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Abstracts from the 53rd European Society of Human Genetics (ESHG) Conference: Interactive e-Posters

Volume 28 | Supplement 1

Virtual Conference

June 6-9, 2020

© European Society of Human Genetics 2020. Modified from the conference website and published with permission.

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”.

Presenting authors are indicated with bold typeface in the contributor lists.

Interactive e-Posters

P01 Reproductive Genetics/Prenatal Genetics


Prenatal diagnostics of 15q11.2 microdeletion syndrome - the possibilities and challenges

K. Belemezova1,2, P. Chaveeva1, V. Stratieva3,4, M. Yankova3,5, V. Peycheva6, R. Bozhilova6, M. Rizov1, S. Yovinska1, M. Hristova-Savova1, R. Kaneva6, I. Dimova1,6

1SAGBAL „Dr Shterev“, Sofia, Bulgaria, 2University Hospital “St. Ivan Rilski”, Department of Immunology, Medical University of Sofia, Sofia, Bulgaria, 3O.S.С.A.R. Clinic, Sofia, Bulgaria, 4First Specialized Hospital of Obstetrics and Gynecology „St. Sofia”, Sofia, Bulgaria, 5University Hospital "Lozenetz", Sofia, Bulgaria, 6Center of Molecular medicine, Medical University of Sofia, Sofia, Bulgaria

Microdeletion in 15q11.2 has been described as a distinct syndrome encompassing an area between two fragile sites in 15q (BP1 and BP2), with approximately 500 bp size, containing NIPA1, NIPA2, TUBGCP5, and CYFIP1 genes. It is a multisystemic disease affecting mostly the nervous system (intellectual deficits, delayed psychomotor development, ataxia, epilepsy, behavioral problems, etc.), followed by congenital heart defects and various dysmorphisms. This microdeletion was found in 0.57-1.27% of pediatric patients targeted for microarray analysis, mainly with developmental delay and intellectual deficits. However, not all of the deletion carriers have a clinical manifestation - it is found in 0.25% of the population of healthy controls. The penetrance of 15q11.2 microdelection syndrome is estimated at 10.4%, which is significantly higher in de novo occurrence. We report 4 cases of prenatally established 15q11.2 microdeletion, in 3 of the cases the microdeletion was inherited from clinically healthy mothers. The indications for aCGH prenatal diagnosis were: 1) ultrasound data for unilateral cleft lip and palate in combination with an increased nuchal translucency of 3.3 mm- mother was the carrier; 2) second pregnancy after the first case; 3) increased risk from the biochemical screening (1:6) and tricuspid regurgitation, brother with neuropsychiatric disability - mother was a carrier; 4) Increased NT of 7.3 mm. Our results highlight the importance of microarray analysis in the diagnostic refinement of pregnancies which are abnormal in early fetal screening. We emphasize the difficulties of genetic counseling in the inheritance and the incomplete penetrance of the aberrations.

K. Belemezova: None. P. Chaveeva: None. V. Stratieva: None. M. Yankova: None. V. Peycheva: None. R. Bozhilova: None. M. Rizov: None. S. Yovinska: None. M. Hristova-Savova: None. R. Kaneva: None. I. Dimova: None.


Improvement of prenatal care in case of fetal anomaly of the corpus callosum using exome sequencing during the pregnancy

S. Heide1, M. Spentchian1, S. Valence2, J. Buratti3, C. Mach3, E. Lejeune3, V. Olin3, M. Massimello1, C. Garel4, E. Blondiaux4, G. Quenum-Miraillet5, S. Chantot-Bastaraud5, M. Milh6, V. des Portes7, M. Spodenkiewic8, V. Layet9, R. Dard10, S. Moutton11, M. Gorce12, M. Nizon13, S. Mercier13, M. Vincent13, C. Beneteau13, J. Jouannic14, M. Moutard2, B. Keren3, D. Héron3

1UF de Génétique Médicale et CRMR « Déficience intellectuelle », Département de Génétique, Groupe Hospitalier Pitié-Salpêtrière, APHP Sorbonne Université, Paris, France, 2Service de Neurologie Pédiatrique, Hôpital Armand Trousseau, APHP Sorbonne Université, Paris, France, 3UF de Génomique du Développement, Département de Génétique, Groupe Hospitalier Pitié-Salpêtrière, APHP Sorbonne Université, Paris, France, 4Service de Radiologie Pédiatrique, Hôpital Armand Trousseau, APHP Sorbonne Université, Paris, France, 5Service de Cytogénétique, Hôpital Armand Trousseau, HUEP, APHP Sorbonne Université, Paris, France, 6Service de Neurologie Pédiatrique, Hôpital La Timone, APHM, Marseille, France, 7Service de Neurologie Pédiatrique, Hospices Civils de Lyon, Bron, France, 8Service de Génétique Clinique, CHU de Reims, Reims, France, 9Service de Génétique Clinique, Hôpital du Havre, Le Havre, France, 10Service de Génétique Clinique, Hôpital de Poissy, Poissy, France, 11Centre de Référence des anomalies du développement, Service de Génétique Médicale, CHU de Dijon, Dijon, France, 12Service de génétique clinique, CHU d’Angers, Angers, France, 13Service de Génétique Clinique, CHU de Nantes, Nantes, France, 14Service de Médecine Fœtale, Hôpital Armand Trousseau, HUEP, APHP Sorbonne Université, Paris, France

Purpose: Anomalies of the corpus callosum (ACC) are usually diagnosed by prenatal ultrasound. This malformation is characterized by its uncertain prognosis especially when isolated which makes the prenatal counseling difficult. Among genetic etiologies, only chromosomal anomalies are investigated during the prenatal period while ACC is due to single gene mutation in most cases and neurodevelopmental prognosis depends on the underlying cause.

Methods: In order to evaluate the feasibility and contribution of prenatal exome sequencing (pES) during the pregnancy, we included 66 fetuses with ACC for trio analysis. Only pathogenic variants in known ACC and/or ID genes were considered.

Results: pES results were available within a median delay of 22 days. A pathogenic SNV was identified in 12 cases (18%). Moreover, a pathogenic CNV was identified in 4 cases. Thus, the genetic cause was determined in 22% of cases. In all cases with diagnoses, pES results enlightened the neurodevelopmental prognosis of the fetus and enabled the parents to choose to continue with or terminate the pregnancy knowingly.

Conclusions: This is the first study of pES focusing on a single malformation characterized by an uncertain prognosis. Our results demonstrate the feasibility of pES in fetuses with prenatal diagnosis of ACC. pES has an evident clinical impact. The impact of pES when negative remains to be evaluated

S. Heide: None. M. Spentchian: None. S. Valence: None. J. Buratti: None. C. Mach: None. E. Lejeune: None. V. Olin: None. M. Massimello: None. C. Garel: None. E. Blondiaux: None. G. Quenum-Miraillet: None. S. Chantot-Bastaraud: None. M. Milh: None. V. des Portes: None. M. Spodenkiewic: None. V. Layet: None. R. Dard: None. S. Moutton: None. M. Gorce: None. M. Nizon: None. S. Mercier: None. M. Vincent: None. C. Beneteau: None. J. Jouannic: None. M. Moutard: None. B. Keren: None. D. Héron: None.


Genetic management of a small deletion in the PWS/AS imprinting center in a prenatal case

C. Pérez1, E. Pestaña2, C. Rivera2, M. Augé1, L. Barranco1, A. Canellas1, M. Costa1, C. de la Iglesia1, D. Fernández1, N. Palau1, B. Méndez1, M. Piqué1, M. Herrero1, D. Yeste1, L. Puig1, R. Ferreti1, M. Punzón1, S. Martín1, J. Mendoza1, N. Pinedo1, M. Rodríguez1, L. Linares1, E. Lloveras1

1Synlab, Esplugues de Llobregat, Spain, 2Synlab, Madrid, Spain

Introduction: Imprinted genes involved in PWS and AS are under control of an imprinting center comprising two regulatory regions, PWS-SRO (around the SNRPN promoter) and the AS-SRO (35 kb upstream). A very few AS patients have small and/or atypical deletions in AS-SRO.

