. Dobbin.

M.O. Woods: None. A. Pirzada: None. G. Zhai: None. B. Fernandez: None.

P03.21D Improving of CRISPR/Cas9 efficacy for F508del mutation correction in cystic fibrosis

S. A. Smirnikhina1, A. Anuchina1, K. Kochergin-Nikitsky1, E. Adilgereeva1, A. Lavrov1,2

1Federal State Budgetary Institution “Research Centre for Medical Genetics” of the Russian Academy of, Moscow, Russian Federation, 2The Russian National Research Medical University Named after N.I. Pirogov, Moscow, Russian Federation

Genome editing using CRISPR/Cas9 seems to be the most promising way for gene therapy to correct F508del mutation in CFTR gene in cystic fibrosis. Design of sgRNA to DNA sequence near mutation is determined by the presence of PAM for Cas9. It is possible to design only one sgRNA for F508del mutation for spCas9 - sgCFTR#1, but in HEK293T cell culture this sgRNA demonstrated low efficiency in indels formation in combination with different spCas9 (13.8%), compared to other sgRNAs to CFTR gene (13-18%), as well as to GFP gene (34.2%). qPCR data showed that sgCFTR#1 and sgGFP_1 expression levels were low and did not differ in 0 and 7 hours after transfection; but expression level of sgGFP_1 became almost 15-fold higher than sgCFTR#1 in 24 hours after transfection; 30 hours after transfection - 22-fold higher. Attempts to increase sgCFTR#1 expression by adding addition expression cassette to the plasmid, fusing sgCFTR#1 with active sgGFP_1 and using hybrid promoter were made, however, increasing of sgCFTR#1 efficacy was not received. Attempts to stabilize sgCFTR#1 by including G-quadruplexes to its sequence, shortening and addition of GG to the 5'-region also failed. Cultivation of the edited cells at lower temperature also did not lead to improved results. Further attempts to enhance sgCFTR#1 expression and its stabilization should be performed, or other Cas9 enzymes, which expand the ability to select sgRNA direct to F508del mutation can be used. The work was partially supported by Russian Science Foundation (agreement 17-75-20095 from 25/07/2017) and Russian Academy of Sciences.

S.A. Smirnikhina: A. Employment (full or part-time); Significant; Research Centre for Medical Genetics. B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Significant; Russian Science Foundation, Russian Academy of Sciences. A. Anuchina: A. Employment (full or part-time); Significant; Research Centre for Medical Genetics. B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Significant; Russian Science Foundation, Russian Academy of Sciences. K. Kochergin-Nikitsky: A. Employment (full or part-time); Significant; Research Centre for Medical Genetics. B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Significant; Russian Science Foundation, Russian Academy of Sciences. E. Adilgereeva: A. Employment (full or part-time); Significant; Research Centre for Medical Genetics. B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Significant; Russian Science Foundation, Russian Academy of Sciences. A. Lavrov: A. Employment (full or part-time); Significant; Research Centre for Medical Genetics. B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Significant; Russian Science Foundation, Russian Academy of Sciences.

P03.22A Diagnosis of monogenetic metabolic hepatopathies by whole-exome sequencing

A. Stalke1, A. Schöner-Heinisch1, B. Auber1, U. Baumann2, T. Illig1,3, B. Skawran1, B. Schlegelberger1, G. Schmidt1, E. Pfister1

1Hannover Medical School, Institute of Human Genetics, Hannover, Germany, 2Hannover Medical School, Paediatric Gastroenterology and Hepatology, Hannover, Germany, 3Hannover Medical School, Hannover Unified Biobank (HUB), Hannover, Germany

Introduction: As neonatal cholestasis is a common infant disease with more than 50 possible etiologies, fast and comprehensive diagnosis is desirable to initiate early therapy. Also for other hepatopathies, especially those with acute liver failure, rapid diagnosis is important, as they could constitute contraindication to liver transplantation or can be treated with specific therapies. We used four different whole-exome-sequencing-(WES)-based in-silico panels to analyze genes associated with different liver-related signs and symptoms.

Methods: DNA was extracted from blood samples of 66 patients (age: 0-46, median 2.50 years, 37 males). Library preparation was performed with TruSeq Nano DNA Library Preparation Kit (Illumina, San Diego, USA) or xGen Exome Research Panel (IDT, Leuven, Belgium). Libraries were sequenced on a NextSeq 500 (Illumina). Data analysis was done with the NGS pipeline megSAP (https://github.com/imgag/megSAP). Variants were filtered using GSvar (University Hospital Tübingen, Germany). Variant classification was done using Alamut Visual (Interactive Biosoftware, Rouen, France) according to standards and guidelines of American College of Medical Genetics and Genomics.

Results: In 25/66 patients we detected (likely) pathogenic variants or variants of unknown significance (VUS). The Table shows details:


signs and symptoms


Number of requests

Number of patients with


(likely) pathogenic variants*

VUS+ pathogenic variant°


heterozygous pathogenic variants or VUS°

no significant findings


Cholestasis with low gamma-glutamyltransferase









Cholestasis with high gamma-glutamyltransferase






Acute liver failure with suspected lysosomal storage disease, mitochondrial DNA depletion syndrome or Wilson disease








Hepatopathies/increased transaminases without primary cholestasis













  1. *Two variants for recessive, one for dominant traits
  2. °for recessive traits

Conclusion:WES-based panel analysis represents a fast and comprehensive tool to diagnose hereditary hepatopathies in order to improve therapy and thus patient’s prognosis. The WES-based approach further offers the possibility to analyze the remaining exome data in patients without clear diagnosis after panel analysis.

A. Stalke: None. A. Schöner-Heinisch: None. B. Auber: None. U. Baumann: None. T. Illig: None. B. Skawran: None. B. Schlegelberger: None. G. Schmidt: None. E. Pfister: None.

P03.23B Genetic linkage analysis identifies candidate modulators of reduced penetrance in heritable pulmonary arterial hypertension

I. Madrigal1,2, P. Puigdevall3, L. Piccari4, I. Blanco4,5, J. Barberà4,5, D. Geiger6, M. Milà1,2, R. Castelo3, C. Badenas1

1Biochemistry and Molecular Genetics Department, Hospital Clinic of Barcelona and IDIBAPS, Barcelona, Spain, 2Centre for Biomedical Research on Rare Diseases (CIBERER), ISCIII, Barcelona, Spain, 3Universitat Pompeu Fabra, Barcelona, Spain, 4Pneumology Department, Hospital Clinic of Barcelona and IDIBAPS, Barcelona, Spain, 5Centro de Investigación Biomédica en Red de Enfermedades Respiratorias (CIBERES), ISCIII, Madrid, Spain, 6Technion Israel Institute of Technology, Haifa, Israel

Introduction: Heritable pulmonary arterial hypertension is a rare disease inherited as an autosomal dominant disease. Mutations in the BMPR2 gene have been detected in 75-80% of the cases. However, only about 20% of all mutation carriers develop the disease, indicating a reduced penetrance.

Material and Methods: We have studied a large family affected by pulmonary arterial hypertension and segregating with the pathogenic missense mutation p.Arg491Gln with a penetrance of ~30%. In order to identify genetic variants that may affect the penetrance in this family, we genotyped 20 mutation carriers (6 affected and 14 asymptomatic) and 12 healthy individuals with the Illumina Infinium CoreExome-24 BeadChip.

Results: Genetic linkage analysis showed two candidate regions in chromosome 2 (q24.2-q31.1 and q33.1-q33.3) to host the modifier and confer susceptibility to the disease among BMPR2 mutation carriers. The modifier is predicted to be common in the population and to be inherited in an autosomal recessive mode. The first region is located 30 Mb upstream from BMPR2, while the second region overlaps the BMPR2 locus itself.

Conclusions: We found a significant enrichment of HPAH-related traits by a regulatory region within q24.2-q31.1, located upstream from the FIGN gene. Full genome sequencing and functional assays will be required to validate the linkage regions, identify the actual variant modulating the penetrance and unravel the molecular mechanism that triggers the disease in the family under study.

Acknowledgments: Instituto de Salud Carlos III (PI15/00483) and ‘fondos FEDER’, AGAUR (2017SGR1134), “CERCA Programme / Generalitat de Catalunya”

I. Madrigal: None. P. Puigdevall: None. L. Piccari: None. I. Blanco: None. J. Barberà: None. D. Geiger: None. M. Milà: None. R. Castelo: None. C. Badenas: None.

P03.24C Search for genetic risk factors in Hirschsprung's disease associated enterocolitis by Whole Exome Sequencing

T. Bachetti1, G. Santamaria1, M. Mosconi2, S. Sartori3, M. De Filippo3, A. Pini Prato4, I. Ceccherini1, F. Lantieri5

1Molecular Genetics Laboratory, G. Gaslini Inst., Genoa, Italy, 2UOC Pediatric Surgery, G. Gaslini Inst., Genoa, Italy, 3Center for Translational Genomics and BioInformatics, IRCCS San Raffaele Scientific Institute, Milan, Italy, 4Children Hospital, AON SS Antonio e Biagio e Cesare Arrigo, Alessandria, Italy, 5Health Science Department (DISSAL) – Biostatistics Unit, University of Genoa, Genoa, Italy

Introduction: Hirschsprung's disease (HSCR) is a congenital gut malformation. The most serious and life-threatening complication is enterocolitis (HAEC), which occurs in one third of the patients. The evident susceptibility to HAEC in HSCR patients suggests a genetic background that can lead to abnormal inflammatory response.

Materials and Methods: To search for genetic factors predisposing to HAEC, we have performed an exome next generation sequencing (NGS) on 24 HSCR patients, 12 with enterocolitis episodes (HAEC) and 12 without (HSCR-only). Patients were selected based on Italian ancestry and absence of additional anomalies. The exomes were sequenced with Illumina at a 50X coverage and variants were filtered based on depth >= 10 and quality >= 10, obtaining 77396 variants. These were further selected based on allele frequency in databases and impact on the protein, and on higher frequency in HAEC than in HSCR-only patients.

Results: We have thus identified 86 variants in 90 genes, which were ranked based on their frequency in the general population, predicted effect, association p-value, and recurrence in the samples. We also explored the gene pathways, biological role, and PubMed citations, particularly in regard to immune and inflammation processes. We are currently validating the five top variants and replicating the results on a larger panel of 25 HAEC and 45 HSCR-only patients.

Conclusions: We have identified a few very promising candidate genes. The most promising variant/s will undergo functional tests to confirm its/their role in HAEC development.

Acknowledgments: Funded by the Italian Ministry of Health (grant GR-2011-02347381)

T. Bachetti: None. G. Santamaria: None. M. Mosconi: None. S. Sartori: None. M. De Filippo: None. A. Pini Prato: None. I. Ceccherini: None. F. Lantieri: None.

P03.25D Whole exome sequencing to detect the cause of early onset nephrocalcinosis

F. PELUSO1, V. Palazzo2, L. Dosa2, G. Traficante2, S. Landini1, R. Artuso2, A. Provenzano1, P. Reho1, E. Bosi1, G. Carignani1, A. La Barbera1, A. Pagliazzi1, G. Forzano1, F. Becherucci3, M. Materassi3, R. Biagiotti4, C. Defilippi5, P. Romagnani3,6, S. Giglio1,2

1Medical Genetics Unit, Department of Biomedical Experimental and Clinical Sciences, Firenze, Italy, 2Medical Genetics Unit, Meyer Children's University Hospital, Florence, Italy, Firenze, Italy, 3Nephrology and dialysis Unit, Meyer Children's Hospital, Florence, Firenze, Italy, 4Prenatal Diagnosis Unit, Meyer Children's University Hospital, Firenze, Italy, 5Department of Diagnostic Imaging, Meyer Children's University Hospital, Firenze, Italy, 6Medical Genetics Unit, Department of Biomedical Experimental and Clinical Sciences "Mario Serio", University of Florence, Florence, Italy

Idiopathic infantile hypercalcemia (IIH) is characterized by severe hypercalcemia with failure to thrive, vomiting, dehydration, and nephrocalcinosis (NC). NC has not a well understood genesis. Monogenic causes were restricted to rare genetic syndromes/tubulopathies.

We examined a 24 month old child for advanced bilateral medullary nephrocalcinosis (accidentally detected at 15 months), failure to thrive, mild hypercalcemia/hypercalciuria, moderate hypophosphatemia without proximal tubulopathy, renal failure and skeletal dysplasia. He’s the firstborn of healthy consanguineous parents and the mother was pregnant; a maternal uncle has had recurrent nephrolitiasis since the age of 22 years old. Whole exome sequencing (WES) was performed in order to clarify the molecular diagnosis and to offer a prenatal test.

WES revealed a novel homozygous SLC34A1 mutation linked to IIH and Fanconi renotubular syndrome 2, and a monoallelic EHHADH mutation, inherited only from the mother that presented bilaterally kidney stones and urinary calculi history, associated with Fanconi renotubular syndrome 3. The same genetic background was identified also in fetal DNA extracted from amniotic fluid.

Treatment of IIH in early age is problematic as vitamin D therapy often exacerbates hypercalcemia. Considering the possibility to check an affected subject from the first month of life since the mother decided to continue the pregnancy, our data could give new insights about the temporal expression profile of SLC34A1 in the kidney and an explanation to the development of hypercalcemia in childhood, where intestinal absorption and renal handling of calcium and phosphate likely differ from older patients with other forms of IIH.

F. Peluso: None. V. Palazzo: None. L. Dosa: None. G. Traficante: None. S. Landini: None. R. Artuso: None. A. Provenzano: None. P. Reho: None. E. Bosi: None. G. Carignani: None. A. La Barbera: None. A. Pagliazzi: None. G. Forzano: None. F. Becherucci: None. M. Materassi: None. R. Biagiotti: None. C. Defilippi: None. P. Romagnani: None. S. Giglio: None.

P03.26A The application of targeted sequencing and whole exome analysis to identify disease-causing variants in familial pulmonary fibrosis

S. Wilkinson1,2, U. Hodgson3, F. Honti1, H. Beckwith1, P. Molyneaux4, W. O. Cookson2, T. Maher4, M. Moffatt2, T. Laitinen3, D. J. Morris-Rosendahl1

1Clinical Genetics and Genomics Laboratory, Royal Brompton and Harefield NHS Foundation Trust, London, United Kingdom, 2Genomic Medicine, National Heart and Lung Institute, Imperial College London, London, United Kingdom, 3Department of Pulmonology, University of Helsinki, Helsinki, Finland, 4Fibrosis Research Group, National Heart and Lung Institute, Imperial College London, London, United Kingdom

Introduction: Idiopathic Pulmonary Fibrosis (IPF) is one of the most common forms of interstitial pneumonia and is irreversible. It has a late age of onset, mostly between 55-75 years (median 66 years) and average survival rate of 3 years post diagnosis. Using next-generation sequencing (NGS) of whole exome (WES) and a targeted respiratory gene panel (Respigene) on Finnish and UK families with familial PF (FPF), we identified many new potentially pathogenic variants causative of FPF.

Materials and Methods: WES and targeted analysis of 42 genes previously associated with interstitial lung disease (ILD) was performed on 25 individuals (21 affected and 4 unaffected) from families with severe, early-onset FPF. Variant assessment was performed according to ACMG guidelines, using an in-house bioinformatics pipeline for classification of SNVs and CNVs, configured to rare respiratory conditions.

Results: Twelve of 25 individuals were found to have variants in ACMG classes 3-5 (VUS to pathogenic), in genes previously associated with FPF and which segregated in families where available. Seven patients were found to have potentially pathogenic variants in TERT, TERC and RTEL1, genes all previously associated with telomerase and telomere integrity. 13/21 (61.9%) patients were positive for the MUC5B SNP, rs35705950, as opposed to 1/4 unaffected individuals. No CNVs were found in any genes related to ILD.

Conclusion: These findings confirm that genes involved in telomere function play a major role in familial pulmonary fibrosis. These findings have potential implications for family members and raise the possibility of predictive testing and early therapeutic intervention in FPF.

S. Wilkinson: None. U. Hodgson: None. F. Honti: None. H. Beckwith: None. P. Molyneaux: None. W.O. Cookson: None. T. Maher: None. M. Moffatt: None. T. Laitinen: None. D.J. Morris-Rosendahl: None.

P03.27B A metabolic biomarker for idiopathic pulmonary fibrosis: a two sample Mendelian randomization study and a case-control study

A. Cerani1,2, S. Ross1,2, D. A. Schwartz3, P. Wolters4, B. Richards1,2

1McGill University, Montreal, QC, Canada, 2Lady Davis Institute for Medical Research, Montreal, QC, Canada, 3University of Colorado, Aurora, CO, United States, 4University of California - San Francisco, San Francisco, CA, Canada

Introduction: Idiopathic pulmonary fibrosis (IPF) is a lethal disease without current effective treatments. There is, therefore, an urgent need to find and validate biomarkers for diagnosis, prognosis, as well as potential drug targets. We propose to combine genomics and metabolomics to meet these objectives.

Methods and Results: To identify potential metabolites associated with IPF, we used an IPF GWAS that identified novel single nucleotide polymorphisms (SNP) associated with isovaleryl dehydrogenase (IVD), whose inhibition is known to increase the metabolite isovalerylcarnitine (IVC). Concurrently, we collaborated in a metabolite GWAS in healthy subjects that found the same IPF-associated SNP to be associated with IVC blood levels. Through 2-sample Mendelian randomization, we observed that genetically decreased IVC levels were strongly associated with increased risk of IPF (p< 2.9x10-10). The non-coding allele that decreased IVC levels increased IVD expression (p< 3.4x10-12). Thus, we hypothesized that low IVC levels are associated with higher risk of IPF. To test our hypothesis, we ran a case-control pilot study and found that IVC levels are decreased in IPF cases relative to controls (P = 2x10-3). Therefore, these data strongly suggest the IVC metabolic pathway could play a role in IPF etiology.

We are currently confirming our results in a larger case-control study.

Conclusion: IPF presents seriously unmet clinical needs. Building on strong preliminary data, we propose that IVC levels are associated with higher risk of IPF. IVC represents a clinically relevant biomarker and its enzyme, IVD, could represent an entirely novel IPF drug target.

A. Cerani: None. S. Ross: None. D.A. Schwartz: None. P. Wolters: None. B. Richards: None.

P03.28C Interallelic interactions mediated by the oligomerization of podocin

E. Balogh1,2, P. Stráner3, G. Schay4, C. Arrondel5, Á. Mikó6,2, G. L’Auné1,2, A. Benmerah5, A. Perczel3, D. K. Menyhárd3, C. Antignac5,7,8, G. Mollet9, K. Tory1,2

1MTA-SE Lendulet Nephrogenetic Laboratory, Budapest, Hungary, 2Semmelweis University, Ist Department of Pediatrics, Budapest, Hungary, 3MTA-ELTE Protein Modeling Research Group and Laboratory of Structural Chemistry and Biology, Eötvös Loránd University, Budapest, Hungary, 4Semmelweis University, Department of Biophysics and Radiation Biology, Budapest, Hungary, 5Laboratory of Hereditary Kidney Diseases, INSERM, UMR 1163, Imagine Institute, Paris, France, 6MTA-SE Lendulet Nephrogenetic Laboratory, Hungarian Academy of Sciences, Budapest, Hungary, 7Université Paris Descartes-Sorbonne Paris Cité, Imagine Institute, Paris, France, 8Assistance Publique – Hôpitaux de Paris, Hôpital Necker-Enfants Malades, Département de Génétique, Paris, France, 9Laboratory of Hereditary Kidney Diseases, INSERM, UMR 1163, Paris, France

NPHS2, the major gene of steroid-resistant nephrotic syndrome, encodes podocin, a membrane-anchored component of the slit diaphragm. We formerly showed that its R229Q variant is pathogenic only when trans-associated to specific 3’ missense mutations secondary to an altered C-terminal dimerization. We aimed to determine the membrane targeting and the oligomerization of podocin in function of its C-terminal integrity. Podocin localization was studied in podocytes co-transfected with GFP- and HA-tagged podocin variants. Oligomerization was assessed by measuring the FRET efficiency between maleimide-stained podocin variants and by size exclusion chromatography. We found the oligomerization to occur exclusively through the C-terminal tail (residues 283-382): principally through the 283-313 (H1) and the 332-348 residues. The interallelic interactions are mediated by oligomerization: while the monomer-forming R286Tfs*17 podocin remained membranous irrespective of the coexpressed podocin variant, podocin variants with an intact H1 significantly influenced each other’s localization (r2=0.68, P = 9.2x10-32). The dominant negative effect resulting in intracellular retention of the pathogenic F344Lfs*4-R229Q heterooligomer occured in parallel with a reduction in the FRET efficiency, suggesting a conformational rearrangement. In contrast, oligomerization with membranous podocin variants prevented the endocytosis of F344Lfs*4 or F344* podocin mutants induced by C-terminal truncation. In conclusion, oligomerization of podocin can mediate both a dominant negative effect and interallelic complementation. Interallelic interactions of 3’ NPHS2 mutations are not restricted to the R229Q variant and have to be considered in compound heterozygous individuals. Supported by MTA-SE Lendulet Research Grant (LP2015-11/2015), NKFIA/OTKA K109718, K116305, KH125566, MedinProt Synergy grant, (ANR-10-IAHU-01), EURenOmics (2012-305608), ANR (GenPod project ANR-12-BSV1-0033.01).

E. Balogh: None. P. Stráner: None. G. Schay: None. C. Arrondel: None. Á. Mikó: None. G. L’Auné: None. A. Benmerah: None. A. Perczel: None. D. K. Menyhárd: None. C. Antignac: None. G. Mollet: None. K. Tory: None.

P03.29D Biallelic Mutations in LRRC56, encoding a protein associated with intraflagellar transport, causes defects in mucociliary clearance and laterality

E. G. Sheridan1, S. Bonnefoy2, C. M. Watson1, K. D. Kernohan3, M. Lemos4, S. Hutchinson12, J. Poulter1, C. O'Callaghan5, R. A. Hirst6, A. Rutman6, L. Huang3, T. Hartley3, D. Grynspan7, C. Li8, I. M. Carr1, D. B. Bonthron1, M. Leroux8, Care4Rare Canada Consortium, K. B. Boycott3, P. Bastin2

1University of Leeds, Leeds, United Kingdom, 2Institut Pasteur, Paris, France, 3University of Ottawa, Ottawa, ON, Canada, 4Institut Pateur, Paris, France, 5University College London, London, United Kingdom, 6University of Leicester, Leicester, United Kingdom, 7Children’s Hospital of Eastern Ontario, Ottawa, ON, Canada, 8Simon Fraser University, Burnaby, BC, Canada

Defects in motile cilia result in conditions characterised by impaired pulmonary mucus clearance, susceptibility to chronic recurrent respiratory infections, male infertility and laterality defects. Here we report that biallelic variants in LRRC56 (also known as ODA8), result in a phenotype comprising laterality defects and chronic pulmonary infections. Investigation of cultured patient epithelial cells revealed severely dyskinetic cilia, but no structural abnormalities on transmission electron microscopy. In human cells, we show that LRRC56 interacts with the intraflagellar transport (IFT) protein IFT88.

In the model organism Trypanosoma brucei, we show LRRC56 is recruited during later phases of flagellar assembly before being removed during flagellum maturation, after cell division. We disrupted anterograde or retrograde IFT in T Brucei by use of RNAi. This showed that LRRC56 acts as an IFT cargo, but only at later stages of flagellar construction.

LRRC56 localisation was unchanged in DNAI1RNAi and ODA7RNAi mutants that are defective in their dynein arm constitution or cytoplasmic preassembly, respectively. Thus LRRC56 does not require the presence of dynein arms to be associated with flagella.

In T. brucei carrying LRRC56 null mutations, or a mutation (p.Leu259Pro) corresponding p.Leu140Pro variant seen in one of the affected families, we observed abnormal ciliary beat patterns and an absence of outer dynein arms restricted to the distal portion of the axoneme. These findings confirm that deleterious variants in LRRC56 result in human disease, and suggest this protein has a likely role in dynein transport during cilia assembly that is evolutionarily important for cilia motility.

E.G. Sheridan: None. S. Bonnefoy: None. C.M. Watson: None. K.D. Kernohan: None. M. Lemos: None. S. Hutchinson1: None. J. Poulter: None. C. O'Callaghan: None. R.A. Hirst: None. A. Rutman: None. L. Huang: None. T. Hartley: None. D. Grynspan: None. C. Li: None. I.M. Carr: None. D.B. Bonthron: None. M. Leroux: None. K.B. Boycott: None. P. Bastin: None.

P03.30A Metformin induced changes in gut microbiome composition in healthy individuals and newly-diagnosed type 2 diabetes patients

I. Elbere1, I. Kalnina1, I. Silamikelis1, I. I. Dindune1, L. Gulbinska1, L. Zaharenko1, I. Radovica-Spalvina1, D. Gudra1, D. Fridmanis1, I. Konrade2, V. Pirags1,3, J. Klovins1

1Latvian Biomedical Research and Study Centre, Riga, Latvia, 2Riga East Clinical University Hospital, Riga, Latvia, 3Pauls Stradins Clinical University Hospital, Riga, Latvia

Metformin is a biguanide class agent widely used as a first-line treatment for type 2 diabetes (T2D). Despite its advantages, metformin has variable therapeutic effects, contraindications, and side effects. Previous findings have led to the hypothesis that both the beneficial and adverse effects of metformin are partially explained by its interaction with the gut microbiome, yet details of these mechanisms remain obscure.

The study was conducted to investigate the effects of metformin treatment on the composition of human gut microbiome. The microbial DNA was extracted from:

(1.) Stool samples obtained from 18 healthy nondiabetic individuals at three time points during metformin treatment: M0 - before metformin treatment, M24h - 24 hours after the first metformin dose, and M7d - after a week-long metformin administration;

(2.) Stool samples acquired from 11 newly-diagnosed T2D patients at two concordant time points - M0 and M7d.

Probable changes in the gut microbiome induced by metformin intake were assessed employing massive parallel sequencing of the 16S rRNA gene V3 region.

A week-long metformin treatment rapidly and significantly decreased alpha diversity of gut microbiome among both groups. Concurrently, significant changes in several taxonomic groups were observed, including an increase in abundance of opportunistic pathogens representing Escherichia-Shigella genus, and decrease in possibly harming family Peptostreptococcaceae. Together these findings support the role of metformin in the modulation of the gut microbiome composition and the hypothesis that there may be some specific interactions further linked with efficacy and side effects of metformin.

This project was supported by ERDF No.:

I. Elbere: None. I. Kalnina: None. I. Silamikelis: None. I.I. Dindune: None. L. Gulbinska: None. L. Zaharenko: None. I. Radovica-Spalvina: None. D. Gudra: None. D. Fridmanis: None. I. Konrade: None. V. Pirags: None. J. Klovins: None.

P03.32C Targeted-NGS panel for inherited nephropathies: an example of clinical utility in a tube

J. Nevado1,2, R. Mena1, V. Gómez del Pozo1, R. Peces3, M. Melgosa4, L. Espinosa4, R. Selgas3, P. D. Lapunzina1,2

1INGEMM-IdiPaz, Madrid, Spain, 2CIBERER, Madrid, Spain, 3Adult Nephrology Department, HULP-IdiPaz, Madrid, Spain, 4Pediatric Nephrology Department, HULP-IdiPaz, Madrid, Spain

Introduction: Kidney diseases can be caused by a wide spectrum of underlying conditions and, in practice, the exact cause of a patient’s renal disease often remains unknown. Knowing the precise genetic defect is important in terms of its diagnostic, prognostic value and appropriate genetic counselling and is likely to become more crucial for directing specific therapy. NGS has opened up a new field allowing the identification of previously undiagnosed hereditary nephropathies. This study aims to investigate the etiology of genetic kidney diseases in the clinical routine of our hospital´s patient population.

Materials and Methods: The Nefroseq® panel V2.0 is a targeted NGS panel containing 350 genes involved in inherited nephropathies, optimizing libraries from Kappa, Target enrichment by SeqCap EZ system (RocheNimblegen), and sequencing on a NextSeq500 platform (Illumina).

Results: Regarding the analysis, it is done differently depending on the patient: restricted to the proband and known genes in patients with a specific phenotype, or open to a trio (including parents) when clinicians suggest an unspecific phenotype. Diagnostic yields are around 51-54%. Remarking is the genetic analysis of the PKD1 (responsible for most ADPKD cases), a diagnostic challenge: the 5′-region of the gene overlapped with a pseudogene. Our approach “in a tube” validated by Long-Rage PCR and Sanger sequencing yielded around 80% and it is cost-effective.

Conclusions: We share our management of the inherited nephropathies cases under a model pointed out to precision medicine, which includes a multidisciplinary committee for discussing the cases included in this customized NGS panel.

J. Nevado: None. R. Mena: None. V. Gómez del Pozo: None. R. Peces: None. M. Melgosa: None. L. Espinosa: None. R. Selgas: None. P.D. Lapunzina: None.

P03.33D Genetic causes of early onset obesity are frequently identified in a tertiary pediatric obesity cohort

L. Kleinendorst1, O. Abawi2, A. E. Brandsma3, M. H. Jongejan4, E. F. C. Van Rossum2, B. Van der Zwaag5, E. L. T. Van den Akker2, M. M. Van Haelst6,1

1Academic Medical Center, Amsterdam, Netherlands, 2Erasmus MC, University Medical Center Rotterdam, Rotterdam, Netherlands, 3Maasstad Hospital, Rotterdam, Netherlands, 4Franciscus Gasthuis, Rotterdam, Netherlands, 5University Medical Center Utrecht, Utrecht, Netherlands, 6VU university medical center, Amsterdam, Netherlands

Obesity is predominantly considered a multifactorial disorder. In unselected patient cohorts, an underlying genetic diagnosis can be established in only a minority of cases. They typically present with early-onset, severe obesity. Establishing a genetic diagnosis can lead to personalized treatment, reduce stigma and support reproductive decision-making. This study provides an overview of obesity-associated mutations and copy number variations (CNV’s) identified in this selected population.

In 174 obese children, referred to the pediatric obesity center Centrum Gezond Gewicht between 2012 and 2017, diagnostic sequencing of 52 obesity-associated genes, CNV detection by SNP-microarray analysis and, on clinical suspicion, specific additional diagnostics were performed to identify genetic causes of obesity.

In 28 patients (16.1%), an underlying genetic cause was identified (table 1). In an additional 22 patients (12.6%), a novel CNV or sequence variant of unknown clinical significance (VUS) was shown in obesity-associated genes, for which the role in the phenotype has yet to be confirmed.

A definitive obesity-associated genetic diagnosis was made in 16.1% of our patients with early-onset obesity. This may increase, if follow-up studies in patients with VUS confirm a causal role for their variants. This diagnostic yield is relatively high compared to similar studies and shows that genetic testing can be highly relevant in selected obese patients, especially when personalized treatment becomes available.

Table 1. Underlying genetic causes identified in our pediatric obesity patient cohort

Genetic disorder

No. of patients


Melanocortin-4 receptor, dominantly inherited obesity


Heterozygous MC4R mutation

Melanocortin-4 receptor deficiency


Homozygous or compound heterozygous MC4R mutation

Leptin receptor deficiency


Homozygous or compound heterozygous LEPR mutation

16p11.2 deletion syndrome


Typical 16p11.2 deletion

Melanocortin-2 receptor accessory protein 2 associated obesity


Heterozygous MRAP2 mutation in 2 sibs

Pseudohypoparathyroidism type 1A


Heterozygous GNAS mutation

Central hypothyroidism


Heterozygous IGSF1 mutation

Cohen syndrome


Homozygous VPS13B mutation

Mental retardation, autosomal dominant 39


Heterozygous MYT1L mutation

Maternal UPD 14


mUPD 14

Proprotein convertase-1 associated obesity


Heterozygous PCSK1 mutation

Proopiomelanocortin associated obesity


Deletion 2p, including POMC gene

Pseudohypoparathyroidism type 1B


Heterozygous STX16 mutation

Single-minded 1 associated obesity


Deletion 6q16.3, including SIM1 gene

Spastic paraplegia type 11


Compound heterozygous SPG11 mutation

L. Kleinendorst: None. O. Abawi: None. A.E. Brandsma: None. M.H. Jongejan: None. E.F.C. Van Rossum: None. B. Van der Zwaag: None. E.L.T. Van den Akker: None. M.M. Van Haelst: None.

P03.35B Isolated oesophageal atresia associated with rare structural variants identified by whole genome sequencing

J. Klar1, H. Engstrand Lilja2, J. Mattisson1, N. Dahl1

1Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden, 2Department of Women's and Children's Health, Section of Paediatric Surgery, Uppsala University Hospital, Uppsala, Sweden

Introduction: Oesophageal atresia (OA) is the most common congenital anomaly of the oesophagus with an incidence of around 1/3500 births. OA with a low tracheoesophageal fistula constitute 85% of all cases and syndromic OA at least 50% of cases. Genetic variants have been identified in a proportion of patients with syndromic OA but the genetics behind isolated forms remains elusive.

Materials and Methods: We identified three families with recurrent and isolated OA suggesting inherited factors behind the malformation. Whole genome sequencing (WGS; Illumina) was performed on DNA from affected individuals (n = 6) and presumed obligate and healthy carriers (n = 4).

Results and Conclusions: We identified single nucleotide variants (SNVs) and small insertions/deletions (indels) using GATK, and structural variants (SVs) using Manta. After stringent filtering (frequency = 0) against the Swedish 1000 genome project (SweGen), only one gene (DOCK4) remained that contains rare SNVs in all 10 individuals. Furthermore, we identified a total of 186 SVs in the affected individuals. Interestingly, we observe a significant enrichment of rare SVs on chromosome 21, suggesting a role for this chromosome in the aetiology of isolated OA in our cohort.

Grants: This work was supported by grants from the Swedish Research Council (2015-02424).

J. Klar: None. H. Engstrand Lilja: None. J. Mattisson: None. N. Dahl: None.

P03.36C Next Generation Sequencing (NGS) differential genetic analysis in polycystic kidney diseases: work in progress

G. Pipitone1, S. Calzavara1, R. Magistroni2,3, S. Merella1, A. Boletta2, M. Ferrari1,4,5, P. Carrera1,4

1Clinical molecular biology laboratory, IRCCS San Raffaele, Milano, Italy, 2Molecular basis of polycystic kidney disease unit, Division of genetics and cell biology, IRCCS San Raffaele, Milano, Italy, 3Division of nephrology and dialysis A.O.U. Policlinico, University of Modena and Reggio Emilia, Modena, Italy, 4Unit of genomics for human disease diagnosis, Division of genetics and cell biology, IRCCS San Raffaele, Milano, Italy, 5University Vita-Salute San Raffaele, Milano, Italy

Polycystic Kidney Diseases are clinically and genetically heterogeneous conditions mainly caused by mutations in PKD1/2. In the absence of a family history and/or in the presence of atypical presentations, other cystic diseases or other clinical conditions should be considered for a differential diagnosis. Thanks to NGS is now possible to modulate genetic test ranging from a few genes to a comprehensive clinical-exome testing, allowing differential genetic analysis based on the clinical suspicion. To evaluate this approach, we applied the illumina Clinical-Exome NGS protocol in 10 patients negative for ADPKD. Analysis of 106 genes, selected due to their relation with atypical form of PKD, was performed using an in-house validated pipeline (100% sensitivity, >94% specificity). Considering a population frequency <1% in the 1000KG and Exac Databases and basing on the consistency with the clinical phenotypes we focused on 16 candidate variants in total. Eight of these variants were private and novel. We found: 4 novel variants in HNF1B, BBS9, REN, GLIS3 and 5 already described variants in BBS9, PKHD1, GLIS3, NPHP3 and WDPCP. Moreover, one novel stop-gained and one start-lost variant were found in INVS and GLIS3 and three intronic and one splice donor variants were found in TMEM138, TMEM67, IFT43 and COL4A1. Interestingly, one frameshift variant was found in PKD1, previously misdiagnosed by Sanger sequencing, indicating a good performance of NGS. All variants, except one (start-lost variant in GLIS3), were confirmed by Sanger sequencing as well as the re-evaluation of clinical phenotypes and the potential for family and functional studies.

G. Pipitone: None. S. Calzavara: None. R. Magistroni: None. S. Merella: None. A. Boletta: None. M. Ferrari: None. P. Carrera: None.

P03.37D A comprehensive genetic testing in children with the clinical diagnosis of ARPKD identifies phenocopies

T. Szabó1, Z. Maróti2, T. Kalmár2, L. Madar3, É. Gombos3, O. Orosz3, K. Tory4,5, I. Balogh3

1Department of Pediatrics, Faculty of Medicine, University of Debrecen, Debrecen, Hungary, 2Department of Pediatrics, Faculty of Medicine, University of Szeged, Szeged, Hungary, 3Division of Clinical Genetics, Department of Laboratory Medicine, Faculty of Medicine, University of Debrecen, Debrecen, Hungary, 4Ist Department of Pediatrics, Semmelweis University, Budapest, Hungary, 5MTA-SE Lendulet Nephrogenetic Laboratory, Budapest, Hungary

Introduction: Autosomal recessive polycystic kidney disease (ARPKD) is genetically one of the least heterogeneous ciliopathies, resulting primarily from mutations of PKHD1. However, 15-40% of patients diagnosed with ARPKD are found not to carry PKHD1 mutations. Aim of this study was to identify whether mutations in other genes might explain these cases.

Materials and Methods:Thirty-six unrelated patients with the clinical diagnosis of ARPKD were tested by PKHD1 sequencing and MLPA. Patients without biallelic mutations were reevaluated and tested for second locus mutations in function of the phenotype, followed, if negative, by clinical exome sequencing.

Results:Twenty-eight patients (78%) carried PKHD1 point mutations, three of whom were heterozygotes for small-scale alterations. Two of the three patients possessed either a duplication of exons 33-35 or a large deletion involving exons 1-55 in trans. All eight patients without PKHD1 mutations had mutations in other genes (PKD1 (n = 2), HNF1B (n = 3), NPHP1, TMEM67, PKD1/TSC2). Perinatal respiratory failure, a kidney length >+4SD and early-onset hypertension were specific features of PKHD1-associated ARPKD.

Conclusions: We found all ARPKD cases without PKHD1 point mutations to be phenocopies, and none to be explained by biallelic PKHD1 copy number variations. Based on this non-consanguineous cohort, screening for copy number variations is indicated only in patients with a heterozygous point mutation. This work was supported by OTKA K109076 and Ministry of National Economy, Hungary, GINOP-2.3.2-15-2016-00039 (to István Balogh, Zoltán Maróti and Tibor Kalmár), MTA-SE Lendulet Research Grant (LP2015-11/2015) of Hungarian Academy of Sciences and NKFIA/OTKA K109718, KH125566 (to Kálmán Tory).

T. Szabó: None. Z. Maróti: None. T. Kalmár: None. L. Madar: None. É. Gombos: None. O. Orosz: None. K. Tory: None. I. Balogh: None.

P03.38A Prader-Willi syndrome: clinical particularities found in 15 patients

S. Calapod1, A. Hrisca2, R. Popescu1, E. Gorduza3, M. Gramescu3, M. Panzaru1, L. Butnariu1, C. Rusu1

1"St.Mary” Children's Hospital - Medical Genetics Center, Iasi, Romania, 2"St.Spiridon” Emergency Clinical Hospital - Department of Endocrinology, Iasi, Romania, 3”Gr. T. Popa” University of Medicine and Pharmacy - Department of Medical Genetics, Iasi, Romania

Prader-Willi syndrome (PWS) is a genetic disorder characterized by severe hypotonia and feeding difficulties in early infancy, followed by excessive eating and morbid obesity in childhood, intellectual disability, small hands and feet and hypogonadism. The genetic defect (microdeletion, uniparental disomy or imprinting defect) involves region 15q11.2-q13. We performed a clinical and genetic study of 15 patients diagnosed with PWS in Iaşi Medical Genetics Center to evaluate the relevance of clinical features for the diagnosis, identify particularities and possible complications. All cases were confirmed using MS-MLPA or FISH testing. We have identified neonatal hypotonia in 11/15 cases, feeding difficulties in infancy in 12/15, excessive weight gain in 9/15 (6/15 being under GH therapy), characteristic facial features in 13/15 and acromicria in 8/15. Micropenis (2/6), cryptorchidism (4/6) and hypoplasia of labia minora (1/9) indicated the presence of hypogenitalism. High pain threshold was seen in 5/15 cases, sleep apnea as well as skin picking in 4/15, ADHD, strabismus and incontinence were associated in 2/15. One case associated central fever. Particular features identified included: skeletal anomalies (5/15), congenital heart defects (4/15), cerebral malformations (3/15), ADHD (2/15), epilepsy (1/15) and multiple allergies (1/15). The correlation of the clinical features with the type of genetic defect will be illustrated in detail. Follow-up revealed: skeletal deformities that contraindicated GH therapy in one case, epilepsy and multiple allergies in another and central fever that led to death in another. In conclusion, we present a clinical study on 15 PWS patients to illustrate particular features and long-time evolution.

S. Calapod: None. A. Hrisca: None. R. Popescu: None. E. Gorduza: None. M. Gramescu: None. M. Panzaru: None. L. Butnariu: None. C. Rusu: None.

P03.39B The importance of comprehensive genomic profiling in differential diagnosis and discovery of novel disease causing genetic variants in patients with pediatric lung diseases

M. Z. Andjelkovic1, P. Minic2,3, M. Vreca1, M. Stojiljkovic1, A. Skakic1, A. Sovtic2, M. Rodic2, V. Skodric-Trifunovic4,3, N. Maric5, J. Visekruna2, V. Spasovski1, S. Pavlovic1

1Institute of Molecular Genetics and Genetic Engineering, Belgrade, Serbia, 2Mother and Child Health Care Institute of Serbia, Belgrade, Serbia, 3School of Medicine, University of Belgrade, Belgrade, Serbia, 4Clinic for Pulmonology, Clinical Center of Serbia, Belgrade, Serbia, 5Clinic for children diseases, Banja Luka, Belgrade, Serbia

Introduction: Primary ciliary dyskinesia (PCD) is a rare inherited autosomal recessive or X-linked disorder that mainly affects lungs. PCD diagnosis requires well-described clinical phenotype combined with the identification of underlying genetic causes since clinical symptoms of PCD overlaps with symptoms of other pediatric lung diseases. The aim of the study was to point out the significance of comprehensive genomic profiling in differential diagnosis of presumed PCD patients and detection of novel disease-causing genetic variants.

Materials and Methods: Using Next Generation Sequencing (NGS) approach and Clinical-Exome Sequencing Panel with 4813 genes, we analysed 74 genes related to PCD and other pediatric lung diseases in a cohort of 21 Serbian patients with clinically suspected PCD.

Results: After variant filtering and prioritization, the molecular diagnosis of PCD was achieved in 14/21 (66.67%) patients. The most frequently mutated gene was DNAH5 (23.33%). We have also genetically diagnosed asthma, NRDS, and bronchiectasis without CF, leading to detection rate of 95.24% (20/21). Identified variants were in homozygous, compound heterozygous and trans-heterozygous state. We also detected a novel homozygous frameshift mutation that leads to premature stop codon in DNAI1 gene (c. 947_948insG, p. Thr318TyrfsTer11), in two affected PCD siblings. For detailed characterization of novel genetic variant we performed RT-qPCR, in silico prediction of variant impact at protein level and protein analysis by Western Blot.

Conclusions: Analysis of the genomic profile of PCD suspected patients improves differential diagnosis and enables prenatal and carrier testing. It also leads to better understanding of the molecular basis of pediatric lung diseases.

M.Z. Andjelkovic: None. P. Minic: None. M. Vreca: None. M. Stojiljkovic: None. A. Skakic: None. A. Sovtic: None. M. Rodic: None. V. Skodric-Trifunovic: None. N. Maric: None. J. Visekruna: None. V. Spasovski: None. S. Pavlovic: None.

P03.40C PLVAP in protein-losing enteropathy: a homozygous missense variant leads to an attenuated phenotype

A. Kurolap1,2, O. Eshach-Adiv1, C. Gonzaga-Jauregui3, K. Dolnikov1, A. Mory1, T. Paperna1, T. Hershkovitz1, J. D. Overton3, M. Kaplan1, Y. Zohar1, A. R. Shuldiner3, G. Berger1, H. N. Baris1

1Rambam Health Care Campus, Haifa, Israel, Haifa, Israel, 2Technion – Israel Institute of Technology, Haifa, Israel, 3Regeneron Genetics Center, Tarrytown, NY, United States

Background: The integrity of the intestinal lymphatics is essential for proper nutrient absorption and tissue homeostasis. Damage to the lymphatic endothelial cells (LECs) leads to an enteric loss of protein-rich lymphatic fluid, i.e. protein-losing enteropathy (PLE). We investigated the genetic cause of PLE in two patients, first-degree cousins once removed, who presented at 22 and 2.5 years.