Patient and Results: Pregnant patient (13 weeks) was referred because a daughter of 4 years old with development delay and peculiar phenotype and a 62Kb copy number loss at 15q11.2, arr[GRCh37] 15q11.2(25119854_25177138)x1. In extended familial studies, mother (pregnant) and grandfather have both the same deletion. MS-MLPA was performed in the daughter and the pregnant. The 15q11.2 deletion was confirmed in both and an altered methylation pattern in the daughter was detected correlating with an AS diagnosis. ArrayCGH analysis (CytoSure™ Constitutional v3 8x60k, OGT, UK) was performed in chorionic villus sample and the same deletion of 62Kb involving U5 exon of AS-SRO (exon 4 according NM sequence of SNRPN gene) was detected. MS-MLPA (PW/AS MS-MLPA Kit, MRC Holland) was performed in amniotic fluid with alteration of the methylation pattern, compatible with hypomethylation of the probes located in the CpG islands region of SNRPN gene was detected. Considering all the results, a prenatal diagnosis of AS was performed.

Comments: IC deletion have been described in a small fraction of AS patients with ID, with no disease-causing variants detected to date.Deletions involving AS-SRO are a molecular defect that must be determined as the recurrence risk in case of a familial IC deletion. Caution should be made in prenatal diagnosis and is appropriate recommending MS-MLPA analysis.

C. Pérez: None. E. Pestaña: None. C. Rivera: None. M. Augé: None. L. Barranco: None. A. Canellas: None. M. Costa: None. C. de la Iglesia: None. D. Fernández: None. N. Palau: None. B. Méndez: None. M. Piqué: None. M. Herrero: None. D. Yeste: None. L. Puig: None. R. Ferreti: None. M. Punzón: None. S. Martín: None. J. Mendoza: None. N. Pinedo: None. M. Rodríguez: None. L. Linares: None. E. Lloveras: None.


START: Thestudy of assistedreproductive technologies

R. Lyle1, C. Page2, Y. Lee2, K. L. Haftorn2, A. Jugessur2, J. Bohlin2, H. K. Gjessing2, P. Magnus2, S. Håberg2

1Oslo University Hospital, Oslo, Norway, 2Norwegian Institute of Public Health, Oslo, Norway

In the last few decades there have been marked changes in patterns of fertility and family structure in rich countries. These include increasing age at childbirth, a lower number of children born to each woman or man, greater use of assisted reproductive technology (ART), higher frequency of family disruptions and increasingly complex family structures. The Centre for Fertility and Health (CeFH; consists of a multidisciplinary research team of epidemiologists, geneticists, demographers, sociologists and economists with research themes including fertility problems and intergenerational transmission of health. The START project aims to understand the causes and health consequences of subfertility and how genetic influences and epigenetic differences are associated with subfertility and Assisted Reproductive Technologies (ART). ART may introduce epigenetic changes in the embryo since the procedures are during a period of extensive epigenetic repatterning. It is unknown which genes may be affected and the potential health consequences for the children. START uses data from the Norwegian Mother, Father and Child Cohort Study (MoBa;, which follows more than 110 000 children and their parents. We have collected genetic and epigenetic data from 1000 trios with children born after ART, and 1000 trios from naturally conceived children analyzed with the Illumina EPIC array and the Illumina HumanCoreExome chip. Investigations are focussed on the effect of ART treatment on imprinted genes and fertility genes, and how epigenetic age acceleration interacts with fertility and ART treatment. Our results show DNA methylation changes due to both parental subfertility and ART procedures.

R. Lyle: None. C. Page: None. Y. Lee: None. K.L. Haftorn: None. A. Jugessur: None. J. Bohlin: None. H.K. Gjessing: None. P. Magnus: None. S. Håberg: None.


Beckwith Wiedemann Syndrome: a prenatal case report with a novel CDKN1C mutation

P. Maia Almeida1,2, S. Moreira Ribeiro1, L. Abreu3, A. Isabel Rei3, M. Branco3, E. Galhano3, J. Saraiva1,4,5, F. Ramos1,3

1Medical Genetics Unit, Centro Hospitalar e Universitário de Coimbra, Coimbra, Portugal, 2Faculty of Health Sciences, University of Beira Interior, Covilhã, Portugal, 3Prenatal Diagnostic Center, Maternidade Bissaya Barreto, Centro Hospitalar e Universitário de Coimbra, Coimbra, Portugal, 4University Clinic of Pediatrics, Faculty of Medicine, University of Coimbra, Coimbra, Portugal, 5Clinical Academic Center of Coimbra, Coimbra, Portugal

Introduction: Beckwith Wiedemann Syndrome (BWS) is a genomic imprinting disorder typically diagnosed after birth. Some ultrasound features that include polyhydramnios, omphalocele, and visceromegaly may raise the clinical suspicion in prenatal. Most reported BWS cases are sporadic, but around 15% may be familial. Delineation of the molecular defects can predict familial recurrence risks and make invasive prenatal diagnosis possible.

Case Report: We report a case of a 41-year-old pregnant woman in whom ultrasound revealed at 24-weeks corpus callosum hypoplasia, macroglossia, omphalocele, short long bones and enlarged kidneys. The pregnancy was terminated and autopsy confirmed the ultrasound findings and also enlarged ovaries and pancreas, cytomegaly of the adrenal cortex and a placental mesenchymal dysplasia. Family history was irrelevant.

Materials and Methods: Clinical and molecular characterization of a prenatal case with diagnosis of BWS. We performed array-CGH, methylation analysis at 11p15.5 BWS locus and Sanger sequencing of CDKN1C gene. The presence of the variant identified in the foetus was tested in the parents.

Results: Array-CGH and 11p15.5 methylation analysis (MS-MLPA) were normal. CDKN1C Sanger sequencing found a heterozygous novel probable pathogenic frameshift variant: NM_000076.2:c.479del p.(Pro160Argfs*112) of maternal origin.

Conclusions: This case demonstrates that search for a clear specific molecular diagnosis is key not only to confirm the diagnosis but also to provide proper genetic counselling. Although present in only 5% of the patients with no family history the identification of a CDKN1C pathogenic variant of maternal origin increases the recurrence risk to 50%.

P. Maia Almeida: None. S. Moreira Ribeiro: None. L. Abreu: None. A. Isabel Rei: None. M. Branco: None. E. Galhano: None. J. Saraiva: None. F. Ramos: None.


Prenatal phenotype in Beckwith-Wiedemann spectrum

D. Carli, C. Bertola, S. Cardaropoli, V. P. Ciuffreda, M. Pieretto, G. B. Ferrero, A. Mussa

Department of Public Health and Pediatrics, University of Torino, Torino, Italy

Introduction: the prenatal phenotype of Beckwith-Wiedemann spectrum (BWSp) is not well characterized. Here we investigate the prenatal features of large series of BWSp patients.

Methods: postnatal phenotype, genotype, prenatal ultrasound (US) features, pregnancy course and biochemical screening tests of 88 patients with BWSp (51 females) were reviewed.

Results: molecular tests had been performed in 86 patients, 80 resulted positives for BWSp-related anomalies, 6 negative with a clinical diagnosis. Seventeen (19.3%) were conceived through artificial reproductive techniques. Twinning occurred in 9 pregnancies and resulted in 13 BWSp newborns. Pregnancy biochemical screening tests showed increased serum markers: PAPP-A at 10-13 weeks of gestational age (n = 44, 1.13 ± 0.61 MoM, p < 0.001), and αFP (1.52 ± 0.80 MoM, p = 0.003), uEstriol (1.41 ± 0.37 MoM, p < 0.001) and total hCG (2.10 ± 2.17 MoM, p < 0.001) at 15-18 weeks (n = 27). Morphology US scan (available in 64) revealed abdominal and head circumferences higher than normal (1.41 ± 1.10 SD, p < 0.001 and 0.44 ± 1.04, p = 0.003, respectively) with normal femur lengths. Fifty-eight patients (66%) had a various combination of pathologic US findings, including enlarged abdominal circumference at 20 weeks’ scan (n = 17), macrosomia at a subsequent scan (n = 35, of these 14 normal at 20 weeks’ scan), omphalocele (n = 12), enlargement of abdominal organs (n = 6), macroglossia (n = 15), adrenal cysts/masses (n = 2), nephroureteral anomalies (n = 11), polyhydramnios (n = 24), placental enlargement (n = 2) or mesenchymal dysplasia (n = 4). Thirty cases had no detected anomaly at any US.