Methods: Whole exome sequencing was performed for two affected and five healthy family members. Variants were filtered based on autosomal recessive inheritance model, population frequencies, and predicted functional effect.

Results: We identified a rare homozygous variant (NM_031310.2:c.101T>C; p.Leu34Pro) in PLVAP, which co-segregated with the disease. The variant is predicted deleterious by bioinformatics algorithms, affecting the protein’s transmembrane domain. Electron microscopy (EM) of the younger patient's duodenal biopsy revealed preserved endothelial fenestral diaphragms.

Discussion: The plasmalemma vesicle-associated protein (PLVAP) is the building block of LEC fenestral diaphragms, which serve as a filtration system that blocks passage of large molecules and pathogens through the intestines. Mouse models of Plvap-deficiency demonstrate PLVAP’s important contribution to LEC barrier function. Recently, a homozygous nonsense variant in PLVAP was reported in a single patient with severe congenital PLE and hypertriglyceridemia. We now show that bi-allelic missense variants in PLVAP can cause an attenuated form of the syndrome, albeit apparently intact endothelial diaphragms on EM. Thus, our report establishes the role of PLVAP in the pathophysiology of PLE, and expands the phenotypic and mutation spectrums of the disorder, underscoring the importance of PLVAP in LEC barrier function in the gut.

A. Kurolap: None. O. Eshach-Adiv: None. C. Gonzaga-Jauregui: A. Employment (full or part-time); Significant; Regeneron Pharmaceuticals Inc. E. Ownership Interest (stock, stock options, patent or other intellectual property); Significant; Regeneron Pharmaceuticals Inc. K. Dolnikov: None. A. Mory: None. T. Paperna: None. T. Hershkovitz: None. J.D. Overton: A. Employment (full or part-time); Significant; Regeneron Pharmaceuticals Inc. E. Ownership Interest (stock, stock options, patent or other intellectual property); Significant; Regeneron Pharmaceuticals Inc. M. Kaplan: None. Y. Zohar: None. A.R. Shuldiner: A. Employment (full or part-time); Significant; Regeneron Pharmaceuticals Inc. E. Ownership Interest (stock, stock options, patent or other intellectual property); Significant; Regeneron Pharmaceuticals Inc. G. Berger: None. H.N. Baris: None.

P03.42A Genes with mutational enrichment in metastatic clear cell kidney cancer associate with patient mortality

A. Mendoza-Alvarez1,2, B. Guillen-Guio2, C. Hernandez-Perez3, A. Baez-Ortega4, J. M. Lorenzo-Salazar1, S. Lakhwani-Lakhwani2, M. D. C. Maeso5, M. Morales3, C. Flores1,2,6

1Genomics Division, Instituto Tecnológico y de Energías Renovables (ITER), Santa Cruz de Tenerife, Spain, 2Research Unit, Hospital Universitario Nuestra Señora de Candelaria, Universidad de La Laguna, Santa Cruz de Tenerife, Spain, 3Service of Medical Oncology, Hospital Universitario Nuestra Señora de Candelaria, Santa Cruz de Tenerife, Spain, 4Transmissible Cancer Group, Department of Veterinary Medicine, University of Cambridge, Cambridge, United Kingdom, 5Department of Pathology, Hospital Universitario Nuestra Señora de Candelaria, Santa Cruz de Tenerife, Spain, 6CIBER de Enfermedades Respiratorias, Instituto de Salud Carlos III, Madrid, Spain

Introduction: Kidney cancer only represents 2-3% of all cancers. However, more than 60% of the patients die after 2-3 years from diagnosis. Here we aimed to evaluate the association of somatic genetic variation with mortality by metastatic kidney cancer.

Materials and Methods:Whole-exome sequencing from paired tumor/normal FFPE tissue obtained from ten patients with metastatic clear-cell renal cell carcinoma was performed with AmpliSeq Exome RDY kit with the Ion Proton System (Thermo Fisher Scientific). Somatypus and VEP were used to identify and annotate single nucleotide variants and small indels. A gene-based burden of putative somatic mutations was calculated, and mutation enrichment assessed by the Fisher’s exact test. Gene-set enrichment analysis (GSEA) was evaluated with EnrichR.

Results: Mean exome-wide target coverage was 127-fold for tumor and 130-fold for matched normal samples. A total of 9,220 putative somatic variants mapping to 5,256 genes were detected across samples. Mutation enrichment was associated with mortality at nominal significance in 138 genes (lowest p = 2.0e-6), and GSEA on this subset evidenced a significant link with renal cancer (p = 2.3e-9), specifically for 49 of the genes.

Conclusions: Mutational burden in 138 genes associate with mortality among metastatic kidney cancer patients, which represents a concise gene set for conducting focused validation and functional studies.

Funding: Fundación CajaCanarias (SALUCAN11); Agreement OA17/008 with ITER to strengthen scientific and technological education, training, research, development and innovation in Genomics, Personalized Medicine and Biotechnology; ACIISI fellowship to BGG (TESIS2015010057) co-funded by European Social Fund; CEDeI fellowship (Cabildo de Tenerife) to AMA.

A. Mendoza-Alvarez: None. B. Guillen-Guio: None. C. Hernandez-Perez: None. A. Baez-Ortega: None. J.M. Lorenzo-Salazar: None. S. Lakhwani-Lakhwani: None. M.D.C. Maeso: None. M. Morales: None. C. Flores: None.

P03.43B Lung metagenomics to predict patient mortality by non-pulmonary sepsis

B. Guillen-Guio1, D. Domínguez2, A. Baez-Ortega3, F. Lorenzo-Diaz4, A. Díaz-de Usera5, R. González-Montelongo5, R. Hernández-Bisshopp2, J. Arias2, L. Soto2, J. Belda6, E. Espinosa2, J. Alcoba-Florez7, J. Villar8,9, C. Flores1,5,9

1Research Unit, Hospital Universitario N.S. de Candelaria, Universidad de La Laguna, Santa Cruz de Tenerife, Spain, 2Department of Anesthesiology, Hospital Universitario N.S. de Candelaria, Santa Cruz de Tenerife, Spain, 3Transmissible Cancer Group, Department of Veterinary Medicine, University of Cambridge, Cambridge, United Kingdom, 4Department of Biochemistry, Microbiology, Cell Biology and Genetics, Universidad de La Laguna, Santa Cruz de Tenerife, Spain, 5Genomics Division, Instituto Tecnológico y de Energías Renovables (ITER), Santa Cruz de Tenerife, Spain, 6Department of Anesthesiology, Hospital Clínico Universitario de Valencia, Valencia, Spain, 7Department of Microbiology, Hospital Universitario N.S. de Candelaria, Santa Cruz de Tenerife, Spain, 8Research Unit, Hospital Universitario Dr. Negrin, Las Palmas de Gran Canaria, Spain, 9CIBER de Enfermedades Respiratorias, Instituto de Salud Carlos III, Madrid, Spain

Introduction: Death by sepsis remains high in patients admitted into intensive care units (ICUs) worldwide. Identifying early biomarkers of disease prognosis remains an urgent necessity. Here we hypothesized that lung microbiome perturbations associate with ICU mortality in septic patients.

Materials and Methods: We analyzed DNA from 41 bronchoalveolar aspirates from non-pulmonary septic ICU patients at sepsis diagnosis (8h), and after 24h and 72h. Bacterial abundance was obtained by DNA sequencing of the 16S rRNA V4. QIIME was used for operative taxonomic units (OTUs) assignment.

Results: The core microbiome (taxa in >50% of patients) was composed by 46 OTUs and its number diminished in deceased compared to surviving patients at all collection times. Diversity differences were evident very early (17 vs 45 OTUs in deceased and survivors at 8h of sepsis diagnosis, respectively; p < 0.01). The four most abundant taxa in non-survivors comprised >89% of the core microbiome, while these only represented 15% of the core microbiome in survivors.

Conclusions: A reduction in bacterial lung diversity within 8h of sepsis diagnosis associates with ICU mortality, providing a novel prognostic biomarker at an early stage of disease.

Funding: ISCIII (PI11/00623, PI16/00049) and co-financed by the European Regional Development Funds, “A way of making Europe” from the European Union; Agreement OA17/008 with ITER to strengthen scientific and technological education, training, research, development and innovation in Genomics, Personalized Medicine and Biotechnology; Fellowships from the ACIISI (TESIS2015010057) co-funded by European Social Fund to BGG and the Spanish Ministry of Education, Culture and Sports (FPU16/01435) to ADU.

B. Guillen-Guio: None. D. Domínguez: None. A. Baez-Ortega: None. F. Lorenzo-Diaz: None. A. Díaz-de Usera: None. R. González-Montelongo: None. R. Hernández-Bisshopp: None. J. Arias: None. L. Soto: None. J. Belda: None. E. Espinosa: None. J. Alcoba-Florez: None. J. Villar: None. C. Flores: None.

P03.45D Searching for causal treatment in patients with Wolfram syndrome

A. Zmyslowska1, M. Borowiec2, E. Polakowska1, A. Lesiak3, W. Mlynarski1

1Department of Pediatrics, Oncology, Hematology and Diabetology, Medical University of Lodz, Lodz, Poland, 2Department of of Clinical Genetics, Medical University of Lodz, Lodz, Poland, 3Department of of Dermatology, Pediatric and Oncology Dermatology, Medical University of Lodz, Lodz, Poland

Introduction: Wolfram syndrome (WFS) is an example of neurodegenerative disease due to increased ER stress coexisting with diabetes mellitus with no causal treatment. WFS is caused by recessive mutations in the WFS1 gene among which are the mutations of premature termination codons (PTCs). Some prospects for the causal treatment of WFS patients can give a readthrough of PTCs. The use of a chemical compound e.g. PTC124 can result in bypassing the PTCs and a continuation of translation. The aim of the study was to evaluate a repairing of a genetic defect by using PTC124 in WFS patients with confirmed mutations of PTCs.

Materials and Methods: Diagnosis of WFS was confirmed by direct sequencing of the WFS1 gene. On fibroblasts from skin biopsies of WFS patients and healthy individuals the in vitro studies were performed involving the induction of ER stress (Tunicamycin) with a subsequent using a compound PTC124 (Ataluren). Evaluation of ER stress induction was performed by mRNA expression analysis of wolframin and markers of the ER stress (7900HT Real Time PCR; Applied Biosystems, USA).

Results: In 12/15 of WFS patients the mutations of PTCs were identified. The most specific markers of ER stress in patients with WFS should be considered: GRP78, GADD153 (CHOP) and ATF4. Thus, a compound PTC124 may unfortunately increase the ER stress.

Conclusions: It seems that PTC124 by ER stress increasing can not be used as a potential causal treatment for the WFS patients.

Supported by the National Science Centre grants No 2014/15/B/NZ5/01579 and 2015/19/B/NZ5/02243.

A. Zmyslowska: None. M. Borowiec: None. E. Polakowska: None. A. Lesiak: None. W. Mlynarski: None.

P03.46A Afifty-year follow-up of familial Hirschsprung disease

E. Passarge1, A. N. Ziegler2, R. J. Hopkin3, H. M. Saal3

1Institut für Humangenetik, Universitätsklinikum Essen, Essen, Germany, 2., Harrison, OH, United States, 3Human Genetics Division, Cincinnati Children´s Hospital Medical Center, Cincinnati, OH, United States

We describe familial Hirschsprung disease (OMIM 142623) with histologically proven congenital intestinal aganglionosis in the proposita, her two affected brothers, her two affected daughters and in the extended family three first cousins (two males and one female). Originally this family was described in 1967 as part of systematic study of the genetics of Hirschsprung disease (Family 21 in E. Passarge, New Eng J Med 1967; 276:138-143). It presumably involves the highest number of affected members documented. In all patients the non-syndromic long segment disease (HSCR type 2) is present. At the age of 8 months the proposita was admitted to Cincinnati Children´s Hospital for ileostomy under the care of one of the authors (EP), then a resident in pediatrics. During the following 15 years she required 41 surgical procedures of the GI tract or the rectum. In spite of the complicated course she is well adjusted to her disorder. This family was re-investigated in November 2017, including two daughters of the proposita also affected with long segment Hirschsprung disease. Both daughters also required several additional surgical procedures. Hirschsprung disease, a genetically heterogeneous and clinically variable disorder, results from the absence or malfunction of intestinal ganglion cells. At least three signal effector pathways are required for normal migration and development of intramural intestinal ganglion cells, (i) the RET tyrosine-kinase receptor and its ligand GDNF (glial-cell-derived neurotrophic factor, OMIM 600837), (ii) endothelin type B receptor (EDNRB, OMIM 600837) and its ligand EDN3 (endothelin 3, OMIM 131244), (iii) SOX10 transcription factor (OMIM 602229).

E. Passarge: None. A.N. Ziegler: None. R.J. Hopkin: None. H.M. Saal: None.

P04 Skeletal, connective tissue, ectodermal and skin disorders

P04.01A Microduplication of 13q31.3 region: clinical and molecular analysis based on a new case

M. Młynek, M. Kucharczyk, D. Wicher, A. Cieślikowska, A. Gutkowska, M. Krajewska-Walasek

Children's Memorial Health Institute, Warsaw, Poland

Microduplication of 13q31.3 is a rare genomic disorder associated with abnormal growth and skeletal development. To date, less than ten comparable submicroscopic duplications involving this region have been described. The most important phenotypic features include developmental delay, digital malformations, growth abnormalities, macrocephaly and facial dysmorphism. MIR17HG (encoding the miR-17~92 polycistronic miRNa cluster) and GPC5 are the major candidate genes for the genotype-phenotype correlation.

Here, we report on the case of a 3 4/12 year-old girl referred for genetic counseling because of excessive height and weight (above the 97th centile) and dysmorphic features (macrocephaly, frontal bossing, hyperthelorism, long palpebral fissures, long philtrum, tilted upper lip, pug nose, widely spaced teeth). In the neonatal period umbilical hernia was observed. At the clinical examination, indistinct speech, laxity of hand joints, long and overlapping toes, café au lait spot on the thigh and hypoplastic nipples were noted.

Conventional G-banding karyotyping revealed a de novo translocation 46,XX,t(1;13)(q21;q13.31). Whole-genome oligonucleotide microarray analysis revealed a 11.47-Mb cryptic interstitial duplication of the 13q31.1q32.1 region associated with the translocation breakpoints (chr13:85583670-97054435; GRCh37). Microarray studies in both parents gave normal results, proving de novo occurrence of this aberration in the child.

In conclusion, this study contributes additional information for the 13q31.3 microduplication and strongly support the involvement of miR-17~92 cluster of miRNA and GPC5 gene in normal growth and skeletal development.

This study was supported by the NCN Grant No. UMO-2016/21/B/NZ5/02541.

M. Młynek: None. M. Kucharczyk: None. D. Wicher: None. A. Cieślikowska: None. A. Gutkowska: None. M. Krajewska-Walasek: None.

P04.02B Generation and validation of the first complete knockout model of abcc6a in zebrafish

M. Van Gils1, A. Willaert1, E. De Vilder1,2, P. Coucke1,2, O. Vanakker1,2

1Center for Medical Genetics, Ghent, Belgium, 2Ghent University Hospital, Ghent, Belgium

Introduction: Pseudoxanthoma elasticum is an ectopic mineralization disease due to biallelic ABCC6 mutations. As no curative therapy is available, development of reliable disease models for compound screening is necessary. Zebrafish, which have a functional abcc6a orthologue, are an excellent candidate model organism, if it has a reliable phenotype. For a morpholino-induced knockdown and a missense mutant model of abcc6a phenotypic discrepancies are reported, questioning their validity. Therefore, we developed a complete CRISPR/Cas9 knockout model and compared its phenotype to a novel mutant (Sa963) and our morpholino model.

Materials and Methods: Both carriers of the CRISPR-induced (c.180delTCGG) and Sa963 (c.2250+1G>A) heterozygotes were out- and incrossed to F4 generations. Morpholino dosage was optimized to 3ng and co-injected with 4.5ng p53-morpholino. Development was monitored during embryonic development into adulthood. Mineralization was assessed via alizarin red S staining and ImageJ analysis.

Results: In all models we noted similar extensive and progressive vertebral hypermineralization (Table 1), starting during embryonic development, with spinal deformities and scoliosis in adults. Contrary to previously reported data, abcc6a was essential for neither embryonic survival nor morphological development.

Conclusions: Observing an identical ossification phenotype in 3 independent models confirms its specificity and indicates for the first time a direct relation between abcc6a deficiency and dysregulated osteogenesis. The phenotype is readily quantifiable and an excellent readout for compound screening.

Table 1: Mineralization semi-quantified at 10 days post fertilization (Mean  ±  SD; *P<0.05)


+/+ or Control

-/- or Morpholino

CRISPR (c.180delTCGG)

100%  ±  9.6

121%  ±  8.8 *

Sa963 (c.2250+1G>A)

100%  ±  15.8

135%  ±  40.2 *

Morpholino abcc6a

100%  ±  19.2

155%  ±  29.6 *

M. Van Gils: None. A. Willaert: None. E. De Vilder: None. P. Coucke: None. O. Vanakker: None.

P04.03C Functional characterization of a predicted regulatory element associated with alopecia areata (AA)

M. M. Mattern1,2, A. Tafazzoli1,2, S. Sivalingam1,2, J. Schultze3, K. Ludwig2, M. M. Nöthen1,2, P. Kokordelis1,2, R. C. Betz1,2

1Institute of Human Genetics, University of Bonn, Bonn, Germany, 2Department of Genomics, Life & Brain Center, University of Bonn, Bonn, Germany, 3Life & Medical Sciences-Institute (LIMES), University of Bonn, Bonn, Germany

Alopecia areata (AA) is a genetic complex hair loss disorder characterized by patchy hair loss. Genetic studies have supported the hypothesis that AA is autoimmune in nature. The identified loci only explain a limited proportion of disease heritability. The goal of the current project is to functionally characterize a regulatory element (RE) and its effect on IL2RA and other genes in close vicinity. Based on data of a recent meta-analysis on AA, we performed a genome-wide in-silico analysis of REs. We cloned a T-cell specific RE, which contains the identified lead SNP, into a pGL4.23 vector to perform dual-luciferase-assay (DLA) in Jurkat cells. Further, we analyzed the effect of the reference and alternative allele on gene transcription in association with a minimal promoter or with a promoter of a predicted target gene.

We identified a region mapping into a predicted T-cell enhancer, which might influence the expression of the nearby AA-susceptibility gene IL2RA. Analysis of CTCF sites revealed co-localization of IL2RA and the RE in the same CTCF flanked domain. DLAs of the cloned RE showed significant decrease of gene expression compared to controls. Furthermore, gene expression was significantly suppressed after integration of the validated IL2RA promoter. However, introduction of the identified SNP led to significantly less suppression.

Taken together, with our experiments we could prove allele-dependent silencing properties of the examined regulatory region. Regulation of the IL2RA gene might play a relevant role in the development of regulatory and effector T-cells in AA.

Funded by the EKFS-Promotionskolleg, Bonn.

M.M. Mattern: None. A. Tafazzoli: None. S. Sivalingam: None. J. Schultze: None. K. Ludwig: None. M.M. Nöthen: None. P. Kokordelis: None. R.C. Betz: None.

P04.04D A homozygous missense change in canine ALPL, an animal model for hypophosphatasia

K. Kyöstilä1,2,3, P. Syrjä1, S. Hundi1,2,3, A. Lappalainen4, R. Viitmaa4, V. Karkamo5, H. Lohi1,2,3

1Department of Veterinary Biosciences, University of Helsinki, Helsinki, Finland, 2Department of Molecular Genetics, Folkhälsan Institute of Genetics, Helsinki, Finland, 3Research Programs Unit, Molecular Neurology, University of Helsinki, Helsinki, Finland, 4Department of Equine and Small Animal Medicine, University of Helsinki, Helsinki, Finland, 5Section of Companion Animal Pathology, Finnish Food Safety Authority Evira, Helsinki, Finland

Introduction: The purebred dogs are affected with similar skeletal disease as humans. In the present study, we have performed clinical, pathological and genetic examinations to characterize a previously unknown, autosomal recessive skeletal disease in the Karelian Bear Dog (KBD) breed.

Materials and Methods: Clinical examinations were performed on six affected dogs and pathological examination on seven dogs. Exome sequencing was carried out on one affected dog.

Results: Altogether seven affected puppies were recognized in the KBDs. The clinical phenotype varied from a failure to thrive and seizures at 2 weeks of age to a growth defect and severe ambulatory difficulties at 12 weeks of age. Serum analysis indicated low alkaline phosphatase activity and hypercalcemia. Radiographic and pathological examinations revealed defective skeletal mineralization and ossification. Exome sequencing identified a homozygous missense variant in a conserved domain of the tissue-nonspecific alkaline phosphatase gene, ALPL. The ALPL variant showed full segregation with the disease in a cohort of 489 KBDs, and was absent from 303 dogs from control breeds.

Conclusions: In humans, near 350 ALPL mutations are known to cause hypophosphatasia, a metabolic bone disease with heterogeneous clinical manifestations. We have identified a recessive ALPL missense change in dogs with clinical and pathological findings compatible with hypophosphatasia. To our knowledge, this is the first time naturally-occurring inherited hypophosphatasia has been described in animals, and we hope our findings contribute to understanding of the disease.

Grants: This study was supported by the Academy of Finland, Jane and Aatos Erkko Foundation and Wisdom Health.

K. Kyöstilä: None. P. Syrjä: None. S. Hundi: None. A. Lappalainen: None. R. Viitmaa: None. V. Karkamo: None. H. Lohi: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Significant; Wisdom Health. F. Consultant/Advisory Board; Modest; Genoscoper Laboratories Oy.

P04.05A Loss of GPNMB causes autosomal recessive amyloidosis cutis dyschromica in humans

C. Yang1, S. Lin2,3, C. Chiang4,5, Y. Wu2,3, W. H'ng1, C. Chang1, Y. Chen1, J. Wu1

1Academia Sinica, Taipei, Taiwan, 2Mackay Medical College, New Taipei City, Taiwan, 3Mackay Memorial Hospital, Taipei, Taiwan, 4Tri-Service General Hospital, Taipei, Taiwan, 5National Defense Medical Center, Taipei, Taiwan

Amyloidosis cutis dyschromica (ACD) is a distinct form of primary cutaneous amyloidosis characterized by generalized hyperpigmentation mottled with small hypopigmented macules on the trunks and limbs. Families and sporadic cases have been reported predominantly in East and Southeast Asian ethnicities; however, the genetic cause has not been elucidated. Homozygous premature nonsense mutations leading to loss of function of GPNMB contribute to the iris pigment dispersion phenotype in mouse pigmentary glaucoma. However, no mutations in GPNMB have been identified in human pigmentary glaucoma and pigment dispersion syndrome. We establish that the compound heterozygosity or homozygosity of GPNMB truncating alleles is the cause of autosomal recessive ACD. Six nonsense or frameshift mutations were identified in nine individuals diagnosed with ACD. Immunofluorescence analysis of skin biopsies showed that GPNMB is expressed in all epidermal cells, with the highest staining observed in melanocytes. GPNMB staining is significantly reduced in the lesional skin of affected individuals. Hyperpigmented lesions exhibited significantly increased amounts of DNA/keratin-positive amyloid deposits in the papillary dermis and infiltrating macrophages compared with hypo-/depigemented macules. Depigmentation of the lesions was attributable to loss of melanocytes. Intracytoplamic fibrillary aggregates were observed in keratinocytes scattered in the lesional epidermis. Thus, our analysis indicates that loss of GPNMB, which has been implicated in melanosome formation, autophagy, phagocytosis, tissue repair, and negative regulation of inflammation, underlies autosomal recessive ACD, and provides insights into the etiology of amyloidosis and pigment dyschromia.

C. Yang: None. S. Lin: None. C. Chiang: None. Y. Wu: None. W. H'ng: None. C. Chang: None. Y. Chen: None. J. Wu: None.

P04.06B MetaXcan gene-based association analysis yields novel insights into male-pattern baldness biology

S. Heilmann-Heimbach1,2, L. M. Hochfeld1,2, V. Schüller3, the MAAN Consortium, M. M. Nöthen1,2

1Institute of Human Genetics, University of Bonn, School of Medicine & University Hospital Bonn, Bonn, Germany, 2Department of Genomics, Life&Brain Center, University of Bonn, Bonn, Germany, 3Institute for Medical Biometry, Informatics, and Epidemiology, University of Bonn, Bonn, Germany

Male-pattern baldness (MPB) is a heritable trait and GWAS have implicated more than 100 genomic regions. The majority of associated variants however are located in noncoding regions and their functional relevance remains unclear. Integrative analyses that link genetic variation with biological function are needed to identify relevant genes and mechanisms. Here, we used MetaXcan (Barbeira et al., bioRxiv) on summary statistics from two published large-scale genetic studies for MPB (Heilmann-Heimbach et al., 2017; Hagenaars et al., 2017) and gene-expression data from human hair follicle (own data) and the GTEx-project (skin, adipose tissue, blood) to test for an association between MPB and the genetically determined regulation of gene-expression in these tissues. The analysis identified associations between MPB and expression levels of 30 (hair follicle) to 637 genes (skin). Twenty-seven genes showed significant association in one or more tissues after Bonferroni correction (P<0.05/27300 gene-tissue pairs). These included previously implicated genes (e.g. WNT10A, IRF4, TWIST1) and novel candidate genes such as SPRR1B and TRADD, which have a reported role in keratinocyte biology and apoptosis, thereby rendering an involvement of these processes in early MPB-pathogenesis likely. The fact that associations with previously implicated genes were detected in subcutaneous adipose tissue (TWIST1) and blood (WNT10A) lends further support to the hypothesis, that cells in the perifollicular environment play a role in MPB. In summary, our analyses provide evidence for an MPB-associated genetic regulation of plausible candidate genes (SPRR1B,TRADD,WNT10A) and biological processes (apoptosis,adipogenesis) and yield novel insights into the functional effects of current GWAS findings.

S. Heilmann-Heimbach: None. L.M. Hochfeld: None. V. Schüller: None. M.M. Nöthen: None.

P04.07C Evidence for a functional interaction between WNT10A and EBF1 in the development of androgenetic alopecia (AGA)

L. M. Hochfeld1,2, D. Broadley3, N. V. Botchkareva3, M. P. Philpott4, S. Schoch5, R. C. Betz1, M. M. Nöthen1,2, S. Heilmann-Heimbach1,2

1Institute of Human Genetics, University of Bonn, School of Medicine & University Hospital Bonn, Bonn, Germany, 2Department of Genomics, Life&Brain Center, University of Bonn, Bonn, Germany, 3School of Chemistry and Bioscience, Centre for Skin Sciences, University of Bradford, Bradford, United Kingdom, 4Centre for Cell Biology and Cutaneous Research, Blizard Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, London, United Kingdom, 5Department of Neuropathology, University of Bonn Medical Center, Bonn, Germany

The WNT10A-locus on 2q35 is a known risk locus for AGA. A reduced WNT10A expression in hair follicle (HF) of risk allele carriers for rs7349332 supports the role of WNT10A as the functionally relevant gene (Heilmann et al., 2013). This lead variant is in high LD (r2=0.96, D’=0.99) with rs3856551, located within a binding site for the transcription factor EBF1. Interestingly, the gene encoding EBF1 is located at the 5q33.3-AGA risk locus, suggesting that changes in EBF1 mediated regulation of WNT10A expression may contribute to AGA. To investigate this potential interaction, we performed luciferase assays by co-transfecting (i) luciferase vectors containing the WNT10A promoter and the 2q35-EBF1 binding site with either the risk or the alternate allele and (ii) an EBF1 expression vector. Our experiments in HEK cells showed that EBF1 activates the WNT10A promoter and that the WNT10A/EBF1 interaction was increased with the risk allele, which together with the previous mRNA expression data suggests that EBF1 acts as a negative regulator of WNT10A. To confirm this interaction in AGA relevant tissue, we tested for co-expression of WNT10A and EBF1 in a published RNA-Seq dataset of mouse HF (Joost et al., 2016) and performed immunofluorescence co-staining in human HF and skin. The analyses revealed the strongest co-expression in HF keratinocytes. Therefore, the initial luciferase assays are repeated in human keratinocytes. In parallel, EBF1 knockout experiments in human HF are under way. Taken together, our data provide the first evidence for a functional interaction between WNT10A and EBF1 in AGA pathobiology.

L.M. Hochfeld: None. D. Broadley: None. N.V. Botchkareva: None. M.P. Philpott: None. S. Schoch: None. R.C. Betz: None. M.M. Nöthen: None. S. Heilmann-Heimbach: None.

P04.08D Genetic investigation of a rare form of severe tooth agenesis: Anodontia

M. Haddaji Mastouri1, P. De Coster2, N. Ben Salah1, A. Touati1, I. Veerecke3, A. Zaghabani4, A. SAAD1, D. H’mida-Ben Brahim1, P. Coucke3

1Laboratory of cytogenetics, molecular genetics and reproductive biology, Farhat HACHED Hospital, Sousse, Tunisia, 2Departement. of Restorative Dentistry and Endodontology, Ghent University Hospital, Ghent, Belgium, 3Department of Medical Genetics, MRB, Ghent University Hospital, Ghent, Belgium, 4Private Practice, Sousse, Tunisia

Introduction: Andontia refers to the total absence of dentition. It can affect permanent and/or temporary dentition. In most cases, anodontia is associated with different clinical manifestations as a part of syndrome but also non-syndromic forms have been described although their etiology is often unclear.

Material and Methods: In this study we explored a Tunisian family with most likely a recessive form of anodontia of permanent dentition in the proband whereas the parents and some other family members show hypodontia. We performed DNA targeted sequencing of candidate genes, PAX9, MSX1, WNT10A and AXIN2 and consequently whole exome sequencing (WES).

Results: Sequencing of the 4 genes did not reveal any causative mutation. Therefore, we performed WES on the anodontia patient. We selected six homozygous mutations and segregation analysis on the remaining family members. One of these is a missense mutation located in NFRKB gene (Nuclear Factor Related to Kappa-B-binding protein) in the proband. Interestingly, in the rest of the family this mutation is compound heterogeneous with most likely a duplication of the NFRKB gene on the other allele. This mutation, predicted to be disease causing, is located at the NFRKB-WHL domain which belongs to a group of helical domains involved in protein-protein interactions. This might suggest that the interaction activity is probably altered in the first stages of development.

Conclusions: This study revealed for the first time the involvement of the NFRKB gene in tooth agenesis. NFRKB might be involved in transcriptional regulation. Further qPCR studies and functional studies will be performed.

M. Haddaji Mastouri: None. P. De Coster: None. N. Ben Salah: None. A. Touati: None. I. Veerecke: None. A. Zaghabani: None. A. Saad: None. D. H’mida-Ben Brahim: None. P. Coucke: None.

P04.09A Novel heterozygous deletion in PIEZO2 identified by NGS in a familial case with distal arthrogryposis type 5

C. Rieubland, S. Gallati, A. Schaller

Division of Human Genetics, Bern, Switzerland

Introduction: Distal arthrogryposis type 5 (DA5) is characterized by multiple congenital contractures of distal extremities associated with ocular abnormalities, facial dysmorphism and restrictive lung disease and caused by heterozygous PIEZO2 gain of function mutations.

Case report: A 4 year old boy presented with severe club foot, camptodactyly, short stature, ophtalmoplegia, deep-set eyes and micrognathia. The family history revealed that his father had congenital arthrogryposis with short stature, keratoconus and astigmatism. The clinical presentation being very suggestive for DA5. Sanger sequencing of PIEZO2 was performed initially and revealed no mutation. NGS panel for distal arthrogryposis did also reveal no mutation in the analyzed genes. As no MLPA is available for PIEZO2, in silico CNV analysis was performed and detected a deletion of exons 32 and 33 in PIEZO2 (c.4634-?_4855+?del). The deletion was confirmed by RT-qPCR.

Discussion: Heterozygous gain of function mutations in PIEZO2 cause DA5, Gordon and Marden-Walker syndrome whereas homozygous loss of function mutations cause muscular atrophy, perinatal respiratory distress, progressive arthrogryposis, scoliosis and loss of proprioception. We hypothesize that the in frame deletion of exons 32 and 33 causes a dominant negative effect resulting in a gain of function of the channel activity.

Conclusion: This is the first report of an intragenic deletion in PIEZO2 in a family with autosomal dominant DA5 diagnosed by NGS. Inframe deletions of PIEZO2 might be a frequent cause of DA5. As no MLPA is available for PIEZO2, in silico CNV analysis using NGS may be a useful tool to diagnose intragenic deletions.

C. Rieubland: None. S. Gallati: None. A. Schaller: None.

P04.10B Camptodactyly-arthropathy-coxa vara-pericarditis syndrome in a large family: A clinical condition with a diagnostic challange

S. Oguz1, P. O. Simsek Kiper2, G. E. Utine2, Y. Alanay3, S. Ozen4, K. Boduroglu2, M. Alikasifoglu1

1Hacettepe University Faculty of Medicine, Department of Medical Genetics, Ankara, Turkey, 2Hacettepe University Faculty of Medicine, Department of Pediatrics, Department of Pediatric Genetics, Ankara, Turkey, 3Acıbadem University Faculty of Medicine, Department of Pediatrics, Department of Pediatric Genetics, Istanbul, Turkey, 4Hacettepe University Faculty of Medicine, Department of Pediatrics, Department of Pediatric Rheumatology, Ankara, Turkey

Introduction:Camptodactyly-arthropathy-coxa vara-pericarditis syndrome (CACP; OMIM 208250) is a rare autosomal recessive disorder caused by PRG4 mutations. PRG4 product is a glycoprotein called lubricin. We report on the clinical and molecular findings of two related families who were initially diagnosed with JIA, and PRG4 mutations were identified in the follow-up.

Materials and Methods: After the referral of the first family with three affected individuals with CACP, a linkage analysis was done and a high LOD score on the long arm of chromosome 1 was found. However, no further studies could be performed, and the family was lost in the follow-up. Years later, and after the discovery of PRG4 mutations in CACP, another patient from another family with a diagnosis of JIA who was not responsive to anti-rheumatic treatment was referred with a suspicion of a genetic disorder of the skeleton. With the help of the pedigree analysis, it was revealed that these two families were actually related, and a recent contact with the first family could be established years after.

Results: Molecular analysis in all patients revealed a homozygous deletion of two nucleotide (c.1910_1911delCT) in the highly repetitive region of exon 6 of PRG4 in all patients.

Conclusions: Patients with CACP syndrome can easily be misdiagnosed as JIA, leading to unnecessary treatment and a delay for the actual diagnosis. In patients with arthropathic conditions, CACP should be kept in mind, especially in the presence of progressive symptoms and parental consanguinity.

S. Oguz: None. P.O. Simsek Kiper: None. G.E. Utine: None. Y. Alanay: None. S. Ozen: None. K. Boduroglu: None. M. Alikasifoglu: None.

P04.11C The effects of calcium-sensing receptor CASR genotypes, treatment duration, gender bone health and mineral metabolism in chronic renal failure patients

P. Ata1, B. Erkal1, D. Gultekin2, B. Altas2, B. Celik2, D. Kayır2, A. Low2, A. Eren3, S. Tuglular4

1Marmara University, School of Medicine Medical Genetics, Istanbul, Turkey, 2Marmara University, School of Medicine, Istanbul, Turkey, 3Koc University, Istanbul, Turkey, 4Marmara University, School of Medicine Nephrology, Istanbul, Turkey

Introduction: The aim of this study is to examine the effect of the CaSR genotype, which significantly affects Ca, P, PTH values on the disease progression in chronic renal failure (CRF) patients.

Materials and Methods: Fortyfıve CRF patients on hemodialysis treatment aged between 15-80yrs who admitted between 1994-2017 were included. Serum PTH, Ca2+ and P values were examined. The control group included 50 healthy patients without CRF, bone disease and osteoporotic risk factors. Genomic DNA isolation was performed using the Gentra Qiagen kit. CaSR allele-specific PCR analysis was performed.Findings: Patients’ average age was 60.5  ± 17.5 years, (male/ female 53.3% / 46.7%) Patients with hemodialysis treatment for less than 3 years had normal CaxP value while this value was increased for patients with hemodialysis over 3 years. The pathological CaxP value was found in 8,3% (n = 2/24) of the male patients and 38,09% (n = 8/21) of the female patients (p = 0,029). In the patient group, the 990th codon AA polymorphism in the CaSR gene was found to be 32.4% (n = 12) while in the control group it was 23.1% (n = 6).

Conclusion: The CaSR kodon 990 AA allele seemed to be a risk factor for bone health in our patient group. Among CRF patients with increased hemodialysis duration osteoporosis and fracture risks were increased. Risk of developing bone disease for female patients was higher than that of males.

P. Ata: None. B. Erkal: None. D. Gultekin: None. B. Altas: None. B. Celik: None. D. Kayır: None. A. Low: None. A. Eren: None. S. Tuglular: None.

P04.12D WES in 42 trios of syndromic and isolated Chiari Malformation type 1: how to define the genetic cause in a high clinical heterogeneous condition

A. La Barbera1, A. Provenzano1, L. Tiberi1, R. Artuso2, V. Palazzo2, P. Reho1, E. Bosi1, A. Pagliazzi1, F. Peluso1, S. Landini1, I. Sani2, L. Giunti2, S. Guarducci2, M. Pantaleo2, B. Lucherini2, M. Scagnet3, L. Genitori3, S. Giglio1

1Medical Genetics Unit, Department of Clinical and Experimental Biomedical Sciences 'Mario Serio', University of Florence, Florence, Italy, 2Medical Genetics Unit, Meyer Children’s University Hospital, Florence, Italy, 3Neurosurgery Unit, Anna Meyer Children's Hospital, University of Florence, Florence, Italy

Chiari malformation type 1 (CM1) is a congenital anomaly of cranio-cerebral junctions characterized by underdevelopment of the occipital bone and posterior fossa (PF) and consequent cerebellar tonsil herniation across the foramen magnum. This condition can impair the normal flow of cerebral spinal fluid (CSF) leading to syringomyelia. Patients display a high degree of clinical variability depending on the compression of the tissue, nerves and on the buildup of CSF pressure. CM1 is also associated with known syndromes, (e.g. craniosynostosis), and is often reported as clinical sign in more complex phenotypes, but the molecular mechanisms of isolated CM1 are not yet known. To understand the molecular basis and which factors could contribute to the high heterogeneity, we performed WES of 42 trios with sporadic and syndromic, without craniosynostosis, CM1. WES of syndromic cases provided a diagnosis of distinct genetic disorders, in which pathogenic variants were associated to bone dysplasia, growth retardation and resistance to gonadotropins. In sporadic cases, variants in genes involved in common molecular pathways have been identified; these are all associated with modeling and deposition of bone matrix and with regulatory processes, all requested in the same pathway. In 75% of trios we found variants in the same recurrent genes, shared both by isolated and syndromic CM1 cases; functional tests on bone biopsies of these patients are underway to demonstrate the pathogenicity of these genes. Our data remark complex interactions among several genes involved in different steps of the same pathways, underlying the high clinical-genetic heterogeneity of CM1.

A. La Barbera: None. A. Provenzano: None. L. Tiberi: None. R. Artuso: None. V. Palazzo: None. P. Reho: None. E. Bosi: None. A. Pagliazzi: None. F. Peluso: None. S. Landini: None. I. Sani: None. L. Giunti: None. S. Guarducci: None. M. Pantaleo: None. B. Lucherini: None. M. Scagnet: None. L. Genitori: None. S. Giglio: None.

P04.13A Exome sequencing of two Italian pedigrees with non isolated Chiari malformation type I reveals candidate genes for cranio-facial development

P. De Marco1, L. Tattini2, A. Accogli1, G. Piatelli1, M. Pavanello1, D. Tortora1, A. Cama1, V. Capra1

1Istituto Giannina Gaslini, Genova, Italy, 2Università di Pisa, Pisa, Italy

Chiari malformation type I (CMI) is a congenital abnormality of the cranio-cerebral junction with an incidence of 1 in 1280. CMI is characterized by underdevelopment of the occipital bone and posterior fossa (PF) and consequent cerebellar tonsil herniation. The presence for a genetic basis to CMI is supported by many lines of evidence. The cellular and molecular mechanisms leading to CM1 are poorly understood. The occipital bone formation is dependent on complex interactions between genes and molecules with pathologies resulting from disruption of this delicate process. Whole-exome sequencing of affected and not affected individuals from two Italian families with non isolated CMI was undertaken. Single nucleotide and short insertion-deletion variants were prioritized using KGGSeq-knowledge-based platform. We identified three heterozygous missense variants: DKK1 c121G>A (p.(A41T)) in the first family, and the LRP4 c.2552C>G (p.(T851R)) and BMP1 c.941G>A (p.(R314H)) in the second family. The variants were located at highly conserved residues, segregated with the disease, but they were not observed in more than 100 unaffected in-house controls. DKK1 encodes for a potent soluble WNT inhibitor that binds to LRP5 and LRP6, and is itself regulated by BMPs. DKK1 is required for embryonic head development and patterning. LRP4 is a novel osteoblast expressed receptor for DKK1 and a WNT and BMP signaling pathways integrator. Screening of DKK1 in a cohort of 65 CMI sporadic patients identified another missense variant, the c.359G>T (p.(R120L)), in two unrelated patients. These findings implicated the WNT signaling in the correct development of the cranial mesenchyme originating the PF.

P. De Marco: None. L. Tattini: None. A. Accogli: None. G. Piatelli: None. M. Pavanello: None. D. Tortora: None. A. Cama: None. V. Capra: None.

P04.14B A small homozygous CHST11 deletion in chondrodysplasia, brachydactyly, overriding digits, clino-symphalangism and synpolydactyly

R. Shabbir1, G. Nalbant2, N. Ahmad3, S. Malik1, A. Tolun2

1Quaid-i-Azam University, Islamabad, Pakistan, 2Bogazici University, Istanbul, Turkey, 3Institute of Biomedical and Genetic Engineering, Islamabad, Pakistan

Introduction: CHST11 is a membrane protein of Golgi that catalyses the transfer of sulphate to position 4 of the N-acetylgalactosamine residue of chondroitin. Chondroitin sulphate is the predominant proteoglycan in cartilage, and its sulphation is important in the developing growth plate of cartilage. A homozygous deletion encompassing part of the gene and the embedded microRNA MIR3922 had been detected in a woman with hand/foot malformation and malignant lymphoproliferative disease. Chst11 deficient mouse has severe chondrodysplasia, congenital arthritis and neonatal lethality. We searched for the causative variant for the unusual combination of limb malformations with variable expressivity accompanied by skeletal defects in a consanguineous Pakistani kindred.

Materials and Methods: We performed detailed clinical investigations in family members. Homozygosity mapping using SNP genotype data was performed to map the disease locus and exome sequencing to identify the underlying molecular defect.

Results: The limb malformations include brachydactyly, overriding digits and clino-symphalangism in hands and feet, and syndactyly and hexadactyly in feet. Skeletal defects include scoliosis, dislocated patellae and fibulae, and pectus excavatum. The disease locus is mapped to a 1.6-Mb region at 12q23, harbouring a homozygous in-frame deletion of 15 nucleotides in CHST11. Novel variant c.467_481del (p.L156_N160del) is deduced to lead to the deletion of five evolutionarily highly conserved amino acids and predicted as damaging to protein by in silico analysis.

Conclusions: Our findings confirm the crucial role of CHST11 in skeletal morphogenesis and show that CHST11 defects have variable manifestations that include a variety of limb malformations and skeletal defects.

(Grant: TUBITAK114Z829)

R. Shabbir: None. G. Nalbant: None. N. Ahmad: None. S. Malik: None. A. Tolun: None.

P04.15C Regenerative approach for treating maxillofacial defects associated with some genetic disorders

H. H. El-Ahmady1, A. F. Abd Elazeem2,3,1, N. E. B. Ahmed2,3, M. A. Abdelrahman2, M. A. Abderazik1

1Al-Azhar Cleft Lip and Palate Treatment Center, Faculty of Dental Medicine for Girls, Al-Azhar university, Cairo, Egypt, 2Department of Oro-dental Genetics, Center of Medical Excellence, National Research Centre, Cairo, Egypt, 3Stem Cell Laboratory, Centre of Excellence for Advanced Sciences, National Research Centre, Cairo, Egypt

Introduction: Cleft palate is one of the most common congenital craniofacial defects that may present alone or in association with various genetic disorders. Repairing such defect is very important to restore oral functions and normal facial features. Regenerative medicine and stem cell therapy are emerging fields that have shown great potentials in treating various diseases. Here, we follow a regenerative approach by introducing a method in which we use autologous bone marrow mononuclear cells (BMMNCs) combined with platelet-rich fibrin (PRF) and nano-hydroxyapatite for bone regeneration in patients with unilateral alveolar clefts.

Materials and Methods: The study included 10 patients with unilateral alveolar cleft defects and of age range 8 to 15 years. Autologous BMMNCs were isolated using the density gradient separation method and seeded on a collagen sponge. The seeded collagen sponge was then used in combination with nano-hydroxyapatite and autologous PRF to repair alveolar cleft defects via the regenerative approach. The effectiveness of the technique was evaluated after 12 months of follow up via clinical and radiographic assessments.

Results: All patients healed probably without any complications. During the 12-month follow-up, no donor site morbidities have been reported. Postoperative pain, bleeding, and swelling were within the normal for same surgical procedures. Cone beam radiographs showed successful complete bone regeneration in all cases.

Conclusion: Results of this study suggest that a mixture of autologous BMMNCs, nano-hydroxyapatite, and PRF greatly promote bone regeneration providing a novel therapeutic strategy for alveolar cleft defects.

H.H. El-Ahmady: None. A.F. Abd Elazeem: None. N.E.B. Ahmed: None. M.A. Abdelrahman: None. M.A. Abderazik: None.

P04.16D Expanded phenotypic spectrum of type I collagenopathy

J. Lee1, B. Lim2, M. Choi3, T. Cho2, J. Chae2

1Gachon University Gil Medical Center, Incheon, Korea, Republic of, 2Seoul National University Children’s Hospital, Seoul, Korea, Republic of, 3Seoul National University College of Medicine, Seoul, Korea, Republic of

It is known that type I collagenopathy has a broad-spectrum phenotypic variability. Here, we report a case of a Korean girl with a heterozygous COL1A1 mutation who had an atypical presentation. A 26-month-old girl presented with delayed motor development and failure to thrive. She had severe growth retardation. She exhibited right-sided plagiocephaly, blue sclerae, and facial dysmorphism, including a small pointed chin, frontal bossing, and a triangular face, but had microcephaly. Whole-exome sequencing revealed a novel de novo heterozygous sequence variant in COL1A1 (p.Gly1127Asp), which was validated by Sanger sequencing. Radiological finding showed generalized osteoporosis with progressive scoliosis of the spine without evidence of platyspondyly related to fractures and bowing of the long bones, and markedly delayed carpal bone age. Muscle pathology showed a marked size variation of myofibers and selective type 1 atrophy. This study expanded the clinical and genetic spectrum of type I collagenopathy with a COL1A1 variant. Therefore, we suggest that type I collagenopathy should be considered in the patients who have some features of osteogenesis imperfecta simultaneously with atypical features such as facial dysmorphism.

J. Lee: None. B. Lim: None. M. Choi: None. T. Cho: None. J. Chae: None.

P04.17A Whole exome sequencing identifies mutations in 10% of familial non-syndromic cleft lip and/or palate patients in genes mutated in well-known syndromes

M. Basha1, B. Demeer2, N. Revencu3, S. Theys4, S. Bou Saba5, O. Boute6, B. Devauchelle7, G. François8, B. Bayet9, M. Vikkula10

1de Duve Institute, Brussels, Belgium, 2Center for Human Genetics, CLAD nord de France, CHU Amiens, Amiens, France, 3Center for Human Genetics, Cliniques universitaires Saint-Luc, University of Louvain, Brussels, Belgium, 4Pediatric Dentistry and Oral Care for Special Needs, Cliniques universitaires Saint-Luc, University of Louvain, Brussels, Belgium, 5Department of Orthodontics and dentofacial orthopedics, Cliniques universitaires Saint-Luc, University of Louvain, Brussels, Belgium, 6Center for Human Genetics, Lille, France, 7Service of Maxillofacial Surgery and Stomatology, CHU Amiens-Picardie, Amiens, France, 8Department of Pediatrics, Cliniques universitaires Saint-Luc, University of Louvain, Brussels, Belgium, 9Centre Labiopalatin, Division of Plastic Surgery, Cliniques universitaires Saint-Luc, University of Louvain, Brussels, Belgium, 10Human Molecular Genetics, de Duve Institute, University of Louvain, Brussels, Belgium

Introduction: Oral clefts i.e. clefts of the lip and/or cleft palate (CL/P) are the most common craniofacial birth defects with an approximate incidence of ~1/700. To date physicians stratify patients with oral clefts into either syndromic CL/P (syCL/P) or non-syndromic CL/P (nsCL/P) depending on whether the CL/P is associated with another anomaly or not. In general, patients with syCL/P follow Mendelian inheritance, while those with nsCL/P, have a complex etiology and as such, do not adhere to Mendelian inheritance. Genome-wide association studies (GWAS) have identified approximately 30 risk loci for nsCL/P, which could explain a small fraction of heritability.

Materials and Methods: To identify variants causing nsCL/P, we conducted Whole Exome Sequencing (WES) on 84 individuals with nsCL/P, drawn from multiplex families (n = 46).

Results: We identified rare damaging variants in four genes known to be mutated in syCL/P: TP63 (1 family), TBX1 (1 family), LRP6 (1 family) and GRHL3 (2 families), and clinical reassessment confirmed the isolated nature of their CL/P.

Conclusion: These data demonstrate that CL/P patients without cardinal signs of a syndrome may still carry a mutation in a gene linked to syCL/P. Rare coding and non-coding variants in syCL/P genes could in part explain the controversial question of “missing heritability” for nsCL/P. Therefore, gene panels designed for diagnostic testing of syCL/P should be used for nsCL/P patients, especially when there is at least 3rd degree family history. This would allow a more precise management, follow-up and genetic counseling. Moreover, stratified cohorts would allow hunting for genetic modifiers.

M. Basha: None. B. Demeer: None. N. Revencu: None. S. Theys: None. S. Bou Saba: None. O. Boute: None. B. Devauchelle: None. G. François: None. B. Bayet: None. M. Vikkula: None.

P04.18B BBS9 as potential tissue-specific key protein in BBSome binding and ciliary trafficking in NCS patients

M. Barba1, L. Di Pietro1, C. Bernardini1, L. Massimi1, G. Tamburrini1, S. Della Longa2, A. Arcovito1, O. Parolini1, S. A. Boyadjiev3, W. Lattanzi1

1Università Cattolica del Sacro Cuore, Rome, Italy, 2University of L'Aquila, L'Aquila, Italy, 3University of California Davis, Sacramento, CA, United States

Nonsyndromic craniosynostosis (NSC) is a congenital malformation due to the premature ossification of calvarial sutures, with an unclear molecular etiopathogenesis. The Bardet-Biedl Syndrome-associated gene 9 (BBS9), already associated to NCS by GWAS, encodes a member of the well-characterized class of BBS proteins that interact through their C-term to form an octameric complex named BBSome, necessary for ciliogenesis and ciliary function. Preliminary data identified a suture specific signature, including BBS9 and several genes involved in primary cilium signaling and assembly. In particular, we showed the overexpression of 5 spliced isoforms of BBS9 in fused suture specimens of single-suture midline NCS patients. The aim of this study is to clarify the mechanism through which BBS9 exerts its regulatory function during the osteogenic commitment and differentiation of somatic stem cells inside skull bone. We confirmed through qPCR the overexpression of the selected BBS9 splice isoforms in mesenchymal stromal cells (CMSC) isolated from fused (p) and unfused (n) sutures of NCS patients. Then, we assessed by immunofluorescence that p-CMSC showed a reduced number of primary cilia compared with same patient-n-CMSC. Computational modeling of the upregulated isoforms showed structural differences in the C-term of the proteins, predicting that their binding affinity within the BBSome may be affected. Taken together, these data suggest that selected BBS9 protein isoforms show a tissue-specific increased expression in osteogenic precursors residing in NCS fused sutures, owing to a less effective assembly of the BBSome, which impairs ciliogenesis. [Funding support: NIH-NIDCR grant R01DE16886, Federazione GENE and Università Cattolica]

M. Barba: None. L. Di Pietro: None. C. Bernardini: None. L. Massimi: None. G. Tamburrini: None. S. Della Longa: None. A. Arcovito: None. O. Parolini: None. S.A. Boyadjiev: None. W. Lattanzi: None.

P04.19C IL11RA-related Crouzon-like autosomal recessive craniosynostosis in ten new patients: which are the differences?

E. Brischoux-Boucher1, A. Trimouille2, G. Baujat3, A. Goldenberg4, E. Schaefer5, B. Guichard6, P. Hannequin7, S. Baer5, C. Cabrol1, E. Weber8, G. Godfrin9, M. Lenoir10, D. Lacombe2, C. Collet11, L. Van Maldergem1

1Centre de genetique humaine CHU Besançon, Besancon, France, 2Service de génétique médicale CHU Bordeaux, Bordeaux, France, 3Institut Imagine CHU Necker-Enfants-Malades, Paris, France, 4Service de genetique CHU Rouen, Rouen, France, 5Service de genetique médicale CHU Strasbourg, Strasbourg, France, 6Service de chirurgie maxillo-faciale CHU Rouen, Rouen, France, 7Service de nrurochirurgie CHU Rouen, Rouen, France, 8Service de chirurgie maxillo-faciale CHU Besançon, Besancon, France, 9Service de neurochirurgie CHU Besançon, Besancon, France, 10Service de radiologie CHU Besançon, Besancon, France, 11Service de biochimie et biologie moléculaire CHU Lariboisière, Paris, France

By describing 10 new patients recruited from a population attending centres for Human Genetics, we further delineate the clinical spectrum of a new syndromic craniosynostosis resembling Crouzon syndrome. Singularly, it is inherited according to an autosomal recessive mode of inheritance. Missense mutations in IL11RA, a gene encoding alpha subunit of interleukin 11 receptor, were at cause in all five families, four of them not reported, including two in the Ig-like C2-type domain. A subset of patients had an associated connective tissue disorder with joint hypermobility and intervertebral discs fragility. Two of our patients had syringomyelia. A lower figure of teeth anomalies than previously reported in the two large series of patients evaluated in dental institutes points towards an ascertainment bias.

E. Brischoux-Boucher: None. A. Trimouille: None. G. Baujat: None. A. Goldenberg: None. E. Schaefer: None. B. Guichard: None. P. Hannequin: None. S. Baer: None. C. Cabrol: None. E. Weber: None. G. Godfrin: None. M. Lenoir: None. D. Lacombe: None. C. Collet: None. L. Van Maldergem: None.

P04.20D Targeted exome sequencing analysis in Turkish non-syndromic craniosynostosis patients

E. Yilmaz, B. Nur, E. Mihci, O. M. Alper


Introduction: Craniosynostosis is described as an early fusion of one or more calvarial sutures. Early closure of these sutures results with new head shape that inhibits the normal growth and development of the brain. Worldwide, the estimated prevalence of the syndrome is 1 in 2500. Craniosynostosis is divided into two sub-goups: syndromic and non-syndromic (nsCRN). More than 70% of the patients have nsCRN. Although more than 50 genes were identified, the genetic cause is mostly unknown. Approximately 24% of cases can be genetically identified.

Method: Unrelated seventeen nsCRN Turkish cases have been sequenced by using Dysmorphia and Dsyplasia Research Panel v2, includes 519 genes, of Ion AmpliSeq. IonS5 sequencing technology was used and data were analyzed with Ingenuity software. Genetic variants in several genes known to cause craniosynostosis were filtered. Clinically pathogenic variants were checked and confirmed by Sanger sequencing.

Results: In six of the cases (35.3%), we identified six different mutations (including three novel ones) in ERF, FREM1 and TCF12 genes. The novel missense mutations are p.P1802L(c.5405C>T) and p.G1493R(c.4477G>A) in FREM1, and frameshift mutation is c.1106_111delCTCTCAC(p.P369fs*26) in TCF12 gene. POLYPHEN, PROVEAN and SIFT analysis revealed that p.P1802L and p.G1493R are predicted to be deleterious and damaging with scores of 0.997; -9.07; 0.003 and 1.000; -3.99; 0.019, respectively.

Conclusion: Our data highlights the importance of increased diagnostic rate (35.3%) with the use of this panel, and help to solve the genotype-phenotype correlations in nsCRN.

Acknowledgement: This research was supported by grant from Scientific Research Project Council of Akdeniz University (#TDK-2015-933).

E. Yilmaz: None. B. Nur: None. E. Mihci: None. O.M. Alper: None.

P04.21A Characterization of the calvarial suture skeletogenic stem cell niche in nonsyndromic craniosynostosis

M. Barba1, L. Di Pietro2, C. Prampolini2, V. Orticelli2, L. Massimi2, P. Frassanito2, S. A. Boyadjiev3, M. Caldarelli2, G. Tamburrini2, O. Parolini2, W. Lattanzi2

1Università Cattolica del Sacro Cuore, Roma, Italy, 2Università Cattolica del Sacro Cuore, Rome, Italy, 3University of California Davis, Sacramento, CA, United States

Nonsyndromic craniosynostosis (NCS) is the congenital premature fusion of skull sutures. The suture mesenchyme houses a skull-specific stem cell niche, plausibly impaired in NCS fused suture sites. To test this hypothesis, we characterized the stem cell niche of open and fused sutures of NCS patients. Lineage-specific markers were analyzed, by qPCR and immunofluorescence, in suture tissues and in calvarial mesenchymal stem cells (CMSC). MSC from alternative tissues served as controls. We analyzed the localization of THY1 (skeletal stemness-marker), GLI1 (putative calvarial stemness-marker) and AXIN2 (mesenchymal cell fate determinant) in suture tissue sections: AXIN2 resulted mainly expressed at the endosteal ossified side, while THY1 and GLI1 were primarily expressed within the trabeculae, enriched with proliferating cells. Both NCS suture tissues and CMSC isolated thereof expressed reduced levels of TEK and ENPEP (bone marrow stem cells differentiation markers) compared with controls. AXIN2 levels were higher in open suture-derived CMSC than in fused suture cells and in controls. Upon in vitro osteogenic induction, the expression of THY1 and GLI1 decreased, whereas AXIN2 levels increased, in both open- and fused- suture derived CMSC. CMSCs isolated from both fused and unfused sutures shared the same marker expression profile, indicating that explant cultures allowed selecting comparable cell populations, THY1+/GLI1+ representing the stem cell population within the human calvarial niche. Our data seem to suggest that in NCS the in vivo tissue microenvironment may cause the enhanced osteogenic differentiation of suture MSCs leading to premature suture closure. Funding support: Federazione GENE and Università Cattolica del Sacro Cuore

M. Barba: None. L. Di Pietro: None. C. Prampolini: None. V. Orticelli: None. L. Massimi: None. P. Frassanito: None. S.A. Boyadjiev: None. M. Caldarelli: None. G. Tamburrini: None. O. Parolini: None. W. Lattanzi: None.

P04.22B The outcome of broad genetic screening in a cohort of 144 patients with craniosynostosis

A. Topa1,2, A. Rohlin2, L. Lovmar2, G. Stenman1,2, L. Kölby3

1Department of Pathology and Genetics, University of Gothenburg, The Sahlgrenska Academy, Gothenburg, Sweden, 2Department of Clinical Pathology and Genetics, Sahlgrenska University Hospital, Gothenburg, Sweden, 3Department of Plastic Surgery, University of Gothenburg, The Sahlgrenska Academy, Gothenburg, Sweden

Approximately 1 in 2000 children are affected by craniosynostosis. During the last few years, mutations in several genes have been identified as cause of early closing of the cranial sutures. The list increases constantly as new genes with relation to craniosynostosis are detected. Craniosynostosis occurs isolated or associated with other symptoms including malformations as part of several syndromic disorders - the most known are represented by Pfeiffer, Crouzon, Jackson-Weiss, Antley-Bixler, Apert, Saethre-Chotzen and Muenke syndromes. The aim of our project was to study the prevalence and spectrum of the genetic disorders which are associated with early suture closing in a unique patient cohort including 144 individuals with mostly uni- or bicoronal synostosis. The mutational screening of our patient cohort has been performed by using a custom-designed NGS-enrichment panel including 63 craniosynostosis-related genes selected from OMIM. In 91 out of 144 screened patients (approximately 63%), either a known previously reported pathogenic variant or a likely pathogenic variant has been detected. As expected, the majority of variants have occurred in the craniosynostosis “core genes”: FGFR2, TWIST1, FGFR3, TCF12, EFNB1 and POR. However, novel likely pathogenic variants have been observed also in IL11RA, KMT2D and SKI-genes. Our study shows that a broad genetic screening using a targeted NGS assay have a high diagnostic yield in a large cohort of patients with craniosynostosis.

A. Topa: None. A. Rohlin: None. L. Lovmar: None. G. Stenman: None. L. Kölby: None.

P04.24D Ultrastructural elastic fiber morphology in cutis laxa reflects the underlying pathogenesis and supports a novel clinical classification

A. Beyens1,2, R. De Rycke3, H. Syryn1, B. Fischer-Zirnsak4, T. Van Damme1, I. Hausser5, M. De Bruyne3, M. Morroni6, S. Nampoothiri7, K. Mahesh8, U. Kornak4, Z. Urban9, S. Hadj-Rabia10, C. Bodemer11, S. De Schepper2, E. C. Davis12, B. Callewaert1

1Center for Medical Genetics, Ghent University Hospital, Ghent, Belgium, 2Department of Dermatology, Ghent University Hospital, Ghent, Belgium, 3Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium, 4Institute of Medical Genetics and Human Genetics, Charité-Universitätsmedizin Berlin, Berlin, Germany, 5Institute of Pathology, Universitätsklinikum Heidelberg, Heidelberg, Germany, 6Department of Experimental and Clinical Medicine, Section of Neuroscience and Cell Biology, School of Medicine, Università Politecnica delle Marche and Electron Microscopy Unit, United Hospitals, Ancona, Italy, 7Department of Pediatric Genetics, Amrita Institute of Medical Sciences and Research Center, Kochi, India, 8Department of Pediatric Cardiology, School of Medicine, Kochi, India, 9Department of Human Genetics, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, PA, United States, 10Service de Dermatologie, CHU Paris - Hôpital Necker-Enfants Malades, Paris, France, 11Centre MAGEC (Maladies rares Génétiques a Expression Cutanée), Service de Dermatologie, CHU Paris - Hôpital Necker-Enfants Malades, Paris, France, 12Department of Anatomy and Cell Biology, McGill University, Montréal, QC, Canada

Background: Cutis laxa (CL) syndromes are a heterogeneous group of multisystem disorders that share a loose, redundant skin as a common feature reflecting elastic fiber (EF) deficiency. The pathogenesis of each of the CL subtypes is different but affects elastogenesis. However, light microscopy of the dermis is non-discriminative and the recently observed vast molecular heterogeneity mitigates the clinical validity and practicality of the current classification, based on the mode of inheritance and systemic involvement.

Aims: We aim to classify the CL subtypes by means of a simple flowchart and to evaluate correlations between EF ultrastructural morphology (as evaluated by transmission electron microscopy) and clinical presentation.

Results: Following literature review, we developed a 7-step flowchart to classify the CL subtypes. As a proof of principle, we systematically evaluated 68 CL patients from our in-house database and could allocate 95% of patients to the right gene. We performed transmission electron microscopy in skin biopsies of all CL subtypes and found discriminative and specific findings that correlate with the main presenting symptoms (emphysema, arterial tortuosity, skeletal defects/mental disability with or without intrauterine growth retardation/cataract). Moreover, EF ultrastuctural morphology reflects the involved molecular pathogenesis and provides new insights in elastic fiber biogenesis.

Conclusion: Our novel nosology of the CL syndromes provides a practical approach to the broad differential diagnosis of CL syndromes. The classification forms a basis to integrate the clinical presentation with the pathogenesis and ultrastructural EF defects and might bode for new management guidelines and therapeutic approaches.

(Grant Reference BOF01N04516)

A. Beyens: None. R. De Rycke: None. H. Syryn: None. B. Fischer-Zirnsak: None. T. Van Damme: None. I. Hausser: None. M. De Bruyne: None. M. Morroni: None. S. Nampoothiri: None. K. Mahesh: None. U. Kornak: None. Z. Urban: None. S. Hadj-Rabia: None. C. Bodemer: None. S. De Schepper: None. E.C. Davis: None. B. Callewaert: None.

P04.25A Diverse mechanisms of germline and somatic mosaicism in CYLD cutaneous syndrome

M. Arefi1, V. Wilson2, D. Bajwa1, S. Zwolinski2, N. Sinclair1, P. Brennan2, N. Bown2, D. Bourn2, M. Santibanez-Koref1, J. Burn1, N. Rajan1

1Institute of Genetic Medicine, Newcastle upon Tyne, United Kingdom, 2Northern Genetics Service, Newcastle upon Tyne, United Kingdom

Six unsolved cases that met clinical diagnostic criteria for CYLD cutaneous syndrome, deemed to be mutation negative following Sanger sequencing, were investigated for clinical and genetic features of mosaicism. One case demonstrated 8% blood mosaicism for a pathogenic mutation using a NGS assay targeting CYLD. We found no causative lesions in the blood of five of the six remaining cases. In two cases, we investigated tumour tissue. In the first case, two tumour samples demonstrated a recurrent 19bp deletion in the skin only. In the second, five distinct mutations resulting in a stop codon in CYLD were detected in five tumours. SNP array analysis of three tumours demonstrated a recurrent 5.5Mb deletion encompassing CYLD and 23 other genes. This patient had a daughter who had developmental delay and unilateral renal agenesis, but no cylindromas. This phenotype has been reported in other patients with large germline deletions including CYLD, suggesting transmission of the deletion. The remaining three cases had clinical features suggesting cutaneous mosaicism. We conclude that, firstly, mosaicism may be detectable in the blood and account for the presence of cylindromas in the skin. Secondly, some patients may only have a unilateral linear arrangement or cluster of tumours, with absence of mosaic mutation in the blood suggesting cutaneous mosaicism alone. Thirdly, mosaic mutations detectable in the blood may affect the germline and be transmissible to offspring; where this is due to a contiguous deletion involving CYLD, the phenotype may involve multiple organ systems.

M. Arefi: None. V. Wilson: None. D. Bajwa: None. S. Zwolinski: None. N. Sinclair: None. P. Brennan: None. N. Bown: None. D. Bourn: None. M. Santibanez-Koref: None. J. Burn: None. N. Rajan: None.

P04.26B Microbial signatures in TIF1γ autoantibody positive dermatomyositis patients

S. Megremis1, T. Walker1, X. He1, J. O’Sullivan1, A. Payton1, N. Pendleton1, L. Hampson1, R. Cooper2, W. Ollier1, H. Chinoy1, I. Hampson1, J. Lamb1

1University of Manchester, Manchester, United Kingdom, 2University of Liverpool, Liverpool, United Kingdom

Introduction: Tripartite motif-containing (TRIM) proteins are involved in innate immunity. Reports support a role for microbial infections in dermatomyositis (DM); TIF1γ (TRIM33) autoantibody positive patients may have reduced ability to restrict pathogen infection.

Material and Methods: Serum total Ig from 20 DM patients and 20 age-matched healthy controls were pooled for competitive panning and clonal expansion of the FliTrxTM bacterial random-peptide surface display system. DNA libraries were sequenced using pair-end high-throughput sequencing. Translated peptide sequences were searched for maximum exact matches against the NCBI microbial database and assigned to taxa.

Results: DM patients exhibited higher number of microbial peptide reads (22x106 vs 14x106) and unique microbial taxa (32x105 vs 28x105). Viral peptides were higher in DM patients (4x106 vs 2x106) and occupied larger space in the complete microbial IgOme (12% vs 9%). Peptides of cellular microbial origin also were increased in DM patients but with no change in the relative abundance (63% vs 64%). Specific differences were observed for dsDNA (Herpesvirales), ssRNA (Orthomyxoviridae), and retro-transcribing viruses (HIV) and for potentially pathogenic bacteria (Streptococcaceae).

Conclusions: This is the first systematic high-throughput investigation of a link between microbial exposures, TIF1γ autoantibodies and dermatomyositis. The increased presence of viral peptides in DM patients, particularly ssRNA and retro-transcribing viruses, agrees with pathways known to be regulated by TRIMs and points towards increased susceptibility for certain viral families. The increased detection of peptides against potentially ‘pathogenic’ but not ‘non-pathogenic’ bacteria from the same families might play a key role in the development of the disease.

S. Megremis: None. T. Walker: None. X. He: None. J. O’Sullivan: None. A. Payton: None. N. Pendleton: None. L. Hampson: None. R. Cooper: None. W. Ollier: None. H. Chinoy: None. I. Hampson: None. J. Lamb: None.

P04.27C A whole exome study identifies novel candidate genes for vertebral bone marrow signal changes (Modic changes)

M. Kraatari1,2,3, S. Skarp1,2,4, J. Niinimäki3,5, J. Karppinen1,3,6, M. Männikkö1,2,4

1Center for Life Course Epidemiology and Systems Medicine, Faculty of Medicine, University of Oulu, Oulu, Finland, 2Center for Cell – Matrix Research, Faculty of Biochemistry and Molecular Medicine, University of Oulu, Oulu, Finland, 3Medical Research Center Oulu, Oulu University Hospital and University of Oulu, Oulu, Finland, 4Biocenter Oulu, University of Oulu, Oulu, Finland, 5Research Unit of Medical Imaging, Physics and Technology, Faculty of Medicine, University of Oulu, Oulu, Finland, 6Finnish Institute of Occupational Health, Health and Work Ability, and the Disability Prevention Center, Oulu, Finland

Lumbar disc degeneration (LDD) is one of the contributing factors behind low back pain (LBP). Modic change (MC) is a phenotype of LDD and is visualized as bone marrow signal intensity changes on magnetic resonance imaging. MC is a heritable trait with heritability estimation around 30%. It is strongly associated with LBP. We studied two families to identify predisposing variants for MC. Nine individuals were chosen for whole exome sequencing. We focused on rare (MAF < 0.01) and private variants with harmful in silico predictions and variants located in regulatory regions. The identified variants were genotyped from additional family members. One rare variant co-segregated with MC in each family. In the Family I, the observed variant was an insertion and deletion mutation in the HSPG2 gene, resulting in a premature stop codon. HSPG2 encodes a heparin sulfate proteoglycan, which is a structural protein expressed in mammalian cartilage and basement membranes. Rare autosomal recessive disorders with osteochondrodysplasia are caused by mutations in the HSPG2 gene. In the Family II, a single nucleotide polymorphism in the MAML1 gene was identified. MAML1 is considered to be a transcriptional coactivator in the Notch signaling pathway which regulates many biological functions including cartilage development and homeostasis. MAML1 has been reported to affect the activity of RUNX2, a transcription factor essential in the osteoblast differentiation. RUNX2 has been reported to be highly expressed in degenerated discs. We identified two promising candidate genes for MC, HSPG2 and MAML1. Our findings are novel in lumbar spine degenerative phenotypes.

M. Kraatari: None. S. Skarp: None. J. Niinimäki: None. J. Karppinen: None. M. Männikkö: None.

P04.28D Intrafamilial phenotypic heterogeneity in dominant dystrophic epidermolysis bullosa associated with G2043R mutation in COL7A1

E. Gökpınar İli1, S. Vural2, C. D. Durmaz1, J. A. McGrath3, P. Ertop2, A. Okçu Heper4, A. Boyvat2, H. Ilgın Ruhi1

1Department of Medical Genetics, Ankara University School of Medicine, Ankara, Turkey, 2Department of Dermatology, Ankara University School of Medicine, Ankara, Turkey, 3St John's Institute of Dermatology, King's College London, Guy's Hospital, London, United Kingdom, 4Department of Pathology, Ankara University School of Medicine, Ankara, Turkey

Introduction: Dystrophic epidermolysis bullosa (DEB) is a rare form of genodermatoses characterized by blistering condition, caused by COL7A1 gene mutations with dominant or recessive inheritance. Autosomal dominant DEB may remain throughout life in a mild form. However, phenotypic aggravation may be seen between patients. Here, we present a family with seven affected members from three-generations with phenotypic heterogeneity.

Materials and Methods: Genomic DNA was isolated from peripheral blood samples of the patients and COL7A1 gene was sequenced.

Results: p.G2043R (c.6127G>A) mutation was found in the index patient and verified in the other affected family members. One patient in the family was diagnosed with DEB while others had severe pruritus and lichenified linear plaques located more prominently on the extensor surface of the arms and shins. Disease onset was early infancy with a significant increase in complaints after puberty. In female patients, who had a more pronounced phenotype, pregnancy was associated with exacerbation of the disease. Nail dystrophy of hands and feet was present in all patients. Index patient had moderately high serum IgE levels. Skin biopsy revealed eosinophilic infiltrate, and the previous diagnosis was in favor of acquired epidermolysis bullosa. Intravenous immunoglobulin (IVIG) treatment was initiated to the Index patient; which decreased her generalized lesions and severe pruritus very effectively.

Conclusions: Genetic and environmental factors as well as hormonal status may be responsible for the intrafamilial phenotypic heterogeneity. Also, we would like to emphasize IVIG as a treatment option in DEB patients with more severe phenotype.

E. Gökpınar İli: None. S. Vural: None. C.D. Durmaz: None. J.A. McGrath: None. P. Ertop: None. A. Okçu Heper: None. A. Boyvat: None. H. Ilgın Ruhi: None.

P04.29A Comparison of the distribution of duplicated regions associated with SHOX gene between LWD/ISS patients and population sample - conclusions of the meta-analysis

R. Solc1, K. HIrschfeldova2

1Charles University in Prague, Faculty of Science, Department of Anthropology and Human Genetics, Prague, Czech Republic, 2Charles University in Prague, First Faculty of Medicine, Institute of Biology and Medical Genetics, Prague, Czech Republic

Introduction: The human SHOX gene encodes an important growth regulating transcription factor. Heterozygous deletions of the gene or deletions of one of its numerous enhancers are responsible for Lėri-Weill dyschondrosteosis (LWD) and small portion of idiopathic short stature (ISS). Effect of reciprocal duplications is less distinct. The aim of our study was to compare frequency, extent and distribution of SHOX gene and associated elements duplications between the LWD/ISS patients and population sample. A preliminary analysis indicated that rather than the difference in frequency it is the difference in distribution of duplicated areas. A meta-analysis of published cases was performed to confirm the consistency of these data.

Material and Methods: For the purpose of meta-analysis, merged groups of published cases were created: LWD patients (31 individuals), ISS patients (29 individuals) and population sample (36 individuals). Only carriers of the duplication with one brakepoint within the chromosomal region chrX:398,000-980,000 (hg19) were included. Extent, distribution and relative frequency of duplications were compared among merged groups.

Results: There was a significant difference in the relative frequency of CNE-9 enhancer duplications (11 vs. 3) and complete SHOX (exon1-6b) duplications (4 vs. 24) (p-value 0.0139 and p-value 0.000014, respectively) between the merged LWD sample and the merged population sample.

Conclusion: We propose, partial duplications of the SHOX gene coding sequences and small duplications encompassing the CNE-9 enhancer are the highly penetrating alleles associated with the increased risk of LWD/ISS development. Acknowledgement: The study was supported by Charles University (UNCE204022) and its Grant Agency (GAUK202615).

R. Solc: None. K. HIrschfeldova: None.

P04.30B Degradation routes of trafficking-defective VLDLR mutants associated with dysequilibrium syndrome

B. R. Ali, P. Kizhakkedath, A. John, L. Al-Gazali

United Arab Emirates University, Al-Ain, United Arab Emirates

Introduction: Endoplasmic reticulum associated degradation (ERAD) of misfolded proteins by the ubiquitin-proteasome system is a recurrent theme in rare genetic disorders and the process crucially involve ER-membrane complexes such as HRD1-SEL1L. Previously, we have reported that missense mutations in the Very Low Density Lipoprotein Receptor gene (VLDLR), causing Dysequilibrium syndrome (DES), disrupt ligand-binding, due to retention of the mutants in ER. This study explores in detail the degradation routes of these ER-retained VLDLR mutants.

Materials and Methods: The missense pathogenic VLDLR variants have been generated by QuikChange site-directed mutagenesis. The constructs were expressed in HEK293 cells and analyzed by immuno-pull down assays and Western blotting. Protein turn-over studies were conducted by translation shut-off assays and inhibition of proteasomal/lysosomal degradation. The HRD1-SEL1L knockout HEK293 cell lines have been generated by CRISPR/Cas9.

Results: We show that VLDLR mutants are retained in the ER for prolonged periods which could be facilitated by association with the ER resident chaperone calnexin. The mutants were found to be aggregation prone and capable of eliciting ER stress. Inhibition studies suggested that these mutants are degraded partially by the proteasomal pathway. Further, the degradation of VLDLR wild type and a mutant were delayed in CRISPR/Cas9 edited SEL1L knock-out cells which could be reversed by exogenous expression of SEL1L.

Conclusions: ER retention of VLDLR mutants involves binding to calnexin, elevated ER stress, and delayed degradation which is dependent on SEL1L. Since LDLR family members share common structural domains, common mechanisms may be involved in their processing and trafficking (31M254).

B.R. Ali: None. P. Kizhakkedath: None. A. John: None. L. Al-Gazali: None.

P04.31C Biallelic homozygous mutation of HSPG2 in a patient with dysssegmental dysplasia, Rolland-Desbuquois type

K. Kurosawa, K. Shono, T. Yokoi, N. Harada, M. Akahira-Azuma, Y. Enomoto, Y. Tsurusaki, N. Aida

Kanagawa Children's Medical Center, Yokohama, Japan

Dyssegmental dysplasia (DD) is a rare autosomal recessive skeletal disorder characterized by congenital short limbed dwarfism. Based on clinical features, it is classified into two types; the severe, lethal Silverman-Handmaker type (DDSH) and milder form Rolland-Desbuquois type (DDRD). Among the molecularly confirmed DD patients, most are DDSD type. There have been no reports in the patients with molecularly confirmed DDRD. Here, we report a case with homozygous biallelic mutation in HSPG2, diagnosed as DDRD based on clinical features in neonatal period. The proband was 15-years-old boy. He was the first child of non-consanguineous and healthy parents. Physical examination revealed cleft lip palate, shortening of limbs, clubfoot, flexion contractures of bilateral knees and elbows, right inguinal hernia, and narrow chest. X-rays showed small thorax and vertebral body size difference, dumbbell-shaped tubular bones. Based on the radiological investigation, he was diagnosed as DDRD. Now, he is 15 years old, with the height 120 cm (-7.5 SD), weight 27.6 kg (-2.8 SD), and had short distance walkable. To confirm the diagnosis molecularly, we performed Mendelian exome using the TruSight One Sequencing Panel (Illumina, Inc., San Diego, CA, USA). Captured DNA was sequenced on a MiSeq platform (Illumina) with 151 bp paired-end reads. Targeted resequencing and Sanger sequencing identified a novel biallelic homozygous variant c.9970G>A, p.G3324R in HSPG2. This is a first case report with molecularly confirmed DDRD. These results suggest genotype-phenotype correlation in the HSPG2 related disorders.

K. Kurosawa: None. K. Shono: None. T. Yokoi: None. N. Harada: None. M. Akahira-Azuma: None. Y. Enomoto: None. Y. Tsurusaki: None. N. Aida: None.

P04.32D Congenital anonychia and uncombable hair syndrome: co-inheritance of homozygous mutations in RSPO4 and PADI3

M. T. Romano1,2, C. K. Hsu3,4,5, A. Nanda6, E. Rashidghamat3, J. Y. W. Lee3, H. Y. Huang4, C. Songsantiphap3,7, J. Y. Y. Lee4, H. Al-Ajmi6, M. A. Simpson8, C. Tziotzios3, R. C. Betz1,2, J. A. McGrath3

1Department of Genomics, Life&Brain, Bonn, Germany, 2Institute of Human Genetics, University of Bonn, Bonn, Germany, 3St John’s Institute of Dermatology, King's College London, London, United Kingdom, 4Department of Dermatology, National Cheng Kung University Hospital, Tainan, Taiwan, 5Institute of Clinical Medicine, National Cheng Kung University, Tainan, Taiwan, 6As’ ad Al-Hamad Dermatology Center, Al Sabah Hospital, Kuwait city, Kuwait, 7Department of Dermatology, Chulalongkorn University, Bangkok, Thailand, 8Division of Genetics and Molecular Medicine, King's College London, London, United Kingdom

Ectodermal dysplasia comprises a heterogeneous group of genetic disorders defined by developmental defects in ectoderm-derived tissues. The great heterogeneity of the symptoms is often an obstacle for the identification of the causative gene, although the use of next generation sequencing has brought new insights. Here, we investigated a 4-year-old Kuwaiti boy showing both congenital anonychia and uncombable hair syndrome. Through whole exome sequencing (WES) we identified mutations in two separate genes, demonstrating that the patient’s phenotype comes from the overlap of two autosomal recessive disorders. With regards to anonychia, we identified a previously known homozygous splice-site mutation in RSPO4. The encoded protein, R-spondin, is expressed in nail mesenchyme and is an activator of the Wnt/β-catenin pathway. For the hair abnormality, we found a novel homozygous missense mutation in PADI3. This gene encodes for peptidylarginine deiminases 3 (PADI3), which is involved in deiminating trichohyalin in the hair follicle, contributing to its structure.All mutations were verified by Sanger sequencing. Furthermore, the PADI3 mutation was investigated through immunoblotting and immunofluorescence in a keratinocyte cell line, showing both lower expression and formation of aggregates for the mutant protein compared to wild-type. In conclusion, our case highlights the value of WES in identifying co-inheritance of two distinct conditions in consanguineous pedigrees, giving rise to an ectodermal dysplasia phenotype.

M.T. Romano: None. C.K. Hsu: None. A. Nanda: None. E. Rashidghamat: None. J.Y.W. Lee: None. H.Y. Huang: None. C. Songsantiphap: None. J.Y.Y. Lee: None. H. Al-Ajmi: None. M.A. Simpson: None. C. Tziotzios: None. R.C. Betz: None. J.A. McGrath: None.

P04.33A A second patient with Classical Ehlers-Danlos Syndrome (cEDS) and congenital hip dislocation caused by the pathogenic variant COL1A1 c. 934C>T, p.Arg312Cys

J. Duong1, A. Rideout2, J. Beis2, S. Parkash2, U. Schwarze3, A. Vandersteen2

1Queens University Medical School, Kingston, ON, Canada, 2IWK Health Centre, Halifax, NS, Canada, 3University of Washington, Seattle, WA, United States

We present an adult woman with cEDS, bilateral hip dislocation and obstetric anesthetic complications. The patient had generalized joint hypermobility, childhood skin fragility and shoulder dislocations. Her bilateral congenital hip dislocations were treated with Plavik harness. She was diagnosed with cEDS at age 15 following evaluation with multiple affected family members. There was an extensive family history of cEDS features in 7 individuals over three generations spanning in age from 7 to 80 years, none were known to have had bone fragility or vascular complications. The patient had a normal echocardiogram during pregnancy. The patient presented with severe post-partum orthostatic headache after failed epidural anesthesia, during which there was unexpected dural puncture. She recovered after epidural blood patch treatment, following unsuccessful sphenopalatine ganglion block. Sequencing of a large panel of EDS genes, identified the COL1A1 variant c. 934C>T, p.Arg312Cys. This variant has been reported with a classical EDS like phenotype. The child of patient 1 reported in Malfait et al. (2007), had congenital hip dislocation (1). The variant is mentioned in the 2017 EDS nosology for both cEDS and vascular type EDS (2). Whilst three patients with this same pathogenic variant have been reported to have had a vascular complication, a recent report of a large family was more reassuring (3). We present further cumulative phenotypic data on a large family with this genotype.

J. Duong: None. A. Rideout: None. J. Beis: None. S. Parkash: None. U. Schwarze: None. A. Vandersteen: None.

P04.34B Molecular genetic analysis of Ehlers-Danlos Syndrome in Northwestern region of Russia

T. I. Kadurina1, E. A. Serebriakova1,2, L. N. Abbakumova3, Y. A. Barbitoff4,2,5, D. E. Polev2, A. S. Glotov2

1North-Western State Medical University named after I.I. Mechnikov, Saint-Petersburg, Russian Federation, 2Biobank of the Research park, Saint-Petersburg State University, Saint-Petersburg, Russian Federation, 3Saint-Petersburg State Pediatric Medical University, Saint-Petersburg, Russian Federation, 4Bioinformatics Institute, Saint-Petersburg, Russian Federation, 5Department of Genetics and Biotechnology, Saint-Petersburg State University, Saint-Petersburg, Russian Federation

Introduction:Ehlers-Danlos Syndrome (EDS) is a heterogeneous group of connective tissue disorder, caused by metabolic imbalance of collagen, the structure and function of myomatrix also the synthesis of proteoglycans. According to a new international classification of EDS (2017), now it is classified in 13 different types and it is associated with mutations in 19 genes. The exception is the hypermobility type EDS (hEDS) the diagnosis of which is based only on clinical criteria.

Materials and Methods: 12 patients with hEDS were examined. NGS was applied to 10 patients with hEDS and 19 genes associated with EDS were analyzed (ADAMTS2, B3GALT6, B4GALT7, C1R, C1S, CHST14, COL1A1, COL1A2, COL12A1, COL3A1, COL5A1, DSE, FLNA, FKBP14, PLOD1, PRDM5, SLC39A13, TNXB, ZNF469). Additionally, direct PCR method was performed to identify del30kb in the TNXB for two patients.

Results: We identified a heterozygous mutation c.2818G>A in one patient in the ADAMTS2 gene with uncertain clinical significance. Mutations in the ADAMTS2 are associated with autosomal recessive, dEDS. In addition, we detected one heterozygous mutation c.3023C>T with uncertain clinical significance in another patient in the COL5A1 gene which is associated with cEDS. A heterozygous del30kb in the TNXB was identified by direct PCR method in two patients.

Conclusions: Our results indicate that heterozygous mutations in genes associated with different types of EDS can be the cause of hEDS, which indicates genetic heterogeneity of this pathology and needs further research.

T.I. Kadurina: None. E.A. Serebriakova: None. L.N. Abbakumova: None. Y.A. Barbitoff: None. D.E. Polev: None. A.S. Glotov: None.

P04.35C Dual diagnosis of Ellis-van Creveld syndrome and hearing loss in a consanguineous family

P. L. Bahena Carbajal1, B. Vona1, R. Maroofian2, G. Mendirattac3, M. Croken3, S. Peng3, S. Peng3, X. Ye3, J. Rezazadeh4, C. Lekszas1, T. Haaf1, L. Edelmannc3, L. Shic3

1Institut of Humangenetics, Würzburg, Germany, 2University of Exeter Medical School, Exeter, United Kingdom, 3Icahn School of Medicine at Mount Sinai, New York, NY, United States, 4Genetic counselling and Rehabilitation Unit, Welfare organization, South Khorasan, Iran, Islamic Republic of

Multilocus analysis of rare or genetically heterogeneous diseases is a distinct advantage of next generation sequencing over conventional single-gene investigations.

We described a female proband from a large consanguineous Iranian family who manifests postlingual, progressive, moderate hearing loss in addition to a disproportionate short-limb dwarfism with distalward shortening of extremities and postaxial hexadactyly. Her brother as well as other members of the family also presented disproportionate short stature and anomalies of the limbs.

Next generation sequencing with a customized skeletal dysplasia panel containing over 370 genes and subsequent bioinformatics analysis disclosed two homozygous mutations in EVC2 (c.2653C>T; p.Arg885*) and COL11A2 (c.966dup; p.Thr323Hisfs*19), respectively. Sanger sequencing showed that both parents were heterozygous carriers for both EVC2 and COL11A2 mutations and the sister was a heterozygous carrier for the COL11A2 mutation but wild type at the EVC2 mutation position.

This study highlights a dual molecular diagnosis in a patient with a blending of two distinct phenotypes and illustrates the advantage and importance of this staple technology to facilitate rapid and comprehensive genetic dissection of a heterogeneous phenotype. The differentiation between phenotypic expansion of a genetic disorder and a blended phenotype that is due to more than one distinct genetic aberration is essential in order to reduce the diagnostic odyssey endured by patients.

P.L. Bahena Carbajal: None. B. Vona: None. R. Maroofian: None. G. Mendirattac: None. M. Croken: None. S. Peng: None. S. Peng: None. X. Ye: None. J. Rezazadeh: None. C. Lekszas: None. T. Haaf: None. L. Edelmannc: None. L. Shic: None.