Conclusions: on the basis of these findings, we propose a new embryo-fetal BWSp scoring system, mirroring the post-natal one, that will implement the prenatal clinical approach of this developmental syndrome.

D. Carli: None. C. Bertola: None. S. Cardaropoli: None. V.P. Ciuffreda: None. M. Pieretto: None. G.B. Ferrero: None. A. Mussa: None.


Prenatal diagnosis of a severe form of BICD2-opathy : further evidence of an extreme phenotypic spectrum

E. Marchionni1, E. Agolini2, G. Mastromoro1, D. Guadagnolo1, F. Di Palma1, C. Pajno3, V. D’Ambrosio3, M. Roggini4, A. Giancotti3, A. Novelli2, A. Pizzuti1

1Department of Experimental Medicine, Sapienza University of Rome, Rome, Italy, 2Laboratory of Medical Genetics, Ospedale Pediatrico Bambino Gesù, Rome, Italy, 3Department of Maternal and Child Health and Urologic Science, Sapienza University of Rome, Rome, Italy, 4Pediatrics and Child Neuropsychiatry Department, Sapienza University of Rome, Rome, Italy

Introduction: Arthrogryposis multiplex congenita (AMC) is characterized by the development of multiple joint contractures. Neurogenic and myogenic-types have been described, resulting in complete or partial movement restriction. In prenatal diagnosis decreased fetal movement is observed, the most severe forms being lethal in utero or after birth. We report a case of a male fetus at 22 gestational weeks with evidence of AMC on ultrasound, presenting fixed extended legs and flexed arms, bilateral clubfoot and absent fetal movements. Frontal oedema was also observed. After amniocentesis, karyotype and SNParray resulted normal.

Materials and Methods: Trio-based Whole Exome Sequencing (WES) was performed on fetus genomic DNA and his unaffected parents.

Results: WES analysis disclosed a de novo heterozygous pathogenic deletion (NM_015250.3: c.1636_1638delAAT; p. Asn546del) in BICD2 gene, associated with autosomal dominant spinal muscular atrophy, lower extremity-predominant 2 (SMALED2). The couple decided to terminate the pregnancy. Babygram showed bilateral genu recurvatum, bilateral clubfoot and clubhand and multiple joints contractures.

Conclusions: BICD2 mutations are associated with severe prenatal-onset forms of spinal muscular atrophy, lower extremity-predominant 2B (SMALED2B) or milder forms with childhood-onset (SMALED2A). In literature few prenatal cases are reported, mostly with early lethal outcome. The same variant was previously described in two children with a severe postnatal phenotype but belated or milder prenatal signs. This observation expands BICD2 phenotypic spectrum, with further evidence of early-onset extreme prenatal forms. To date, the localization of the disease-causing mutations does not seem to correlate with disease severity, other molecular mechanisms should be investigated to elucidate genotype-phenotype correlations.

E. Marchionni: None. E. Agolini: None. G. Mastromoro: None. D. Guadagnolo: None. F. Di Palma: None. C. Pajno: None. V. D’Ambrosio: None. M. Roggini: None. A. Giancotti: None. A. Novelli: None. A. Pizzuti: None.


Development and validation of an expanded pan-ethnic carrier screening test

S. Kyriakou, A. Achilleos, M. Georgiadou, C. Loizides, M. Nicoalou, V. Soteriou, C. Kkoufou, L. Constantinou, G. Billioud, K. Tsangaras, E. Kypri, M. Ioannides, G. Koumbaris, P. C. Patsalis

NIPD Genetics, Nicosia, Cyprus

Introduction: Carrier screening increasingly becomes more prevalent as prospective parents become more aware that the cumulative risk of having an affected child with an autosomal or X-linked condition can be higher than that for common aneuploidies. The changing admixture of populations, and the increase in gamete donation in assisted reproductive technologies have led professional organizations and professional bodies to recommend expanded carrier screening to the general population.

Materials and Methods: The test was developed using targeted in-solution hybridization enrichment followed by next generation sequencing covering all gene exons, and intron-exon boundaries, associated with 229 autosomal recessive and X-linked conditions. The sensitivity and specificity for single nucleotide variant (SNV), indel and copy number variant (CNV) detection was ascertained in a blind validation study.

Results: SNVs and indels were detected at high sensitivity (>99.0%) and specificity (>99.9%). The algorithm was also designed to detect CNVs to a single or a few exons with high sensitivity and specificity. Each positive call was confirmed with an orthogonal method.

Conclusions: We have developed and validated an expanded pan-ethnic carrier screening test which includes 229 autosomal recessive and X-linked conditions. The test provides individuals and prospective parents with clinical information, helping them understand and evaluate their reproductive options.

S. Kyriakou: A. Employment (full or part-time); Significant; NIPD Genetics. A. Achilleos: A. Employment (full or part-time); Significant; NIPD Genetics. M. Georgiadou: A. Employment (full or part-time); Significant; NIPD Genetics. C. Loizides: A. Employment (full or part-time); Significant; NIPD Genetics. M. Nicoalou: A. Employment (full or part-time); Significant; NIPD Genetics. V. Soteriou: A. Employment (full or part-time); Significant; NIPD Genetics. C. Kkoufou: A. Employment (full or part-time); Significant; NIPD Genetics. L. Constantinou: A. Employment (full or part-time); Significant; NIPD Genetics. G. Billioud: A. Employment (full or part-time); Significant; NIPD Genetics. K. Tsangaras: A. Employment (full or part-time); Significant; NIPD Genetics. E. Kypri: A. Employment (full or part-time); Significant; NIPD Genetics. M. Ioannides: 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.


CGH-array as first-tier test in all undergoing invasive prenatal samples: an experience for a reference Centre.

M. A. Mori1, J. Nevado1, E. Mansilla1, F. A. Garcia-Santiago1, E. Antolín2, C. Peña1, C. Adam1, P. D. Lapunzina1

1INGEMM-IdiPaz, Madrid, Spain, 2Sº Fisiopatología Fetal, HULP, Madrid, Spain

Introduction: In the last years microarrays (CMA) has demonstrated to be a significant tool useful in the prenatal area, mainly in fetuses with sonography anomalies. However, using array-CGH in an invasive prenatal context is still under a significant and controversial debate especially regarding the use of CMA in advance maternal age or maternal anxiety samples. Previous studies supported the evidence on the feasibility of introducing a-CGH as a first-line diagnostic testing routine prenatal diagnosis practice (1). Indeed, others and our laboratory have reported significant diagnostic yields also in low-risk pregnancies, between 1.6-4% (2,3).

Methods: We used customized Karyoarray®v3.0 platform (4) to study consecutive invasive prenatal samples along two-year period. QF-PCR positive cases were excluded.

Results: We analyzed 800 samples and we found globally 6% of pathogenic CNVs and 2% of VUS. We segregated low-risk pregnancy samples (positive biochemical screening, soft markers, maternal anxiety, and antenatal death without sonographic anomalies). Diagnostic yield in this group was 4.5% (17/379).