P04.37A Application of a machine learning approach to identify miRNA fingerprint signatures in recessive dystrophic epidermolysis bullosa

R. Zauner1, M. Wimmer1, T. Lettner1, S. Atzmüller2, J. Pröll2, C. Guttmann-Gruber1, E. M. Murauer1, J. Reichelt1, D. Strunk3, J. W. Bauer4, V. Wally1

1EB House Austria, Research Program for Molecular Therapy of Genodermatoses Department of Dermatology, University Hospital of the Paracelsus Medical University, Salzburg, Austria, 2Red Cross Transfusion Service for Upper Austria, Linz, Austria, 3Institute for Experimental and Clinical Cell Therapy, Paracelsus Medical University, Salzburg, Austria, 4Department of Dermatology, University Hospital of the Paracelsus Medical University, Salzburg, Austria

Recessive dystrophic epidermolysis bullosa (RDEB) represents one of the most severe subforms of a rare genodermatosis and is caused by mutations within the COL7A1 gene. Absent or dysfunctional type VII collagen impedes the structural integrity of the molecular link between epidermis and underlying dermis, which manifests in severe blister formation sublamina densa and erosions of skin and mucous membranes. RDEB patients suffer from chronically impaired wound healing and an extraordinary high risk of developing a particularly aggressive form of squamous cell carcinoma (SCC). So far there is only very limited data available on non-coding RNA events in RDEB cancer progression.

In order to widen the therapeutic spectrum for RDEB cancer patients we aim to improve our understanding on the impact of differentially expressed small non-coding miRNAs on disease progression and to explore their potential as biomarkers. Therefore, Illumina miRNA-seq was performed on RDEB-SCC, UV-SCC, RDEB and nonEB keratinocytes derived from skin biopsies. Fingerprint miRNAs able to discriminate RDEB-SCCs from the other experimental groups were derived applying a neural network machine learning approach. Specific self-organizing maps were trained on our miRNA-seq data sets and co-expression modules of miRNAs with differential expression profiles extracted. In a guilt-by-association assessment based on microarray expression data, we could demonstrate an enrichment of target mRNAs with strong correlation to signature miRNAs, in hallmark signatures like epithelial-to-mesenchymal transition, which is related to the observed aggressive phenotype in RDEB-SCCs.

Eventually, our set of identified fingerprint miRNAs may provide the foundation for the identification of biomarkers and drugable targets.

R. Zauner: None. M. Wimmer: None. T. Lettner: None. S. Atzmüller: None. J. Pröll: None. C. Guttmann-Gruber: None. E.M. Murauer: None. J. Reichelt: None. D. Strunk: None. J.W. Bauer: None. V. Wally: None.

P04.38B Major player in miRNA processing appear altered in recessive dystrophic epidermolysis bullosa squamous cell carcinoma

M. Wimmer1, R. Zauner1, A. Waldmann1, H. Bodocian1, M. Ablinger1, S. Atzmueller2, J. Proell2, D. Strunk3, J. W. Bauer1, J. Reichelt1, V. Wally1

1EB House Austria, Research Program for Molecular Therapy of Genodermatoses, Department of Dermatology, University Hospital of the Paracelsus Medical University, Salzburg, Austria, 2Red Cross Transfusion Service for Upper Austria, Linz, Austria, 3Institute for Experimental and Clinical Cell Therapy, Paracelsus Medical University, Salzburg, Austria

Recessive dystrophic epidermolysis bullosa (RDEB) is characterized by loss-of-function mutations in the COL7A1 gene, resulting in impaired or absent type VII collagen protein, a major anchoring molecule at the dermal-epidermal juction. Clinically, patients suffer from extraordinary sensitivity of the skin to mechanic friction or trauma and present with an increased risk of infections. One major concern in this patient population relates to the high incidence of developing a particularly aggressive form of squamous cell carcinomas (SCCs), resulting in a highly increased mortality due to frequent metastatic spreading. Recent studies have demonstrated a significantly deregulated miRNome in RDEB-SCC in vitro. In order to investigate the cause of the observed altered miRNA abundance, we analyzed expression levels of major components of the miRNA biogenesis pathway via microarray, sqRT-PCR and Western blot. Sanger sequencing of mutational hotspots was performed for major components of the miRNA processing machinery. Cultured primary skin cells derived from patient SCCs and keratinocytes and healthy controls served as experimental groups.

We found significantly increased RNA and protein expression levels of DROSHA in SCC cells as compared to healthy controls, without indication of mutagenic events in reported hotspots. Karyotyping of RDEB SCC cells confirmed chromosomal abberations in regions of interest concerning DROSHA.

A dysregulation of canonical miRNA biogenesis is associated with many diseases, particularly cancer, and could prove instrumental in further deepening our understanding of triggers and promoters of SCC in RDEB.

M. Wimmer: None. R. Zauner: None. A. Waldmann: None. H. Bodocian: None. M. Ablinger: None. S. Atzmueller: None. J. Proell: None. D. Strunk: None. J.W. Bauer: None. J. Reichelt: None. V. Wally: None.

P04.39C Correction of type XVII collagen using spliceosome-mediated RNA trans-splicing

M. Reisenberger, P. Schlager, J. Reichelt, J. W. Bauer, U. Koller, S. Hainzl, M. Ablinger, T. Lettner, V. Wally

EB House Austria, Research Program for Molecular Therapy of Genodermatoses, salzburg, Austria

Distinct subtypes of junctional EB (JEB) are caused by mutations within the COL17A1 gene, encoding type XVII collagen, which leads to fragility of the skin and blister formation within the lamina lucida upon minor friction. So far, there is no causal therapy available for this EB subtype. SMaRT is a post-transcriptional therapeutic approach, which uses the cellular splicing machinery to replace mutation-bearing exons. The aim of this study was to apply SMaRT therapy for the correction of any mutation downstream from COL17A1 exon 33. We engineered a set of RNA trans-splicing molecules (RTMs), which bind COL17A1 pre-mRNA, thereby inducing a trans-splicing reaction that results in the generation of a hybrid mRNA, containing RTM-derived and endogenous gene portions. In a minigene-based approach we first studied the impact of various RTM sequence modifications on trans-splicing efficiency and specificity. The modifications included binding domain optimization, removal of the COL17A1 3’UTR and removal of potential cryptic splice sites. Retroviral transduction of a JEB keratinocyte line harbouring a compound heterozygous mutation (exon 52 and 53) with the most functional RTM (RTM135mSU), coding for the COL17A1 wild type exons 34-56, led to restoration of type XVII collagen expression at mRNA level and protein level assessed by immunofluorescence. We found that a binding domain covering the intron/exon junction and the removal of splice sites in the RTMs coding region resulted in an increased trans-splicing efficiency. Our results indicate that the SMaRT technology is a promising tool for the development of an RNA therapy for JEB patients.

M. Reisenberger: None. P. Schlager: None. J. Reichelt: None. J.W. Bauer: None. U. Koller: None. S. Hainzl: None. M. Ablinger: None. T. Lettner: None. V. Wally: None.

P04.40D Seven additional families with spondylocarpotarsal synostosis syndrome with novel biallelic deleterious variants in FLNB

S. Salian1, A. Shukla1, H. Shah1, S. N. Bhat1, V. R. Bhat1, S. Nampoothiri2, R. Shenoy3, S. R. Phadke4, S. V. Hariharan5, K. M. Girisha1

1Kasturba Medical College, Manipal, Udupi, India, 2Department of Pediatric Genetics, Amrita Institute of Medical Sciences and Research Centre, Cochin, India, 3Department of Pediatrics, KS Hegde Medical Academy, Mangalore, India, 4Department of Medical Genetics, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, India, 5Department of Pediatrics, Sree Avittom Thirunal Hospital, Government Medical College, Thiruvananthapuram, India

Introduction: Location and/or type of variants in FLNB result in a spectrum of osteochondrodysplasias: spondylocarpotarsal synostosis syndrome and Larsen syndrome that are milder and atelosteogenesis I and III, and Boomerang dysplasia that are perinatal lethal. So far, nine bi-allelic loss-of-function variants in FLNB are reported to cause spondylocarpotarsal synostosis syndrome in nine families. We aimed to identify pathogenic variants in FLNB in ten patients from seven families with spondylocarpotarsal synostosis syndrome.

Material and Methods: As FLNB is a large gene with 46 exons, we preferred exome sequencing index patients from all the seven families as this is cost-efficient at our center followed by Sanger validation and segregation analysis.

Results: Clinical features of ten patients (six females and four males) included short stature (9/9), short neck (6/10), pectus carinatum (5/10), facial dysmorphism (2/10) and cleft lip and palate (1/10). Radiological features were fused vertebrae (9/10), carpal fusion (10/10), tarsal fusion (5/5), scoliosis (9/10), lumbar lordosis (2/10) and crowding of ribs (9/10). Seven novel homozygous variants were identified in FLNB: c.6317del [p.(Pro2106ArgfsX12)] in patient 1, c.1493del [p.(Glu498GlyfsX4)] in patient 2, c.1243C>T [p.(Arg415X)] in patient 3, c.1204del [p.(Val402TryfsX88)] in patient 4, c.28G>T [p.(Glu10X)] in patient 5, c.1429delinsCT [p.(Val477Leufs*2)] in patients 6 and 7, and c.1592dup [p.(His532ThrfsX9)] in patients 8, 9 and 10.

Conclusion: Our work demonstrates that spondylocarpotarsal synostosis syndrome has a unique pattern of anomalous vertebral segmentation and all the reported patients have truncating variants in FLNB. The study is funded by Indian Council of Medical Research.

S. Salian: None. A. Shukla: None. H. Shah: None. S.N. Bhat: None. V.R. Bhat: None. S. Nampoothiri: None. R. Shenoy: None. S.R. Phadke: None. S.V. Hariharan: None. K.M. Girisha: None.

P04.42B Novel clinical features in frontometaphyseal dysplasia 2 caused by a recurrent mutation in MAP3K7

A. Costantini1, C. Wallgren-Pettersson2, O. Mäkitie1,3,4,5

1Department of Molecular Medicine and Surgery and Center for Molecular Medicine, Karolinska Institutet, Stockholm, Sweden, 2Folkhälsan Institute of Genetics and Department of Medical and Clinical Genetics, University of Helsinki, Helsinki, Finland, Helsinki, Finland, 3Folkhälsan Institute of Genetics and University of Helsinki, Helsinki, Finland, 4Children's Hospital, University of Helsinki and Helsinki University Hospital, Helsinki, Finland, 5Department of Clinical Genetics, Karolinska University Hospital, Stockholm, Sweden

Frontometaphyseal dysplasia 2 (FMD2) is a skeletal dysplasia with supraorbital hyperostosis combined with undermodeling of the bones, joint contractures and some extraskeletal features. It is caused by heterozygous mutations in MAP3K7, encoding the Mitogen-Activated Protein Kinase 7. MAP3K7 is activated by TGF-β and plays an important role in osteogenesis. Less than 20 patients with FMD2 and MAP3K7 mutations have been described thus far. Three out of four patients harbor a recurrent missense mutation, NM_003188.3: c.1454C>T, p.(Pro485Leu), which leads to a more severe phenotype than mutations in other domains. Here we describe an additional patient with FMD2 caused by the recurrent c.1454C>T MAP3K7 mutation, identified as a de novo variant by whole-genome sequencing. The 17-year-old boy has the characteristic skeletal and facial features of FMD2. However, several novel features were also observed, including multiple hemangiomas, hand and foot abnormalities, growth retardation, spina bifida, Sprengel deformity, Chiari malformation and ocular manifestations. He also showed keloid scars but, in contrast to other patients harboring the same mutation, he does not have intellectual disability. This report expands the clinical spectrum of FMD2 caused by the recurrent c.1454C>T, p.(Pro485Leu) mutation in MAP3K7.

A. Costantini: None. C. Wallgren-Pettersson: None. O. Mäkitie: None.

P04.44D MicroRNA profiling in human neural crest cells and integration of GWAS data suggest involvement of miRNA149 in craniofacial disease

L. G. Stuessel1,2, L. M. Hochfeld1,2, J. Schroeder1,2, F. Thieme1,2, T. Hess1,2, J. Gehlen1,2, S. Heilmann-Heimbach1,2, M. Knapp3, E. Mangold2, A. Rada-Iglesias4,5, K. U. Ludwig1,2

1Department of Genomics, Life&Brain Center, Bonn, Germany, 2Institute of Human Genetics, University of Bonn, Bonn, Germany, 3Institute of Medical Biometry Informatics and Epidemiology, University of Bonn, Bonn, Germany, 4Center for Molecular Medicine Cologne (CMMC), University of Cologne, Cologne, Germany, 5Cologne Excellence Cluster for Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany

GWAS for nonsyndromic cleft lip with/without cleft palate (nsCL/P) have identified 40 risk loci for this human craniofacial malformation. The vast majority of these associated loci map to non-coding genomic regions. One possible mechanism by which variants at these loci might exert their regulatory effect is the control of microRNA (miRNA) expression and miRNA-mediated gene regulation, in disease-relevant tissue. In the present study, we sought to identify candidate miRNAs in human neural crest cells (hNCCs), an early precursor cell population of facial tissue, and integrate GWAS data to identify potential susceptibility miRNA candidates that might be involved in nsCL/P.

MiRNA profiling was performed in four independent hNCC samples, previously generated from induced pluripotent stem cells, using the Affymetrix miRNA 4.0 array platform. After stringent filtering, 152 miRNAs were identified as expressed in hNCCs, none of which mapped to the known nsCL/P loci. However, positional integration of GWAS summary statistics revealed 25 variants (at P-value <0.01) that were located within 1kb of a candidate miRNA. Association of one variant, a low-frequency variant at chr. 2q37.3 close to miRNA-149, was confirmed in a replication analysis of an independent nsCL/P cohort. Target gene prediction with miRWalk2.0 and literature research revealed that miRNA149 likely binds known nsCL/P candidate genes such as FGFR1, BMP9 and RUNX2, and has been shown to differentially interact with MTHFR upon folate deficiency. Although further functional characterization is required and currently ongoing, our study suggests that miRNA-149 might be involved as regulatory mechanism in facial development.

L.G. Stuessel: None. L.M. Hochfeld: None. J. Schroeder: None. F. Thieme: None. T. Hess: None. J. Gehlen: None. S. Heilmann-Heimbach: None. M. Knapp: None. E. Mangold: None. A. Rada-Iglesias: None. K.U. Ludwig: None.

P04.45A Genetic analysis of genodermatoses in domestic animals

A. Bauer1,2, P. Balmer1,3, M. A. T. Brunner1,4, M. Caduff1,2, M. De Lucia5, C. Drögemüller1,2, M. Drögemüller1,2, V. Jagannathan1,2, J. Nimmo6, L. Murgiano1,2,7, B. S. Sayar1,8, N. Tarasova9, K. Timm10, R. E. Towers11,12, G. Zur13, E. Müller1,8,14, P. Roosje1,3, M. M. Welle1,4, T. Leeb1,2

1Dermfocus, University of Bern, Bern, Switzerland, 2Institute of Genetics, Vetsuisse Faculty, University of Bern, Bern, Switzerland, 3Division of Clinical Dermatology, Department of Clinical Veterinary Medicine, Vetsuisse Faculty, University of Bern, Bern, Switzerland, 4Institute of Animal Pathology, Vetsuisse Faculty, University of Bern, Bern, Switzerland, 5San Marco Veterinary Clinic and Laboratory, Padova, Italy, 6ASAP Laboratory, Mulgrave, Victoria, Australia, 7Section of Ophthalmology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA, United States, 8Department of Biomedical Research, Molecular Dermatology and Stem Cell Research, University of Bern, Bern, Switzerland, 9Russian Akhal-Teke Association, Moscow, Russian Federation, 10Vetderm, Hünenberg, Switzerland, 11Institute of Medical Genetics, Cardiff University, Cardiff, United Kingdom, 12Tees, Esk and Wear Valleys NHS Foundation Trust, United Kingdom, 13Veterinary Teaching Hospital, The Koret School of Veterinary Medicine, The Hebrew University of Jerusalem, Rehovot, Israel, 14Clinic for Dermatology, Inselspital, Bern University Hospital, Bern, Switzerland

Introduction: Spontaneous mutants in domestic animal species are valuable models to study heritable human disorders. Purebred animals are kept in closed populations necessitating a certain degree of inbreeding, which favors the expression of recessive alleles. Due to the unique population structure of purebred animals, identification of disease causing genetic variants is often more straightforward than in humans.

Materials and Methods: Genetic mapping and whole genome sequencing approaches were used.

Results: We identified candidate causative variants for several genodermatoses including variants in genes that had not previously been associated with disease phenotypes in humans. As an example, we discovered genetic variants in SUV39H2 in dogs with hereditary nasal parakeratosis, which revealed a role for SUV39H2 in keratinocyte differentiation.

Selected domestic animal models for human genodermatoses




Human disorder (MIM#)






X-linked hypohidrotic ectodermal dysplasia

dog, cattle



hereditary footpad hyperkeratosis


palmoplantar keratoderma and exuberant scalp hair


incontinentia pigmenti




brindle 1


300918, 308205, 308800


congenital cornification disorder


308050, 300831


oculocutaneous albinism, type 2


203200, 227220


naked foal syndrome




hereditary nasal parakeratosis




streaked hairlessness



Conclusions: The study provides new candidate genes for genodermatoses in human and veterinary medicine and a better understanding of the genotype-phenotype correlation.

Grant: Swiss National Science Foundation CRSII3_160738/1

A. Bauer: None. P. Balmer: None. M.A.T. Brunner: None. M. Caduff: None. M. De Lucia: None. C. Drögemüller: None. M. Drögemüller: None. V. Jagannathan: None. J. Nimmo: None. L. Murgiano: None. B.S. Sayar: None. N. Tarasova: None. K. Timm: None. R.E. Towers: None. G. Zur: None. E. Müller: None. P. Roosje: None. M.M. Welle: None. T. Leeb: None.

P04.46B Novel case with a double “apparently” balanced rearrangement disrupting EXT1 in a patient with hereditary multiple exostoses

A. Alexandrou1, N. Salameh1, I. Papaevripidou1, N. Nicolaou2, P. Myriathopoulos1, A. Ketoni1, P. Evangelidou1, G. A. Tanteles2, C. Sismani1,3

1Cytogenetics and Genomics Department, The Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus, 2Clinical Genetics Clinic, The Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus, 3The Cyprus School of Molecular Medicine, The Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus

Hereditary multiple exostoses (HME) is an autosomal dominant skeletal disorder characterized by the development of multiple, circumscript, occasionally painful and usually symmetric bony protuberances called osteochondromas. HME is caused by EXT1 and EXT2 loss of function mutations. Most pathogenic mutations are nonsense followed by missense mutations and deletions. We report on a patient with a rare and complex genotype resulting in a classical HME phenotype.

Mutations in EXT1 and EXT2 were excluded by Sanger sequencing. The patient was subsequently referred for karyotype and array-CGH analyses. Results obtained were validated with FISH and qRT-PCR and parental studies determined the mode of inheritance.

Chromosomal analysis revealed a de novo “apparently” double balanced rearrangement: a balanced translocation between chromosomes 2 and 3 at breakpoints 2q22 and 3q13.2 and a pericentric 8p23.1q24.1 inversion both of which were confirmed by FISH analysis. Subsequently, array-CGH analysis revealed a novel heterozygous deletion within the EXT1 gene at the inversion breakpoints, rendering the inversion as unbalanced. The inheritance mode as well as the size of the deletion was further investigated by qRT-PCR and the deletion was characterized as a de novo 3.1kb deletion removing exon 10. The inversion in combination with the 8p23.1 deletion most likely abolishes the transcription of EXT1 downstream of exon 10 hence resulting in a truncated protein.

To conclude, a rare and novel pathogenic cause of HME is presented in this study, highlighting the importance of additional comprehensive cytogenetic investigation when EXT1 and EXT2 mutation analysis is negative.

A. Alexandrou: None. N. Salameh: None. I. Papaevripidou: None. N. Nicolaou: None. P. Myriathopoulos: None. A. Ketoni: None. P. Evangelidou: None. G.A. Tanteles: None. C. Sismani: None.

P04.47C An insertion mutation in HOXC13 underlies pure hair and nail ectodermal dysplasia with lacrimal duct obstruction

A. Humbatova1,2,3, R. Maroofian4, M. Romano1,2, A. Tafazzoli1,2, M. Behnam5, N. Dilaver6, N. Nouri5, M. Salehi5,7, S. Wolf1,2, J. Frank8, P. Kokordelis1,2, R. Betz1,2

1Institute of Human Genetics, University of Bonn, School of Medicine & University Hospital Bonn, Bonn, Germany, 2Department of Genomics, Life&Brain Center, Bonn, Germany, 3Institute of Genetic Resources, Azerbaijan National Academy of Sciences, Baku, Azerbaijan, 4Molecular & Clinical Sciences Research Institute, St. George's University of London, Cranmer Terrace, London, United Kingdom, 5Medical Genetics Laboratory of Genome, Isfahan, Iran, Islamic Republic of, 6Swansea University Medical School, Swansea University, Wales, United Kingdom, 7Division of Genetics and Molecular Biology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran, Islamic Republic of, 8Department of Dermatology, Venereology and Allergology, University Medical Center Göttingen, Göttingen, Germany

Ectodermal dysplasias (EDs) encompass a large group of clinically and genetically heterogeneous hereditary disorders that are defined by abnormal development of at least two ectodermal structures, which include but are not limited to hair, nails, teeth and sweat glands. A rare form of autosomal recessive ED that is usually characterized by hypotrichosis and nail dystrophy only is known as pure hair and nail ectodermal dysplasia (PHNED). To date, mutations in KRT85, KRT74, and HOXC13 have been reported in patients and families of different ethnic origin with PHNED. Here, we studied two sisters from a consanguineous Iranian marriage who were not only affected by hypotrichosis and nail dysplasia but also lacrimal duct obstruction (LDO). Homozygosity mapping and GeneDistiller analysis suggested linkage of the disease to chromosome 12q13.13. In this region, KRT85, KRT74, and HOXC13 were the most plausible candidate genes. Sanger sequencing of HOXC13 revealed a hitherto undescribed homozygous 28 bp insertion mutation (c.837_838insACTTGCGGCTAGCAAGTTCATCACCAAA; p. A280Tfs*4) in exon 2 in both affected children that was present in the heterozygous state in the parents.

LDO has not previously been described in association with PHNED although this symptom has been frequently observed in other types of ED. Therefore, LDO might have been neglected or underdiagnosed in earlier reports of PHNED. The clinical and molecular genetic findings in the family expand the phenotypic and mutation spectrum of PHNED and suggest that LDO should be examined for when clinically evaluating individuals and families with this rare variant of ED.

A. Humbatova: None. R. Maroofian: None. M. Romano: None. A. Tafazzoli: None. M. Behnam: None. N. Dilaver: None. N. Nouri: None. M. Salehi: None. S. Wolf: None. J. Frank: None. P. Kokordelis: None. R. Betz: None.

P04.48D Italian validation of the functional difficulties questionnaire (FDQ-9) and its correlation with major determinants of quality of life in adults with hypermobile Ehlers-Danlos syndrome/hypermobility spectrum disorders

S. Morlino1, C. Dordoni2, I. Sperduti3, C. Piedimonte4, M. Ritelli2, M. Colombi2, P. Grammatico1, M. Castori5

1Lab. Medical Genetics, Dep. Molecular Medicine, Sapienza University, S.Camillo-Forlanini Hospital, Rome, Italy, 2Div. Biology and Genetics, Dep. Molecular and Translational Medicine, University of Brescia, Brescia, Italy, 3Biostatistics, IRCCS San Gallicano Dermatologic Institute, Rome, Italy, 4Department of Experimental Medicine, Sapienza University, "Umberto I" Hospital, Rome, Italy, 5Div. Medical Genetics, IRCCS Casa Sollievo della Sofferenza, San Giovanni Rotondo (FG), Italy

The 2017 EDS International Classification defined the new criteria for hypermobile Ehlers-Danlos syndrome (hEDS), which is now considered one end of a continuous spectrum originating from isolated, non-syndromic joint hypermobility (JH) and passing through hypermobility spectrum disorders (HSD). Preliminary data indicate a link between JH and neurodevelopmental disorders, and the strongest evidence is the non-causal association with developmental coordination disorder (DCD) in children. Assessing DCD in adults is difficult and the recently described functional difficulties questionnaire 9 (FDQ-9) is one of the few available tools. The aims of this study were: (i) to validate FDQ-9 in Italian and to normalize its values in 230 Italian non-clinical patients; and (i) to explore the relationship of FDQ-9 with the brief pain inventory, composite autonomic symptom score 31, multidimensional fatigue inventory, ADHD self-report version 1.1, and the SF-36 for quality of life in 105 Italian adults with hEDS/HSD. Validity of FDQ-9 was assessed by the Pearson test in 10 bilingual individuals and 5 bilingual hEDS/HSD patients who completed the questionnaire in English and Italian. In the hEDS/HSD group, 67% patients had a value of FDQ-9 above the cut-off and, therefore, a high probability of DCD in their developmental age. Multivariate analysis was carried out comparing FDQ-9 values with features of pain, fatigue, autonomic dysfunction, ADHD and quality of life, and demonstrated an influence of a past history of coordination troubles on chronic symptoms in adults with hEDS/HSD. Our preliminary data open wider management and therapeutic perspectives for coordination troubles in hypermobile individuals.

S. Morlino: None. C. Dordoni: None. I. Sperduti: None. C. Piedimonte: None. M. Ritelli: None. M. Colombi: None. P. Grammatico: None. M. Castori: None.

P04.49A Detection of mosaic Copy-Number Variations from Whole-Exome Sequencing in mosaic pigmentation disorders using XHMM and a custom SNP approach

A. SORLIN1,2,3, É. Tisserant2,3, J. Thevenon1,3,2, Y. Duffourd2,3, P. Kuentz2,3,4, V. Carmignac2,3, V. Cormier-Daire5, C. Michot5, V. Malan6, M. Beaujard6, F. Morice-Picard7, C. Rooryck-Thambo8, C. Vincent-Delorme9, T. Smol10, É. Boudry-Labis10, S. Hadj Rabia11, A. Phan12, M. Cordier13, M. Till13, D. Sanlaville13, J. St-Onge3,2,14, C. Thauvin-Robinet1,2,3, A. Mosca Boidron15,2,3, R. Olaso16, A. Boland16, J. Deleuze16, B. Keren17, L. Faivre1,2,3, J. Rivière2,3,14,18, P. Callier2,3,15, P. Vabres2,3,19

1Centre de Génétique, CHU Dijon Bourgogne, Dijon, France, 2INSERM 1231, Génétique des Anomalies du Développement, Université Bourgogne Franche-Comté, Dijon, France, 3Fédération Hospitalo-Universitaire Médecine Translationnelle et Anomalies du Développement, CHU Dijon Bourgogne, Dijon, France, 4Génétique Biologique Histologie, CHRU de Besançon, Besançon, France, 5AP-HP, Hôpital Necker-Enfants malades, Genetics Departement, Centre of Reference for Skeletal Dysplasia, INSERM UMR 1163, Institut Imagine, University Paris Descartes-Sorbonne Paris Cité, Paris, France, 6Service d'Histologie-Embryologie-Cytogénétique, Hôpital Universitaire Necker-Enfants Malades, Paris, France, 7Service de génétique médicale, CHU de Bordeaux-GH Pellegrin, Bordeaux, France, 8Laboratoire de génétique moléculaire, CHU de Bordeaux-GH Pellegrin, Bordeaux, France, 9Service de génétique clinique, CHRU de Lille - Hôpital Jeanne de Flandre, Lille, France, 10Laboratoire de Génétique médicale, CHRU de Lille - Hôpital Jeanne de Flandre, Lille, France, 11Service de dermatologie, Hôpital Universitaire Necker-Enfants Malades, Paris, France, 12Pediatric Dermatology Department, Hôpital Femme Mère Enfant, Hospices Civils de Lyon, Lyon, France, 13Service de génétique, GH Est-Hôpital Femme Mère Enfant, Hospices Civils de Lyon, Lyon, France, 14Child Health and Human Development Program, Research Institute of the McGill University Health Centre, Montreal, QC, Canada, 15Laboratoire de génétique moléculaire et de Cytogénétique, Plateau Technique de Biologie, CHU Dijon Bourgogne, Dijon, France, 16Centre National de Recherche en Génomique Humaine, Evry, France, 17Département de Génétique et Centre de Référence Déficiences Intellectuelles de Causes Rares, Hôpital de la Pitié-Salpétrière, AP–HP, Paris, France, 18Department of Human Genetics, Faculty of Medicine, McGill University, Montreal, QC, Canada, 19Service de Dermatologie, CHU Dijon Bourgogne, Dijon, France

Whole exome sequencing (WES) is as a powerful tool for deciphering the genetic basis of developmental disorder, either single nucleotides variants (SNV), indels, or recently copy number variants (CNV). In mosaic development disorders involving the skin, it has allowed detection of post-zygotic mutations (mSNV) in various genes, but detection of mosaic CNV (mCNV) still relies on conventional cytogenetic studies, such as array-CGH. We sought to develop an all-in-one strategy for patients with mosaic disorders, using trio-WES (lesional skin versus parent’s blood). We combined a SNV detection pipeline with a CNV detection approach, based on both a read-depth approach and a custom SNP-based approach.

We performed WES in 74 patients with cutaneous mosaic disorders. We detected 6 mCNV, all in a subgroup of 21 patients with hypomelanosis of Ito without known pathogenic SNV: 3 mosaic trisomy (chromosomes 7, 12, 15) and 3 smaller mCNV. Blood and skin karyotypes were previously negative, due either to the absence of mosaic cells in blood or to their elimination from cultured fibroblasts. For the 3 mosaic trisomies, SNP inheritance and b-allele frequency provided information on the parental origin of extra chromosomes and clues to understand the underlying mechanism. We have confirmed that chromosomal mosaicism is associated with mosaic pigmentation disorders (6/21, 29% in our cohort). An appropriate fresh tissue sample is essential. Our combined approach showed a good efficiency to detect both (m)SNV and (m)CNV in a single one-step assay, doubling our diagnostic rate, and offering new perspectives in the study of mosaic developmental disorder.

A. Sorlin: None. É. Tisserant: None. J. Thevenon: None. Y. Duffourd: None. P. Kuentz: None. V. Carmignac: None. V. Cormier-Daire: None. C. Michot: None. V. Malan: None. M. Beaujard: None. F. Morice-Picard: None. C. Rooryck-Thambo: None. C. Vincent-Delorme: None. T. Smol: None. É. Boudry-Labis: None. S. Hadj Rabia: None. A. Phan: None. M. Cordier: None. M. Till: None. D. Sanlaville: None. J. St-Onge: None. C. Thauvin-Robinet: None. A. Mosca Boidron: None. R. Olaso: None. A. Boland: None. J. Deleuze: None. B. Keren: None. L. Faivre: None. J. Rivière: None. P. Callier: None. P. Vabres: None.

P04.50B Results of diagnostics of ichthyoses and epidermolysis bullosa using dedicated next generation sequencing panel

K. Wertheim-Tysarowska1, D. Śniegórska1, A. Grabarczyk1, S. Radomska1, A. Kutkowska-Kaźmierczak1, J. Sawicka1, M. Jackiewicz1, P. Bialik1, A. Kujko1, A. M. Rygiel1, K. Niepokoj1, L. Ruszkowska2, K. Osipowicz3, R. Smigiel4, B. Wawrzycki5, K. Wozniak3, A. Jazela-Stanek6, A. Jakubiuk- Tomaszuk7, A. Eckersdorf-Mastalerz8, A. Barczyk1, D. Marańska9, I. Dąbrowska-Wójciak10, K. Ebner11, J. Castaneda1, N. Bezniakow1, M. Firek-Pędras12, E. Obersztyn1, M. Pasinska13, A. Pietrzyk14, K. Szczałuba15, J. Wierzba16, P. Wlasienko1, C. Kowalewski3, J. Bal1

1Medical Genetic Department, Institute of Mother and Child, Warsaw, Poland, 2Department of Paediatric Dermatology, Specialist Hospital Miedzyleski, Warsaw, Poland, 3Department of Dermatology and Immunodermatology, Warsaw Medical University, Warsaw, Poland, 4Department of Paediatrics and Rare Disorders,Wroclaw Medical University, Warsaw, Poland, 5Department of Dermatology, Venereology and Pediatric Dermatology, Medical University of Lublin, Lublin, Poland, 6The Children’s Memorial Health Institute, Warsaw, Poland, 7Podlaskie Medical Center "Genetics", Bialystok, Poland, 8Department of Clinical Genetics, Medical University of Lodz, Lodz, Poland, 9Department of Adult Dermatology, Specialist Hospital Miedzyleski, Warsaw, Poland, 10Department of Neonatology, SALVE ZOZ, Lodz, Poland, 11Department of Intensive Care and Anaesthesiology for Children, Medical University of Lodz,, Lodz, Poland, 12Clinical Hospital No. 6 of the Silesian Medical University in Katowice, Katowice, Poland, 13Department of Clinical Genetics - Collegium Medicum Nicolaus Copernicus University in Bydgoszcz, Bydgoszcz, Poland, 14Department of Genetics and Pathology, Pomeranian Medical University, Szczecin, Poland, 15MedGen Medical Center, Warsaw, Poland., Warsaw, Poland, 16Departments of Pediatrics, Hematology, Oncology and Department of General Nursery, Medical University of Gdansk, Gdansk, Poland

Introduction: Ichthyoses (I) and epidermolysis bullosa (EB) are rare, monogenic skin comprising several dozens of clinical entities, caused by mutations in over 36 and 21 genes, in I and EB, respectively. Low incidence, genetic diversity, overlapping phenotypes and age-dependent disease course negatively influence the detection rate in traditional diagnostic procedure based on phenotype to genotype approach. The aim of the study was to elaborate the cost effective genodermatoses-dedicated next generation sequencing (NGS) panel.

Materials and Methods: We enrolled 103 and 8 patients presenting clinical symptoms of I and EB, respectively. A self-designed panel of 86 genes (Roche NimbleGen SeqCap EZ System) for library preparation and MiSeq sequencer (Illumina) were used, followed by Sanger sequencing/MLPA for mutations verification.

Results: We identified full genotype in 86/103 (83%) of I and 7/8 (87%) of EB patients. In 12/103 (12%) of I patients we didn’t detect any mutation, while in 6 (5 I and 1 EB) we found mutation in one allele only. Overall, we detected mutations in 19 distinct genes. Furthermore, in 10 patients, in addition to their primary disease-causing mutations, harbored also another possibly pathogenic mutation in one allele of the other gene, including semi-dominant mutations in FLG.

Conclusions: In conclusion, our panel proved to be an efficient and sensitive first-line diagnostic tool. Our data provide further information regarding molecular epidemiology of I and EB and also focus on the presence of secondary mutations, which have important impact on genetic counseling and, presumably, may affect therapy. Supported by grant NCN 2014/13/D/NZ5/03304

K. Wertheim-Tysarowska: None. D. Śniegórska: None. A. Grabarczyk: None. S. Radomska: None. A. Kutkowska-Kaźmierczak: None. J. Sawicka: None. M. Jackiewicz: None. P. Bialik: None. A. Kujko: None. A.M. Rygiel: None. K. Niepokoj: None. L. Ruszkowska: None. K. Osipowicz: None. R. Smigiel: None. B. Wawrzycki: None. K. Wozniak: None. A. Jazela-Stanek: None. A. Jakubiuk- Tomaszuk: None. A. Eckersdorf-Mastalerz: None. A. Barczyk: None. D. Marańska: None. I. Dąbrowska-Wójciak: None. K. Ebner: None. J. Castaneda: None. N. Bezniakow: None. M. Firek-Pędras: None. E. Obersztyn: None. M. Pasinska: None. A. Pietrzyk: None. K. Szczałuba: None. J. Wierzba: None. P. Wlasienko: None. C. Kowalewski: None. J. Bal: None.

P04.52D Ichthyosis prematurity syndrome identified for the first time in the Spanish population. Unprecedented molecular characterization of a FATP4 splicing variant in humans

U. Esperón Moldes1, M. Ginarte Val2, M. Santamariña Pena1, B. Rordríguez Lage1, L. Rodríguez Pazos3, A. Vega Gliemmo1

1Fundación pública galega de medicina xenómica, Santiago de compostela, Spain, 2Servicio de dermatología del complexo hospitalario universitario de santiago de compostela, Santiago de compostela, Spain, 3Servicio de dermatología del complexo hospitalario universitario de vigo, Vigo, Spain

Introduction: Ichthyosis prematurity syndrome (IPS) is a rare syndromic form of autosomic recessive ichthyosis caused by mutations in FATP4. To date, three different FATP4 splice site mutations have been associated with IPS, but none of them has been yet characterized at RNA level in humans.

Materials and Methods: Two 26 and 27 years-old Spanish siblings who presented with congenital ichthyosis clinical manifestations, and tested negative for autosomal recessive congenital ichthyosis (ARCI) related genes, were sent to our service. Clinical features were carefully assessed and genetic analysis was performed in both patients and their parents. In silico predictions of mutational effects and further characterization by RNA study was performed for one putative splicing variant identified.

Results: Two novel FATP4 mutations were found in both patients: one frameshift variant, c.1322dup, p.Gly442Argfs*2 and one intronic substitution, c.988-19A>G. Parents were heterozygous for each of the variants. Molecular characterization of c.988-19A>G showed that this variant creates one aberrant transcript that lacks the first 45bp of exon 8, which encodes the end of the protein ATP/AMP motif, and it also leads to a deregulation of naturally occurring isoforms. In silico analyses predicted the variant to alter the recognition sites for splicing regulatory proteins.

Conclusions: This study describes, for the first time, two cases of genetically diagnosed IPS in the Spanish population, adding new mutations to the current list of FATP4 pathogenic variants. It also characterizes the non canonical splice-site c.988-19A>G mutation, being the first splicing study in FATP4 in humans.

Grant references: Fundación Ramón Areces

U. Esperón Moldes: None. M. Ginarte Val: None. M. Santamariña Pena: None. B. Rordríguez Lage: None. L. Rodríguez Pazos: None. A. Vega Gliemmo: None.

P04.54B Novel mutations and clinical variability in Van der Woude Syndrome


1Ankara Yildirim Beyazit Universi̇ty, Department of Medical Genetics, Ankara, Turkey, 2Hacetepe University, Faculty of Medicine, Department of Plastic Reconstructive and Aesthetic Surgery, Ankara, Turkey

Van der Woude Syndrome (OMIM 119300) is an autosomal dominant clinical condition characterized by cleft palate, cleft lip, and lower lip pits. vWS is the most common cause of syndromic cleft lip-palate, and also 2% of all cleft lip and palate cases. vWS presents with variable phenotypic features and high penetrance. Interferon Regulatory Factor 6 (IRF6) gene mutations have been reported as the cause of vWS. The 3th and 4th exons of the gene encode DNA binding domain; 7,8, and 9th exons encode protein binding domain. More than 300 IRF6 mutations have been reported as the cause of vWS syndrome. Mutations are mostly clustered in exons encoding functional domains. Mutations in the IRF6 gene are also associated with the Popliteal Pterygium Syndrome(OMIM 119500), which has similar orofacial features with vWS; however, it also presents with popliteal webs, syndactyly, and genital anomalies. Here we report, 43 patients with vWS at 6 different families and two of them have novel mutations, which have not reported before. A novel c.841-2A>C mutation was identified in family A and a novel c.881T>A mutation was identified in family B. These mutations will provide a better understanding of the phenotypic effect of exon 7 mutations. Due to variable expression, the same mutation can lead to different clinical manifestations. However, different mutations in the same genes can also be presenting with several phenotypes. For this reason, the clinical effect of new mutations help us for better understanding the causes of the disease. It contributes to the genetic counselling.

A.C. Ceylan: None. I. Vargel: None.

P04.55C Do homozygous mutations in the upstream of Ig-like C2-type 2 domain of FGFR1 result in isolated ectrodactyly?

B. Cavdarli, V. Topcu, A. Bakir

Department of Medical Genetics, Ankara Numune Education and Research Hospital, Ankara, Turkey

Introduction: Ectrodactyly, also known as split hand/foot malformation, is seen in 1 of 8,500-25,000 newborn as isolated or part of a syndrome. There are various types of ectrodactyly that are inherited with autosomal recessive, dominant and X linked manner. Ectrodactyly is also a component of Hartsfield syndrome resulting in homozygous or heterozygous mutations in Ig-like C2-type 2 domain (conserved) of FGFR1 gene. Our purpose for presenting this work is to discuss homozygous mutations in the upstream region of FGFR1 gene conserved region may be a cause of isolated ectrodactyly.

Materials and Methods: We performed next-generation sequencing (NGS) panel (Trusight one sequencing panel) for a 2 years old female with left-hand ectrodactyly. Neuromotor and growth development of the patient were normal and no abnormality was observed in cranial MRI and echocardiography. All laboratory tests including biochemical and metabolic screenings were also normal.

Results: A homozygous mutation (c.386A>C; p.Asp129Ala) was detected with NGS panel in the upstream of Ig-like C2-type 2 domain of FGFR1 gene. p.Asp129Ala hasn’t been reported before and insilico tools predicted the mutation as deleterious. The mutation is detected as heterozygous in unaffected parents and expression analysis currently continues.

Conclusion: Hartsfield syndrome, characterized by ectrodactyly and holoprosencephaly, is the result of FGFR1 gene conserved domain mutations. Up to now, FGFR1 mutations have not been reported in patients with isolated ectrodactyly. This report is unique in terms of showing that homozygous mutations in upstream of Ig-like C2-type 2 domain of FGFR1 gene are related with isolated ectrodactyly.

B. Cavdarli: None. V. Topcu: None. A. Bakir: None.

P04.57A Further delineation of the linkeropathy syndrome due to glucuronyltransferase I-deficiency in a family with B3GAT3 compound heterozygosity for two novel mutations revealed by whole exome sequencing

M. Ritelli1, C. Dordoni1, E. Giacopuzzi1, N. Chiarelli1, V. Cinquina1, M. Venturini2, M. Colombi1

1Division of Biology and Genetics, Department of Molecular and Translational Medicine, University of Brescia, Brescia, Italy, 2Division of Dermatology, Department of Clinical and Experimental Sciences, Spedali Civili University Hospital, Brescia, Italy

Linkeropathies are a group of heterogeneous syndromes along a spectrum of skeletal and connective tissue disorders (CTDs) with the full disease range yet to be defined. Linkeropathy genes encode for enzymes branching glycosaminoglycan chains onto proteoglycans via a common tetrasaccharide linker; XYLT1 and XYLT2 encode for xylosyltransferases, B4GALT7 and B3GALT6 for galactosyltransferases, and B3GAT3 for a glucuronyltransferase. XYLT1 and XYLT2 are associated respectively with Desbuquois dysplasia type 2 and with spondylo-ocular syndrome, B4GALT7 and B3GALT6 with spondylodysplastic Ehlers-Danlos syndrome (spEDS). For B3GAT3, 23 patients, all but one from 9 consanguineous families with homozygous missense variants, have been described ranging in phenotypic severity from mild to severe and resembling Larsen-, Antley-Bixler-, Shprintzen-Goldberg-, and Geroderma osteodysplastica-like syndromes. Here, we report on a 16-year-old girl with a clinical suspicion of spEDS. She was born to non-consanguineous parents and presented with facial dysmorphism (dolichocephaly, prominent forehead, enophthalmos, midface hypoplasia, micrognathia, low-set ears), short stature, muscle hypotonia, severe kyphoscoliosis, joint laxity with recurrent dislocations, pectus carinatum, bilateral radio-ulnar synostosis, atlanto-occipital instability, and bilateral pes planovalgus. Medical history included severe low bone density, congenital hip dislocation, atrial septal defect, and anterior ectopic anus. Trio analysis by WES revealed compound heterozygosity for two novel missense mutations in B3GAT3, thus expanding its allelic repertoire. We provide a comparative overview of the phenotypic features of linkeropathies headlining the extended phenotypic range of B3GAT3 mutations that overlaps with skeletal dysplasias and other CTDs including EDS, hence offering future perspectives for EDS nosology and clinical research in this field.

M. Ritelli: None. C. Dordoni: None. E. Giacopuzzi: None. N. Chiarelli: None. V. Cinquina: None. M. Venturini: None. M. Colombi: None.

P04.58B Higher diagnostic yield can be achieved by using multigene NGS panel in testing patients with Marfan-related disorders

A. karamzade1, Z. Golchehre1, M. Keramatipour1, M. Saberi1, A. Nasrollahzadeh1, M. Arabpour1, P. Nourmohammadi2, R. Behdad3

1Tehran university of medical sciences, Tehran, Iran, Islamic Republic of, 2Pishgam Biotech Company, NGS Department,North Kargar Street, Tehran, Iran, Islamic Republic of, 3Watson Genetic Laboratory, North Kargar Street, Tehran, Iran, Islamic Republic of

Introduction: Marfan syndrome is a life threatening condition with estimated prevalence of 1:5,000-1:10,000. All cases appear to be due to heterozygous mutation in FBN1 gene. However, there are other heritable conditions with partially overlapping phenotypes caused by other genetic defects. Here, we compare the diagnostic yield of using NGS multigene panel with FBN1-only testing, in 34 patients.

Methods: Targeted NGS was applied to analyze 34 samples from individuals with clinical presentation of Marfan-related disorders. Twenty samples were analyzed for FBN1 gene only, and 14 samples were analyzed for a panel of 14 genes named as “Marfan, Aneurysm and Related Disorders”. Target regions captured with Nimblegen chip, followed by NGS on Illumina platform. Candidate variants were interpreted according to ACMG-guideline for variant interpretation 2015.

Results: In 10 samples, out of 20 samples, that were analyzed for FBN1 gene only, a strong candidate causative variant with pathogenic, likely pathogenic or VUS classification were detected. Other 10 samples end up with a negative result. On the other hand, all 14 samples tested for the panel of 14 genes, resulted strong candidate causative variants. 9 out of 14 carried a causative variant in FBN1 gene (64%), while 5 samples had possible causative variants in five other genes. Missense variants were the most common causative variants and in total 7 novel variants were also identified.

Conclusion: This study showed using NGS panel of genes associated with Marfan-related disorders, gives a higher diagnostic yield for such patients. Nearly 30% found in this study were novel variants.

A. karamzade: None. Z. Golchehre: None. M. Keramatipour: None. M. Saberi: None. A. Nasrollahzadeh: None. M. Arabpour: None. P. Nourmohammadi: None. R. Behdad: None.

P04.61A Two novel probands with Myhre syndrome identified through whole exome sequencing

I. Meerschaut1,2, A. Beyens1, W. Steyaert1, R. De Rycke3, B. Menten1, K. Bonte4, T. De Backer5, S. Janssens1, F. Malfait1, J. Panzer6, F. Plasschaert7, P. Coucke1, D. De Wolf6, B. Callewaert1

1Center for Medical Genetics, Ghent University Hospital, Ghent, Belgium, 2Department of Pediatrics, Ghent University Hospital, Ghent, Belgium, 3Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium, 4Department of Head, Neck and Maxillofacial Surgery, Ghent University Hospital, Ghent, Belgium, 5Department of Cardiology, Ghent University Hospital, Ghent, Belgium, 6Department of Pediatric Cardiology, Ghent University Hospital, Ghent, Belgium, 7Department of Orthopedic Surgery, Ghent University Hospital, Ghent, Belgium

Introduction: Myhre syndrome (MYHRS) (MIM 139210) is a rare autosomal dominant multisystem connective tissue disorder, characterized by mental retardation, cardiopathy, skeletal abnormalities, short stature, scleroderma and laryngeal stenosis. So far, all reported cases were due to de novogain-of-function missense mutations in SMAD4, encoding the SMAD protein commonly required for both transforming growth factor-beta and bone morphogenic proteins signal transduction.

Materials and Methods: We report on two adult probands with MYHRS identified through whole exome sequencing and performed transmission electron microscopy (TEM) on a skin biopsy.

Results: The first proband presented with a congenital heart defect and vertebral anomalies, but normal skin, stature and intelligence. Later-on, she developed severe laryngeal stenosis. She has two similarly affected children. The second proband presented with visual impairment following lensectomy in childhood, short stature, brachydactyly, stiff skin and decreased peripheral sensitivity. Both patients harbor the recurrent heterozygous c.1486C>T (p.Arg496Cys) mutation in SMAD4 (NM_005359). TEM of the dermis of the second proband shows dens collagen and irregular elastin cores with globular deposits and almost absent surrounding microfibrils.

Conclusions: We report on two novel probands with MYHRS. To our knowledge our data represent the first familial case of MYHRS and further widens the clinical spectrum of the disorder. TEM analysis implicates both collagen and elastic fiber anomalies and may shed novel insights on the relation between growth factor signaling and extracellular matrix homeostasis.

Grants: FWO: G028415N to PC and BC

I. Meerschaut: None. A. Beyens: None. W. Steyaert: None. R. De Rycke: None. B. Menten: None. K. Bonte: None. T. De Backer: None. S. Janssens: None. F. Malfait: None. J. Panzer: None. F. Plasschaert: None. P. Coucke: None. D. De Wolf: None. B. Callewaert: None.

P04.62B CALMPlex: a multi-gene panel for variant detection in patients with Phakomatoses and overlapping disorders

T. Giugliano1, C. Santoro2, A. Torella1,3, G. Esposito1,3, S. Perrotta2, V. Nigro1,3, G. Piluso1

1Dipartimento di Medicina di Precisione, Università degli Studi della Campania “Luigi Vanvitelli”, Napoli, Italy, 2Dipartimento della Donna, del Bambino e di Chirurgia Generale e Specialistica, Università degli Studi della Campania “Luigi Vanvitelli”, Napoli, Italy, 3TIGEM (Telethon Institute of Genetics and Medicine), Pozzuoli, Italy

Phakomatoses, characterized by pigmentary manifestations, are phenotypically overlapping disorders often difficult to distinguish only on the basis of clinic in early childhood. Nevertheless, a proper diagnosis is essential for an appropriate clinical management. We developed CALMPlex, a targeted NGS platform for molecular diagnosis of Phakomatoses. It comprises 70 disease-causing genes, including genes responsible for Phakomatoses, RNF135 and SUZ12 as NF1 flanking genes involved in 17q11 microdeletion, SPRED2 and SPRED3 as SPRED1 homologous genes and all the genes of RASopathies. It also includes 25 candidate genes identified as direct interactors of the selected disease genes with different bioinformatics tools (i.e. STRING, GeneMANIA). We enrolled 149 children (0-18 years) with clinical diagnosis of Phakomatoses. Additional 13 samples with already known mutations were used as training set to validate CALMPlex. 10/149 patients were clinically diagnosed as NF1 not confirmed by Sanger sequencing at RNA analysis. CALMPlex detected all disease-causing mutations in the training set and a NF1 mutation in 100% (10/10) of molecularly unresolved NF1 cases. In the other 139 cases, CALMPlex identified the molecular defect in 57%(80/139) of them: 77 had mutations in genes fitting the clinical diagnosis (NF1, NF2, SPRED1, PTPN11, KIT, TSC1, TSC2, LZTR1 and PPP1CB), while 3 had mutations in an unexpected gene respect to initial clinical suspicion. Pathogenic variants in candidate genes have also been detected in some of patients remained undiagnosed but a further characterization is necessary. CALMPlex is a useful first-tier test for genetic evaluation of these phenotypically overlapping conditions especially when only pigmentary manifestations occur.

T. Giugliano: None. C. Santoro: None. A. Torella: None. G. Esposito: None. S. Perrotta: None. V. Nigro: None. G. Piluso: None.

P04.63C Genetic diagnosis of bone mineralisation disorders with Next Generation Sequencing and definition of five novel pathogenic variations

H. Tozkır, S. Demir, H. Gürkan, D. Eker, E. Atli

Trakya University, Medical Faculty, Department of Medical Genetics, Edirne, Turkey

Introduction: Bone mineralisation disorders are a very heterogenious group of bone disorders both in terms of clinical manifestations and genetic background. Next Generation Sequencing (NGS) is a powerfull technology allowing analysis of a lot of genes of a number of patients simultaneusly with a low cost in a short time. We aimed to report six novel variations of bone mineralisation disorders-related genes in 6 different patients directed to our department with clinical diagnosis of abnormal bone mineralisation.

Materials and Methods: Genomic DNA samples were isolated from EDTA-blood samples of 11 patients. Libraries were prepared with Osteo-GeneSGKit DensidadOsea, IVD -CE kit according to instructions of manufacturer’s. Fastq generation was performed by MiSeq Reporter (v2.5.1; Illumina Inc.) Genomize Seq. Platform was used for variant calling and filtering. Varsome was used for in silico analysis of variants and ClinVar and HGMD databases were accepted as refference for known variant interpretation,

Results: We defined 5 novel and 4 known mutations in 9 (4 variants in COL1A1, 2 variants in COL1A2, 1 variant in PHEX, 1 variant in TGFB1 and 1 variant in SLC34A1) out of 11 patients (81.81 %).

Conclusion: We suggest that sequencing of the genes associated with bone mineralisation simultaneously with Next Generation Sequencing offers a practical approach to define genetic background of this type of heterogenious diseases.

H. Tozkır: None. S. Demir: None. H. Gürkan: None. D. Eker: None. E. Atli: None.

P04.64D Large-scale resequencing study of nsCL/P candidate genes in 1061 nsCL/P cases and 1591 controls

N. Ishorst1,2, L. Henschel1,2, F. Thieme1,2, D. Drichel3, S. Sivalingam1,2, S. L. Mehrem1,2, A. C. Fechtner1,2, A. Heimbach1,2, M. Alblas1,2, K. Keppler1,2, A. Hoischen4,5,6, K. Aldhorae7, B. Braumann8, M. Martini9, L. Gölz10, H. Reutter1,11, S. Nowak1, M. Knapp12, M. M. Nöthen1,2, M. Nothnagel3, T. Becker13, K. U. Ludwig1,2, E. Mangold1

1Institute of Human Genetics, University of Bonn, Bonn, Germany, 2Department of Genomics, Life&Brain Center, University of Bonn, Bonn, Germany, 3Cologne Center for Genomics, University of Cologne, Cologne, Germany, 4Department of Human Genetics, Radboud University Medical Center, Nijmegen, Netherlands, 5Department of Internal Medicine, Radboud University Medical Center, Nijmegen, Netherlands, 6Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, Nijmegen, Netherlands, 7Orthodontic Department, College of Dentistry, Thamar University, Thamar, Yemen, 8Department of Orthodontics, University of Cologne, Cologne, Germany, 9Department of Oral and Maxillo-Facial-Plastic Surgery, University of Bonn, Bonn, Germany, 10Department of Orthodontics, University of Bonn, Bonn, Germany, 11Department of Neonatology, Children's Hospital, University of Bonn, Bonn, Germany, 12Institute of Medical Biometry, Informatics and Epidemiology, University of Bonn, Bonn, Germany, 13Institute for Community Medicine, University of Greifswald, Greifswald, Germany

Non-syndromic cleft lip with or without cleft palate (nsCL/P) is one of the most common congenital malformations and has a multifactorial etiology. To date, a number of common risk variants have been identified for nsCL/P, explaining about 25% of the genetic heritability. We hypothesize that some of the remaining genetic liability is explained by rare dominant de novo mutations. In order to identify such rare de novo events, we performed whole exome sequencing (WES) in 50 nsCL/P patients and their unaffected parents. The analysis resulted in 33 rare de novo events in 33 genes. To find further support for these candidate genes and to strengthen our hypothesis of dominant de novo events adding to nsCL/P etiology, the candidate genes were subjected to a multiplex resequencing study with single molecule molecular inversion probes (smMIPs) in a multiethnic case/control sample of Arabian, Mexican and Central European ancestry (ncases=1,061, ncontrols=1,591). The assay was designed using the standard MIPgen pipeline and was successful for 32 genes. Libraries were sequenced on an Illumina HiSeq2500 2x125bp using Illumina v4 paired-end chemistry. Raw reads were aligned with BWA and variants were called with UnifiedGenotyper. Downstream analysis included filtering for CADD ≥ 15 and MAF ≤ 0.1% followed by a manual inspection of reads and a validation step including segregation analysis. Our preliminary results of the resequencing approach show the presence of further rare variants/rare de novo events in our candidate genes in nsCL/P patients. Further results will be presented at the conference.

N. Ishorst: None. L. Henschel: None. F. Thieme: None. D. Drichel: None. S. Sivalingam: None. S.L. Mehrem: None. A.C. Fechtner: None. A. Heimbach: None. M. Alblas: None. K. Keppler: None. A. Hoischen: None. K. Aldhorae: None. B. Braumann: None. M. Martini: None. L. Gölz: None. H. Reutter: None. S. Nowak: None. M. Knapp: None. M.M. Nöthen: None. M. Nothnagel: None. T. Becker: None. K.U. Ludwig: None. E. Mangold: None.

P04.66B Opsismodysplasia - report on long-term follow-up of a previously described and two new Portuguese cases

P. Maia Almeida1,2, H. G. Santos3, J. M. Saraiva1,4, J. Seabra5, C. Reis1, M. Venâncio1,6, K. Heath7, V. Cormier-Daire8, S. B Sousa1,6

1Medical Genetics Unit, Hospital Pediátrico, Centro Hospitalar e Universitário de Coimbra, Coimbra, Portugal, 2Faculty of Health Sciences, University of Beira Interior, Covilhã, Portugal, 3Serviço de Genética Médica, Departamento de Pediatria, Centro Hospitalar Lisboa Norte, Hospital de Santa Maria, Centro Académico de Medicina de Lisboa, Lisboa, Portugal, 4University Clinic of Pediatrics, Faculty of Medicine, University of Coimbra, Coimbra, Portugal, 5Serviço de Ortopedia Pediátrica, Hospital Pediátrico, Centro Hospitalar e Universitário de Coimbra, Coimbra, Portugal, 6University Clinic of Genetics, Faculty of Medicine, University of Coimbra, Coimbra, Portugal, 7Institute of Medical and Molecular Genetics (INGEMM), IdiPAZ and Skeletal dysplasia multidisciplinary Unit (UMDE), Hospital Universitário La Paz, Madrid, Spain and CIBERER, ISCIII, Madrid, Madrid, Spain, 8Department of Medical Genetics, INSERM U1163, Université Paris-Descartes, Institut Imagine, Hôpital Necker-Enfants Malades, Paris, France, Paris, France

Introduction: Opsismodysplasia (OPSM) is a rare autosomal recessive spondyloepimetaphyseal dysplasia caused by biallelic mutations in INPPL1 gene, which encodes SHIP2, one of the phosphatases that catalyse dephosphorylation at the 5-position of phosphoinositides. OPSMD is characterised by severe pre and post-natal micromelia with extremely short hands and feet, large fontanel, craniofacial dysmorphisms and typical radiographic features such as major delay in bone maturation, platyspondyly, squared metacarpals and metaphyseal cupping.

Case Reports: We report on three unrelated Portuguese patients, two male and one female, with the diagnosis of OPSM based on their clinical, radiographic and molecular findings. Patient 1 was the 6th case described in the literature and is now 25 years, likely one of the oldest known patients alive. From his follow-up we highlight: adult height of 1.05m; spinal surgery for severe scoliosis (4 years); severe atlantoaxial instability; bilateral cryptorchidism; mitral valve prolapse; noninvasive ventilation during sleep (since 15 years); without other significant problems. Patient 2 (5 years) has also typical features while Patient 3 (8 years) has a milder phenotype, in accordance with his genotype: compound heterozygous for c.1497+5G>C and c.1649T>C(p.Phe550Ser) in INPPL1 gene. Patients 1 and 2 are homozygous for null mutations: c.2719C>T(p.Arg907*) and c.768-769del(p.Glu258Alafs*45), respectively. Neurodevelopmental assessments showed normal cognitive function and marked motor delay.

Discussion: Once thought to be a lethal condition, OPSM turned out to have a wider phenotypic variability, well-illustrated in the description of these 3 patients. Despite the growing number of reported surviving cases, information is lacking on natural history and long-term follow-up.

P. Maia Almeida: None. H. G. Santos: None. J. M. Saraiva: None. J. Seabra: None. C. Reis: None. M. Venâncio: None. K. Heath: None. V. Cormier-Daire: None. S. B Sousa: None.

P04.67C Whole exome sequencing in Finnish families identifies new candidate genes for osteoarthritis

S. Skarp1,2, O. Kämäräinen2, G. Wei2, E. Jakkula2, I. Kiviranta3,4, H. Kröger5, J. Auvinen1, P. Lehenkari6, L. Ala-Kokko7, M. Männikkö1,2

1Center for Life Course Health Research, Faculty of Medicine, University of Oulu, Oulu, Finland, 2Faculty of Biochemistry and Molecular Medicine, University of Oulu, Oulu, Finland, 3Department of Orthopaedics and Traumatology, University of Helsinki and Helsinki University Hospital, Helsinki, Finland, 4Department of Orthopaedics and Traumatology, Jyväskylä Central Hospital, Jyväskylä, Finland, 5Department of Orthopaedics and Traumatology, Kuopio University Hospital and Kuopio Musculoskeletal Research Unit, University of Eastern Finland, Kuopio, Finland, 6Department of Anatomy and Cell biology and Surgery Clinic, Medical Research Center, University of Oulu and Oulu University Hospital, Oulu, Finland, 7Connective Tissue Gene Tests, Allentown, PA, United States

Introduction: Osteoarthritis (OA) is the most common degenerative joint disease characterized by progressive degradation of the joint cartilage. OA is a complex disease that has a strong genetic background with heritability estimations of 39% and 60% for knee and hip OA, respectively. The objective of this study was to identify rare variants predisposing subjects to OA in three Finnish families.

Materials and Methods: Eight subjects from three families with hip and knee OA were studied using whole exome sequencing. We focused on rare exonic variants with predicted pathogenicity and variants located in active promoter or strong enhancer regions. The software tool ANNOVAR was used for functional annotation and minor allele frequencies were obtained from the Exome Aggregation Consortium (ExAC) and Sequencing Initiative Suomi (SISu) database. Expression of identified genes in human bone and cartilage tissue was studied using PCR.

Results: Two rare variants co-segregated with OA in two families. In Family 8 a missense variant was observed in the OLIG3 gene that encodes a transcription factor known to be associated with rheumatoid arthritis and inflammatory polyarthritis. In Family 12 the observed variant was located in the transcription start site of the FIP1L1 gene. FIP1L1 participates in the regulation of polyadenylation. Both FIP1L1 and OLIG3 were observed to be expressed in human bone and cartilage tissues.

Conclusion: The exome sequencing revealed novel candidate genes for OA. OLIG3 and FIP1L1 may participate in the regulatory events leading to OA.

S. Skarp: None. O. Kämäräinen: None. G. Wei: None. E. Jakkula: None. I. Kiviranta: None. H. Kröger: None. J. Auvinen: None. P. Lehenkari: None. L. Ala-Kokko: None. M. Männikkö: None.

P04.68D Mutation spectrum, genotype-phenotype correlation & digenic inheritance in a large Indian cohort of osteogenesis imperfecta

G. S. Bhavani1, J. Mrosk2, H. Shah1, J. Hecht3, U. Krüger3, A. Shukla1, U. Kornak2,3,4, K. M. Girisha1

1Kasturba Medical College, Manipal, India, 2Institute of Medical Genetics and Human Genetics, Berlin, Germany, 3Berlin-Brandenburg Center for Regenerative Therapies, Berlin, Germany, 4Max Planck Institute for Molecular Genetics, Berlin, Germany

Introduction: Osteogenesis Imperfecta (OI) is a clinically and genetically heterogeneous disorder. Till date, pathogenic variants in approximately twenty genes are known to cause OI with mutations in the type 1 collagen genes (COL1A1 and COL1A2) being the most common cause.

Materials and Methods: In our study, we evaluated the clinical features of OI in 50 Indian index patients. Grouping according to phenotypic and radiographic features revealed four individuals with Bruck syndrome, three patients with hypertrophic callus and twenty with extreme bone bowing. The molecular evaluation was done by a small custom designed gene panel or exome sequencing.

Results: In all but two patients, pathogenic variants in known disease genes were detected. We observed a total of 24 novel mutations and 24 known OI mutations, of which several were recurrent. In one patient no relevant mutation was found, and another patient harboured a class III COL1A1 intronic variant. The percentage of autosomal recessive forms due to mutations in BMP1, FKBP10, LEPRE1, SERPINF1, and WNT1 was unusually high (48%). Cases with FKBP10, IFITM5, and WNT1 mutations could best be distinguished clinically. Most severe forms were due to IFITM5 and LEPRE1 mutations, followed by qualitative COL1A1, SERPINF1, and WNT1 mutations. Quantitative COL1A1 mutations and COL1A2 mutations had milder effects. In one family we found evidence for a digenic inheritance pattern due to heterozygous variants in COL1A1 and COL1A2.Conclusions: The findings in our large cohort demonstrate the clinical utility of gene panel testing for OI.

Grant:DBT-BMBF Cooperative Science Program (BT/IN/Germany-BMBF/05/GK/2015-16)

G.S. Bhavani: None. J. Mrosk: None. H. Shah: None. J. Hecht: None. U. Krüger: None. A. Shukla: None. U. Kornak: None. K.M. Girisha: None.

P04.69A Sclerostin and bone: The role of the SOST gene in osteoporosis and fragility fractures in Malta

D. Scerri, A. Xuereb Anastasi, M. M. Formosa

Department of Applied Biomedical Science, Faculty of Health Sciences, Msida, Malta

Introduction: Sclerostin is an important regulator of the bone remodelling cycle acting as an inhibitor of the canonical Wnt signalling pathway, resulting in reduced bone mass. The aim of the study was to identify known or novel SOST variants associated with osteoporosis and fracture susceptibility in the Malta Osteoporotic Fracture Study (MOFS).

Materials and Methods: Sanger sequencing of the SOST exons and their intronic flanking regions, together with the promoter and 3’untraslated region (UTR) was performed in 200 individuals having normal and low bone mineral density (BMD).

Results: A total of 10 known and 3 novel variants were identified including: rs851055, rs140960915, rs117857467, rs59613373, rs17882143, rs768384322, rs199560099, rs17883310, rs17881550, rs17886183, c.220+134(T>C), c*331(A>G) and c*390(C>A). Preliminary logistic regression with regards to the rs851055 promoter variant (G>A), indicate that homozygosity for the A allele was associated with a protective effect on BMD at the lumbar spine, LS (Age adjusted Odds ratio: 0.1 [95% confidence interval 0.03-0.8]) and total hip, TH (OR: 0.3 [0.1-1.0]). The rs17881550 3’ UTR insertion (-/G) also showed a protective effect on LS BMD (OR 0.1 [0.03-0.7]), TH (OR 0.2 [0.04-0.9]), and also with fracture risk (OR 0.2 [0.05-0.9]) in women with the homozygous mutant genotype compared to women with the homozygous wild-type genotype.

Conclusion: Observations suggest that the rs851055 and rs17881550 variants might be affecting transcriptional activation or epigenetic mechanisms resulting in altered Sclerostin function. All variants will be replicated in the entire MOFS collection to determine association with BMD and fracture susceptibility at different anatomical sites.

D. Scerri: None. A. Xuereb Anastasi: None. M.M. Formosa: None.

P04.70B A novel recurrent mutation (c.373delC) in HPGD gene is responsible for Pachydermoperiostosis

K. Bernatowicz1, A. Kashyap1, C. Wleczyk2, S. Zajączek1

1Department of Genetics and Pathology, Pomeranian Medical University, Szczecin, Poland, 2Cytogenetic Unit, Department of Laboratory Diagnostics, Pomeranian Medical University, Szczecin, Poland

Pachydermoperiostosis (primary hypertrophic osteoarthropathy, Touraine-Solente-Gole syndrome, MIM 167100) is a rare genetic disorder characterized by digital clubbing, pachydermia and periostosis. Genetic background of this disease has been recently revealed. Homozygous and compound heterozygous mutations in HPGD gene cause insufficiency of 15-hydroxyprostaglandin dehydrogenase, an enzyme responsible for PGE2 catabolism. Elevated levels of PGE2 were reported in all homozygous patients, thus leading to chronic inflammation process, most strikingly affecting joints.

We recently diagnosed two unrelated patients from non-consanguineous families, age 7 and 23, both with digital clubbing, prominent sweating of hands and feet and joints pain. In older patient pachydermia and excessive sweating was noticeable, however in younger patient the skin is not yet affected. HPGD gene analysis was performed and in both of them the same two truncating mutations were found – c.175_176delCT and c.373delC. Biparental origin of these mutations was confirmed. First mutation (c.175_176delCT) is the most common mutation of HPGD gene in Caucasian population, whereas second mutation (c.373delC) has not been reported previously.

We assume that the c.373delC truncating mutation of HPGD gene, together with the c.175_176delCT mutation, might be the founder mutations in Polish population, responsible for more cases of Pachydermoperiostosis syndrome.

K. Bernatowicz: None. A. Kashyap: None. C. Wleczyk: None. S. Zajączek: None.

P04.71C Polymorphisms in genes involved in the base excision repair (BER) pathway are associated with susceptibility to Paget´s disease of bone

C. Gutiérrez-Cerrajero1,2, R. Usategui-Martín1,2, S. Jiménez-Vázquez2, I. Calero-Paniagua1,3,4, J. García-Aparicio1,5, L. Corral-Gudino1,6, J. del Pino-Montes1,4, R. González-Sarmiento1,2,7

1Instituto De Investigación Biómedica De Salamanca (IBSAL), Salamanca, Spain, 2Unidad de Medicina Molecular, Facultad de Medicina, Universidad de Salamanca, Salamanca, Spain, 3Servicio de Reumatología, Hospital Universitario, Salamanca, Spain, 4Servicio de Medicina Interna, Hospital Virgen de la Luz, Cuenca, Spain, 5Servicio de Medicina Interna Hospital Universitario, Salamanca, Spain, 6Servicio de Medicina Interna, Hospital del Bierzo, Ponferrada, Spain, 7Laboratorio 14, Instituto de Biología Molecular Y Celular del Cáncer, Salamanca, Spain

Introduction: Paget´s disease of bone (PDB) is a chronic bone metabolic disorder. Currently, PDB is the second most frequent bone disorder. PDB is a focal disorder affecting the skeleton segmentally. Its cause is unknown, but it has been hypothesised that somatic mutations could be responsible for the mosaicism described in PDB patients. Therefore, our hypothesis is that defective response to DNA damage may lead to somatic mutations, which in turn increase the risk of PDB.

Materials and Methods: We analysed polymorphisms in DNA repair genes involved in the BER, NER and DSBR pathways in order to evaluate the role of these variants in modulating PDB risk.

Results: We found statistically significant differences in genotypic and allelic distribution for polymorphisms in genes involved in the BER pathway. Our results showed that carrying the allele T of the XRCC1 rs1799782 polymorphism and the allele G of the APEX rs1130409 polymorphism increased the risk of developing PDB.

Conclusions: These polymorphisms could cause a lower DNA repair efficiency and this might lead to local somatic mutations favouring the bone metabolic alterations characteristic of PDB. This is the first report showing an association between polymorphism in genes involved in the BER pathway and PDB.

This work was supported by a grant from Instituto de Salud Carlos III (Ministry of Economy and Competitiveness) (ISC IIII-FEDER: PI16/01920)

C. Gutiérrez-Cerrajero: None. R. Usategui-Martín: None. S. Jiménez-Vázquez: None. I. Calero-Paniagua: None. J. García-Aparicio: None. L. Corral-Gudino: None. J. del Pino-Montes: None. R. González-Sarmiento: None.

P04.72D Screening a French series of Ehlers-Danlos syndrome patients with periodontal manifestations reveals new variants in C1R and C1S and refines the clinical features of periodontal Ehlers-Danlos syndrome

A. Legrand1,2, M. Devriese1, S. Adham1, K. Benistan3, E. Schaefer4, R. Jaussaud5, M. Frank1, X. Jeunemaitre1,2, J. Albuisson1,2

1Département de génétique et Centre de Référence des Maladies Vasculaires, Hôpital Européen Georges Pompidou, Assistance Publique-Hôpitaux de Paris, Paris, France, 2INSERM, U970, Paris Centre de Recherche Cardiovasculaire, Paris, France, 3Département de Génétique, Hôpital Raymond Poincaré, Assistance Publique-Hôpitaux de Paris, Garches, France, 4Service de Génétique Médicale, Hôpitaux Universitaires de Strasbourg, Strasbourg, France, 5Département de Médecine Interne et Immunologie Clinique, CHU de Nancy-Hôpitaux de Brabois, Vandoeuvre-Lès-Nancy, France

Background : Pathogenic variants in C1R and C1S were recently discovered as a cause for periodontal Ehlers-Danlos syndrome (pEDS, previously EDS VIII). EDS are connective tissue disorders defined by major criteria: joint laxity and skin alterations. pEDS share several features with vascular EDS (vEDS), like acrogeria, gum fragility and arterial or gastrointestinal ruptures.

Methods : C1R and C1S were screened by Sanger Sequencing in a series of 20 cases with periodontal manifestations and addressed to the French National Reference Centre for vEDS (Georges Pompidou European Hospital, Paris, France).

Results : We found 3 unrelated cases harboring C1R or C1S missense variants: p.Tyr302Cys (previously reported) and p.Cys309Phe in C1R, p.Cys321Ser in C1S. The two last variants affect cysteines in CCP1 domains, like most described pathogenic variants. These affected cases had three characteristic features: early-onset periodontitis with complete tooth loss, easy bruising and pretibial hyperpigmentation. Two were sporadic cases and one had a family history of periodontitis. The negative cases had unusual periodontal manifestations and severe joint hypermobility. We are currently building a database of confirmed pEDS patients to study the role of pathogenic variants in the immune response because of recurrent infections (40% of cases).

Conclusions : We confirmed pEDS is a rare condition with a well-defined phenotype including the three features abovementioned. The clinical diagnosis should lead to genotype C1R and C1S. The pathophysiology remains to be discovered, including the impact in the immune response system.

A. Legrand: None. M. Devriese: None. S. Adham: None. K. Benistan: None. E. Schaefer: None. R. Jaussaud: None. M. Frank: None. X. Jeunemaitre: None. J. Albuisson: None.

P04.73A PLS3 deletions lead to severe spinal osteoporosis and disturbed bone matrix mineralization

A. J. Kämpe1, A. Costantini1, Y. Levy-shraga2,3, L. Zeitlin4, P. Roschger5, F. Taylan1, A. Lindstrand1,6, E. P. Paschalis5, S. Gamsjaeger5, A. Raas-Rothschild7, M. Hövel8, H. Jiao9, K. Klaushofer5, C. Grasemann10, O. Mäkitie1,6,11

1Department of Molecular Medicine and Surgery and Center for Molecular Medicine, Stockholm, Sweden, 2Pediatric Endocrinology Unit, Edmond and Lily Safra Children's Hospital, Sheba Medical Center, Tel-Hashomer, Israel, 3Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv, Israel, 4Pediatric Orthopedic Department, Dana-Dwek Children's Hospital, Sourasly Medical Center, Tel-Aviv, Israel, 5Ludwig Boltzmann Institute of Osteology at the Hanusch Hospital of WGKK and AUVA Trauma Centre Meidling, 1st Medical Department Hanusch Hospital, Vienna, Austria, 6Department of Clinical Genetics, Karolinska University Hospital, Stockholm, Sweden, 7Institute for Rare Diseases, The Danek Gertner Institute of Human Genetics, Sheba Medical Center, Tel-Hashomer, Israel, 8Department of Orthopedics and Trauma Surgery, University Hospital Essen and the University of Duisburg-Essen, Essen, Germany, 9Department of Biosciences and Nutrition, and Science for Life Laboratory, Karolinska Institutet, Stockholm, Sweden, 10Klinik für Kinderheilkunde II, University Hospital Essen and the University of Duisburg-Essen, Essen, Germany, 11Children's Hospital, University of Helsinki and Helsinki University Hospital, Helsinki, Finland

Introduction: PLS3 is one of the genes associated with X-linked osteoporosis in children and pathogenetic variants have thus far been reported in 16 families. However, our understanding of PLS3’s specific role in bone metabolism and how it exerts its effects is still poor. To study this further we investigated 3 children from 2 different families with PLS3 deletions.

Material and Methods: Family 1 consisted of 2 brothers, 11 and 7 years old, and their healthy parents. For the older brother, a transiliac bone biopsy was taken and extensively analyzed using histomorphometry, Quantitate Backscattered Electron Imaging and Raman microspectroscopy. Family 2 consisted of a 12-year-old boy, his osteoporotic mother and healthy father. Massive parallel sequencing and array-CGH was used to genetically evaluate all subjects.

Results: In both families the affected boys had childhood-onset osteoporosis with severe spinal involvement and multiple compression fractures. Both brothers in Family 1 had a deletion of exon 4-16 in PLS3, which was inherited from their mother. The index boy in Family 2 had a larger deletion, also inherited from his mother, which spanned the entire PLS3 gene. All children had a normal biochemical profile. Results from the bone tissue analyses showed a strong hypomineralization of the bone and a striking increase in osteoid volume, osteoid thickness and mineralizing lag time.

Conclusion: Our results indicate that PLS3 deletions lead to severe spinal osteoporosis and defective bone matrix mineralization, and suggest that PLS3 is directly involved in the mineralization process.

A.J. Kämpe: None. A. Costantini: None. Y. Levy-shraga: None. L. Zeitlin: None. P. Roschger: None. F. Taylan: None. A. Lindstrand: None. E.P. Paschalis: None. S. Gamsjaeger: None. A. Raas-Rothschild: None. M. Hövel: None. H. Jiao: None. K. Klaushofer: None. C. Grasemann: None. O. Mäkitie: None.

P04.74B Post axial polydactyly: isolated symptom or part of a syndrome. A case of BBS8 with two novel molecular anomalies


1CHU Liège, Liège, Belgium, 2CHL KannerKlinik, Luxembourg, Luxembourg

Background. Polydactyly can be isolated or encountered in more than 300 syndromes, mostly ciliopathies.

Methods. A young girl aged 5 was investigated for dysmorphic features, speech delay, obesity, strabismus and post axial polydactyly of the four limbs.

Results. Array CGH revealed a de novo heterozygous duplication of uncertain significance in the 13q31.3 region including the gene GPC6. A panel analysis for Bardet Biedl Syndrome (BBS) revealed a new maternally inherited pathogenic splice site mutation in intron 13 of the TTC8 gene (c.1317+1G>A) and a likely pathogenic splice site variant in the exon 5 of the TTC8 gene inherited from the father (c.459G>A (p.Thr153Thr)).

Discussion. A previous publication of a case of PAP-A2 with a similar duplication has postulated GPC6 as a candidate gene for PAPA-A2. For our patient, the additional symptoms have led to test for BBS. This analysis showed a never described mutation: a new pathogenic splice site mutation in intron 13. According to the algorithms, the variant c.1317+1G>A leads to a loss of the natural splice site leading to exon skipping, inclusion of intronic sequences or usage of a cryptic splice site.

Conclusion. We describe a girl with a BBS8 with two novel molecular anomalies: a new splice site mutation in intron 13 of TTC8 and a duplication including GPC6, a candidate gene in the pathogenesis of PAP-A2. The patient’s polydactyly is a feature of BBS, but the duplication of GPC6 has probably influenced the phenotype of the patient, with polydactyly of all four limbs.

J. Harvengt: None. U. Schierloh: None. S. Bulk: None. G. Pierquin: None.

P04.76D The inflammasome pathway is involved in PXE through IL1B upregulation in patients with a severe cardiovascular PXE phenotype

E. Y. G. De Vilder1,2,3, L. Martin4, G. Lefthériotis5, P. Coucke1, A. De Paepe1, F. Van Nieuwerburgh6, O. M. Vanakker1

1Center for Medical Genetics, Ghent University Hospital, Ghent, Belgium, 2Department of Ophthalmology, Ghent University Hospital, Ghent, Belgium, 3PhD Fellow of the Research Foundation - Flanders, Brussels, Belgium, 4Department of Dermatology, Angers University Hospital, Angers, France, 5Department of Vascular Investigations, Nice University Hospital, Nice, France, 6Department of Pharmaceutics, Laboratory of Pharmaceutical Biotechnology, Ghent University, Ghent, Belgium

Introduction: Pseudoxanthoma elasticum (PXE), an autosomal recessive ectopic mineralization disorder, causes skin, eye and cardiovascular symptoms. The disease shows striking phenotypic variability without underlying genotype-phenotype correlations. Therefore, we evaluated the collective modifying effect of rare variants on the PXE cardiovascular disease severity.

Methods: Whole Exome Sequencing and burden tests (SKAT-O and C-alpha) were performed in 12 PXE patients with an extreme cardiovascular phenotype (severe/mild). Functional validation included inflammasome stimulation with LPS/ATP in dermal fibroblasts of PXE patients with extreme cardiovascular phenotypes (4 severe/6 mild) and 2 healthy controls. Readout comprised qPCR analysis for NLRP1 and inflammasome outcome parameter IL1B.

Results: Sixteen (SKAT-O) and 74 (C-alpha) genes were identified as significant modifiers of the PXE cardiovascular disease and were enriched for 3 pathways: calcium homeostasis, vascular disease and apoptosis. One modifier, NLRP1, is linked to vascular disease and apoptosis, and was withheld for functional validation. IL1B was upregulated in dermal fibroblasts of PXE patients with a severe versus mild cardiovascular phenotype (fold change (FC) +/- 8; Mann-Whitney (MW) test: p < 0.001) and a severe phenotype versus healthy controls (FC +/- 51; MW test: p < 0.001). In addition, baseline IL1B expression was significantly higher in PXE patients versus healthy controls (FC +/- 10; MW test: p < 0.001).

Conclusion: This study provides functional validation for a predicted modifier gene in the PXE cardiovascular phenotype, and for the first time implicates inflammasome involvement in the PXE pathophysiology. This will help to direct future research on the mechanisms underlying PXE and related soft tissue calcification disorders (FWO14/ASP/084).

E.Y.G. De Vilder: None. L. Martin: None. G. Lefthériotis: None. P. Coucke: None. A. De Paepe: None. F. Van Nieuwerburgh: None. O.M. Vanakker: None.

P04.78B Association analyses of functional NCF1 variants in psoriatic arthritis and psoriasis vulgaris

U. D. Hüffmeier1, S. Löhr1, A. B. Ekici1, S. Uebe1, M. Köhm2, F. Behrens2, B. Böhm2, M. Sticherling3, G. Schett4, R. Mössner5, A. Nimeh6, G. Assmann7, J. Rech4, V. Oji8, R. Holmdahl9, H. Burkhardt2, A. Reis1

1Human Genetics, University of Erlangen, Erlangen, Germany, 2Division of Rheumatology and IME, Fraunhofer Project Group Translational Medicine and Pharmacology, Johann Wolfgang Goethe University, Frankfurt am Main, Germany, 3Department of Dermatology, University Hospital Erlangen, Erlangen, Germany, 4Department of Internal Medicine 3 - Rheumatology and Immunology, Friedrich-Alexander-Universität Erlangen-Nürnberg and Universitätsklinikum Erlangen, Erlangen, Germany, 5Department of Dermatology, Georg-August-University Göttingen, Göttingen, Germany, 6Department of Rheumatology, Fachklinik Bad Bentheim, Bad Bentheim, Germany, 7Department of Internal Medicine I, José-Carreras Centrum for Immuno- and Gene Therapy, University of Saarland Medical School, Homburg/Saar, Germany, 8Department of Dermatology, University of Münster, Münster, Germany, 9Division of Medical Inflammation Research, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden

Psoriatic arthritis (PsA) and psoriasis vulgaris (PsV) are common chronic inflammatory disorders of complex etiology. In a mouse-model, psoriasis-like skin and joint symptoms were aggravated in a NADPH oxidase deficient strain, indicating regulation by reactive oxygen species (ROS). A subunit of the human NADPH oxidase is encoded by NCF1, a gene located in a structurally complex genomic region on chromosome 7q11.23 including two homologous pseudogene copies. The considerable homology of the genomic region comprising NCF1, its pseudogenes and further genes predisposes to genomic rearrangements resulting in variability of copy number (CN). A reduced NCF1 CN and a functional missense variant (c.269G>A/p.Arg90His) in NCF1, causing a reduced ROS production, have recently been identified as strong genetic risk factors in other autoimmune diseases. Those associations combined with the animal model prompted us to analyze both variants in 1,248 PsA, 1,157 PsV patients and 932 controls. NCF1 and pseudogene CNs were determined by qPCR, the functional variant was genotyped with a nested PCR strategy. We did not observe evidence for association with the NCF1 CN nor with the functional variant in carriers of the most frequent CN ratio (4 pseudogenes : 2 gene copies) despite 97% power to detect nominally significant association with PsA or PsV with c.269G>A/p.Arg90His. The negative findings make a role of these functional NCF1 variants in psoriasis unlikely, but since oxidative burst seems to play a role in autoimmune disorders, other pathways resulting in deviant ROS production might play a role in the pathogenesis of the disease.


U.D. Hüffmeier: None. S. Löhr: None. A.B. Ekici: None. S. Uebe: None. M. Köhm: None. F. Behrens: None. B. Böhm: None. M. Sticherling: None. G. Schett: None. R. Mössner: None. A. Nimeh: None. G. Assmann: None. J. Rech: None. V. Oji: None. R. Holmdahl: None. H. Burkhardt: None. A. Reis: None.

P04.79C Evidence of common and differential genetic biomarkers for Ps and PsA

V. Caputo1, V. Errichiello1, C. Strafella1,2, A. Mazzotta3, G. Novelli1, F. Sangiuolo1, E. Campione4, R. Cascella1,5, E. Giardina1,6

1Department of Biomedicine and Prevention, “Tor Vergata” University, Rome, Italy, 2Emotest Laboratory, Pozzuoli, Italy, 3UOSD Dermatology, San Camillo Forlanini Hospital, Rome, Italy, 4Department of Systems Medicine, Division of Dermatology, University of Rome “Tor Vergata”, Rome, Italy, 5Department of Chemical Pharmaceutical and Biomolecular Technologies, Catholic University “Our Lady of Good Counsel” Laprakë, Rruga Dritan Hoxha, Tirana, Albania, 6Laboratory of Genomic Medicine UILDM, IRCCS Santa Lucia Foundation, Rome, Italy

Introduction: Collagens provide stability and resilience to extracellular matrix of connective tissues, including dermis, blood vessels and bone. Therefore, collagen genes could be involved in the etiopathogenesis of Psoriasis (Ps, OMIM#177900) and Psoriatic Arthritis (PsA, OMIM#607507), which display dysfunction and instability of the connective tissues. COL10A1 (rs3812111, A/T), COL6A5 (rs12488457, A/C), COL8A1 (rs13081855, G/T) and the miR-146a (rs2910164, G/C) were selected as potential biomarkers for Ps and PsA susceptibility.

Materials and Methods: 393 Ps, 424 PsA and 600 controls were genotyped by Real Time-PCR and subjected to biostatistic analysis using chi-square test and evaluation of ORs. The potential pathogenetic impact of the associated genes was then investigated by bioinformatic tools.

Results: rs12488457 (A/C, COL6A5), rs13081855 (G/T, COL8A1) and rs2910164 (G/C, miR-146a) were associated with both Ps [rs12488457: p = 2.97*10-9;, OR (C): 1.75, CI95%: 1.44-2.13; rs13081855: p = 0.001, OR (T): 1.79, CI95%:1.24-2.59; rs2910164: p=0.01, OR (G): 1.29, CI95%:1.04-1.61] and PsA [rs12488457: p = 1.24*10-5, OR (C): 2.46, CI95%: 2.03-2.97; rs13081855: p=9.06*10-6, OR (T): 2.17, CI95%:1.53-3.06; rs2910164: p=0.04, OR (G): 1.23 CI95%:1.0-1.51], while rs3812111 (A/T, COL10A1) showed significant association with PsA only [p = 0.008, OR (T):1.29, CI95%:1.07-1.57]. The bioinformatic analysis reported that COL6A5, COL8A1 and miR-146a may be potentially involved in alteration of the proliferation, neovascularization and inflammation pathways leading to Ps and PsA. On the other hand, COL10A1 was implicated in bone metabolism mechanisms which are dysregulated in PsA.

Conclusion: COL6A5, COL8A1, miR-146a and COL10A1 represent new susceptibility biomarkers for Ps and PsA, reflecting thereby the existence of differential mechanisms underlying the etiopathogenesis of these pathologies.

V. Caputo: None. V. Errichiello: None. C. Strafella: None. A. Mazzotta: None. G. Novelli: None. F. Sangiuolo: None. E. Campione: None. R. Cascella: None. E. Giardina: None.

P04.80D Analysis of the genetic background of psoriasis in a Hungarian cohort

A. Penyige1, E. A. Janka2, I. Sawhney2, A. Csordás2, Z. Kovács2, A. Szegedi2, P. Holló3, S. Fiatal4, R. Ádány4, D. Törőcsik2, É. Remenyik2

1Univ. of Debrecen, Faculty of Medicine, Department of Human Genetics, Debrecen, Hungary, 2Univ. of Debrecen, Faculty of Medicine, Department of Dermatology, Debrecen, Hungary, 3Semmelweis University, Department of Dermatovenereology and Dermatooncology, Budapest, Hungary, 4Univ. of Debrecen, Faculty of Public Health, Department of Preventive Medicine, Debrecen, Hungary

Introduction: Psoriasis is chronic, inflammatory disorder of the skin, often associated with arthritis and systemic co-morbidities. Previous genetic studies established that psoriasis susceptibility has strong genetic components beside environmental risk factors.