Discussion: In conclusion, the use of the array-CGH in the prenatal context demonstrated a greater diagnostic capacity not only in fetuses with ultrasound abnormalities but also in low-risk pregnancies. Therefore, we definitively recommend the possibility of using the array-CGH in all those gestations that undergo invasive prenatal diagnosis, after excluding by QF-PCR the most common aneuploidies. (1)Kan AS. et al., 2014. PLoS One. 9(2):e87988. (2)Wapner RJ. et al., 2012. N Engl J Med.367:2175-2184. (3)Armengol L. et al. 2012. Hum Genet. 131:513-523. (4)Vallespin E. et al,. 2013. Am J Med Genet A.161A:1950-60.

M.A. Mori: None. J. Nevado: None. E. Mansilla: None. F.A. Garcia-Santiago: None. E. Antolín: None. C. Peña: None. C. Adam: None. P.D. Lapunzina: None.



M. Kadkhoda zadeh, s. seyedhassani, f. harrazi zadeh yazdi, a. danafar, m. mirjalili, f. saeb

yazdgenetic, yazd, Iran, Islamic Republic of

Introduction:A 39- year- old mother was referred with GA=16 w + 5 d and high risk pregnancy screening test for T 21. The first child of this patient was a 14- years old suffering from developmental delay and mental retardation. On the other hand, this family had two spontaneous abortion. These evidences revealed enough reasons for probability of chromosomal abnormalities such as Down syndrome, monosomy X, and other chromosomal disorder in fetus. Amniocentesis was suggested for complementary investigation.

Methods and materials: Twenty metaphases cells were studied on the basis of G-Banding technique from culture of amniocytes in two flasks. Then, karyotypig was done on the basis of G-Banding for the parents.

Result: In this study, we described a paternal chromosomal aberration of twins. Through a combination of cytogenetic assessment of fetus and parents 46, XY, der (9) t(2;9) (p23;p22)pat was shown. Mother’s karyotype was 46XX but father’s karyotype revealed an abnormal chromosome complement in all cells with balanced 46,XY,t(2;9)(p23;p22).

Conclusions: Paternal chromosomal balanced translocation can explain repeated abortions in this family. On the other hand, unbalanced chromosomal translocation resulting from paternal inheritance lead to developmental delay in his son. This study was highly emphasized the priority of genetic counseling and attention to the family history and genetic basis of parents in evolution of repeated spontaneous abortion. Legal abortion was recommended to unbalance chromosomal abnormalities.

M. Kadkhoda zadeh: None. S. seyedhassani: None. F. harrazi zadeh yazdi: None. A. danafar: None. M. mirjalili: None. F. saeb: None.


Karyotype analysis of 8531 pregnancies in prenataly identified cases with amniocentesis from south of Turkey

A. Pazarbasi1, D. Alptekin1, N. S. Ilgaz1, I. N. Uslu1, G. Ay1, S. Kocaturk-Sel1, O. Demirhan1, U. Luleyap1, M. B. Yilmaz1, N. Çetinel1, G. Evyapan1, G. Comertpay-Tufan1, S. Buyukkurt2

1Çukurova University, Faculty of Medicine, Dept. of Medical Biology, Adana, Turkey, 2Çukurova University, Faculty of Medicine, Dept. of Obstetrics and Gynecology, Adana, Turkey

Amniocentesis is a very crucial diagnostic procedure for preventing the birth of genetically defective fetuses in order to decrease the prevalence of genetic diseases in populations. A retrospective review of our amniocentesis database for the period from January 2000 to February 2020 was carried out. The karyotyping of 8531 fetuses was carried out in Department of Medical Biology from the samples of amniotic fluids which were sent from Department of Gynecology and Obstetrics of Balcali Hospital. A standart nomenclature has been developed to describe each of types of abnormality found in human chromosomes. A total of 8531 amniocentesis specimens were processed during the study period. 620 fetuses (7.26%) had various chromosomal abnormalities. 54.51% of abnormal karyotypes (338 cases) were numerical and 43.38% (269 cases) were structural. Both numerical and structural chromosomal aberrations were observed in 13 cases (2.09%). The ratios were as: trisomy 21 (49.70%), trisomy 18 (17.75%), monosomy X (9.46%), trisomy 13 (6.80%), Triploidy (4.73%), Klinefelter Syndrome (3.25%), Trisomy X (1. 18%), XYY Syndrome (0.88%), and the others in all numerical abnormalities. The frequent structural abnormalities were as: 46,XX/XY, inv(9) (p11;q12)/(p11;q13)(28.25 %), 46,XX/XY, 1qh(+)(11.89%), 46,XY, Yqh(-)(7.43%), 46,XX/XY, 16qh(+)(7.06%), 46,XX/XY, 9qh(+)(4.46%) and 46,XY, Yqh(+)(4.08%). Balanced and unbalanced translocations, deletions and duplications were also found in less ratio. According to the literature and our results, advanced maternal age is the main cause of fetal chromosomal abnormalities. Fetal chromosomal abnormality ratio that we found was 7.26%. This ratio emphasize the importance of prenatal diagnosis.

A. Pazarbasi: None. D. Alptekin: None. N.S. Ilgaz: None. I.N. Uslu: None. G. Ay: None. S. Kocaturk-Sel: None. O. Demirhan: None. U. Luleyap: None. M.B. Yilmaz: None. N. Çetinel: None. G. Evyapan: None. G. Comertpay-Tufan: None. S. Buyukkurt: None.


Microarray testing in pregnancies with nuchal translucency 3-3.4 mm - time to change the cut-off.

L. Sagi-Dain1, I. Maya2, A. Singer3

1Carmel Medical Center, Haifa, Israel, 2Rabin Medical Center, Petach Tikva, Israel, 3Sheba Medical Center, Ramat Gan, Israel

Objective: To examine the risk for chromosomal microarray analysis (CMA) aberrations in fetuses with nuchal translucency (NT) of 3 to 3.4 mm. Methods: The results of all CMA tests performed due to an NT of 3 to 3.4 mm were retrospectively retrieved from the Ministry of Health computerized database. The rates of clinically significant (pathogenic and likely pathogenic) microarray findings were compared to a previously published local control population, encompassing 5541 fetuses with normal ultrasound. Results: Overall, 619 CMA analyses were performed due to a sonographic indication of NT of 3 to 3.4 mm. Of these, 29 (4.7%) clinically significant copy number variants were detected, a considerably increased risk compared to control pregnancies. Fetal karyotyping would have missed 31% of the abnormal cases, while non-invasive prenatal screening (NIPS) would have overlooked 31-49% of the anomalies, depending on the method. Conclusions: The outcomes of our study, representing the largest number of CMA results in fetuses with NT of 3 to 3.4 mm, suggest that the rate of abnormal CMA findings in these pregnancies is significantly higher compared to pregnancies with normal ultrasound. Our results support the consideration to perform invasive prenatal testing for CMA in pregnancies with NT above 3 mm. In addition, as several of the CMA findings in these pregnancies are not associated with major sonographic anomalies, it seems that NIPS with later sonographic survey cannot adequately replace NT measurement.

L. Sagi-Dain: None. I. Maya: None. A. Singer: None.


Three fetuses with achondrogenesis type 2 explained by maternal mosaicism

S. Z. Jan1, R. F. Suijkerbuijk1, Y. J. Vos1, P. M. Grootscholten1, L. Zijlstra1, K. Bouman1, M. T. M. Franssen2, M. H. Schoots3, N. Corsten-Janssen1, B. Sikkema-Raddatz1

1Univeristy Medical Center Groningen, Department of Genetics, Groningen, Netherlands, 2Univeristy Medical Center Groningen, Department of Obstetrics, Groningen, Netherlands, 3Univeristy Medical Center Groningen, Department of Pathology and Medical Biology, Groningen, Netherlands

Background: Adding whole exome sequencing (WES) to the standard diagnostic procedure in fetuses with ultrasound anomalies improves the diagnostic yield. Identifying a diagnosis is important for parental choice and informed reproductive decision making, amongst other factors. Here we describe three fetuses in whom a diagnosis of achondrogenesis type 2 (COL2A1 variant) due to maternal mosaicism was made using WES.