Materials and Methods: 19 SNPs in 15 candidate genes were genotyped in 782 psoriasis patients and 2000 controls and their association with psoriasis was assessed in a case-control study. Departures from HWE were tested using Fischer exact test. Allelic and genotypic association tests were performed by using either χ2 test or Fisher’s exact test, then allelic and genotypic odds ratios with 95% confidence intervals were calculated, the model was adjusted to confounding factors, too. Association of inferred haplotypes with psoriasis was assessed by a log-additive model. Pathway and network analysis was also carried out.

Results: 12 SNPs in 11 candidate genes showed significant association with psoriasis susceptibility. Six SNPs have protective effect and six confer increased risk for psoriasis revealing modest effect on phenotype. The same variants were found to be significantly associated with the early onset of psoriasis. Protecting and sensitizing haplotypes were identified on chromosomes 1, 5, 6 and 7.

Conclusion: Network analysis of disease associated genes - LCE3D, PLCL2, IL12B, TNF, TRAF3IP2, TNFAIP3, IL6, PON1, FTO and SPATA2 - identified six functional modules centered around TNF, TNFAIP3, IL12B/IL6, SPATA2, PON1 and PLCL2. These results underlie the importance of TNF and NFκB signaling pathways, cytokine production and inflammatory response in psoriasis patients and hints connections to obesity, asthma, atherosclerosis, and IBD among others.

A. Penyige: None. E.A. Janka: None. I. Sawhney: None. A. Csordás: None. Z. Kovács: None. A. Szegedi: None. P. Holló: None. S. Fiatal: None. R. Ádány: None. D. Törőcsik: None. É. Remenyik: None.

P04.81A Expanding the clinical and mutational spectrum of Roberts Syndrome with previously unreported endocrine findings

N. Guleray1, P. O. Simsek Kiper2, G. E. Utine2, K. Boduroglu2, M. Alikasifoglu1

1Department of Medical Genetics, Hacettepe University Faculty of Medicine, Ankara, Turkey, 2Department of Pediatric Genetics, Department of Pediatrics, Hacettepe University Faculty of Medicine, Ankara, Turkey

Introduction: Roberts/SC Phocomelia syndrome (MIM 268300,269000) is a rare autosomal recessive disorder characterized by limb anomalies, craniofacial dysmorphism, pre- and postnatal growth failure, developmental delay and a variety of visceral anomalies. Roberts syndrome is part of a spectrum named ‘’cohesinopathies’’ with varying phenotypic expression, and is caused by mutations in ESCO2. ESCO2 encodes a protein that establishes sister chromatid cohesion during S phase. In this study, we report on 4 new patients with previously unreported endocrine findings.

Material and Methods: Three patients clinically and radiologically diagnosed with Roberts syndrome, were screened for ESCO2 variations using Sanger sequencing analysis while one patient diagnosed with FATCO syndrome underwent whole exome sequencing.

Results: Sanger sequencing revealed 3 pathogenic mutations, including a novel mutation identified in a conserved region of ESCO2. While the patient with novel c.1433_1437delCTTTT mutation had severe intellectual disability, the other two patients with previously reported c.1131+1G>A splice donor mutation presented with generalized decreased bone mineral density and hypothyroidism, respectively. Finally in the patient with clinical findings resembling FATCO syndrome, WES analysis revealed c.879_880delAG. In this individual premature adrenarche without significant hormonal imbalance was found and this finding was attributed to the Roberts syndrome.

Conclusion: This study demonstrates that premature adrenarche and hypothyroidism may accompany Roberts syndrome. ESCO2 expression in thyroid and adrenal glands as well as the previous description of thyroid agenesis, hypothyroidism and osteoporosis in another ‘’cohesinopathy’’ Cornelia de Lange syndrome further support this observation. This study provides further evidence that patients with Roberts syndrome might be evaluated endocrinologically during their follow-up.

N. Guleray: None. P.O. Simsek Kiper: None. G.E. Utine: None. K. Boduroglu: None. M. Alikasifoglu: None.

P04.82B Skeletal abnormalities in SATB2-associated syndrome

M. Rio1, M. Mouille1, S. Breton2, J. Souberbielle3, G. Maruani3, A. Afenjar4, J. Amiel1,5, Y. Capri6, M. Fouillet7, A. Goldenberg8, C. Michot1,9, C. Mignot4, L. Perrin6, A. Putoux10, C. Quelin11, J. Van Gils12, V. Cormier Daire1,5,9

1Département de génétique, Hôpital Necker-Enfants Malades, APHP, paris, France, 2Centre de référence maladies osseuses constitutionnelles, Hôpital Necker-Enfants Malades, paris, France, 3Laboratoire d'explorations fonctionnelles, Hôpital Necker-Enfants Malades, APHP, paris, France, 4Département de génétique, Hôpital Trousseau, APHP, paris, France, 5INSERM UMR1163, Institut Imagine, Université Paris Descartes Sorbonne Paris Cité, Paris, France, 6Département de génétique, Hôpital Robert debré, APHP, paris, France, 7Service de pédiatrie, CHU Lyon, Lyon, France, 8Département de génétique, CHU Rouen, Rouen, France, 9Centre de référence maladies osseuses constitutionnelles, Hôpital Necker-Enfants Malades, Paris, France, 10Service de génétique, CHU Lyon, Lyon, France, 11Service de génétique, CHU Rennes, Rennes, France, 12service de génétique, CHU Bordeaux, Bordeaux, France

The SATB2-associated syndrome (SAS) has been recently proposed as a new clinically recognizable syndrome that results from deleterious alterations of the SATB2 gene in humans. The characteristic features are intellectual disability with absent or limited speech development, behavioral problems, craniofacial abnormalities with cleft or high palate, and dental abnormalities. About fifty patients have been reported in the literature. Skeletal abnormalities such as tibial bowing, bone fragility or osteoporosis have been reported in patients, suggesting a higher frequency of skeletal complications in SAS. The role of SATB2 in the regulation of skeletal development has recently been demonstrated. Indeed, SATB2 is a regulator of the Osx promoter, itself responsible for the differentiation of mesenchymal cells into osteoblasts. In this context, we decided to perform a non-interventional, multicentric research to understand mechanisms that lead to osteopenia in SAS patients. For all patients, we carried out a complete phosphocalcic assessment including markers of bone formation and bone resorption. We also reviewed skeletal radiographies and bone densitometry when available. We report here our data of 23 patients with SAS (21 with intragenic pathogenic variant and 2 with a microdeletion) aged from 4 to 33 years. A clinically significant fracture history was present in 10 patients. Osteopenia was present in skeletal radiographies of 10/10 patients. Low mineral density ascertained by osteodensitometry was present in 4/10 patients. We hope that ungoing results will allow better clinical management of SAS patients.

M. Rio: None. M. Mouille: None. S. Breton: None. J. Souberbielle: None. G. Maruani: None. A. Afenjar: None. J. Amiel: None. Y. Capri: None. M. Fouillet: None. A. Goldenberg: None. C. Michot: None. C. Mignot: None. L. Perrin: None. A. Putoux: None. C. Quelin: None. J. Van Gils: None. V. Cormier Daire: None.

P04.83C Identification of novel candidate genes for idiopathic short stature using whole exome sequencing

C. T. Thiel1, N. N. Hauer1, C. Vogl1, R. Ahmadian2, P. S. Dhandapany3, B. Popp1, C. Büttner1, S. Ube1, H. Sticht4, F. Ferrazzi1, A. B. Ekici1, A. de Luca5, E. Schöller1, S. Schuhmann1, K. E. Heath6, A. Hisado-Oliva6, P. Klinger7, S. Boppudi8, J. Kelkel9, A. M. Jung9, C. Kraus1, U. Trautmann1, A. Wiesener1, K. Kutsche10, A. Rauch11, D. Wieczorek12, T. Rohrer9, M. Zenker8, H. Dörr13, A. Reis1

1Institute of Human Genetics, FAU Erlangen-Nürnberg, Erlangen, Germany, 2Institute of Biochemistry and Molecular Biology II, Medical Faculty, Heinrich-Heine University, Düsseldorf, Germany, 3The Mindich Child Health and Development Institute Icahn School of Medicine at Mount Sinai, New York, NY, United States, 4Institute of Biochemistry FAU Erlangen-Nürnberg, Erlangen, Germany, 5Mendel Laboratory Casa Sollievo della Sofferenza Hospital IRCCS San Giovanni Rotondo, Rome, Italy, 6Institute of Medical and Molecular Genetics and Skeletal dysplasia Multidisciplinary Unit Hospital Universitario La Paz Universidad Autónoma de Madrid IdiPAZ and CIBERER, Madrid, Spain, 7Department of Orthopaedic Rheumatology, FAU Erlangen-Nürnberg, Erlangen, Germany, 8Institute of Human Genetics Otto‐von‐Guericke University Magdeburg, Magdeburg, Germany, 9Division of Pediatric Endocrinology Department of Pediatrics and Neonatology Saarland University Hospital, Homburg/Saar, Germany, 10Institute of Human Genetics University Medical Center Hamburg-Eppendorf, Hamburg, Germany, 11Institute of Medical Genetics University of Zurich, Zürich, Switzerland, 12Institute of Human-Genetics University Duesseldorf, Duesseldorf, Germany, 13Department of Pediatrics and Adolescent Medicine FAU Erlangen-Nürnberg, Erlangen, Germany

Short stature affects 3% of the population. We recently demonstrated that exome sequencing is able to identify disease causing mutations in known short stature genes in 19% of patients. We now performed exome sequencing in 211 clinically characterized families without mutations in known short stature genes to identify potential candidate genes. Variants were assessed for a potential effect on the gene and its product using multiple lines of evidence including expression in chondrocytes. We found 21 genes mutated in at least two patients. Six were especially strong candidates (CPZ, EDEM3, FBRS, RASA3, SLC7A8 and USP45) with mutations in at least two patients. The resulting proteins participate in protein degradation, transcriptional regulation and protein transport. One outstanding candidate gene, RASA3, is a member of the RAS-MAPK pathway. Mutations in other members of this pathway are known to cause diseases of the RASopathy complex, with short stature as a main symptom. The 2 patients carrying de novo mutations in RASA3 share some clinical characteristics with other RASopathy patients. We recently identified a third patient with a de novo mutation in RASA3. As GH treatment is discussed for some RASopathy patients, successful application in one of our patients suggested that GH might be indicated in the treatment of these patients. In conclusion, using exome analyses in patients with idiopathic short stature, we found 21 strong candidate genes in 40 patients. Thus, Exome sequencing can therefore be of great value to identify the underlying genetic cause in these individuals.

C.T. Thiel: None. N.N. Hauer: None. C. Vogl: None. R. Ahmadian: None. P.S. Dhandapany: None. B. Popp: None. C. Büttner: None. S. Ube: None. H. Sticht: None. F. Ferrazzi: None. A.B. Ekici: None. A. de Luca: None. E. Schöller: None. S. Schuhmann: None. K.E. Heath: None. A. Hisado-Oliva: None. P. Klinger: None. S. Boppudi: None. J. Kelkel: None. A.M. Jung: None. C. Kraus: None. U. Trautmann: None. A. Wiesener: None. K. Kutsche: None. A. Rauch: None. D. Wieczorek: None. T. Rohrer: None. M. Zenker: None. H. Dörr: None. A. Reis: None.


F. Fernandez1, G. Pi2, M. Carmona1, L. Pedrola1, M. Evole3, A. Zuñiga1, J. Cervera1

1Unidad de Genética. HUP La Fe., Valencia, Spain, 2Serv. Pediatria. Hospital de la Ribera., Alzira, Spain, 3Serv. Dermatología. HUP La Fe., Valencia, Spain

Introduction: Sjögren-Larsson syndrome is a neurocutaneous disease due to an inborn anomaly of lipid metabolism and characterized by congenital ichthyosis, intellectual deficit and spasticity. Half of the patients approximately are not able to walk. It is due to mutations of the ALDH3A2 gene (17p11.2), which encodes the enzyme required in the oxidation of fatty alcohols in fatty acids. Transmission is autosomal recessive. They usually live to adulthood requiring care. Neurological symptoms and intellectual deficit do not evolve after puberty. Early symptomatology suggests more serious involvement. GEN: ALDH3A2

Case report: Our index case was a 20-year-old female patient with neurological problems (seizures and spasticity) and congenital bone malformations. She presented a dry, rough, and scaly with a brownish or yellowish tone skin in the trunk and extremities, hyperpigmented hyperkeratotic plaques predominantly in flexures and abundant scalp scaling. She had a 15-year-old sister with the same symptomatology, but who also has a spastic, scissor gait, mental retardation and most severe skin lesions. A genetic study was carried out, detecting the variant c.471 + 1delG in the ALDH3A2 gene in homozygous state. This variant is described as pathological with an autosomal recessive pattern of inheritance, which confirms at the genetic level the clinical suspicion of SJOGREN-LARSSON SYNDROME. The variant detected is also present in sister of the patient with the same clinical diagnosis but with a most severe phenotype. Genetic confirmation is of great importance in these patients, due to the clinical follow-up they require, and offers the possibility of assessing genetic counseling.

F. Fernandez: None. G. Pi: None. M. Carmona: None. L. Pedrola: None. M. Evole: None. A. Zuñiga: None. J. Cervera: None.

P04.85A SHOX haploinsufficiency presenting with isolated short long bones in the second and third trimester

S. Ramachandrappa1, A. Kulkarni1, H. Gandhi2, C. Ellis3, R. Hutt4, L. Roberts4, R. Hamid5, A. Papageorghiou6, S. Mansour1

1South West Thames Regional Genetics Unit, London, United Kingdom, 2Department of Obstetrics and Gynaecology, Surrey and Sussex Healthcare NHS Trust, Surrey, United Kingdom, 3Department of Obstetrics and Gynaecology, Epsom and St Helier University Hospitals NHS Trust, Epsom, United Kingdom, 4Department of Obstetrics and Gynaecology, Royal Surrey County Hospital NHS Foundation Trust, Guildford, United Kingdom, 5Department of Obstetrics and Gynaecology, Croydon Health Services NHS Trust, Croydon, United Kingdom, 6Fetal Medicine Unit, St George’s University of London, London, United Kingdom

Haploinsufficiency of the transcription factor Short Stature Homeobox (SHOX) manifests as a spectrum of clinical phenotypes, ranging from disproportionate short stature and Madelung deformity to isolated short stature. Here, we describe five infants with molecularly confirmed diagnoses of SHOX haploinsufficiency who presented in-utero with short long bones during routine antenatal scanning from as early as 19 weeks gestation. Other fetal growth parameters were normal. The molecular basis of SHOX haploinsufficiency was distinct in each case. In four cases SHOX haploinsufficiency was inherited from a previously undiagnosed parent. In our de novo case, SHOX haploinsufficiency reflected the formation of a derivative sex chromosome during paternal meiosis. Final adult height in the SHOX deficient parents ranged from -1.9 to -1.2 SDS. All affected parents had disproportionately short limbs and two affected mothers had bilateral Madelung deformity. To our knowledge, SHOX haploinsufficiency has not previously been reported to present in-utero. Our experience illustrates that SHOX deficiency should form part of the differential diagnosis of fetal short long bones and suggests a low threshold for genetic testing. This should be particularly targeted at, but not limited to, families with a history of features suggestive of SHOX deficiency. Data on the postnatal growth of our index cases is presented which demonstrates that antenatal presentation of SHOX haploinsufficiency is not indicative of severe postnatal growth restriction. Early identification of SHOX deficiency will enable accurate genetic counselling reflecting a good postnatal outcome and facilitate optimal initiation of growth hormone therapy.

S. Ramachandrappa: None. A. Kulkarni: None. H. Gandhi: None. C. Ellis: None. R. Hutt: None. L. Roberts: None. R. Hamid: None. A. Papageorghiou: None. S. Mansour: None.

P04.86B Functional missense and splicing variants in the retinoic acid catabolizing enzyme CYP26C1 in idiopathic short stature

G. A. Rappold1, A. Montalbano1, L. Juergensen2, M. Fukami3, C. T. Thiel4, N. H. Hauer4, R. Roeth1, B. Weiss1, Y. Naiki5, T. Ogata6, D. Hassel2

1Department of Human Molecular Genetics, Institute of Human Genetics, Heidelberg University, Heidelberg, Germany, 2Department of Internal Medicine III, Cardiology, Heidelberg University, Heidelberg, Germany, 3Department of Molecular Endocrinology, National Research Institute for Child Health and Development, Tokyo, Japan, 4Institute of Human Genetics, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany, 5Division of Endocrinology and Metabolism, National Center for Child Health and Development, Tokyo, Japan, 6Department of Pediatrics, Hamamatsu University School of Medicine, Hamamatsu, Japan

Height is a complex quantitative trait with a high heritability. Short stature is diagnosed when height is significantly below the average of the general population for that person´s age and sex. We have recently found that the retinoic acid degrading enzyme CYP26C1 is a modifier for SHOX deficiency phenotypes towards more severe clinical manifestations. Here, we asked whether damaging variants in CYP26C1 alone could lead to short stature. We performed exome and Sanger sequencing to analyze 856 individuals with short stature where SHOX deficiency was previously excluded. Three different damaging missense variants and one splicing variant were identified in six independent individuals; the functional significance of the identified variants was tested in vitro or in vivo using Zebrafish as a model. The genetic and functional data reported here indicate that CYP26C1 represents a novel gene underlying growth disorders and that damaging variants in the absence of SHOX mutations can lead to short stature.

G.A. Rappold: None. A. Montalbano: None. L. Juergensen: None. M. Fukami: None. C.T. Thiel: None. N.H. Hauer: None. R. Roeth: None. B. Weiss: None. Y. Naiki: None. T. Ogata: None. D. Hassel: None.

P04.87C Clinical, demographic and nosologic characterisation of the genetic disorders of the skeleton in Turkey: The skeletal dysplasia registry

P. O. Simsek-Kiper1, G. E. Utine1, E. Z. Taskiran2, C. Kosukcu3, U. Arslan4, Y. Alanay5, M. Alikasifoglu2, K. Boduroglu1

1Hacettepe University School of Medicine Pediatric Genetics Unit, Ankara, Turkey, 2Hacettepe University School of Medicine Medical Genetics Department, Ankara, Turkey, 3Hacettepe University Institute of Health Sciences,Department of Bioinformatics, Ankara, Turkey, 4Hacettepe University Institute of Public Health, Ankara, Turkey, 5Acıbadem University Faculty of Medicine Department of Pediatric Genetics, İstanbul, Turkey

Introduction: We aimed to determine the clinical, demographic and nosologic characteristics of the genetic disorders of the skeleton in Turkey over the past 13 years (February 2005-February 2018).

Materials and Methods: “The Skeletal Dysplasia Registry” was established in 2005 at Hacettepe University Pediatric Genetics Department, a tertiary reference center for all pediatric subspecialties. The registry mainly included patients with the genetics disorders of the skeleton. Age at diagnosis, geographical region of referral, family history, parental consanguinity, clinical diagnosis, molecular diagnosis and inheritance patterns when available were reviewed respectively. The study was approved by Hacettepe University Noninterventional Clinical Research Ethics Board (Ref No: GO 17/321).

Results: The findings of a total of 884 patients were reviewed. The overall consanguinity rate in our study is 52% with some regional differences. A comparison is made in two time periods, 2005-2009 and 2009-2018. The registry has enabled scientific contributions through clinical, radiographic and molecular delineation of different clinical diagnosis including identification of founder mutations in FKBP10 in autosomal recessive OI and SFRP4 mutations in Pyle disease. Additional patients with recently identified genes including RSPRY1, EXTL3, XYLT2, EXOC6 and PGAP3 were also diagnosed and registered during this time period.

Conclusions: The present study provides further information about the clinical and nosological characteristics of the genetic disorders of the skeleton in Turkey. Despite limitations this registry still represents the first of its own kind from Turkey and around, and is a useful tool not only for the physicians but also for the families.

P.O. Simsek-Kiper: None. G.E. Utine: None. E.Z. Taskiran: None. C. Kosukcu: None. U. Arslan: None. Y. Alanay: None. M. Alikasifoglu: None. K. Boduroglu: None.

P04.88D Duplication of 10q24 locus: broadening the clinical and radiological spectrum

M. Holder-Espinasse1, A. Jamsheer2, F. Escande3, J. Andrieux3, F. Petit4, A. Sowinska-Seidler2, M. Socha2, A. Jakubiuk-Tomaszuk5, M. Gerard6, M. Matthieu-Dramard7, V. Cormier-Daire8, A. Verloes9, A. Toutain10, G. Plessis6, P. Jonveaux11, C. Baumann12, A. David13, C. Farra14, E. Colin15, S. Jacquemont16, A. Rossi17, S. Mansour18, N. Ghali19, A. Moncla20, N. Lahiri21, J. Hurst22, E. Pollina23, C. Patch1, A. Valat24, A. Mezel25, P. Bourgeot26, S. Manouvrier-Hanu4

1Clinical Genetics Department, Guy’s Hospital, London, United Kingdom, 2Department of Medical Genetics, Poznan, Poland, 3CHU Lille, Institut de Biochimie et Genetique Moleculaire, Lille, France, 4CHU Lille, Clinique de Genetique Guy Fontaine, Lille, France, 5Medical Genetics Unit, Bialystok, Poland, 6Service de Genetique, Caen, France, 7Service de Genetique, Amiens, France, 8Institut Imagine, Paris, France, 9Service de Genetique, Hopital Robert-Debre, Paris, France, 10Service de Genetique, Tours, France, 11Service de Genetique, Nancy, French Guiana, 12Service de Genetique, Hopital Robert Debre, Paris, France, 13Service de Genetique, Nantes, France, 14American University, Beirut, Lebanon, 15Service de Genetique, Angers, France, 16Department of Paediatrics, Montreal, QC, Canada, 17Laboratoire de Cytogenetique, Bois-Guillaume, France, 18Clinical Genetics Department, Saint George's Hospital, London, United Kingdom, 19North West Genetics Service, Harrow, United Kingdom, 20Laboratoire de genetique chromosomique, Marseille, France, 21Clinical Genetics Department, Saint George’s Hospital, London, United Kingdom, 22Clinical Genetics Department, Great Ormond Street Hospital, London, United Kingdom, 23Pathology Department, Queen Elizabeth Hospital, Woolwich, United Kingdom, 24CPDPN, CHU Lille, Lille, French Guiana, 25Service de chirurgie orthopedique, CHU Lille, Lille, France, 26CPDPN, CHU Lille, Lille, France

Split hand-split foot malformation (SHFM) is a rare condition that occurs in 1 in 8500-25000 newborns and accounts for 15% of all limb reduction defects. SHFM is heterogeneous and can be isolated, associated with other malformations or syndromic. The mode of inheritance is mostly autosomal dominant with incomplete penetrance, but can be X-linked or autosomal recessive. Seven loci are currently known: SHFM1 at 7q21.2q22.1 (DLX5 gene), SHFM2 at Xq26, SHFM3 at 10q24q25, SHFM4 at 3q27 (TP63 gene), SHFM5 at 2q31 and SHFM6 as a result of mutations in WNT10B (chromosome 12q13). Duplications at 17p13.3 are seen in SHFM when isolated or associated with long bone deficiency. Tandem genomic duplications at chromosome 10q24 involving at least the DACTYLIN gene are associated with SHFM3. No point mutation in any of the genes residing within the region has been identified so far, but duplication of exon 1 of the BTRC gene may explain the phenotype, with likely complex alterations of gene regulation mechanisms that would impair limb morphogenesis. We report on 32 new index cases identified by array-CGH and/or by qPCR, including some prenatal ones, leading to termination for the most severe. Twenty-three cases were presenting with SHFM and 7 with monodactyly only. Two had an overlapping phenotype. Additional findings were identified in 5 (renal dysplasia, cutis aplasia, hypogonadism and agenesis of corpus callosum with hydrocephalus). We present their clinical and radiological findings and review the literature on this rearrangement that seems to be one of the most frequent cause of SHFM.

M. Holder-Espinasse: None. A. Jamsheer: None. F. Escande: None. J. Andrieux: None. F. Petit: None. A. Sowinska-Seidler: None. M. Socha: None. A. Jakubiuk-Tomaszuk: None. M. Gerard: None. M. Matthieu-Dramard: None. V. Cormier-Daire: None. A. Verloes: None. A. Toutain: None. G. Plessis: None. P. Jonveaux: None. C. Baumann: None. A. David: None. C. Farra: None. E. Colin: None. S. Jacquemont: None. A. Rossi: None. S. Mansour: None. N. Ghali: None. A. Moncla: None. N. Lahiri: None. J. Hurst: None. E. Pollina: None. C. Patch: None. A. Valat: None. A. Mezel: None. P. Bourgeot: None. S. Manouvrier-Hanu: None.

P04.89A A Syrian patient with Steel syndrome due to compound heterozygous COL27A1 mutations with hitherto undescribed colobomas extending the clinical spectrum

L. Pölsler1, B. Simma2, U. Schatz1, J. Zschocke1, S. Rudnik1

1Division of Human Genetics; Medical University Innsbruck, Innsbruck, Austria, 2Department of Pediatrics and Adolescent Medicine; Academic Teaching Hospital LKH Feldkirch, Feldkirch, Austria

Introduction: The combination of short stature, bilateral congenital dislocation of the hip, carpal coalition, dislocation of the radial head, cavus deformity, scoliosis, and vertebral anomalies was first described in 1993 by Steel et al. (OMIM #615155) in twenty-three children from Puerto Rico. It is caused by deficient matrix protein collagen 27 alpha 1 expressed in cartilage, skin, and tendons and is inherited as an autosomal recessive trait. Causative mutations in the gene COL27A1 have been identified primarily as a possible founder effect in Puerto Ricans and in only two more families, in one of which the patient also presented hearing loss.

Here we report a girl aged 9 years born to non-consanguineous Syrian parents with characteristic features of Steel syndrome (short stature, massive malalignment of large joints, kyphoskoliosis, hearing loss) and matching facial dysmorphism (large, laterally extended palpebral fissures, arched eyebrows, flat midface, long philtrum, and short nose with low hanging columella) who also showed bilateral colobomas of the irides, retinae, and uveae with unilateral affection of the macula, which have not been described before. Her intelligence seemed normal, as was an MRI of the brain.

Results: Exome sequencing identified two novel compound heterozygous variants in COL27A1: c.93del, p.(Phe32Leufs*71) in exon 2 and c.3075del, p.(Lys1026Argfs*33) in exon 26 in this child. Her parents were confirmed heterozygous carriers.

Conclusions: Our findings extend the clinical spectrum of this exceptionally rare disorder and provide evidence of developmental defects of the eye caused by COL27A1 mutations.

Abstract ESHG 2018 Milano. Version 1.2 02.02.2018

L. Pölsler: None. B. Simma: None. U. Schatz: None. J. Zschocke: None. S. Rudnik: None.

P04.90B Distinct phenotypes within TRPV4-associated disorders in the infantile period

B. Tüysüz1, N. Güneş1, G. Yeşil2, E. Özer1, D. Uludağ-Alkaya1, D. Pehlivan3, J. Lupski3

1İstanbul University, Cerrahpasa Medical Faculty, İstanbul, Turkey, 2Bezmialem Vakif University, Department of Medical Genetics,, İstanbul, Turkey, 3Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, United States

Introduction: TRPV4 is a calcium permeable non-selective cation channel expresses in different tissues. TRPV4 mutations have been implicated in autosomal dominant diseases of skeletal and peripheral nervous system. Here, we report TRPV4 mutations in three patients with spondylometaphyseal dysplasia Kozlowski type (SMDK), metatropic dysplasia (MD) and scapuloperoneal spinal muscular atrophy (SPSMA).

Metod and Result:Patient-1 was a 3-year-old girl. She had normal height, short trunk, lumbar scoliosis, generalized severe platyspondyly, left proximal femoral metaphysis irregularity, short femoral necks and delayed carpal ossification which were compatible with SMDK. Progressive scoliosis, waiddling gait and metaphyseal irregularity of right distal ulna and radial bones were developed with aging. At 8.5 years of age her height was -2.5 SDS. Patient-2, a 2-year-old boy had torticollis (noticed in 3rd month), narrow chest with normal stature (-1.7SD), was diagnosed as MD especially shortening of long tubular bones, tail-like sacral appendage and radiological features including defective ossification of servical bodies, platyspondyly, metaphyseal flaring, and delayed epiphyseal ossification. His height is -1.7 SD at 4.5 years of age. Heterozygous TRPV4 mutations c.1781G>A and c.2396C>G were detected by Sanger sequencing in Patient 1 and 2, respectively. Patient-3, a 1-year-old boy had laryngomalacia, torticollis, hip dysplasia, muscle weakness, bilateral pes equinovarus, and scoliosis. Mild platyspondyly and acetabular irregularity were present radiologically. Using exome sequencing, we identified heterozygous novel mutation in TRPV4 (c.806G>A). The patient was diagnosed SPSMA phenotype.

Conclusion: This study showed both neuromuscular diseases and skeletal dysplasia due to TRPV4 mutation in infantile period should be kept in mind.

B. Tüysüz: None. N. Güneş: None. G. Yeşil: None. E. Özer: None. D. Uludağ-Alkaya: None. D. Pehlivan: None. J. Lupski: None.

P04.91C Transcriptome analysis of skin fibroblasts with dominant negative COL3A1 mutations provides insights into the molecular pathology of vascular Ehlers-Danlos syndrome

N. Chiarelli, G. Carini, N. Zoppi, M. Ritelli, M. Colombi

Division of Biology and Genetics, Department of Molecular and Translational Medicine, University of Brescia, Brescia, Italy

Vascular Ehlers-Danlos syndrome (vEDS) is a dominant inherited connective tissue disorder caused by mutations in the COL3A1 gene encoding type III collagen (COLLIII), the major expressed collagen in blood vessels and hollow organs. The majority of COL3A1 causative variants are glycine substitutions and in-frame splice mutations in the triple helix domain that through a dominant negative effect are associated with the severe clinical spectrum characterized by fragility of soft connective tissues with arterial and organ ruptures. Herein, we performed gene expression profiling in cultured skin fibroblasts from three patients with different structural COL3A1 mutations. Transcriptome analysis revealed significant expression changes of several genes involved in maintenance of endoplasmic reticulum (ER) homeostasis, COLLs folding, extracellular matrix (ECM) organization, proteasome complex, and cell cycle regulation. Protein analyses showed that aberrant COLLIII expression causes the disassembly of many structural ECM constituents, such as fibrillins, EMILINs, elastin, perlecan, decorin, and versican, all playing a crucial role in the vascular system. Furthermore, the altered distribution of the ER marker protein disulfide isomerase PDI and the strong reduction of the COLLs-modifying enzyme FKBP22 are consistent with the disturbance of ER-related homeostasis and COLLs post-translational modifications, indicated by microarray. Our findings provide a picture of the gene expression changes in vEDS skin fibroblasts and highlight that dominant negative mutations in COL3A1 affect maturation and deposition into the ECM of several structural proteins crucial to the integrity of soft connective tissues, and that ER dysfunction might play an important role in the etiology of this severe vascular disorder.

N. Chiarelli: None. G. Carini: None. N. Zoppi: None. M. Ritelli: None. M. Colombi: None.

P05 Cardiovascular disorders

P05.01A Genetic variants in familial abdominal aortic aneurysms

A. IJpma1,2, D. Heijsman1,3, H. T. Bruggenwirth1,3, D. Majoor-Krakauer1,3

1Erasmus MC, Rotterdam, Netherlands, 2Clinical Bioinformatics, Rotterdam, Netherlands, 3Department of Clinical Genetics, Rotterdam, Netherlands

Introduction: Abdominal Aortic Aneurysm (AAA) has a prevalence of 5% in the elderly population. An AAA occurs when the aorta below the renal arteries expands to a diameter of 3cm or more. In the Netherlands, each year approximately 5000 AAA patients are hospitalized and around 750 people die due to AAA rupture.

Hypothesis: Approximately 20% of AAA patients are familial and our hypothesis is that genetic predisposition is a significant cause for abdominal aneurysm pathology. Our goal is to identify the genes that play a role in the formation of AAA.

Methods: Our study population consists of approximately 1250 AAA patients. So far we sequenced 548 of these AAA patients. Complete Genomics whole genome sequencing (WGS) was performed in 3 families (15 individuals)and whole exome sequencing (WES) on the illumina platform using Agilent Haloplex and CRE sureselect exome capturing technology was performed in 71 families (175 individuals) and 358 single familial AAA patients. Burden analysis was used to identify genes enriched in our AAA population.

Results: We present the detailed workflow of the analysis of the genomics data. We will discuss several candidate genes identified such as the enrichment for variants we identified in the COL4A2 gene.

Conclusions: In 118 out of 512 families a variant in a diagnostic AAA gene was found. Analysis of all genes in the exome dataset led to the identification of several candidate genes that show variants in more than one AAA family and that have not been linked to AAA before.

A. IJpma: None. D. Heijsman: None. H.T. Bruggenwirth: None. D. Majoor-Krakauer: None.

P05.02B Targeted massively parallel sequencing of a representative cohort of Czech patients with various rare aortopathies demonstrates the clinical utility of genetic testing and the need for a multidisciplinary approach to at risk families

P. Votypka1, A. Krebsova2, P. Norambuena1, V. Zoubkova1, M. Vlckova1, M. Nemcikova1, M. Havlovicova1, A. Puchmajerova3, M. Balascakova1, M. Macek, Jr.1

1Department of Biology and Medical Genetics, 2nd Faculty of Medicine, Charles University and Motol, Prague, Czech Republic, 2Department of Cardiology, Institute for Clinical and Experimental Medicine, Prague, Czech Republic, 3GENNET, Prague, Czech Republic

Introduction: Aortopathies represent heterogeneous group of rare inherited disorders with a variable phenotype ranging form of aortic aneurysm/dissection with/without associated cardiac valvular disease. We studied the distribution of variants within selected candidate genes in a representative cohort of Czech paediatric-/adult patients.

Materials and Methods: Massively parallel sequencing was performed in 120 unrelated individuals (average age 42,5 years) using a custom-made panel comprising either 136 or 229 cardiac/aortic conditions-related genes (NimbleGen/Illumina). Detected variants were validated by Sanger DNA sequencing and segregation analysis. In 40 “sequencing-negative” cases CNVs in the FBN1, TGFBR1 and TGFBR2 genes were examined by MLPA (MRC-Holland).

Results: Pathogenic/likely pathogenic DNA variant (Class ≥ 4) were found in 10/120 (8.3%) cases, while VUS (Class 3) were detected in 40/120 (33.3%) patients comprising genes FBN1, NOTCH1, FBN2, MYH11 and others. Majority of pathogenic variants were observed in FBN1 (20.0%), while CNVs were not identified. Interestingly, pathogenic variants were also observed in 7/120 (5,8%) patients within aortopathy-unrelated genes conferring other cardiovascular risks.

Conclusions: As expected majority of variants were identified in connective tissue-related genes. The overall lower variant detection rate corresponds to published data. The detection of pathogenic or potentially pathogenic variants for other cardiac conditions (e.g. arrhythmias) demonstrates the diagnostic usefulness of broader gene panels. Segregation analysis together with clinical examination of positive cases increases the utility of DNA sequencing, thereby underscores the multidisciplinary character of our approach and usefulness of cooperating with compliant at risk families. Supported by 00064203, CZ.2.16/3.1.00/24022, LD14073, IGA NT13770.15-27682A and IKEM: 00023001; 9039.

P. Votypka: None. A. Krebsova: None. P. Norambuena: None. V. Zoubkova: None. M. Vlckova: None. M. Nemcikova: None. M. Havlovicova: None. A. Puchmajerova: None. M. Balascakova: None. M. Macek, Jr.: None.

P05.03C MicroRNAs in Arrhythmogenic Cardiomyopathy: from tissue-profile to circulating-signature

M. Bueno Marinas1, R. Celeghin1, M. Cason1, E. Lazzarini1, D. Paladin2, G. Thiene1, C. Basso1, K. Pilichou1

1Department of Cardiac, Thoracic and Vascular Sciences. University of Padua, Padua, Italy, 2Ditta Dr. Dino Paladin, Padua, Italy

Background: Arrhythmogenic cardiomyopathy (AC) is a clinically and genetically heterogeneous disease, characterized by progressive myocardial fibro-fatty replacement and high risk of sudden cardiac death. Half of AC patients harbor private desmosomal gene mutations. MicroRNAs (miRNA) have been associated with numerous pathophysiological conditions, as gene-expression regulatory molecules. Their role in AC is largely unknown.

Methods: The study cohort comprised 59 genotype-positive AC-subjects and 24 healthy controls (Ctrl). 84-miRNA array analysis was carried out on frozen right-ventricle myocardial tissue samples derived from 8 AC heart-transplanted patients; 9 AC-whole blood samples, and 6 Ctrls. miRNA validation was performed by qPCR (ΔΔCt method) on 42-AC and 18-Ctrl.

Results: miRNA profiling on AC-tissue samples displayed a genotype-related profile showing 19 differentially expressed miRNAs in PKP2 carriers, 15 in DSP carriers and 14 in DSG2 carriers. A common signature was identified between PKP2 and DSP carriers (PKP2/DSP profile), different from DSG2 profile. In silico target prediction of both profiles marked Hippo Signaling Pathway (p-value 1.6e-9 and 6.4e-6). Analysis of AC-tissue sample-data as a unique group confirmed 26 miRNAs (AC-tissue profile) with predicted targets in the AC pathway (p-value 0.01). AC-blood miRNA profiling showed a 14-miRNA signature, of which 10 miRNAs were found also in AC-tissue profile.

Conclusions: A genotype-related miRNA profile was observed on AC-tissue samples, as to reflect clinical variability. In addition, 10 miRNAs in common were identified between AC-tissue and AC-blood profiles, demonstrating a specific AC-miRNA signature. In silico analysis highlighted pathways involved in AC pathogenesis demonstrating a key role of miRNAs in AC.

M. Bueno Marinas: None. R. Celeghin: None. M. Cason: None. E. Lazzarini: None. D. Paladin: None. G. Thiene: None. C. Basso: None. K. Pilichou: None.

P05.04D Differential gene expression analysis in Arrhythmogenic Cardiomyopathy

M. Cason1, M. Bueno Marinas1, R. Celeghin1, E. Lazzarini1, S. Rizzo1, K. Ludwig1, C. R. Bezzina2, C. A. Remme2, B. Bauce1, G. Thiene1, C. Basso1, K. Pilichou1

1Department of Cardiac, Thoracic and Vascular Sciences, University of Padua, Padua, Italy, 2Department of Clinical and Experimental Cardiology, University of Amsterdam, Amsterdam, Netherlands

Background. Arrhythmogenic cardiomyopathy (AC) is an inherited myocardial disease characterized by fibro-fatty replacement of the myocardium and life-threatening arrhythmias caused mainly by low penetrant mutations in desmosome-encoding genes. The molecular mechanism underlying disease pathogenesis is still unclear. To determine molecular pathways underlying disease onset, gene expression profiling was performed on AC patients and transgenic mice with a desmoglein-2 (dsg2) mutation.

Methods. RNA-Seq was carried out, separately, on the right (RV) and the left (LV) ventricle of 9 AC heart-transplanted patients carrying pathogenic desmosomal mutations paired with 6 age-matched nondiseased donors. RNA-Seq was carried out also on 8 transgenic mice over-expressing NS-dsg2 mutation (TgNS) at 2 different age-groups (<2weeks and >3weeks), to deduct genetic/epigenetic interference-factors and on a paired age-matched control group comprised 6 over-expressing wild type dsg2 (TgWt) and 2Wt mice.

Results. 1136 and 822 differentially expressed genes (DEGs) were respectively identified in the RV and LV of AC patients. Further, 143 DEGs were identified comparing TgNS<2weeks and TgNS>3weeks gene expression profiling. Finally, 82 DEGs were shared comparing human and murine (TgNS>3weeks) expression-profiling among which genes most linked to the suppression of the canonical WNT/β-catenin and the activation of TGF-β signaling pathways. However, 29 DEGs were identified in TgNS<2weeks comparing them to age-matched controls (WT<2weeks and TgWt < 2weeks).

Conclusions. Transcriptome profiling enabled the identification of the culprit molecule aiding cell-cell contact detachment under stress conditions and wound healing repair through suppression of the WNT/β-catenin signalling pathway.

M. Cason: None. M. Bueno Marinas: None. R. Celeghin: None. E. Lazzarini: None. S. Rizzo: None. K. Ludwig: None. C.R. Bezzina: None. C.A. Remme: None. B. Bauce: None. G. Thiene: None. C. Basso: None. K. Pilichou: None.

P05.06B The effect of the expression level of tissue inhibitor of metalloproteinase-3 on the development of atherosclerosis in patients with myocardial infarction

G. Celebi1, F. Guclu-Geyik1, D. Ozsoy2, C. Yildiz2, M. Yildiz3, D. Oksen3, E. Komurcu-Bayrak1

1Aziz Sancar Institute of Experimental Medicine,Istanbul University, Istanbul, Turkey, 2Institute of Cardiology,Istanbul University, Istanbul, Turkey, 3Anesthesiology and Reanimation,Istanbul University Cardiology Institute, Istanbul, Turkey

TIMP3, a member of the Tissue Inhibitors of Metalloproteinases (TIMPs) binds to components of the extracellular matrix and forms insoluble complex. TIMP3 has a crucial regulatory role in adipose tissue. Our aim is to reveal the relationship between TIMP3 expression and atherosclerosis pathogenicity in peri-coronary epicardial adipose tissue (EAT) and circulating leukocytes of patients with myocardial infarction (MI). Methodologically, the expression levels of TIMP3 were investigated in patients with MI (n = 46) who had atherosclerosis severity determined by SYNTAX and GENSINI scores that compared with the control group (n = 24). The expression levels of TIMP3 in the leukocytes (n = 69) and peri-coronary EAT (n = 23) obtained from coronary artery bypass graft surgery using qRT-PCR. The expression results were analyzed using the comparative CT method, and results were statistically evaluated. Previously unpublished findings showed that TIMP3 expression levels were significantly lower in post-mortem advanced atherosclerotic plaques than in normal arteries (p < 0.05) and that TIMP-3 protein was present in plaque enriched with macrophages of the coronary artery sections. In this study, it was determined that expressions of TIMP3 increased 1.25 fold in peri-coronary EAT and decreased 4.5 fold in circulating leukocytes as compared to control samples (p > 0.05). The data were evaluated in detail according to conventional risk factors. In conclusion, TIMP3 expression levels in leukocytes and fat tissue affecting the development of atherosclerosis were evaluated in patients with MI. These analyses are still ongoing in more patient samples. This study was supported by Scientific Research Projects Coordination Unit of Istanbul University (Project numbers:28473 and 21496)

G. Celebi: None. F. Guclu-Geyik: None. D. Ozsoy: None. C. Yildiz: None. M. Yildiz: None. D. Oksen: None. E. Komurcu-Bayrak: None.

P05.07C Identification of novel loci for heart rate response to exercise and recovery

J. Ramírez1, S. van Duijvenboden2, I. Ntalla1, B. Mifsud1, H. R. Warren1, E. Tzanis1, M. Orini3, A. Tinker1, P. D. Lambiase2, P. B. Munroe1

1William Harvey Research Institute, London, United Kingdom, 2Institute of Cardiovascular Science, University College London, London, United Kingdom, 3Barts Heart Centre, St Bartholomews Hospital, London, United Kingdom

Introduction: Reduced heart rate (HR) responses to exercise (ΔHRex) and to recovery (ΔHRrec) are associated with higher cardiovascular mortality rates, possibly due to abnormalities in autonomic balance. We aimed to discover single-nucleotide polymorphisms associated with both indices and to identify associated biological pathways.

Methods: A total of 67,257 participants in an exercise test from the UK Biobank study were included. We calculated differences in HR at peak exercise (ΔHRex) and at 1-minute post-peak exercise (ΔHRrec) with respect to resting HR. Next, we randomly divided participants into discovery (N = 40,000) and replication (N = 27,257) cohorts and we performed a genome-wide association study (GWAS) for each trait in the discovery dataset and validated the findings in the replication cohort. We finally conducted a combined meta-analysis of GWAS using the full cohort for both traits.

Results: We robustly validated six and eight independent SNPs for ΔHRex and ΔHRrec, respectively. The combined analysis revealed a further eight and seven SNPs for each respective trait that were genome-wide significant (P < 5x10-8). In total, 14 and 15 SNPs were identified for ΔHRex and ΔHRrec, respectively, with eight SNPs being common across traits. Bioinformatics analyses highlighted neural development and adrenergic modulation by the autonomic nervous system pathways.

Conclusion: Our results demonstrate that ΔHRex and ΔHRrec are genetically modulated. Our biological findings support the potential link between genetics and autonomic modulation and highlight several plausible candidate genes for both traits. Future studies will confirm the contributions of our identified genetic variants to cardiovascular risk.

J. Ramírez: None. S. van Duijvenboden: None. I. Ntalla: None. B. Mifsud: None. H.R. Warren: None. E. Tzanis: None. M. Orini: None. A. Tinker: None. P.D. Lambiase: None. P.B. Munroe: None.

P05.08D Gene expression signature in human immortalized venous endothelial cells, human coronary artery and human internal thoracic artery endothelial cells exposed to different types of mineral-organic nanoparticles

M. Sinitsky1,2, E. Velikanova1, D. Shishkova1, M. Asanov1, A. Ponasenko1, A. Kutikhin1

1Research Institute for Complex Issues of Cardiovascular Diseases, Kemerovo, Russian Federation, 2Federal Research Center of Coal and Coal Chemistry, Kemerovo, Russian Federation

Introduction:Mineral-organic nanoparticles (bions) are a result of increasing concentration of precipitating ions or failure in the mechanism of their excretion from human body. It was found that the atherosclerosis and heart valves calcification risk factors are similar to ones of bions formation. This indicates that bions play role in the pathogenesis of cardiovascular calcification.

Materials and Methods: Confluent cultures of human immortalized venous endothelial cells (EA.hy 926), human coronary artery (HCAEC) and human internal thoracic artery endothelial cells (HITAEC) exposed to different types of bions (magnesium phosphate bions [MFB], spherical calcium phosphate bions [SCFB], and needle-like calcium phosphate bions [ICPB]) were used in this study. Expression of LDLR, VLDLR, SCARF1, NOS3, PXDN genes was evaluated by RT-qPCR. Results were normalized by three housekeeping genes (ACTB, GAPDH, B2M). Expression level was calculated by Pfaffl method.

Results:2-fold decreasing of SCARF1 expression was detected in EA.hy 926 culture exposed to SCFB. In HCAEC culture we found no significant differences in gene expression signature. In HITAEC culture the exposure by all types of bions caused an increase in VLDLR gene expression. HITAEC cultures exposed by all types of mineral-organic nanoparticles are characterized by enhanced expression of all studied genes compared to HCAEC.

Conclusions: Exposure by mineral-organic nanoparticles can lead to changing in gene expression signature in different types of endothelial cells depending on type of bions. HITAEC are more sensitive to such exposure. The reported study was funded by Russian Foundation for Basic Research according to the research project № 17-04-00570.

M. Sinitsky: None. E. Velikanova: None. D. Shishkova: None. M. Asanov: None. A. Ponasenko: None. A. Kutikhin: None.

P05.09A Assessment of CNVs in dilated cardiomyopathies by whole exome sequencing

L. Piherova1, F. Majer1, A. Krebsova2, P. Melenovska1, V. Stranecky1, T. Palecek3, M. Kubanek2, S. Kmoch1

1Research Unit for Rare Disease, Charles University, Prague, Czech Republic, 2Department of Cardiology, Institute of Clinical and Experimental Medicine, Prague, Czech Republic, 32nd Internal Clinic, Charles University, Prague, Czech Republic

Genetic testing for inherited cardiomyopathies has improved in the last decade using next generation sequencing (NGS). Although CNV of large segments of DNA may significantly affect transcription and translation of cardiomyopathic genes, these abnormalities remain often unrecognized even with the use of NGS.

We have performed whole exome sequencing in a cohort of 323 patients with dilated cardiomyopathy. We used a read-depth coverage strategy to call CNVs from the short-read sequence data. Detected CNVs were then validated by q-PCR, RT-PCR and western blot analysis. The prevalence of major CNVs in the cohort was 2%. We have found large deletions in DMD, LAMP2, FLNC and MYH7 genes, which were predicted to cause major structural and functional abnormalities of the affected genes. The corresponding pedigrees and clinical phenotypes will be presented. Assessment of CNVs with whole exome sequencing elucidates genetic architecture in a substantial proportion of patients with dilated cardiomyopathy. It should be a routine part of NGS bioinformatics. Supported by the research grant: AZV-MZ 15-27682A and IKEM 00023001.

L. Piherova: None. F. Majer: None. A. Krebsova: None. P. Melenovska: None. V. Stranecky: None. T. Palecek: None. M. Kubanek: None. S. Kmoch: None.

P05.10B Rare variants in cardiomyopathy related genes - a Portuguese cohort population

A. M. Coutinho1, J. Tavares1, M. Carmo-Fonseca2, D. Antunes1,3

1GenoMed - Diagnósticos de Medicina Molecular SA, Lisbon, Portugal, 2Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Lisbon, Portugal, 3Medical Genetics Department, Hospital de Dona Estefânia, Centro Hospitalar Lisboa Central, Lisbon, Portugal

Introduction: With the latter advances in high throughput sequencing technologies there was an increasing demand for broader comprehensive Next-Generation Sequencing (NGS) gene panels to screen for mutations in genetic diseases with heterogeneous etiology. In cardiology, genetic testing has been routinely offered to patients to improve prognosis through appropriate lifestyle and medical interventions. There are now 340 gene entries retrieved under “cardiomyopathy” search in OMIM and 375 genes under the HPO superclass “Abnormal myocardium morphology” (HP:0001637).

Methods: The study population comprised 150 unrelated patients diagnosed with cardiomyopathies, namely Hypertrophic Cardiomyopathy (HCM), Dilated Cardiomyopathy (DCM) and Arrhythmogenic Right Ventricular Cardiomyopathy (ARVC). Up to 57 genes associated with cardiomyopathies were sequenced, mostly by NGS.

Results: Pathogenic variants were detected in 39 patients and in further 50 patients it was found an uncertain significance/likely pathogenic variant, namely novel variants in 17 patients (11.3%). One novel variant was detected in two unrelated DCM patients in the LMNA gene - c.490G>A, p.(Asp164Asn), suggesting an increased frequency in the Portuguese population.

Conclusions: Although genetic analysis through comprehensive multigene NGS approach potentially increases diagnostic rate, it also raises new challenges. Rare benign specific population polymorphisms add extra difficulty regarding pathogenicity classification of variants. Further characterization studies of specific populations could improve classification variants algorithm, preferably within public databases with the possibility to identify variants by nationality.

A.M. Coutinho: None. J. Tavares: None. M. Carmo-Fonseca: None. D. Antunes: None.

P05.11C Identification of gene mutations in pediatric cardiomyopathy by whole exome sequencing

K. Rojnueangnit1, B. Sirichongkolthong1, R. Wongwandee1, T. Khetkham1, S. Noojarern2, A. Khongkraparn2, D. Wattanasirichaigoon2

1Thammasat University, Klong-Luang, Thailand, 2Ramathibodi Hospital, Mahidol University, Bangkok, Thailand

Introduction: Primary cardiomyopathy in children is rare but serious condition with a high mortality rate. Hypertrophic and dilated cardiomyopathies are the most common presentations. Etiology has been mainly idiopathic; however, with the use of next generation sequencing techniques, it has been noted that up to nearly half of idiopathic pediatric cases arose from a specific genetic mutation. Therefore, this study aimed to identify genetic causes of primary unexplained cardiomyopathy.

Materials and Methods: Children with primary unexplained cardiomyopathy, ranging from newborns to 20-year olds, were recruited during March 2016 to May 2017 at Thammasat University Hospital. Complete patient history and physical examination data were collected by a geneticist; cardiac examination and echocardiogram was performed by pediatric cardiologists. Whole exome sequencing was performed in all cases.

Results: Fourteen patients were enrolled in our study: 8 cases were dilated type, and 6 were hypertrophic. Two were excluded given identification causes during period of follow-up (hypocalcemia and pace-maker induced dilated cardiomyopathy). 118 gene lists for cardiomyopathy were analyzed in 12 included cases. In cases of syndromic cardiomyopathy, specific genes were added to aid the analysis, but none was detected. Pathogenic and likely pathogenic mutations were identified in 5 patients: SOS1, HRAS, TTN, FLNC and TXNRD2.

Conclusion: Our cohort demonstrated that 41% of our cases were not actually idiopathic. In light of this revelation and despite its high cost, genetic testing is also useful in determining genetic risk in the family as well as helping to predict the prognosis of the cardiomyopathy.

K. Rojnueangnit: None. B. Sirichongkolthong: None. R. Wongwandee: None. T. Khetkham: None. S. Noojarern: None. A. Khongkraparn: None. D. Wattanasirichaigoon: None.

P05.13A Targeted Next-Generation Sequencing in Patients with Non-syndromic and syndromic Congenital Heart Disease

A. Chirita-Emandi1, N. Andreescu1, R. Jurcut2, G. Doros3, A. Popoiu3, A. Lacatusu4, A. Dobrescu5, M. Puiu1

1Center of Genomic Medicine, University of Medicine and Pharmacy “Victor Babes” Timisoara, Timisoara, Romania, 2Institute of Emergency for Cardiovascular Diseases "Prof.dr.C.C.Iliescu", University of Medicine and Pharmacy "Carol Davila" Bucharest, Bucharest, Romania, 3Clinical Emergency Hospital for Children “Louis Turcanu” Timisoara, University of Medicine and Pharmacy “Victor Babes” Timisoara, Timisoara, Romania, 4Emergency Clinical County Hospital, Timisoara. Department of Pediatrics, University of Medicine and Pharmacy “Victor Babes” Timisoara, Timisoara, Romania, 5Department of Genetics, University of Medicine and Pharmacy of Craiova, Craiova, Romania

Congenital heart diseases are genetically heterogeneous. Targeted next-generation sequencing (NGS) can identify the genetic causes in a significant proportion of the population.

Aim: We tested a targeted NGS specific gene panel in patients, adults and children, with syndromic and non-syndromic cardiac involvement.

Results: The patient cohort included 77 patients(52 males;28.4  ±  22.6years), 41 people with hypertrophic/dilated cardiomyopathy, 3 with arrhythmogenic cardiomyopathy, and 33 with syndromic cardiac involvement (including RASopathies and fibrillinopathies). Amplicon libraries for 174 related genes were generated using the TruSightCardio®kit(Illumina,CA) and sequenced using the Illumina MiSeq platform. Sequence data were processed using ANNOVAR software. Thirty-six variants (30 missense, 4 frameshift, 2 splice-site variants) were considered pathogenic or likely pathogenic and 10 were variants of unknown significance (VUS) in MYH7;MYBPC3;MYL2; MYO6;MYPN;ACTC1;BAG3;CSRP3;KCNH2;DSC2;CACNA1C;FBN1;PTPN1;SOS1;BRAF;LPL;ABCC9 and APOE genes. Twenty-five variants present in public databases, with very rare allele frequency, have been previously linked to cardiomyopathy or relevant syndromic phenotypes. Twenty-four were novel mutations, currently not found in public databases, of which 9 were classified as VUS. Two patients carried pathogenic variants for two diseases (heterozygotes in different genes), with possibly synergistic deleterious effects.

Conclusion:First-line targeted genetic NGS testing identified a significant variant (pathogenic or likely pathogenic) for the phenotype, in 51,5%(17/33) of the cases with syndromic cardiac involvement, and in 40.9%(18/44) of the cases with non-syndromic cardiomyopathies, providing the opportunity for diagnostic, risk stratification and prevention, along with genetic counselling.

Funding: Development of Existing Infrastructure and Creation of New Infrastructure POSCCE-A2-O2.2.1-2013-1, Center of Genomic Medicine of the University of Medicine and Pharmacy ‘Victor Babes’ Timisoara.

A. Chirita-Emandi: None. N. Andreescu: None. R. Jurcut: None. G. Doros: None. A. Popoiu: None. A. Lacatusu: None. A. Dobrescu: None. M. Puiu: None.

P05.14B The association of genes of fibrogenesis to the development of cardiovascular continuum comorbidity

I. A. Goncharova1,2, M. S. Nazarenko1,2, T. B. Pecherina2, V. V. Kashtalap2, N. B. Tarasenko1, O. L. Barbarash2, V. P. Puzyrev1

1Scientific Research Institute of Medical Genetics, Tomsk, Russian Federation, 2Research Institute for Complex Problems of Cardiovascular Diseases, Kemerovo, Russian Federation

The aim of this study was to assess the genetic structure of cardiovascular continuum comorbidity. The study included 531 patients with myocardial infarction (MI) and 285 Russian inhabitants of Siberia. Рatients were divided into groups: 113 MI patients without risk factors (аrterial hypertension (HT), dyslipidemia (DLE), type 2 diabetes mellitus (T2D)); 146 MI patients with HT; 96 MI patients with HT and HDL; 96 MI patients with HT, HDL and T2D designated as “syntropy of cardiovascular continuum”. Genotyping of 58 SNPs was performed on Sequenom MassARRAY (USA). These SNPs are localized in the genes involved in fibrogenesis and/or associated with cardiovascular diseases and atherosclerotic plaque stability. Statistical data analysis was performed in the software environment R. Isolate MI showed the association with genes of various biological pathways: immune response CD79A (rs3810153), IFNGR1 (rs17181457), endothelial dysfunction KIAA1462(rs3739998), reparations LIG1(rs20579), fibrogenesis (ADAMDEC1(rs3765124). In groups with risk factors, it was found that: MI and HT associations with genes involved in fibrogenesis ITGA4(rs1143674), ITGB5(rs6778643, rs1007856), ADAMDEC1(rs3765124), CDKN2BAS1(rs1333049) and immune response IFNGR1 (rs17181457); MI, HT and DLE associations with genes involved in fibrogenesis (ITGA4(rs1143674), ITGB5(rs6778643), ADAMDEC1(rs3765124), CDKN2BAS1(rs1333049)), immune response IFNGR1 (rs17181457), homeostasis of glucose and low-density lipoproteins (TAS 2R38(rs1726866), LDLR(rs2738446)). “Syntropy of cardiovascular continuum“ associations with genes involved in fibrogenesis (CDKN2BAS1(rs1333049), (MTAP(rs7023329)), endothelial dysfunction KIAA1462(rs3739998), lipid metabolism APOA2(rs5082). In conclusion, isolate MI and MI with risk factors had different genetic susceptibility profiles. Cardiovascular continuum comorbidity was characterized by genes involved in various biological processes. The study was conducted with the support of the RFBR (№16-04-00840\16).

I.A. Goncharova: None. M.S. Nazarenko: None. T.B. Pecherina: None. V.V. Kashtalap: None. N.B. Tarasenko: None. O.L. Barbarash: None. V.P. Puzyrev: None.

P05.15C KRIT1 loss of function induces a chronic Nrf2-mediated adaptive homeostasis that sensitizes cells to oxidative stress: implication for Cerebral Cavernous Malformation disease

E. Trapani1, C. Antognelli2, S. Delle Monache3, A. Perrelli1, C. Fornelli1, V. Benedetti1, S. Sarri1, G. Costantino1, F. Geddo1, A. Zotta1, L. Goitre1, S. F. Retta1

1Department of Clinical and Biological Sciences, Orbassano, TO, Italy, 2Department of Experimental Medicine, Perugia, Italy, 3Department of Biotechnological and Applied Clinical Sciences, L'Aquila, Italy

KRIT1 (CCM1) is a disease gene responsible for Cerebral Cavernous Malformations (CCM), a major cerebrovascular disease of proven genetic origin affecting 0.3-0.5% of the population. Previously, we demonstrated that KRIT1 loss-of-function is associated with altered redox homeostasis, suggesting a novel pathogenic mechanism for CCM disease and raising the possibility that KRIT1 loss exerts pleiotropic effects on multiple redox-sensitive mechanisms. To address this possibility, we investigated major redox-sensitive pathways and enzymatic systems that play critical roles in fundamental cytoprotective mechanisms of adaptive responses to oxidative stress, including the master Nrf2 antioxidant defense pathway and its downstream target Glyoxalase 1 (Glo1), a pivotal stress-responsive defense enzyme involved in cellular protection against glycative and oxidative stress through the metabolism of methylglyoxal (MG). Experimental outcomes showed that KRIT1 loss-of-function induces a redox-sensitive sustained upregulation of Nrf2 and Glo1, and a drop in intracellular levels of major apoptosis-protective proteins, including MG-modified heat shock protein 70 (Hsp70) and 27 (Hsp27), leading to a chronic adaptive redox homeostasis that counteracts intrinsic oxidative stress but increases susceptibility to oxidative DNA damage and apoptosis. While supporting and extending the pleiotropic functions of KRIT1, these findings shed new light on the mechanistic relationship between KRIT1 loss-of-function and enhanced cell sensitivity to oxidative stress, thus providing valuable new insights into CCM pathogenesis and novel options for the development of preventive and therapeutic strategies.

E. Trapani: None. C. Antognelli: None. S. Delle Monache: None. A. Perrelli: None. C. Fornelli: None. V. Benedetti: None. S. Sarri: None. G. Costantino: None. F. Geddo: None. A. Zotta: None. L. Goitre: None. S.F. Retta: None.

P05.16D Quantification of DNA copy number variations in patients with coronary artery disease by digital droplet PCR

A. A. Sleptsov1, M. S. Nazarenko1,2,3, N. R. Valiakhmetov1, A. N. Kazantsev2, O. L. Barbarash2, V. P. Puzyrev1

1Research Institute of Medical Genetics, Tomsk NRMC, Tomsk, Russian Federation, 2Institute for complex issues of cardiovascular diseases, Kemerovo, Russian Federation, 3Siberian State Medical University, Tomsk, Russian Federation

Introduction: Recently we performed the genome-wide analysis of CNVs in patients with coronary artery disease (CAD) by using array-CGH. We found 90 CNVs, and 13% of them were novel. The aim of this study was to determine the frequencies of the several candidate CNVs (SFMBT1, PRKRA, and SIRPB1).

Materials and Methods: We have extracted DNA specimens from white blood cells of 100 patients with CAD, 100 patients with both CAD and diabetes mellitus 2 type (DM2), and 130 persons without any clinical and laboratory features of atherosclerosis, at the same ages. Digital droplet PCR (QX200) along with TaqMan Assays was used to identify CNVs.

Results: We detected the loss in the 3p21.1 region (SFMBT1) in 11.48% patients and 8.5% in control group. However, patients with CAD and DM2 had more frequently loss of SFMBT1 (16%) than patients with CAD (8%). The frequency of gain 2q31.2 (PRKRA) was 6% in patients with CAD whereas in control group we identified it in one person only. The frequency of loss 20p13 (SIRPB1) was 67% in both groups.

Conclusion: It is likely that the identified CNV loss in the 3p21.1 region (SFMBT1) plays a certain role in the risk of developing CAD and DM2 (p = 0.006). At the same time, we detected a trend towards increased frequency of the gain in the 2q31.2 region (PRKRA) in patients with CAD.

A.A. Sleptsov: None. M.S. Nazarenko: None. N.R. Valiakhmetov: None. A.N. Kazantsev: None. O.L. Barbarash: None. V.P. Puzyrev: None.

P05.18B Multivariate analysis of comorbidities in congenital heart disease: Making sense of phenotypic heterogeneity

R. Cabrera1, M. Miranda-Fernández1, V. Huertas-Quiñones1, G. Alberto1, N. Sandoval1, C. M. Restrepo2, P. Laissue2, C. Silva2, K. Moreno Medina1, R. Dennis Verano1

1Fundacion Cardioinfantil - Instituto de Cardiología, Bogota, Colombia, 2Universidad de la Rosario, Bogota, Colombia

Introduction: The clinical presentation of congenital heart disease (CHD) is often accompanied by diverse comorbidities within and outside the cardiovascular system. There is significant heterogeneity in the presence of comorbidities and this can make genetic diagnosis and identification of new CHD-associated genes challenging, especially for rare forms of CHD, where cohorts are usually small. This is evident in low diagnostic yields of standard criteria for suspicion of 22q11-deletion syndrome (22q11DS), and limited identification of genes associated with Ebstein Anomaly (EA).

Materials and Methods: Taking advantage of a concentration of cases of rare forms of CHD in a single center in Colombia, unusually large cohorts of patients with suspicion of 22q11DS and EA were assembled. A comorbidity database was constructed and multivariate statistical analysis was carried out to identify correlations between comorbidities in different subgroups in each cohort.

Results: The data show phenotypic heterogeneity in CHD can mask the existence of identifiable subgroups which can be used to improve diagnosis and identify genetic variants in CHD. In patients meeting criteria for 22q11.2DS, multivariate analysis of comorbidities can improve the specificity of clinical evaluations without sacrificing sensitivity. In patients with EA, multivariate analysis revealed a distinct homogenous subgroup with a likely distinct genetic etiology, presenting with pre-excitation arrhythmias with reduced outflow tract obstruction and improved survival.

Conclusions: Multivariate analysis can reveal clinically relevant patterns in comorbidity datasets of phenotypically heterogeneously disease cohorts. This approach can be used in the identification of novel genetic causes and the improvement of clinical diagnostic criteria.

R. Cabrera: None. M. Miranda-Fernández: None. V. Huertas-Quiñones: None. G. Alberto: None. N. Sandoval: None. C.M. Restrepo: None. P. Laissue: None. C. Silva: None. K. Moreno Medina: None. R. Dennis Verano: None.

P05.19C Integration of large-scale genomic data sources to identify novel genetic loci for congenital heart disease

E. Fotiou, S. Williams, D. Page, K. Hentges, B. Keavney

Division of Cardiovascular Science, Manchester, United Kingdom

Background: Small nucleotide variants and copy number variants (CNV) have been found to affect congenital heart disease (CHD) risk. Yet, the identification of the genetic causes of CHD remains quite challenging.

Purpose: To integrate data on both classes of variation associated with non-syndromic CHD cases as a better means for identification of candidate genes predisposing to CHD.

Methods: Here, we have updated a previously published CHD case CNV list and generated a control CNV list using: a) DECIPHER database, b) ISCA database, c) ECARUCA database, d) 1000 Genome phase 3 dataset, e) DGV database and f) published literature.

Results: Analysing deleted (del) and duplicated (dup) CNVs independently resulted in unique case CNV regions not present in the controls. Further filtering led to the identification of 54 novel candidate protein-coding genes in del CNVs present only in non-syndromic CHD cases and with high/medium impact variants in exome data from our cohort of Tetralogy of Fallot patients. Moreover, we have identified 50 genes in those unique case CNV regions that were previously shown to be associated with CHD such as GATA4 for atria septal defects and NKX2-6 for conotruncal heart malformations.

Conclusions: We demonstrate a promising new strategy with the integration of large-scale genomic data sources to identify novel candidate genes for CHD and their contribution in heart development. We are currently performing functional work with our strongest candidate gene for which there is evidence that knockout mouse has ventricular septal defects and hypoplastic aortic valve.

Grant reference: British Heart Foundation

E. Fotiou: None. S. Williams: None. D. Page: None. K. Hentges: None. B. Keavney: None.

P05.20D Molecular autopsy reveals clues for genetic basis of congenital valve defect

F. A. R. Madia1, A. T. Dias2, É. A. Zanardo2, J. G. Damasceno2, A. M. Nascimento1, T. V. M. M. Costa2, S. N. Chehimi1, G. M. Novo-Filho2, M. M. Montenegro2, Y. G. Oliveira2, A. B. Freitas2, L. L. Vieira2, R. Schultz3, F. T. Gonçalves4, C. Fridman4, C. A. Kim5, L. D. Kulikowski1,2

1Laboratorio de Citogenomica, Departamento de Pediatria, Faculdade de Medicina FMUSP, Universidade de Sao Paulo, Sao Paulo, Brazil, 2Laboratorio de Citogenomica, Departamento de Patologia, Faculdade de Medicina FMUSP, Universidade de Sao Paulo, Sao Paulo, Brazil, 3Servico de Anatomia Patologica, Departamento de Patologia, Faculdade de Medicina FMUSP, Universidade de Sao Paulo, Sao Paulo, Brazil, 4Departamento de Medicina Legal, Etica Medica e Medicina Social e do Trabalho, Faculdade de Medicina FMUSP, Universidade de Sao Paulo, Sao Paulo, Brazil, 5Unidade de Genetica, Departamento de Pediatria, Instituto da Crianca, Hospital das Clinicas HCFMUSP, Universidade de Sao Paulo, Sao Paulo, Brazil

Introduction: Congenital heart defect (CHD) consists in a large set of functional and structural anomalies that arise during the cardiac embryogenesis including septal defects, valve defects, and outflow tract anomalies. Valve defect is an important cause of mortality and morbidity. However, the genetic basis of congenital valve defect is unclear.

Materials and Methods: We investigate the contribution of genomic alterations in the valve defect`s pathogenesis using molecular methods in 18 cases postmortem of stillbirth and infant from Serviço de Verificação de Óbitos, HC-FMUSP. DNA samples from skin, diaphragm, and heart tissues were evaluated using AmpFℓSTR® MiniFiler™ PCR Amplification Kit (Life Technologies™, California, USA) and Multiplex Ligation-dependent Probe Amplification (MLPA) with different kits (MCR-Holland, Amsterdam, the Netherlands).

Results: In 8 out of 18 stillbirth and infant (44,4%) show alterations in the genome, including trisomy 18 (5 cases), trisomy 21 (2 cases) and duplication of 4p16 (1 case). The tricuspid valve defect was reported in all syndromes describe above. Besides that, the mitral valve defect was associated with deletion of 4p16, and abnormalities of aortic and pulmonary valve was associated with trisomy 18.

Conclusion: Molecular autopsy is a significant tool for the characterization of the basis of cardiac valve defect and also become vital for an accurate genetic counseling.

Grants: FAPESP: 09/53105-9 and FINEP-CT INFRA 0160/12 SP8.

F.A.R. Madia: None. A.T. Dias: None. É.A. Zanardo: None. J.G. Damasceno: None. A.M. Nascimento: None. T.V.M.M. Costa: None. S.N. Chehimi: None. G.M. Novo-Filho: None. M.M. Montenegro: None. Y.G. Oliveira: None. A.B. Freitas: None. L.L. Vieira: None. R. Schultz: None. F.T. Gonçalves: None. C. Fridman: None. C.A. Kim: None. L.D. Kulikowski: None.

P05.21A Somatic DNA methylation and copy number landscape of coronary artery disease patients

M. S. Nazarenko1,2,3, A. V. Markov1, A. A. Sleptcov1, A. N. Kazancev3, O. L. Barbarash3, V. P. Puzyrev1

1Research Institute of Medical Genetics, Tomsk, Russian Federation, 2Siberian State Medical University, Tomsk, Russian Federation, 3Research Institute for Complex Problems of Cardiovascular Diseases, Kemerovo, Russian Federation

DNA methylation and copy number variations (CNVs) of affected and healthy vascular tissues of patients with atherosclerosis had not been investigated in detail. The Illumina HumanMethylation27 BeadChip and Agilent SurePrintG3 HumanCGH+SNP 2×400K microarrays were used for DNA testing from right coronary arteries in the area of advanced atherosclerotic plaques and atherosclerotic-resistant internal mammary arteries of six patients with coronary artery disease. The atherosclerotic plaques to compare with the healthy arteries were characterized by predominate DNA hypermethylation changes. These genes were annotated with muscle system process and positive regulation of cytosolic calcium ion concentration in Gene Ontology terms. In contrast, hypomethylated genes encode molecules belonging to different biological processes such as development, immune/inflammation responses, lipid storage, and programmed cell death. In atherosclerotic plaques the most pronounced hypomethylation was registered in 2q31.1 (HOXD4/HOXD3/MIR10B), 7p15.2 (HOXA7) and 11p11.2 (ALX4). Moreover, methylation changes at 2q31.1 in blood cells were consistently associated with smoke and ischemic stroke. We identified 90 high-confidence CNVs that were present in matched arteries studied. Gene Ontology analysis revealed enrichments in the immune/inflammation responses and olfactory transduction. Furthermore, two patients contained the gain in 10q24.31 (ERLIN1) that affected only the blood DNA but not arteries. There was not overlap between both DNA methylation and copy number changes in arteries. In conclusion, although DNA methylation differences do not appear to be linked to the copy number changes in arteries of patients with coronary heart disease both mechanisms may be important in the disease.

M.S. Nazarenko: None. A.V. Markov: None. A.A. Sleptcov: None. A.N. Kazancev: None. O.L. Barbarash: None. V.P. Puzyrev: None.

P05.22B Monogenic and complex risk factors for ischemic heart disease in Finland - Elucidating the role of severe hypercholesterolemia

N. Junna1, P. Ripatti1, I. Surakka1,2, S. Ripatti1,3, E. Widén1

1FIMM, Institute for Molecular Medicine, University of Helsinki, Helsinki, Finland, 2Department of Internal Medicine, University of Michigan, Ann Arbor, MI, United States, 3Department of Public Health, Clinicum, Faculty of Medicine, University of Helsinki, Helsinki, Finland

Severe hypercholesterolemia is one of the most significant risk factors for coronary artery disease (CAD), and it has a strong and complex genetic predisposition. Currently, not even patients with inherited forms of severe hypercholesterolemia are adequately identified in Finland, and only a part of their mutation load is known.

To unravel the underlying genetic architecture, we screened 206 individuals with severe hypercholesterolemia (LDL-cholesterol >= 5mmol/l) in the prospective GeneRISK study, encompassing 7,328 subjects, aged 45-64 years, from Southern Finland. Exome-sequencing identified a novel, likely causal mutation in LDLR (R574L), but surprisingly we didn’t identify any of the hypercholesterolemia-associated LDLR-mutations previously shown to be enriched in Finland. Neither did we identify potentially causal mutations in APOB or PCSK9. Polygenic modeling (106 LDL-cholesterol-associated SNPs) explained 17% of the LDL-cholesterol variation in the cohort but suggested only slight clustering of polygenes with severe hypercholesterolemia.

Evaluating the use of lipid-lowering medication in the GeneRISK-cohort, we found that only 4.3% of individuals with severe hypercholesterolemia were on lipid-lowering treatment at baseline. This is considerably less than the 10.1% that were treated in the full cohort. Our preliminary data further show that at follow-up 1.5 years later, lipid-lowering medication had been initiated only to an additional 3.3% of the individuals with severe hypercholesterolemia. Given that patients with hypercholesterolemia are poorly identified and treated, there is an unmet need not only to further characterize causal variants underlying severe hypercholesterolemia but also to raise awareness among caretakers to take action to reduce the disease burden.

N. Junna: None. P. Ripatti: None. I. Surakka: None. S. Ripatti: None. E. Widén: None.

P05.23C Polygenic hyperlipidemias and coronary artery disease risk

P. Ripatti1, J. T. Rämö1, S. Söderlund2,3, I. Surakka1,4, A. S. Havulinna1,5, N. B. Freimer6, V. Salomaa5, A. Palotie1,7,8, M. Taskinen2,9, S. Ripatti1,10

1Institute for Molecular Medicine Finland, Helsinki, Finland, 2Research Programs Unit, Diabetes & Obesity, University of Helsinki, Helsinki, Finland, 3Department of Internal Medicine, Helsinki University Hospital, Helsinki, Finland, 4Department of Internal Medicine, University of Michigan, Ann Arbor, MI, United States, 5Department of Public Health Solutions, National Institute for Health and Welfare, Helsinki, Finland, 6Center for Neurobehavioral Genetics, Semel Institute for Neuroscience and Human Behavior, University of California, Los Angeles, CA, United States, 7The Stanley Center for Psychiatric Research, The Broad Institute of MIT and Harvard, Cambridge, MA, United States, 8Psychiatric & Neurodevelopmental Genetics Unit, Massachusetts General Hospital, Boston, MA, United States, 9Clinical Research Institute HUCH, Ltd., Helsinki, Finland, 10Department of Public Health, Clinicum, Faculty of Medicine, University of Helsinki, Helsinki, Finland

Hyperlipidemia, particularly increased LDL-cholesterol (LDL-C) or triglycerides (TGs), is a treatable risk factor for coronary artery disease (CAD). In addition to high-penetrant mutations in genes like LDLR, APOB and PCSK9, hyperlipidemias can be a consequence of polygenic burden. Whereas monogenic familial hypercholesterolemia (FH) increases CAD risk considerably due to high lifelong exposure to LDL-C, it is unclear whether a high polygenic load of LDL-C or TG-increasing variants increases CAD risk.

In this prospective cohort study, we constructed incident CAD events (n=970) from the genotyped FINRISK population cohort (n=20499) linked with healthcare registries. We tested if high (>90th percentile) polygenic scores for LDL-C or TG increased CAD risk.

Finnish LDLR FH mutations increased LDL-C 3.5 mmol/l on average, and CAD risk considerably (HR 3.67 [1.18-11.43]). In contrast, high LDL-C score increased LDL-C 0.8 mmol/l and CAD risk only marginally (HR 1.22 [1.00-1.49]).

No monogenic large-effect TG-increasing variants were present. High TG score, however, increased TG 0.6 mmol/l on average, and CAD risk significantly (HR 1.26 [1.04-1.54]). Comparing hypertriglyceridemic individuals (TG >2.5 mmol/l) to individuals with TG <1.7 mmol/l, those with high TG score had higher CAD risk (HR 2.11 [1.58-2.81]) than those without high TG score (HR 1.61 [1.33-1.94]) despite comparable TG levels (p=0.022). Only 5.2% of the hypertriglyceridemic individuals received fibrate treatment.

The CAD risk associated with a high polygenic load of LDL-C or TG-increasing variants depends directly on their effect on lipid levels. The clinical utility of polygenic scores needs further study.

P. Ripatti: None. J.T. Rämö: None. S. Söderlund: None. I. Surakka: None. A.S. Havulinna: None. N.B. Freimer: None. V. Salomaa: None. A. Palotie: F. Consultant/Advisory Board; Modest; Pfizer Genetics Scientific Advisory Panel. M. Taskinen: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Modest; Amgen, Novo Nordisk, Merck, Sharpe & Dohme. Other; Modest; Amgen, Novo Nordisk, Sanofi Aventis, Chiesi, Astra Zeneca, Pfizer. S. Ripatti: None.

P05.24D Refinement of coronary artery disease risk assessment by genomic information

J. Partanen1, P. Ripatti1, N. Mars1, P. Pöllänen2, K. Hotakainen3,4, J. Partanen5, E. Widén1, S. Ripatti1,6

1Institute for Molecular Medicine Finland, University of Helsinki, Helsinki, Finland, 2Carea – Kymenlaakso Social and Health Services, Kotka, Finland, 3Department of Clinical Chemistry and Hematology, Medicum, Faculty of Medicine, University of Helsinki, Helsinki, Finland, 4Mehiläinen Oy, Helsinki, Finland, 5Finnish Red Cross Blood Service, Helsinki, Finland, 6Department of Public Health, Clinicum, Faculty of Medicine, University of Helsinki, Helsinki, Finland

Introduction: Current cardiovascular disease (CVD) risk assessment relies on clinical risk scores, which fail to identify a large proportion of individuals who develop CVD. Novel biomarkers may complement such scores. Genomic information has improved risk estimation in several prospective cohort studies and is therefore the most promising candidate to enhance clinical risk assessment.

Materials and Methods: We established a novel cohort of 7439 coronary artery disease (CAD)-free 45-65-year-old individuals from southern Finland. We estimated their CAD risk using 1) an established national clinical risk score, and 2) the clinical score combined with a genetic risk score (GRS) for CAD. We returned both estimates to the participants. We aimed to 1) evaluate the CAD risk spectrum of the cohort, 2) assess how combining the GRS to the clinical score refines risk estimation, and 3) identify high-risk individuals with actionable clinical characteristics.

Results: Genotyped participants numbered 5125. Those with high CAD risk (10-year risk ≥10%) increased from 406 (7.9%) to 452 (8.8%) with the combined score; 113 were reclassified to a lower risk category and 159 to the high-risk category. The combined score refined CAD risk clinically meaningfully in 950 (18.5%) participants. Of the 452 high-risk participants based on the combined score, 197 (44%) smoked, 165 (37%) were obese, 80 (18%) had diabetes, 310 (69%) had LDL-C >3 mmol/l, 98 (22%) used statins and 205 (45%) used antihypertensives.

Conclusions: Incorporating the CAD GRS to the clinical risk score considerably refined CAD risk estimation. High-risk individuals presented multiple risk factors and intervention opportunities.

J. Partanen: None. P. Ripatti: None. N. Mars: None. P. Pöllänen: None. K. Hotakainen: A. Employment (full or part-time); Significant; Mehiläinen Oy. J. Partanen: None. E. Widén: None. S. Ripatti: None.

P05.25A A novel candidate frameshift mutation for Catecholaminergic Polymorphic Ventricular Tachycardia

F. B. Isik, M. D. Sozuguzel, N. Genc, E. F. Caralan, Z. Dogru, C. Akdeniz, V. Tuzcu, H. Cangul

Medipol University, istanbul, Turkey

Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT) is a cardiac disorder which characterized by arrhythmias, sudden cardiac arrest after physical activity or emotional stress which could result from CASQ2 gene mutations which follow autosomal recessive pattern. CASQ2 encodes the protein Calsequestrin-2 which is a high-capacity calcium-binding protein acting as an internal calcium store in muscle. Therefore, it plays a pivotal role in excitation-contraction coupling which regulates the rate of heart beats. In this study, we identified a novel mutation on CASQ2 (NM_001232.3) gene in two, non-related patients with overlapping symptoms such as repetitive syncope and polymorphic ventricular tachycardia. Molecular analysis of the 2 patients’ by next generation sequencing showed a homozygous splice variant p.L79X (c.237delT) which is not currently present in clinical databases. We evaluated this novel variant as a likely pathogenic variant because of its potential nonsense effect. In vivo functional studies at transcriptional and translational level are underway to demonstrate the causative role of this mutation in pathogenesis of CPVT.

F.B. Isik: None. M.D. Sozuguzel: None. N. Genc: None. E.F. Caralan: None. Z. Dogru: None. C. Akdeniz: None. V. Tuzcu: None. H. Cangul: None.

P05.27C Variation in the LPL gene, low density lipoprotein-cholesterol lowering alleles and risk of coronary disease and type 2 diabetes

L. A. Lotta1, I. Stewart1, S. Sharp1, F. Day1, S. Burgess1, J. Luan1, L. Cai1, L. Wittemans1, N. Kerrison1, K. Khaw1, M. McCarthy2, S. O'Rahilly1, R. Scott1, D. Savage1, J. Perry1, C. Langenberg1, N. Wareham1

1University of Cambridge, Cambridge, United Kingdom, 2University of Oxford, Oxford, United Kingdom

Background: Several pharmacological enhancers of lipoprotein lipase (LPL) are in preclinical/early-clinical development for dyslipidemia, but it is unknown if they will reduce cardio-metabolic disease risk when added to existing lipid-lowering drugs. Human genetics can be used to study if genetic-differences in LPL-mediated lipolysis contribute to these disease independent of pathways targeted by existing drugs.

Methods: Using individual-level genetic data from 390,470 individuals and a “factorial” design, we investigated the independent and combined consequences on coronary disease and type 2 diabetes risk of triglyceride-lowering LPL-alleles and LDL-C-lowering alleles at different genes, including those encoding targets of current LDL-C-lowering therapy (HMGCR, NPC1L1 and PCSK9).

Results: People carrying a higher than median load of both triglyceride-lowering LPL-alleles and LDL-C lowering alleles had an odds ratio (OR) for coronary disease of 0.73 compared to people below the median of both exposures (95% confidence interval [CI], 0.70 to 0.76; p = 2.8×10-52), which was a greater effect than that observed in people with higher than median load of either exposure. Triglyceride-lowering LPL-alleles were strongly associated with lower diabetes risk (odds ratio per standard deviation genetically-lower triglycerides, 0.69; 95% CI, 0.62 to 0.76; p = 2.6×10-13). In factorial analyses, this protective association neutralized the association of LDL-C lowering alleles with a higher diabetes risk (pheterogeneity in effect estimates = 0.0094).

Conclusions:Triglyceride-lowering LPL-alleles and LDL-C-lowering genetic mechanisms have independent contributions to a lower risk of coronary disease. These findings provide human genetics evidence to support the development of agents that enhance LPL-mediated lipolysis for use in the context of LDL-C-lowering therapy.

L.A. Lotta: None. I. Stewart: None. S. Sharp: None. F. Day: None. S. Burgess: None. J. Luan: None. L. Cai: None. L. Wittemans: None. N. Kerrison: None. K. Khaw: None. M. McCarthy: Other; Significant; Grants from Eli Lilly, Roche, AstraZeneca, Merck, Janssen, Servier, Novo Nordisk, Sanofi-Aventis, Boehringer Ingelheim, Pfizer, and Takeda; and honoraria from Novo Nordisk and Pfzier. S. O'Rahilly: Other; Significant; Personal fees from Pfizer, AstraZeneca, iMed, and ERX Pharmaceuticals for serving on advisory boards and scientific panels. R. Scott: A. Employment (full or part-time); Significant; Full-time employment with GSK.. D. Savage: None. J. Perry: None. C. Langenberg: None. N. Wareham: None.

P05.28D Digenic mutations of MYH7 and RYR2 in siblings manifesting with severe cardiac dysfunction

M. Nagasaka1,2, M. Taniguchi-Ikeda1,3,4, H. Inagaki4, I. Morioka1, H. Kurahashi4, K. Iijima1

1Department of Pediatrics, Kobe University Graduate School of Medicine, Kobe, Japan, 2Department of Pediatrics and Neonatology,Takatsuki General Hospital, Takatsuki, Japan, 3Division of Genetic Counseling, Kobe University Hospital, Kobe, Japan, 4Division of Molecular Genetics, Institute for Comprehensive Medical Science, Fujita Health University, Toyoake, Japan

Introduction: Mutations in the Myosin heavy chain 7 (MYH7) are associated with inherited cardiomyopathies. On the other hand, mutations in the Ryanodine receptor 2 (RYR2) are associated with fatal arrhythmias such as catecholaminergic polymorphic ventricular tachycardia. Both of them exhibit autosomal dominant patterns of inheritance.

Case Report: The proband was born at 34 weeks of gestation with a birth weight of 2452g. He presented with severe cardiac dysfunction at birth and needed strict intensive care. At 3 years-old, he had intractable epilepsy and severe neurodevelopmental impairment. An elder brother who was born at 39 weeks of gestation died shortly after birth because of cardiac failure. There were strong family history of sudden cardiac death in the father’s trait. The mother manifested with heart failure after the delivery of the proband and presented with tachycardia during treadmill stress test. Whole-exome sequencing identified two variants in the siblings, a heterozygous missense variant in MYH7 (c.728G>A) inherited from the father, and in RYR2 (c.5428G>C), inherited from the mother. The variant in MYH7 was reported as a pathogenic allele. The variant in RYR2 was considered likely to be pathogenic since PHRD-like scaled Combined Annotation-Dependent Depletion (CADD) score suggested deleteriousness (25.9).

Conclusions: We report sibling cases manifesting with severe cardiac dysfunction possibly due to digenic MYH7 and RYR2 mutations. Digenic mutations may cause severer clinical manifestation than that caused by each mutation.

M. Nagasaka: None. M. Taniguchi-Ikeda: None. H. Inagaki: None. I. Morioka: None. H. Kurahashi: None. K. Iijima: None.

P05.29A Increasing the number of genes in the genetic screening of dilated cardiomyopathy: is more actually more?

E. Vanhoutte, J. Verdonschot, G. Claes, A. van den Wijngaard, A. Helderman-van den Enden, M. Hazebroek, S. Heymans, H. Brunner, I. Krapels

Maastricht University Medical Center, Maastricht, Netherlands

Background: Dilated cardiomyopathy (DCM) is characterized by systolic dysfunction with dilatation of the left ventricle and/or right ventricle. The Dutch Society for Clinical Genetic Laboratory Diagnostics (VKGL) previously recommended genetic testing in DCM patients using a core panel of 47 cardiomyopathy-associated genes (including TTN, MYH7, PLN and LMNA). However, DCM is a complex multifactorial disorder involving multiple genes. Other genes are probably involved in the pathogenesis. Therefore, a panel containing 347 genes involved in cardiac disorders was composed.

Aims: To determine the yield of genetic variants in gene panel testing involving 347 heart related genes in 100 genetically unsolved DCM patients in a Dutch population.