Materials & Methods: A couple expecting dichorionic diamniotic twins, both with severe limb shortening and fetal hydrops, was referred to our department. A previous pregnancy had been terminated for the same reasons. QF-PCR and SNP array were performed on DNA of chorionic villi from both fetuses of the current pregnancy. WES was performed on parental DNA (extracted from blood) and fetal DNA.

Results: QF-PCR and SNP-array showed no abnormalities for both fetuses. WES revealed a pathogenic, apparently de novo, COL2A1 variant c.3062_3079del p.(Pro1021_Gly1026del) in both samples, which explained the ultrasound abnormalities. Retrospective analysis confirmed the presence of the same COL2A1 variant in the previous pregnancy. Re-analysis of parental WES data revealed the variant in less than 1% of maternal reads, implying an extremely low grade of mosaicism, which was confirmed by Sanger sequencing.

Conclusions: This case study highlights the added value of WES in prenatal diagnostics, including the capacity to identify extremely low-grade parental mosaicism that profoundly impact the recurrence risk of a genetic disease. It also suggests that the possibility of very low-grade parental mosaicism should be closely investigated when an apparently de novo variant is identified.

S.Z. Jan: None. R.F. Suijkerbuijk: None. Y.J. Vos: None. P.M. Grootscholten: None. L. Zijlstra: None. K. Bouman: None. M.T.M. Franssen: None. M.H. Schoots: None. N. Corsten-Janssen: None. B. Sikkema-Raddatz: None.


Prenatal diagnostics in Estonia: a 2-year prospective study assessing the performance of combined first-trimester screening, chromosomal microarray analysis, and next-generation sequencing

K. Ridnõi1,2, K. Muru1,3, M. Keernik1, S. Pajusalu1,3,4, E. Ustav5, P. Tammur1, T. Mölter-Väär1, T. Kahre1,3, U. Šamarina1, K. Asser5, F. Szirko2, T. Reimand1,3,6, K. Õunap1,3

1Department of Clinical Genetics, United Laboratories, Tartu University Hospital, Tartu, Estonia, 2Centre for Perinatal Care, Women’s Clinic, East-Tallinn Central Hospital, Tallinn, Estonia, 3Department of Clinical Genetics, Institute of Clinical Medicine, University of Tartu, Tartu, Estonia, 4Yale University School of Medicine, Department of Genetics, New Haven, CT, United States, 5Women’s Clinic, Tartu University Hospital, Tartu, Estonia, 6Department of Biomedicine, Institute of Biomedicine and Translational Medicine, University of Tartu, Tartu, Estonia

Introduction: The implementation of cell-free fetal DNA (cff-DNA) testing in maternal blood opened possibilities to improve performance of combined first trimester screening (cFTS). On the other hand, use of new molecular methods, such as chromosomal microarray analysis (CMA) and next-generation sequencing (NGS), have shown benefits in diagnosis of chromosomal and genetic diseases, which are not detectable with cff-DNA screening, but require invasive procedure. We prospectively evaluated performance of cFTS in Estonia during 2 years using national guidelines with application of CMA and NGS in high-risk pregnancies.

Material and methods: We investigated 14,566 singleton pregnancies (60% of all pregnancies). We selected 334 high-risk pregnancies for CMA diagnostic performance evaluation and 28 cases of highly dysmorphic fetuses for NGS analysis. CMA study group was divided into two groups based on the indications for testing: group A patients with high-risk for trisomies after cFTS, but normal ultrasound and group B patients who met criteria for CMA as a first-tier diagnostic test.

Results: We found the first trimester detection rate 94%, 100% and 100% for trisomies 21, 18 and 13, respectively, with false-positive rate of 3.1%. The diagnostic yield of CMA was overall 3.6% (1.7% in Group A and 6.0% in Group B). In NGS analysis group we report diagnostic yield of 17.9%. Combining these three methods, overall detection rate of all genetic anomalies was 101 (0.7%).

Conclusions: the use of CMA and NGS in high-risk pregnancies after cFTS is justified and increase diagnostic yield. Funding: Estonian Research Council grants PUT355, PRG471, and PUTJD827.

K. Ridnõi: None. K. Muru: None. M. Keernik: None. S. Pajusalu: None. E. Ustav: None. P. Tammur: None. T. Mölter-Väär: None. T. Kahre: None. U. Šamarina: None. K. Asser: None. F. Szirko: None. T. Reimand: None. K. Õunap: None.


Higher incidence of congenital anomalies in children born after assisted reproduction in the Czech Republic: Population based study

A. Sipek Jr1,2,3, V. Gregor2,4, J. Klaschka5,6, M. Maly5,7, A. Sipek Sr2,4,8

1Institute of Biology and Medical Genetics, First Faculty of Medicine, Charles University, Prague, Czech Republic, 2Department of Medical Genetics, Thomayer Hospital, Prague, Czech Republic, 3Institute of Medical Genetics, Third Faculty of Medicicne, Charles University, Prague, Czech Republic, 4Department of Medical Genetics, Pronatal Sanatorium, Prague, Czech Republic, 5Institute of Computer Science of the Czech Academy of Sciences, Prague, Czech Republic, 6Institute of Biophysics and Informatics, First Faculty of Medicine, Charles University, Prague, Czech Republic, 7National Institute of Public Health, Prague, Czech Republic, 8GENNET, Prague, Czech Republic

Introduction: The number of children conceived by assisted reproduction techniques (ART) is increasing in the Czech Republic. The main goal of this study was to evaluate the incidence of congenital anomalies in ART conceived children.

Methods: The retrospective epidemiological analysis was performed using data from the National Registry of Congenital Anomalies and National Registry of Newborns, run by the Institute of Health Information and Statistics of the Czech Republic. All diagnoses of congenital anomalies (Q00-Q99) were included. We compared the incidences of congenital anomalies in naturally conceived children and ART conceived children born in the Czech Republic during 5 years period (2013-2017). Statistical analysis was performed by Fisher’s exact test.

Results: During the selected period there were 547 675 children born in the Czech Republic (531 064 were naturally conceived children and 16 611 were ART conceived children). The incidence of congenital anomalies was 3.90% in naturally conceived children group and 4.35% in ART conceived children. This difference is statistically significant (p = 0.003). Analyzing different subgroups of congenital anomalies we have found a significantly increased incidence of anomalies in two subgroups: Congenital malformations of the circulatory system (Q20-Q28) and Congenital malformations of the respiratory system (Q30-Q34).

Discussion: The increased risk of congenital anomalies in ART conceived children was highlighted in many studies however the results are often diverse. In our population-based study, we confirmed a significantly higher general incidence of congenital anomalies in ART conceived children.

The study is supported by the RVO project: “Thomayerova nemocnice - TN, 00064190”.

A. Sipek Jr: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Modest; Research grant provided by Ministry of Health of the Czech Republic, RVO project: “Thomayerova nemocnice TN, 00064190”. V. Gregor: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Modest; Research grant provided by Ministry of Health of the Czech Republic, RVO project: “Thomayerova nemocnice TN, 00064190”. J. Klaschka: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Modest; Research grant provided by Ministry of Health of the Czech Republic, RVO project: “Thomayerova nemocnice TN, 00064190”. M. Maly: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Modest; Research grant provided by Ministry of Health of the Czech Republic, RVO project: “Thomayerova nemocnice TN, 00064190”. A. Sipek Sr: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Modest; Research grant provided by Ministry of Health of the Czech Republic, RVO project: “Thomayerova nemocnice TN, 00064190”.