Methods: 100 DCM patients without (likely) pathogenic mutations in the 47 core cardiomyopathy genes were offered genetic analysis through whole exome sequencing using a targeted filter consisting of 347 heart related genes.

Results: In 51 patients, genetic analysis of the 347-heart gene panel has been completed. Variants of unknown significance, likely pathogenic or pathogenic variants were detected in 15 (29%), 3 (6%) and 0 (0%) patients respectively. So far, likely pathogenic variants were identified in ANK2, KCNQ1 and FLNC. The gene with the highest yield is FLNC. Mutations in FLNC are associated with myopathy and cardiomyopathies. ANK2 and KCNQ1 are associated with inherited primary arrhythmias.

Conclusions: It is likely that FLNC has a major role in the pathogenesis of DCM. Therefore, FLNC should be part of the core panel of cardiomyopathy genes. Furthermore, variants in primary arrhythmia genes might explain a small number of patients.

E. Vanhoutte: None. J. Verdonschot: None. G. Claes: None. A. van den Wijngaard: None. A. Helderman-van den Enden: None. M. Hazebroek: None. S. Heymans: None. H. Brunner: None. I. Krapels: None.

P05.30B A targeted 18 gene panel approach detects a likely genetic cause in 25% of Scottish Dilated Cardiomyopathy patients

J. Dean1, E. Cleary2, C. McWilliam3, R. McGowan4, W. Lam2, D. O'Sullivan1, S. Tennant1

1NHS Grampian, Aberdeen, United Kingdom, 2NHS Lothian, Edinburgh, United Kingdom, 3NHS Tayside, Dundee, United Kingdom, 4NHS Greater Glasgow, Glasgow, United Kingdom

Dilated cardiomyopathy (DCM) is aetiologically heterogeneous, and up to 35% of cases are found to be familial after investigation of first degree relatives. Amongst those with genetic DCM, truncating mutations in TTN are thought to be the most common cause, being found in around 30% of familial cases in most series. In Scotland, patients referred for genetic investigation of dilated cardiomyopathy are tested using 2 gene panels, one with 13 genes encoding components of the sarcomere including TTN, and the other including 5 genes also associated with cardiac disorders with an arrhythmia phenotype (SCN5A, ABCC9, PLN, DES and LMNA). The 5 gene arrhythmia panel came into use in 2017. We report an audit of the outcome of this approach. In 2017, 106 patients with DCM were referred for genetic testing in Scotland (population ~5.4 million). Only 25% had a genetic variant likely to cause DCM, 8% being associated with truncating mutations in TTN. The majority (17%) had variants in other cardiac genes (including 5% in TNNT2, 4% in ABCC9 and 2% in MYH7, other genes contributed a smaller number to the total). This real world data from the Scottish Laboratory Consortium Molecular Diagnostic service suggests that in Scotland, TTN accounts for fewer cases than might be expected from the literature. A 25% overall detection rate nevertheless suggests that targeted gene panel testing using genes for which good genotype-phenotype data is available remains a useful way to investigate for inherited DCM.

J. Dean: None. E. Cleary: None. C. McWilliam: None. R. McGowan: None. W. Lam: None. D. O'Sullivan: None. S. Tennant: None.

P05.31C Mortality Risk associated with Truncating Founder Mutations in Titin

M. Jansen1, A. F. Baas1, K. Y. van Spaendonck-Zwarts2, A. S. Ummels1, A. van den Wijngaard3, J. D. H. Jongbloed4, M. A. van Slegtenhorst5, R. H. Lekanne Deprez6, M. W. Wessels5, M. Michels7, A. C. Houweling8, E. T. Hoorntje9, P. J. T. M. Helderman-van den Enden3, D. Q. M. C. Barge-Schaapveld10, J. van Tintelen6, M. P. van den Berg4, A. A. M. Wilde6, H. Ploos van Amstel1, E. A. M. Hennekam1, F. W. Asselbergs1, E. J. G. Sijbrands5, D. Dooijes1

1University Medical Center Utrecht, University Utrecht, Utrecht, Netherlands, 2Academic Medical Centre, Amsterdam, Netherlands, 3Maastricht University Medical Centre, Maastricht, Netherlands, 4University Medical Centre Groningen, Groningen, Netherlands, 5Erasmus Medical Center, Rotterdam, Netherlands, 6Academic Medical Center, Amsterdam, Netherlands, 7Erasmus Medical Centre, Rotterdam, Netherlands, 8VU University Medical Centre, Amsterdam, Netherlands, 9University Medical Centre Groningen, University of Groningen, Groningen, Netherlands, 10Leiden University Medical Centre, Leiden, Netherlands

Background: Truncating Titin variants (TTNtv) are a major cause of dilated cardiomyopathy (DCM), a major cause of heart failure. TTNtv are also frequently present in the general population and associated with a variable phenotype. The prognosis of asymptomatic TTNtv carriers is poorly understood. Objectives: To assess the clinical relevance of TTNtv by analysing TTNtv associated all-cause mortality in historical pedigrees and in present-day patients.

Methods: Haplotype analysis identified five recurrent A-band TTNtv as founder mutations. Three multigenerational pedigrees were traced back to 18th century ancestors. Using the Family Tree Mortality Ratio method, standardized mortality ratios (SMR, standardized for sex, age and calendar-period) were calculated. Similarly, SMR was calculated in parents of 128 present-day DCM patients with a TTNtv using the reverse parent-offspring method. Subgroups were compared with Poisson regression.

Results: In 20,522 person-years, overall mortality was not significantly increased in three historical pedigrees compared to the general population (SMR 1.06, 95% CI 0.95-1.18, p = 0.162). However, mortality was significantly increased in subjects living after 1965 (SMR 1.27, 95% CI 1.04-1.53, p = 0.009), and subjects aged ≥60 (SMR 1.17, 95% CI 1.01-1.35, p = 0.02). Mutation-specific differences in mortality were observed. Reverse parent-offspring analysis showed excess mortality (SMR 1.26, 95% CI 1.07-1.48, p = 0.003), driven by subjects aged ≥60.

Conclusion: The natural history of TTNtv showed a relatively mild phenotype with significant excess mortality after 1965 and in subjects aged ≥60 years. Increased mortality above 60 years was also seen in parents of present-day patients. With increasing life expectancy, TTNtv-associated mortality will likely become more prevalent.

M. Jansen: None. A.F. Baas: None. K.Y. van Spaendonck-Zwarts: None. A.S. Ummels: None. A. van den Wijngaard: None. J.D.H. Jongbloed: None. M.A. van Slegtenhorst: None. R.H. Lekanne Deprez: None. M.W. Wessels: None. M. Michels: None. A.C. Houweling: None. E.T. Hoorntje: None. P.J.T.M. Helderman-van den Enden: None. D.Q.M.C. Barge-Schaapveld: None. J. van Tintelen: None. M.P. van den Berg: None. A.A.M. Wilde: None. H. Ploos van Amstel: None. E.A.M. Hennekam: None. F.W. Asselbergs: None. E.J.G. Sijbrands: None. D. Dooijes: None.

P05.32D DNA methylation changes in destabilization of carotid atherosclerotic plaque

A. V. Markov1, M. S. Nazarenko1,2,3, A. Zarubin1,2, D. V. Sharysh2, A. N. Kazantsev3, O. L. Barbarash3, V. P. Puzyrev1,2

1Research Institute of Medical Genetics, Tomsk NRMC, Tomsk, Russian Federation, 2Siberian State Medical University, Tomsk, Russian Federation, 3Research Institute for Complex Issues of Cardiovascular Diseases, Kemerovo, Russian Federation

Introduction: Destabilization of carotid atherosclerotic plaque (CAP) is known to be the cause of cerebrovascular ischemia and stroke, but epigenetic framework for this complex process is poorly investigated. Our study aimed to compare genome-wide DNA methylation profiles in cells of stable and unstable atherosclerotic plaques of carotid arteries.

Materials and Methods: CAPs were obtained from 16 patients (10 males, 6 females, aged 65  ±  6 years) and divided into histologically stable (n = 8) and unstable (n = 8) samples. CpG methylation in specimens was estimated using Infinium MethylationEPIC BeadChip (Illumina) and analyzed in R/Bioconductor.

Results: After discard of sex chromosome-mapped, polymorphic, cross-hybridized and poorly detected microarray probes, of remained 775836 sites, 1829 CpG-sites showed differential methylation (|Δβ|>0.15) according to CAP instability (p < 0.05) and located commonly beyond CpG-islands. Hypomethylated 1194 sites were overrepresented genes functioning in immune cells and participating in different biological processes associated with immune response (i.e. leukocyte activation, T-cell differentiation, etc.). Other 635 sites with elevated methylation level in unstable plaques resided within genes largely involved in morphogenesis and developmental processes (i.e. cytoskeleton organization, cell adhesion, etc.). Unsupervised analysis revealed moderate correlation of the first principal components with measures of lipid core and cap of CAPs, and also with heart failure severity and ACE inhibitors intake.

Conclusions: DNA methylation changes in unstable CAPs are associated with activation of immune response and impaired remodeling of artery wall, which can reflect the underlying processes of plaque destabilization on epigenetic level. The study is supported by a grant of Russian Scientific Foundation (16-15-10150).

A.V. Markov: None. M.S. Nazarenko: None. A. Zarubin: None. D.V. Sharysh: None. A.N. Kazantsev: None. O.L. Barbarash: None. V.P. Puzyrev: None.

P05.33A FABP4 gene expression in epicardial adipose tissue is related to obesity associated coronary heart disease

V. Miroshnikova1, A. Panteleeva2, E. Polyakova3, I. Pobozheva4, N. Razgildina4, O. Belyaeva3, E. Baranova3, O. Berkovich3, S. Pchelina4

1Petersburg nuclear physics institute, Gatchina, Russian Federation, 2Petersburg nuclear physics institute, NRC, Gatchina, Russian Federation, 3First Pavlov State Medical University, St. Petersburg, Russian Federation, 4Petersburg nuclear physics institute, NRC "Kurchatov Institute", Gatchina, Russian Federation

Objective: The fatty acid-binding protein 4 (FABP4) has been described as a biomarker for adiposity. Being expressed by both adipocytes and macrophages it takes part in intracellular lipid traffic and acts as an important adipocytokine. Increased circulating FABP4 level is associated with obesity, insulin resistance and atherosclerosis. However, little is known about FABP4 gene expression in epicardial adipose tissue (EAT) during obesity and its potential influence on coronary atherosclerosis development.

The aim of this study was to evaluate FABP4 mRNA expression in EAT in patients with obesity and coronary heart disease (CHD).

Methods: We analyzed: 1) group of 50 patients with CHD which consisted of 26 obese persons and 24 normal weight persons; 2) control group of 12 individuals without CHD which consisted of 6 obese persons and 6 normal weight persons. EAT samples were obtained from CHD patients during coronary bypass surgery and from control persons during heart valve surgery. Atherosclerosis severity was evaluated by coronary angiography. FABP4 and macrophage marker CD68 mRNA levels in EAT were determined by real-time RT-PCR.

Results: FABP4 mRNA levels were significantly reduced in obese CHD patients when compared with obese controls (p < 0.01). Among normal weight persons there was no difference in FABP4 expression between patients with CHD and corresponding controls without CHD. FABP4 mRNA levels within obese subgroups were highly correlated with proinflammatory macrophage marker CD68 mRNA levels (r = 0.602, p < 0.01).

Conclusions: Our results indicate that FABP4 gene expression in EAT is associated with coronary heart disease developed on obesity background.

V. Miroshnikova: None. A. Panteleeva: None. E. Polyakova: None. I. Pobozheva: None. N. Razgildina: None. O. Belyaeva: None. E. Baranova: None. O. Berkovich: None. S. Pchelina: None.

P05.34B The incidence and risk of cardiovascular disease outcomes in patients with Familial Hypercholesterolaemia: a matched survival analysis using a large UK primary care prospective cohort

B. Iyen1, S. Weng1, R. Akyea1, J. Kai1, J. Leonardi-Bee2, S. E. Humphries3, P. Roderick4, N. Qureshi1

1Division of Primary Care, University of Nottingham, Nottingham, United Kingdom, 2Division of Epidemiology and Public Health, University of Nottingham, Nottingham, United Kingdom, 3Cardiovascular Genetics, Institute of Cardiovascular Science, University College London, London, United Kingdom, 4Faculty of Medicine, Primary care and Population sciences, University of Southampton, Southampton, United Kingdom

Introduction: Heterozygous familial hypercholesterolemia (FH) is the most common inherited cause of raised LDL-cholesterol and is associated with an increased risk of coronary heart disease (CHD). This study aimed to determine if FH is associated with other cardiovascular diseases (CVD) including transient ischaemic attack (TIA), stroke and peripheral vascular disease (PVD).

Material and methods: Using routine primary care data from the UK Clinical Practice Research Datalink (CPRD), we used primary care disease codes to select subjects with clinical FH diagnoses who were free from CVD at baseline, and matched them with population controls according to age, sex and general practice. Cox proportional hazards regression models stratified on the matched pairs, were used to determine hazard ratios (HR) for CVD (up to 10 years follow-up) between FH patients and their matched controls.

Results: The incidence rates of CVD in patients with FH (n = 14073; 4,481 CVD events) and in the controls (n = 42,130; 1,767 CVD events) were 25.8 and 3.1 per 1000 person-years respectively. Compared with controls, the hazard ratio for CVD among those with clinical FH was 8.87 (95% CI: 8.31-9.47, p < 0.0001). The hazard ratios for coronary heart disease (CHD), TIA/stroke and PVD were 10.34 (95% CI: 9.56-11.17; p < 0.0001), 6.51 (95% CI: 5.65-7.51; p < 0.0001) and 6.93 (95% CI: 5.89-8.16; p < 0.0001) respectively. These hazard ratios remained significant after adjusting for known CVD risk factors.

Conclusion: Individuals with clinical FH have significantly higher risk of coronary heart disease, stroke/transient ischaemic attack and peripheral vascular disease over time.

Funder: NIHR HTA Programme Grant

B. Iyen: None. S. Weng: None. R. Akyea: None. J. Kai: None. J. Leonardi-Bee: None. S.E. Humphries: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Modest; Steve E Humphries was funded by the British Heart Foundation (BHF PG08/008) and by the NIHR UCLH BRC. F. Consultant/Advisory Board; Modest; Steve E Humphries was a member of the NICE Familial Hypercholesterolaemia Guideline Development group (2016–2017). Other; Modest; Medical Director and minority shareholder of a UCL spin-out company called StoreGene, which uses a 20 SNP genetic test in combination with the classical risk profile, to estimate individual CVD risk. P. Roderick: None. N. Qureshi: F. Consultant/Advisory Board; Modest; Nadeem Qureshi was a member of the NICE Familial Hypercholesterolaemia Guideline Development group (2016–2017).

P05.35C Improving identification of familial hypercholesterolaemia in primary care using FAMCAT (Familial Hypercholesterolaemia Case Ascertainment Tool): validation in a large population database

W. Stephen, J. Kai, R. Akyea, N. Qureshi

University of Nottingham, Nottingham, United Kingdom

Introduction: Familial Hypercholesterolaemia (FH) is a common inherited cause of raised cholesterol. However, over 80% of people with FH are still not identified, leading to many avoidable heart attacks and early deaths. This study assessed the validity of a new FH case-finding algorithm (FAMCAT), intended for application to patients’ primary health care records, in a large population cohort.

Material and Methods: Analysis of 747,194 patients’ data from QRESEARCH, a UK primary care database, was conducted by applying FAMCAT regression equations to predict probability of FH. There were 1,397 patients diagnosed with definite FH. Prediction accuracy of FAMCAT, determined by the area under the receiver operating characteristics curve (AUC), was compared to use of established Simon-Broome and Dutch Lipid Clinic criteria for FH.

Results: FAMCAT resulted in an overall AUC of 0.805 (95% confidence interval [CI] 0.793 - 0.817), performing significantly better in identifying FH than Simon-Broome (SB) criteria (0.692, 95% CI 0.680 - 0.705) or Dutch Lipid Clinic (DLC) criteria (0.716, 95% CI 0.703 - 0.729). Prediction of FH by minority ethnic group resulted in AUCs ranging from 0.755 (95% CI 0.626 - 0.885) for Asian/Asian British to 0.871 (95% CI 0.808 - 0.934) for Black/Black British/African.

Conclusions: The FAMCAT algorithm offers significant improvement for identifying FH compared to DLC or SB criteria in UK primary care practice. FAMCAT may require recalibration in, and for use other international and ethnically diverse population groups. Further research assessing the clinical utility of FAMCAT with genetic test diagnosis is recommended.

W. Stephen: F. Consultant/Advisory Board; Modest; roadtohealth ltd. J. Kai: None. R. Akyea: None. N. Qureshi: None.

P05.36D Genetics, lifestyle and cholesterol in young women

A. Rimbert1, J. Balder1,2, X. Zhang3, M. Viel4, R. Kanninga4, F. van Dijk4, P. Lansberg1, R. J. Sinke4, J. Kuivenhoven1

1Department of Pediatrics, Section Molecular Genetics, University Medical Center Groningen, University of Groningen, Groningen, Netherlands, 2Department of Vascular Medicine, University Medical Center Groningen, University of Groningen, Groningen, Netherlands, 3Department of Vascular Medicine, Academic Medical Center, Amsterdam, Netherlands, 4Department of Genetics, University Medical Center Groningen, University of Groningen, Groningen, Netherlands

Introduction:Low-density lipoprotein cholesterol (LDL-c), a causal risk factor for atherosclerosis, is mostly studied upon clinical events. Women are usually affected later in life than men and are underdiagnosed and undertreated in cardiovascular investigations. This study addresses genetic and lifestyle factors affecting LDL-c in young women.

Methods: Premenopausal women with LDL-c ≤ 1st percentile (≤ 50 mg/dl; n = 119) and ≥ 99th percentile (≥ 186 mg/dl; n = 121) were selected from a Dutch population-based cohort (Lifelines). We applied a one-step, novel NGS-based gene-panel to establish genetic origins of hypo- and hypercholesterolemia. A “healthy lifestyle score” was extracted from questionnaires.

Results: 15,7% of women with low LDL-c carried mutations causing monogenic hypocholesterolemia and 49,6% were genetically predisposed to low LDL-c based on extremely low weighted genetic risk-scores. A healthier lifestyle was not associated with low LDL-c in women without genetic predisposition. 16,8% of women with high LDL-c carried mutations causing familial hypercholesterolemia, whereas 21% were predisposed to high LDL-c based on polygenic risk-scores. Women without such genetic predisposition exhibited a significantly unfavorable lifestyle.

Conclusions: Our study demonstrates the need for early assessment of cardiovascular risk profiles in apparently healthy young women to identify those with LDL cholesterol levels above the 99th percentile for their age: 1) 17% of the cases appeared molecularly diagnosed with familial hypercholesterolemia; 2) our data indicate that an unfavorable lifestyle is significantly associated with severe hypercholesterolemia in genetically unaffected women, which may need further evaluation and advice to prevent future cardiovascular complications.

Grants: CVON2011-2016; CVON2017-2020; FP7-603091; NHS 2015T068

A. Rimbert: None. J. Balder: None. X. Zhang: None. M. Viel: None. R. Kanninga: None. F. van Dijk: None. P. Lansberg: None. R.J. Sinke: None. J. Kuivenhoven: None.

P05.37A Fibromuscular dysplasia: Identifying potential genetic causal variants by whole-exome sequencing

E. Vanhoutte, G. Claes, I. Krapels, P. de Leeuw, H. Brunner, A. Kroon

Maastricht University Medical Center, Maastricht, Netherlands

Background: Fibromuscular dysplasia (FMD) is a noninflammatory, nonartherosclerotic disorder of medium-sized arteries. FMD leads to arterial stenosis, occlusion, aneurysms and dissection, most commonly involving renal and internal carotid arteries. Clinical manifestations commonly include hypertension, dizziness and pulsatile tinnitus.

The etiology of FMD remains unknown. Since FMD occurs in families in approximately 10%, a genetic origin seems plausible.

Aims: To identify genetic variants involved in FMD.

Methods: So far, genetic analysis through whole exome sequencing was performed in five patients with FMD. Only rare and novel truncating, missense and deletion variants predicted to be conserved and deleterious are considered to be of interest.

Results: In one patient, we identified a missense variant in COL4A1, a nonsense variant in COL4A2 and an 85kb deletion in ACTA2. In two other patients, a missense variant in JAG1 and a missense variant in COL4A2 were identified respectively.

The collagen IV network seems to be critical for structural integrity in the basement membrane. COL4A1 and COL4A2 mutations are associated with multisystem disorders with abnormalities in the vasculature and other organs.

Mutations in JAG1 causes Alagille syndrome in which vascular anomalies including bilateral renal artery stenosis leading to hypertension and internal carotid artery aneurysms have been reported.

Literature reports one mutation in ACTA2 causing dilatation of proximal internal carotid artery, occlusive disease of terminal internal carotid artery, and abnormally straight course of intracranial arteries.

Conclusions: FMD is likely to be a multifactorial disorder, however vascular and connective tissue genes might be involved in the pathogenesis of FMD.

E. Vanhoutte: None. G. Claes: None. I. Krapels: None. P. de Leeuw: None. H. Brunner: None. A. Kroon: None.

P05.38B Discovery and fine-mapping of type 2 diabetes susceptibility loci in diverse populations using more than a million individuals

A. Mahajan1,2, H. Kitajima1, X. Sim3, M. C. Y. Ng4, W. Zhang5, J. E. Below6, K. J. Gaulton7, A. P. Morris1,8, on behalf of the DIAMANTE Consortium

1Wellcome Centre for Human Genetics, Oxford, United Kingdom, 2Oxford Centre for Diabetes, Endocrinology and Metabolism, Radcliffe Department of Medicine, University of Oxford, Oxford, United Kingdom, 3Saw Swee Hock School of Public Health, National University of Singapore, Singapore, Singapore, 4Center for Genomics and Personalized Medicine Research, Wake Forest School of Medicine, Winston-Salem, Winston-Salem, NC, United States, 5School of Public Health, Imperial College London, London, United Kingdom, 6Human Genetics Center, University of Texas Health Science Center at Houston, Houston, TX, United States, 7Department of Pediatrics, University of California San Diego, La Jolla, CA, United States, 8Department of Biostatistics, University of Liverpool, Liverpool, United Kingdom

To discover type 2 diabetes (T2D) loci and enhance fine-mapping resolution, we conducted the largest meta-analysis of genome-wide association studies of the disease to date by aggregating 171,262 cases and 1,075,072 controls from diverse populations (45% non-European ancestry). We identified 208 loci at genome-wide significance (p<5x10-8), including 41 mapping outside regions previously implicated in T2D (accounting for those discovered in European-specific component of this study). Across these loci, conditional analyses revealed a total of 342 distinct signals of association (locus-wide significance, p<10-5). We observed strong evidence of heterogeneity in allelic effects on T2D (pHET<1.5x10-4, Bonferroni correction) correlated with ancestry at 16% of signals, including LEP (rs7778167, pHET=4x10-25, East Asian specific) and multiple associations at/near KCNQ1 and TCF7L2 (representing ethnic-specific/-differentiated effects). T2D-associated variants showed significant enrichment (odds-ratio range 1.90-6.63; p<0.05) in coding exons, pancreatic islet enhancers and promoters, adipose enhancers, and binding sites for transcription factors, including NKX2.2 and FOXA2. Increased sample size, population diversity, and annotation-informed fine-mapping substantially improved localisation of potential causal variants compared with previous efforts, and highlighted 76 signals with a single variant accounting for >80% of the posterior probability of association (PPA); of these 35 signals had PPA of >99%. Clustering of signal-specific annotation enrichment highlighted distinct clades of T2D associations driven by different underlying molecular processes. These analyses represent the most comprehensive view of the genetic contribution to T2D to date and, through integration with expression quantitative trait loci in disease-relevant tissues, point to previously unreported effector genes and mediating molecular mechanisms at several loci.

A. Mahajan: None. H. Kitajima: None. X. Sim: None. M.C.Y. Ng: None. W. Zhang: None. J.E. Below: None. K.J. Gaulton: None. A.P. Morris: None.

P05.39C Copy number variation analysis in cardiac congenital septal defects

G. Crauciuc1, F. Tripon1, L. Gozar2, R. Togănel2, C. Bănescu3

1University of Medicine and Pharmacy, Tîrgu Mureş, Romania, 2Emergency Institute for Cardiovascular Diseases and Transplantation, Tîrgu Mureş, Romania, 3Genetics Laboratory, Center for Advanced Medical and Pharmaceutical Research, University of Medicine and Pharmacy, Tîrgu Mureş, Romania

Congenital cardiac septal defects (CCSD) are the most frequently type of congenital heart malformation, often occurs sporadically, without an obvious cause in most cases. Recent studies demonstrated the involvement of several genes variation in cardiac development process, factors that have been underestimated in the past. The aim of our study was to evaluate if the CCSD patients present copy number variations (CNVs) and establish whether the CNVs investigation should be performed in management of this patients. A number of 26 pediatric patients were enrolled. The CNVs were determined using the multiplex ligation-dependent probe amplification (MLPA) technique and P234-A3 SALSA MLPA probemix which contain probe for GATA 3 and GATA 4 gene analysis. The MLPA analysis revealed CNVs in two patients. Both of them present a duplication located in 10p14 (GATA 3 gene, exons 4 and 5). Several patients present a high ratio for 8p23.1 (GATA 4 gene) and a low ratio for 10p14 (GATA 3 gene) but the signals were statistically insignificant. Six patients present low ratio for flanking probe (CELF2 probe, according to the manufacturer CNVs of flanking probes are unlikely to be related to the condition tested). Twenty patients received recommendation for target sequencing of different exons. Based on our results, we may consider the mentioned MLPA kit as a first step in CCSD patient’s management.

G. Crauciuc: None. F. Tripon: None. L. Gozar: None. R. Togănel: None. C. Bănescu: None.

P05.41A Next Generation Sequencing (NGS) panel revealed new candidate genes and variants in 25 Hypertrophic Cardiomyopathy patients

B. Turkgenc1, S. G. Temel2,3, F. Uysal4, S. Ugan Atik5, F. Oztunc5, A. Sulu6, F. Ekici7, C. Ayabakan8, E. Odemis9, A. Saygili10, A. Koka5, I. Ozkan Akinci11, Y. Alanay12,13, A. Celiker14, A. Ozer15, M. C. Yakicier16

1Acibadem Genetic Diagnostic Center, Istanbul, Turkey, 2Department of Medical Genetic, Faculty of Medicine, Uludag University, Bursa, Turkey, 3Department of Histology and Embryology, Faculty of Medicine, Uludag University, Bursa, Turkey, 4Department of Pediatric Cardiology, Faculty of Medicine, Uludag University, Bursa, Turkey, 5Department of Pediatric Cardiology, Cerrahpasa Medical Faculty, Istanbul, Turkey, 6Department of Pediatric Cardiology, Faculty of Medicine, Gaziantep University, Gaziantep, Turkey, 7Department of Pediatric Cardiology, Faculty of Medicine, Akdeniz University, Antalya, Turkey, 8Department of Pediatric Cardiology, Baskent University Istanbul Hospital, Istanbul, Turkey, 9Department of Pediatric Cardiology, Acibadem University, Acibadem Atakent Hospital, Istanbul, Turkey, 10Department of Pediatric Cardiology, Medical Faculty of Acibadem University, Istanbul, Turkey, 11Department of Anesthesiology and Reanimation, Istanbul Medical Faculty, Istanbul, Turkey, 12Department of Medical Genetics, Acibadem University, Istanbul, Turkey, 13Department of Pediatric Genetic, Acibadem Maslak Hospital, Istanbul, Turkey, 14Department of Pediatric Cardiology, Faculty of Medicine, Koç University, Istanbul, Turkey, 15Department of Medical Biology and Genetics, Marmara University, Istanbul, Turkey, 16Department of Molecular Biology and Genetic, Acibadem University, Faculty of Science, Istanbul, Turkey

Introduction: Familial Hypertrophic cardiomyopathy (HCM) is a multigenic heart condition characterized by hypertrophy of the cardiac muscle. The prevalence of HCM is estimated at 1:500 in the general population.The aim of our study was evaluation of complex genetic profile of next generation sequencing (NGS) results in Turkish patients with HCM.

Methods: We designed a targeted cardiac panel of 68 genes using NGS technology on Iontorrent PGM.

Results: Analyses of 25 HCM patients revealed a total of 54 variants in cardiac genes of which 9 (16,7%) were pathogenic, 5 (%9,2) were potentially pathogenic, 34 (63%) were variant of unknown significance (VUS) and 6 (11,1%) were likely benign. Only one of the patient (4%) had no suspicious variant in examined genes. 10 patients (40%) carried at least one pathogenic/potentially pathogenic mutations in MYH6, MYH7, MYBPC3, TNNI3, TNNT2, DSP, DPP6, JUP, KCNQ1 and SCN5A genes, while other 14 patients (56%) had at least one VUS variant. A total of 12 novel variants were identified, which consist of 1 frameshift insertion in JUP gene, 1 frameshift deletion in DPP6 gene and 9 missense variants in DPP6, MYH7, MYH6, GJA5, NKX2-5, TMPO and TRDN genes. An interesting finding is co-occurrence of dominant pathogenic/likely pathogenic variants in different genes, as in the three cases explains the complexity and severity of the observed phenotypes.

Conclusion: Identification of pathogenic/potentially pathogenic variants and novel candidate variants/genes has led to new opportunities for prevention and therapy of lethal HCM. Acknowledgements: The study was supported by SANTEZ Grant (0253. STZ.2013-2), Turkey.

B. Turkgenc: None. S.G. Temel: None. F. Uysal: None. S. Ugan Atik: None. F. Oztunc: None. A. Sulu: None. F. Ekici: None. C. Ayabakan: None. E. Odemis: None. A. Saygili: None. A. Koka: None. I. Ozkan Akinci: None. Y. Alanay: None. A. Celiker: None. A. Ozer: None. M.C. Yakicier: None.

P05.43C Identification of a novel homozygous loss-of-function variant in JPH2 in two unrelated families affected by lethal Neonatal hypertrophic cardiomyopathy

R. Maroofian1, N. Mazaheri2,3, M. Zamani2,3, G. Shariati2,3, A. Sedaghat2,3, Y. Jamshidi1, H. Galehdari2,3

1Molecular and Clinical Sciences Institute, St George's University of London, London, United Kingdom, 2Department of Genetics, Faculty of Science, Shahid Chamran University of Ahvaz, Ahvaz, Iran, Islamic Republic of, 3Narges Medical Genetics and Prenatal Diagnosis Laboratory, Ahvaz, Iran, Islamic Republic of

Pediatric cardiomyopathies represent a clinically and genetically heterogeneous group of disorders affecting the ventricular myocardium, with an annual incidence of ~ 1.5 per 100,000 children. They are associated with substantial morbidity and mortality: up to 40% die or undergo transplantation. Here we studied two unrelated consanguineous families from the same geographic region of Iran, with 4 children who died due to infantile cardiomyopathy. Affected offspring presented with severe hypertrophic cardiomyopathy and right atrium enlargement in utero, at birth, or in early childhood. Whole exome sequencing (WES) of DNA from the proband of the first family led to identification of a novel homozygous frameshift variant (p.Glu641*) in JPH2. The parents of the proband and a healthy sibling were heterozygous. Echocardiography revealed no abnormalities in the carriers. Due to unavailability of samples WES could only be carried out for the parents of the second family, however the same heterozygous frameshift variant was identified in both parents. The variant is novel and absent from population databases including gnomAD, the ethnically-matched GME variome, Iranome, and in ~1000 WES in-house control subjects from the same geographic region as the families investigated. The variant occurs in the protein C-terminal region of JPH2, just upstream of a 22-amino acid transmembrane anchor responsible for binding of the protein to the sarcoplasmic/endoplasmic reticulum, thereby potentially disrupting binding. Our findings add to the growing evidence that mutations in JPH2 play a role in HCM; and suggest that this novel biallelic truncating mutation can give rise to severe, early-onset pediatric cardiomyopathy.

R. Maroofian: None. N. Mazaheri: None. M. Zamani: None. G. Shariati: None. A. Sedaghat: None. Y. Jamshidi: None. H. Galehdari: None.

P05.44D Functional analysis of six novel LDLR mutations in the Argentinian population

A. Gómez1, G. Giunta2,3, L. Helman2, A. Pontoglio4, L. Kaeser1, U. Toscanini1, R. Colombo4,5, L. Cuniberti3

1PRICAI-FUNDACIÓN FAVALORO, Buenos Aires, Argentina, 2Unidad Metabólica, Hospital Universitario Fundación Favaloro (HUFF), Buenos Aires, Argentina, 3Laboratorio de Lípidos y Aterosclerosis, IMETTYB‒Universidad Favaloro‒CONICET, Buenos Aires, Argentina, 4Center for the Study of Rare Hereditary Diseases, Niguarda Ca' Granda Metropolitan Hospital, Milan, Italy, 5Institute of Clinical Biochemistry, Faculty of Medicine, Catholic University and Policlinico Agostino Gemelli, Rome, Italy

Introduction: Mutations in the gene encoding the low-density lipoprotein receptor (LDLR) are involved in the molecular etiology of autosomal dominant familial hypercholesterolemia (FH), an hereditary condition associated with coronary heart disease and the risk of premature death. Based on FH prevalence among Caucasians, it is expected that >80,000 cases are present in Argentina, less than 1% of which have been identified so far. Previously, in a cohort of Argentinian FH patients we detected eight unreported LDLR single nucleotide variants (SNVs). Six of them are located in the coding regions of the gene and lead to an amino acid substitution. To assess the pathogenicity of each SNV, we performed a functional investigation of the corresponding variant of the receptor proteins.

Materials and Methods: Binding, uptake, and degradation of 125-I-labeled LDL by cultured CHO cells expressing wild-type and mutant LDLR proteins were compared in the presence and absence of an excess of unlabelled LDL. Levels of LDLR mRNA expression were determined by qRT-PCR 48 h after transfection with plasmids.

Results: Four out of the six LDLR mutants showed a markedly reduced in vitro receptor activity, suggesting their role in the pathogenesis of FH.

Conclusions: According to in silico predictions and conservation analysis in multiple species, two of the six identified LDLR variants are likely benign SNVs. The remaining four variants can be categorized as “pathogenic variants” that expand the spectrum of FH-linked LDLR mutations in the Argentinian population.

A. Gómez: None. G. Giunta: None. L. Helman: None. A. Pontoglio: None. L. Kaeser: None. U. Toscanini: None. R. Colombo: None. L. Cuniberti: None.

P05.45A Exome sequencing in Russian families with noncompaction cardiomyopathy

A. N. Meshkov1, O. V. Kulikova1, R. P. Myasnikov1, N. V. Shcherbakova1, A. A. Zharikova1, S. N. Koretsky1, A. V. Kiseleva1, A. I. Ershova1, M. S. Kharlap1, E. N. Basargina2, N. A. Sdvigova2, E. A. Mershina3, V. E. Sinitsyn3, O. M. Drapkina1, S. A. Boytsov1

1National Medical Research Center for Preventive Medicine, Moscow, Russian Federation, 2National Medical Research Center of Children's Health, Moscow, Russian Federation, 3Federal Center of Treatment and Rehabilitation, Moscow, Russian Federation

Introduction: Left ventricular noncompaction cardiomyopathy (LVNC), a relatively rare cardiomyopathy, is characterized by a high incidence of serious complications and early death. Over 60 genes associated with the development of family cases of LVNC are described, in most cases the inheritance type is autosomal dominant. However, causal variants in these genes are detected in less than 50% of families. The search for new genes is actual.

Materials and Methods: We formed the cohort of patients with LVNC and their relatives of 1 and 2 degrees of kinship. The enrollment of participants was done by cascade and reverse-cascade methods in the heart failure center. Probands should have met echocardiographic and cardiac MRI criteria for noncompaction. Molecular testing was performed using exome sequencing for members of 14 families (43 participants). We searched for pathogenic and probably pathogenic variants in 66 genes associated with LVNC and additionally in 122 genes associated with other cardiomyopathies.

Results: In 8 out of 14 families pathogenic or probably pathogenic variants of the nucleotide sequence were identified. Only 4 variants were found in genes associated with LVNC - 2 pathogenic variants in TTN and 2 probably pathogenic in MYBPC3 and TPM1. One pathogenic variant was found in VCL and 3 probably pathogenic variants were found in DES, TBX1 and FHOD3 associated with other cardiomyopathies.

Conclusions: The data obtained may indicate the detection of 4 new genes (DES, VCL, TBX1, FHOD3) associated with LVNC. The reported study was funded by RFBR according to the research project #17-04-00521.

A.N. Meshkov: None. O.V. Kulikova: None. R.P. Myasnikov: None. N.V. Shcherbakova: None. A.A. Zharikova: None. S.N. Koretsky: None. A.V. Kiseleva: None. A.I. Ershova: None. M.S. Kharlap: None. E.N. Basargina: None. N.A. Sdvigova: None. E.A. Mershina: None. V.E. Sinitsyn: None. O.M. Drapkina: None. S.A. Boytsov: None.

P05.46B Long QT syndrome mutation spectrum in Russians

E. G. Okuneva1, A. A. Kozina1,2, K. Tsukanov1, A. Krasnenko1, P. Shatalov3, T. A. Trofimova3, V. M. Solovyev3, V. V. Ilinsky1

1Genotek Ltd.(Genotek IT.), Moscow, Russian Federation, 2Institute of Biomedical Chemistry, Moscow, Russian Federation, 3The Research and Clinical Institute for Pediatrics named after Academician Yuri Veltischev of the Pirogov Russian National Research Medical University of the Russian Ministry of Health, Moscow, Russian Federation

Mutations in more than 16 genes can cause long QT syndrome (LQT). Detection of variants in these genes is necessary for diagnostics, targeted gene therapy, behavioral management, family screening and prophylaxis of sudden cardiac death. We report frequencies of disease-causing variants from 73 patients with LQT. For all patients we did exome sequencing using Agilent Focused Exome enrichment panel and Illumina HiSeq2500. For variant calling and pathogenicity scoring we followed ACMG guidelines. We also confirmed all mutations by Sanger sequencing. No disease-causing mutations were identified in 14 cases suggesting new genes might be involved in pathogenesis. 59 patients had 91 disease-causing variants in LQT and cardiac arrhythmias associated genes. 27 patients had more than one variant in those genes. We discovered 22 new variants, that were not reported previously in dbNSFP, Clinvar, OMIM, HGMD, 1000Genomes and ExAC . In almost half cases (46) we discovered pathogenic variants in KCNE1, KCNH2, KCNQ1 or SCN5A genes. In remaining cases (45) we found pathogenic variants in 18 other genes, including 7 variants in ANK2, which are rarely reported in LQT patients.This work was funded by Fundamental Scientific Research Program of the Russian Academy of Sciences for 2013-2020.

E.G. Okuneva: None. A.A. Kozina: None. K. Tsukanov: None. A. Krasnenko: None. P. Shatalov: None. T.A. Trofimova: None. V.M. Solovyev: None. V.V. Ilinsky: None.

P05.47C Lymphedema distichiasis syndrome: dominant FOXC2 mutations causing protein aggregation and loss of transcriptional activity

D. Tavian1, S. Missaglia1, P. E. Maltese2, S. Michelini3, M. Bertelli2

1University, Milan, Italy, 2Magi-onlus, Rovereto, Italy, 3San Giovanni Battista Hospital, Rome, Italy

Introduction: Forkhead transcription factor FOXC2 is essential for the correct development and maintenance of lymphatic system. Mutations in the FOXC2 gene are associated with primary lymphedema-distichiasis syndrome (LD), a congenital autosomal dominant disorder that causes a dysfunction of the lymphatic vessels. It has been demonstrated that FOXC2 variations can either reduce or increase protein function. To clarify the molecular mechanism through which FOXC2 mutations can affect transcriptional activity, we analyzed two missense mutations (p.L80F and p.R121H), two frameshift variations (p.H199Pfs264* and p.M276Dfs186*) and one nonsense mutation (p.Y109*), previously identified in LD patients.

Material and Methods: To investigate subcellular localization, HeLa cells have been transfected with FOXC2 mutant plasmids, expressing FOXC2 with GFP at the N-terminus, and immunofluorescence analysis has been performed. Subsequently, transactivation activity has been evaluated by a Luciferase assay. Finally, the effects of missense and nonsense mutations on FOXC2 protein structure have been evaluate using bioinformatics tools.

Results: p.L80F, p.R121H, p.H199Pfs264* and p.M276Dfs186* were able to correctly localize in the nucleus, but they produced FOXC2 protein aggregates characterized by partial or total loss of transcriptional activity. Moreover, p.H199Pfs264* and p.M276Dfs186 protein aggregation involved also chromatin structure. The p.Y109* also caused the formation of protein aggregates, even though it was mainly localized in the cytoplasm. Finally, the analysis of FOXC2 protein structure revealed that missense and nonsense mutations dramatically modified the DNA binding-site conformation.

Conclusions: In Lymphedema-distichiasis, FOXC2 mislocalization, structure modification and nuclear or cytoplasmic aggregates formation can be considered the main cause of loss of protein function

D. Tavian: None. S. Missaglia: None. P.E. Maltese: None. S. Michelini: None. M. Bertelli: None.

P05.48D Induction of angiogenesis by M13 bacteriophage-RGD nanofibrous surfaces on human umbilical vein endothelial cells

Z. Safari, M. Sadeghizadeh

TMU, Tehran, Iran, Islamic Republic of

Introduction: The ability to create, remodeling and regulate the human blood system holds wide range of medical utilization and carries possible therapeutic effects on patients with variety of cardiovascular diseases. Commonly, Clinical use of autologous vessels has been reported; However, transplant rejection unfortunately occurs in most patients. Likewise, biomaterial tissue engineering on vascular replacement devices has been recently improved.

Materials and Method: In the following study, comparing the ability of M13 Bacteriophage, M13 bacteriophage-RGD and Gelatin surfaces to enhance the angiogenic potential of human umbilical vein endothelial cells (HUVECs) was investigated, The response of HUVECs cells to surfaces were studied through cell apoptosis, cell proliferation, growth factors secretion (VEGF), and angiogenic-endogenic-associated genes (Matrix metallopeptidase 9 (MMP9), Endothelial nitric oxide synthase eNOS). Real Time-PCR, ELISA, Nitric Oxide assay and Zymography was operated on samples. After 2 days of cell seeding, Gene and Protein expression data were analyzed and compared by two-way analysis of variance.

Result: Our results show that M13 phage-RGD appeared Non-cytotoxic to the cells. Furthermore, these result was associated with a markedly increase on cell proliferation and migration. M13 phage-RGD characterized a strong increase in MMP-9, eNOS and VEGF-A mRNAs as well as leading to higher expression level of VEGF-A protein, MMP-9 activity and NO release in HUVECs in comparison to other surfaces.

Conclusion: Beneficial effect of M13 phage-RGD propose these nanostructures as an advantageous and potential biomaterial in order to promote angiogenesis, which are becoming feasible therapeutic applications for variety of ailments.

Z. Safari: None. M. Sadeghizadeh: None.

P05.50B The role of genetic variation in phenotype variability and response to treatment in Marfan syndrome

B. Loeys1, J. Meester1, E. Franssen1, G. Vandeweyer1, A. Verstraeten1, H. C. Dietz2, L. Van Laer1, Marfan trial - Pediatric Heart Network investigators

1Center for Medical Genetics, University of Antwerp/Antwerp University Hospital, Antwerp, Belgium, 2McKusick Nathans Institute for Genetic Medicine, Johns Hopkins University, Baltimore, MD, United States

In a large cohort of 300 clinically diagnosed MFS patients, we addressed the following questions: Which proportion of classic MFS patients has an FBN1 mutation? Are there genotype-phenotype correlations? Does the nature of the FBN1 mutation predict cardiovascular treatment outcome? Next-generation targeted resequencing of FBN1 and related genes was followed by deletion/duplication testing. Pathogenic FBN1 mutation was identified in 94% of patients (including 15 del/dups). Three percent of MFS patients had non-FBN1 mutations. A similar small fraction had no causal variant but was clinically indistinguishable from the FBN1 mutation positives. We confirm prior literature that mutations creating or deleting cysteines are significantly associated with lens dislocation (p = 10-7). Premature termination codons are more often associated with skeletal findings, but not with cardiovascular severity. We did not observe a correlation between mutations in the middle region of FBN1 and phenotypical severity. Mutations at the C- and N-terminal (exons 1-16/60-66) end tend to lead to a milder cardiovascular phenotype (p = 0.047). We observed no difference in aortic root size or progression nor in treatment outcome comparing dominant negative with haploinsufficient mutations. No effect of mutation location on treatment outcome could be detected