A low-cost microsatellite marker method for quick andA low-cost microsatellite marker method for quick and reliable prenatal 22q11.2DS screening and detection in fetuses with suspected congenital heart defects

L. Torres-Juan1, M. Coll-Ferrer1, M. Prado-Farnos1, R. Martorell-Riera1, E. Amengual-Cloquell1, A. Perez-Granero1, A. Tubau2, M. Juan2, J. Rosell1, I. Martinez-Lopez1, D. Heine-Suñer1

1Institute of Health Research of the Balearic Islands (IDISBA) and Hospital Son Espases, Palma, Spain, 2Institute of Health Research of the Balearic Islands (IDISBA) and Hospital Son Llatzer, Palma, Spain

Congenital heart defects (CHD) are the most common form of birth defects and affect 1% of newborns and an even higher percentage of unborn fetuses. CHDs are a frequent finding during routine ultrasound prenatal screening. Once detected, it is important to determine their etiology. The 22q11.2 deletion syndrome (22q11.2 DS) is the most frequent known genetic cause of CHDs (1.9% prevalence). We present a multiplex marker PCR kit to detect 22q11.2 deletions and duplications which we prospectively used in 1630 individuals and 450 fetal cases with a congenital heart defect referred by an obstetrician. Important features: 1) Low cost (there are 2 mixes of 11 pairs of primers and 2 conventional PCR reactions). 2) Fast, results can be given within hours of receiving the sample (DNA extraction -> PCR 1.5 hours -> automatic sequencer). 3) Safely rules out sample contamination by maternal DNA. 4) Uses small amounts of DNA (10 ng) and can be performed starting from amniotic fluid or non-cultured chorion biopsy or any other tissue. 5) In prenatal samples, it would be complementary to the QF-PCR (negative samples for trisomy 13, 18 or 21). 6) The parental origin of the deletion/duplication is determined when the fetus is analyzed with the parents. 7) Detects atypical and typical deletions, extending the analysis far beyond that given by the FISH probe that has been historically used (TUPLE1). 8) We have developed a panel of 31 additional markers, not included in the multiplex, to be used in case of lack of informativity.

L. Torres-Juan: None. M. Coll-Ferrer: None. M. Prado-Farnos: None. R. Martorell-Riera: None. E. Amengual-Cloquell: None. A. Perez-Granero: None. A. Tubau: None. M. Juan: None. J. Rosell: None. I. Martinez-Lopez: None. D. Heine-Suñer: None.


The effects of the CFTR genotype on the reproductive system in adult male patients with cystic fibrosis

S. Repina1, S. Krasovskiy1,2, M. Shtaut1, G. Shmarina1, T. Adyan1, V. Chernykh1

1Research Centre for Medical Genetics, Moscow, Russian Federation, 2Pulmonology Research Institute, Moscow, Russian Federation

Background: Commonly male patients with Cystic Fibrosis (CF) is infertile due to obstructive azoospermia (OA), at that an influence of CFTR genotype on reproductive system is poor understood.

Materials and methods: We examined 93 Russian male CF patients (17-45 y.o., mean - 24.9 ± 5.7 y.o.). Physical examination, scrotal ultrasonography, hormonal, biochemical, and semen analysis were done. Standard and biochemical semen examination were done according to the WHO’s (2010) recommendations. The CFTR gene variants were analyzed using by AFPL, MLPA and DNA sequencing (Sanger’s method and MPS).

Results: CFTR mutations were detected in 100% CF alleles. Folowing genotypes were found: F508del/F508del (20.4%); F508del/3849+10kbC>T (15.1%); F508del/2789+5G>A (4.3%); F508del/CFTRdele2.3 (4.3%); F508del/E92K (3.2%); F508del/L138ins (3.2%); F508del/3272-16T>A (2.2%); F508del/W1282X (2.2%); others (45.3%). The spectrum of CFTR gene pathogenic variants and genotypes showed high frequency of “mild” pathogenic variants (25.8%) and genotypes (50.5%), as well as a relatively high frequency of 3849+10kbС>T variant (12.4%), which was identified in 8.2% patients with OA and 94.4% with no OA (p < 0.00001). Statistically significant difference was found between patients with F508del/3849+10kbC>T and F508del/F508del genotypes for a prevalence of OA/seminal ducts obstruction (14.3% vs 100%, p < 0.05) and testicular hypoplasia (26.9% vs 66.7%, p = 0.03).

Conclusions: CF dramatically affects reproductive organs, male fertility and semen parameters in CF patients, at that 3849+10kbС>T mutation in the CFTR genotype is major genetic factor of male fertility.

S. Repina: None. S. Krasovskiy: None. M. Shtaut: None. G. Shmarina: None. T. Adyan: None. V. Chernykh: None.


First and second trimester biochemical screening: additional value in the era of NIPS

R. Michaelson-Cohen1,2,3, A. Lahad3,4

1Medical Genetics Institute, Shaare Zedek Medical Center, Jerusalem, Israel, 2Department of Obstetrics and Gynecology, Shaare Zedek Medcial Center, Jerusalem, Israel, 3Hebrew University, Jerusalem, Israel, 4Clalit Health Services, Jerusalem, Israel

Background: Since implementation of NIPS, first and second trimester biochemical tests may no longer be necessary for Down Syndrome screening. However, studies suggest their ability to predict pregnancy complications. We sought to evaluate whether screening tests have value for this purpose.

Methods: Our cohort included all women who had first and/or second trimester screening from 2015-2018 and subsequently delivered in our center. Data was collected on demographics, medical history, biochemical screening and maternal-fetal pregnancy outcomes. Logistic regression was performed to examine correlations between biochemical markers and pregnancy complications using SPSS version 25.0 (SPSS, Inc).

Results: Over 3.5 years, 5400 women had screening tests and later delivered in our center. Decreased PAPP-A was associated with preeclampsia, gestational diabetes, IUGR and preterm delivery (PTD), OR=1.792, 1.364, 1.403 and 1.277, respectively, p < 0.005. Elevated AFP was associated with PTD, maternal blood administration, IUGR and low 5-minute Apgar-score, OR=2.044, 1.95, 1.452 and 1.22, respectively, p < 0.033. Decreased HCG in second trimester was correlated with IUGR, OR=1.346, p < 0.05. Elevated HCG in second trimester was correlated with PTD and newborn NICU admission, OR=1.192, 1.285, respectively, p < 0.015. In both first and second trimesters, elevated HCG was associated with preeclampsia, OR= 1.169, 1.309, respectively, p < 0.05. Low estriol was correlated with IUGR, OR=3.597, p < 0.001. After controlling for confounders (age, ethnicity, IVF, parity, etc.) most associations remained significant.

Discussion: Despite superiority of NIPS for Down Syndrome screening, biochemical screening is still valuable for prediction of pregnancy complications. Identifying women with high-risk pregnancies has clinical implications for prenatal care and risk-reducing management strategies.

R. Michaelson-Cohen: None. A. Lahad: None.


Use of prenatal exome sequencing in fetuses with ultrasound anomalies

M. Segura-Puimedon, B. Campos, J. Luna, C. Sintas, M. de Castro-Miró, H. Díez, S. B. Estruch, R. Garcia, L. Quintana, J. Rodríguez, L. Armengol

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

Introduction: Whole exome sequencing (WES) is an established diagnostic tool in postnatal settings for individuals with a suspected genetic condition. Recently, it is increasingly used as a diagnostic tool in prenatal settings as well. We present here our experience using this technology in fetuses with ultrasound anomalies.

Material and Methods: WES was performed in 166 fetal samples with ultrasound anomalies. 104 samples were from evolutive pregnancies and 62 were from legal interruptions or stillbirths. In 61 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 specific syndrome suspicion. Pathogenic or likely pathogenic variants were identified in n = 42 (25%) of samples. In n = 40 (24%) cases, variants of unknown significance were identified, and in 13 of them inheritance was consistent with expected pattern, including a probable mosaicism case. In half of the cases (50,6%) no candidate variant was identified. Diagnostic yield was higher in fetuses with skeletal anomalies, where pathogenic or likely pathogenic variants were identified in (n = 10) 31% of cases, and in fetuses with increased nuchal translucency or hidrops (Noonan syndrome suspicion), where a pathogenic variant was found in 24% (n = 6) of the samples. No pathogenic variants were found in fetuses with cardiovascular anomalies (n = 9).

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

M. Segura-Puimedon: A. Employment (full or part-time); Significant; Quantitative Genomic Medicine Laboratories, qGenomics, Barcelona, Spain. B. Campos: A. Employment (full or part-time); Significant; Quantitative Genomic Medicine Laboratories, qGenomics, Barcelona, Spain. J. Luna: A. Employment (full or part-time); Significant; Quantitative Genomic Medicine Laboratories, qGenomics, Barcelona, Spain. C. Sintas: A. Employment (full or part-time); Significant; Quantitative Genomic Medicine Laboratories, qGenomics, Barcelona, Spain. M. de Castro-Miró: A. Employment (full or part-time); Significant; Quantitative Genomic Medicine Laboratories, qGenomics, Barcelona, Spain. H. Díez: A. Employment (full or part-time); Significant; Quantitative Genomic Medicine Laboratories, qGenomics, Barcelona, Spain. S.B. Estruch: A. Employment (full or part-time); Significant; Quantitative Genomic Medicine Laboratories, qGenomics, Barcelona, Spain. R. Garcia: A. Employment (full or part-time); Significant; Quantitative Genomic Medicine Laboratories, qGenomics, Barcelona, Spain. L. Quintana: A. Employment (full or part-time); Significant; Quantitative Genomic Medicine Laboratories, qGenomics, Barcelona, Spain. J. Rodríguez: A. Employment (full or part-time); Significant; Quantitative Genomic Medicine Laboratories, qGenomics, Barcelona, Spain. L. Armengol: A. Employment (full or part-time); Significant; Quantitative Genomic Medicine Laboratories, qGenomics, Barcelona, Spain.


AnDDI-Prenatome: The French exome sequencing study in prenatal diagnosis: causal diagnosis identified in nearly half of fetuses

F. Tran Mau Them1,2, A. Bruel1,2, A. Vitobello1,2, S. Nambot3, S. Moutton3, A. Sorlin1,2,3, J. Delanne3, A. Ziegler4, E. Colin4, A. Guichet4, M. Legendre5, C. Colson6,7, C. Abel8, A. Putoux9, J. Amiel10,11, A. Lavillaureix12, C. Quelin12, A. Guerrot13, A. Brehin13, L. Faivre3,2, C. Philippe1,2, V. Couturier1,2, Y. Duffourd2, C. Thauvin-Robinet1,2,14

1Unité Fonctionnelle d’Innovation diagnostique des maladies rares, FHU-TRANSLAD, Dijon, France, 2Inserm – UB UMR 1231 GAD « Génétique des Anomalies du Développement », FHU-TRANSLAD, Dijon, France, 3Centre de Référence Maladies rares « Anomalies du Développement et syndromes malformatifs », Centre de Génétique, FHU-TRANSLAD, Dijon, France, 4Biochemistry and Genetics Department, University Hospital of Angers, Angers, France, 5Service de Génétique Médicale, CHU de Bordeaux – Hopital Pellegrin, Bordeaux, France, 6CHU Lille, Clinique de Génétique Guy Fontaine, Lille, France, 7EA7364, RADEME, Université de Lille, Lille, France, 8Service de génétique et centre de diagnostic anténatal, Lyon, France, 9Service de Génétique, Centre de Référence Anomalies du Développement et Syndromes Malformatifs, Lyon, France, 10Service de Génétique Médicale et Clinique, Hôpital Necker-Enfants Malades, Paris, France, 11Equipe "Embryologie et Génétiques des malformations congénitales", Institut Imagine - INSERM U1163, Paris, France, 12Centre de Référence Maladies Rares CLAD-Ouest, CHU Hôpital Sud, Rennes, France, 13Normandie Univ, UNIROUEN, Inserm U1245 and Rouen University Hospital, Department of Genetics and Reference Center for Developmental Disorders, F 76000, Normandy Center for Genomic and Personalized Medicine, Rouen, France, 14Centre de Référence « Déficiences Intellectuelles de causes rares », Centre de Génétique, FHU-TRANSLAD, Dijon, France

Introduction: Exome sequencing (ES) is the most powerful and current test for patients with developmental abnormalities (DA). It has considerably been deployed in postnatal indications with a diagnostic yield ranging from 30 to 40%. Several prenatal studies report a diagnostic yield ranging from 15% to 35%, depending on the exome strategy and the inclusion criteria. The French national network for rare diseases with AD deployed a pilot project of feasibility for rapid prenatal exome sequencing.

Materials and methods: We aimed to include 61 fetuses and both parents. The main inclusion criteria were: 2 major malformations or, 1 major and 1 minor malformation or, 1 malformation and a strong genetic suspicion. The feasibility aim was to emit a diagnostic report in 5-6 weeks. Array-CGH and ES-trio were concomitantly performed. ES-trio was captured with the TWIST-v2 kit and sequenced on a NovaSeq6000 device with a 70X average depth.

Results: We included 23 patients. The turnaround time from receipt of samples to the diagnostic report was on average of 27 days for the negative patients and of 40 days for the positive patients. The diagnostic yield was of 44% with 7 SNV and 3 CNV, including a tetrasomy 12p. Array-CGH allowed a diagnosis in 13% and exome in 44%. Results were concordant for CNV detection between array-CGH and ES-trio.

Discussion: Prenatal trio-ES appears feasible in a useful time and strongly helpful for decision in 65 % and genetic counseling was impactful in at least 44%.

F. Tran Mau Them: None. A. Bruel: None. A. Vitobello: None. S. Nambot: None. S. Moutton: None. A. Sorlin: None. J. Delanne: None. A. Ziegler: None. E. Colin: None. A. Guichet: None. M. Legendre: None. C. Colson: None. C. Abel: None. A. Putoux: None. J. Amiel: None. A. Lavillaureix: None. C. Quelin: None. A. Guerrot: None. A. Brehin: None. L. Faivre: None. C. Philippe: None. V. Couturier: None. Y. Duffourd: None. C. Thauvin-Robinet: None.


Preconception carrier screening in consanguineous couples by whole exome sequencing

A. Abulí, I. Valenzuela, M. Codina, E. Rovira, D. Palau, S. Ourani, A. Cueto-González, I. Paramonov, E. García-Arumí, E. Tizzano, I. Cusco

Hospital Vall d’Hebron, Barcelona, Spain

Consanguineous couples have an increased risk of severe abnormalities and mortality in offspring due to autosomal recessive disorders. Exome-sequencing provides the possibility of wide preconception carrier screening (PCS) in consanguineous couples who may be at risk of rare genetic disorders. The efficacy of this strategy has been poorly explored. We retrospectively analyzed exome sequencing data from 27 affected children and 3 fetuses (after termination of pregnancy) of consanguineous couples. We explored diagnostic yield and carrier status for recessive disorders. Overall diagnostic yield was 67% (20/30 patients). We detected homozygous causative variants in 19 cases and one de novo heterozygous variant. Out of 20 causal variants, 18 were classified as pathogenic or likely pathogenic and two as unknown significance, which were reclassified as likely pathogenic because of highly specific phenotype. Based on our results and considering diagnostic yield and variant classification, causative pathogenic recessive mutations would have been detected in at least 60% of consanguineous couples by preconception exome-sequencing. Most of these conditions (16/20) would not have been detected in PCS panels for most prevalent diseases considering a carrier frequency of 1/250. Most of the patients were carriers of one additional heterozygous recessive mutation (70%, 21/30) and a smaller number were carriers of two (17%, 5/30) or more (13%, 4/30). The average carrier burden of autosomal recessive conditions was of 1.7 pathogenic mutations per patient. PCS by exome-sequencing increases the detection rate of rare recessive conditions in consanguineous couples and could potentially increase their reproductive autonomy and facilitate informed decision making.

A. Abulí: None. I. Valenzuela: None. M. Codina: None. E. Rovira: None. D. Palau: None. S. Ourani: None. A. Cueto-González: None. I. Paramonov: None. E. García-Arumí: None. E. Tizzano: None. I. Cusco: None.


Does maternal blood exosomal DNA effective for the fetal RHD and SRY genotyping?

B. Yaşa1, O. Şahin2, S. Sözer1

1Aziz Sancar Institute of Experimental Medicine, Istanbul, Turkey, 2Clinic of Obstetrics and Gynecology, Okmeydanı Training and Research Hospital, Istanbul, Turkey

Introduction: Recent advances have led to the development of important techniques in extracellular fetal DNA derived from maternal blood for prenatal diagnosis. Exosomes’ DNA content is not affected structurally by nuclease activity and have a potential clinical application. Determination of fetal Rhesus D(RhD) and sex is important for prenatal diagnosis in some diseases.

Material And Method: Fetal RHD and SRY genotyping was performed using exosomal DNA(exoDNA) from peripheral blood of 81 RhD negative pregnant women with exosome isolation kit. Specific primers were applied for the SRY and RHD genes amplifications by Real-Time polymerase chain reaction. The average age of the cases in the study was 28.5 ± 5.5(year ± SD). The mean gestational week of the cases was 25.81 ± 9.02. The exoDNA concentrations obtained were between 1.8-21.9 ng/μL with an average of 11.65 ± 8.08 ng/μL.

Results: The results were confirmed with the hospital’s New Born Follow Up System. After analysis of the 81 cases, no amplification was observed in 16 samples, including the GAPDH gene. 100% accuracy was achieved in the RHD and SRY detection from the remaining 65 samples.

Conclusions: It was determined that fetal RHD and SRY genotyping could be performed with exoDNA from maternal plasma. The isolation technique of exoDNA could be enhanced and improved for high concentration with more sensitive detection covering early pregnancy age. This technique might provide a new perspective to the prenatal diagnosis as well as to the treatment options all which require more detailed studies. The present work was supported by the Research Fund of Istanbul University. Project No. 29083

B. Yaşa: None. O. Şahin: None. S. Sözer: None.


Population-based preconception expanded carrier screening for severe recessive disorders: Does a GP-provided test offer with couple results meet criteria for responsible implementation?

J. Schuurmans1,2, M. Plantinga1, E. Birnie1, K. M. Abbott1, A. D. Diemers1, D. M. Van der Kolk1, L. M. Van den Heuvel1, M. Y. Berger1, M. Verkerk1, A. Fenwick2, A. V. Ranchor1, A. Lucassen2, I. M. Van Langen1

1University Medical Center Groningen, Groningen, Netherlands, 2University of Southampton, Southampton, United Kingdom

Introduction: Expanded carrier screening (ECS) enables the identification of couples who are at increased risk of having children with recessive conditions. Based on preliminary research, we conducted a pilot study of GP-provided population-based preconception ECS (2016-2018) to evaluate whether this approach would meet criteria for responsible implementation as specified by Henneman et al., EJHG, 2016. The free test-offer consisted of 50 severe early-onset childhood conditions. Only couple-results were reported. We discuss key insights to guide large-scale implementation.

Methods: We used a mixed-methods approach. Trained GPs from nine practices invited their female patients (n = 4295, aged 18-40) and partners. We addressed uptake, feasibility (e.g. pre-test counselling possible within 20 minutes), informed-choice, psychological outcomes (e.g. anxiety, decisional conflict) and reproductive intentions using a longitudinal survey (couples) and semi-structured interviews (GPs).

Results: 190 couples were included. 130 (approximately 15% of the eligible population) accepted the test-offer within one month and attended pre-test counselling. 117 (90%) proceeded with testing. More than 90% of test-participants made an informed-choice. GPs felt confident to conduct counselling, although having to make a GP-appointment with both partners was a barrier. Most participants reported low anxiety and decisional conflict; this was relatively higher in test-offer decliners than acceptors.

Conclusions: Population-based preconception ECS for a specific set of severe conditions provided by motivated and trained GPs is feasible. We will discuss how responsible implementation can best be facilitated. The possibility of prenatal ECS, ECS to high-risk couples or which HCPs should offer this (e.g. midwives, fertility clinics) should now also be investigated.

J. Schuurmans: None. M. Plantinga: None. E. Birnie: None. K.M. Abbott: None. A.D. Diemers: None. D.M. Van der Kolk: None. L.M. Van den Heuvel: None. M.Y. Berger: None. M. Verkerk: None. A. Fenwick: None. A.V. Ranchor: None. A. Lucassen: None. I.M. Van Langen: None.


Donor oocyte pregnancies and fetal fraction: managing patient expectations and providing accurate information

M. Pastrick, M. K. Maisenbacher, G. Goldring, W. DiNonno, A. Ryan

Natera, Inc, San Carlos, CA, United States

Objective: Retrospective analysis to determine if differences in fetal fraction (FF) are observed in donor oocyte pregnancies compared to the general population.

Methods: Noninvasive prenatal testing samples from singleton pregnancies were analyzed at a single reference lab. NIPT was performed using a SNP-based method with FF measured as previously described.1 FF from 1611 donor oocytes was analyzed and compared to a large set of reference cases matched for maternal weight (MW) and gestational age (GA). A z-score was calculated for each donor oocyte compared to its reference data. If no impact to FF from the use of donor or in vitro fertilization (IVF), the average z-score is expected to be zero. Statistical analysis was performed using a z-test to establish if this was the case.

Results: The average z-score for the donor oocyte cases was -0.4. A z-test determined this deviation from normal to be significant (p < 0.00001), indicating lower FF in donor oocyte cases vs. corresponding reference data.

Conclusions: Previous studies have reported lower FF in patients undergoing IVF and in donor oocyte populations.2,3 Lower FF has also been associated with increased MW, early GA, certain maternal health conditions, and abnormal fetal results (T18/T13/triploidy).2,3,4 Our results reveal statistically significant lower FF in donor oocyte pregnancies compared to matched reference data.

References 1. Zimmerman et al., Prenatal Diagnosis 2012; 32: 1-9 2. Revello R. et al., Ultrasound Obstet Gynecol. 2016 Jun;47(6):698-704. 3. Yanaihara A. et al., J Assist Reprod Genet. 2018 Mar;35(3):431-434. 4. Ebbing C. et al., PLoS One. 2013 Jul 30;8(7):e70380.

M. Pastrick: A. Employment (full or part-time); Significant; Natera, Inc. M.K. Maisenbacher: A. Employment (full or part-time); Significant; Natera, Inc. G. Goldring: A. Employment (full or part-time); Significant; Natera, Inc. W. DiNonno: A. Employment (full or part-time); Significant; Natera, Inc. A. Ryan: A. Employment (full or part-time); Significant; Natera, Inc.


Repeat Associated Non-AUG (RAN)-Translation in fragile X associated Premature Ovarian Insufficiency (FXPOI): FMRpolyG as predictive tool?

X. Nguyen1, B. Messmer1, K. Hinderhofer2, T. Strowitzki3, J. Rehnitz1,3

1Division of Reproduction Genetics, Department of Gynecological Endocrinology and Fertility Disorders, University Women’s Hospital, Heidelberg, Germany, 2Institute of Human Genetics, University Heidelberg, Laboratory of Molecular Genetics, Heidelberg, Germany, 3Department of Gynecological Endocrinology and Fertility Disorders, University Women’s Hospital, Heidelberg, Germany

Fragile-X-associated-primary-ovarian-insufficiency (FXPOI) is characterized by oligo/amenorrhea and hypergonadotropic hypogonadism by reason of the expansion of CGG-repeats in the 5′UTR of FMR1, called permutation (PM) (n: 55-200). RAN-translation depending on variable CGG-repeat length is also hypothesized to cause FXPOI due to the production of a polyglycine-containing FMR1-protein, FMRpolyG. FMRpolyG expression in granulosa cells (GCs) of FXPOI-model-mice leads to perturbed ovarian function. However, the expression of FMRpolyG in leukocytes and GCs of women with different ovarian response and variable CGG repeat length has not been investigated yet. We performed Western-blot (WB) to analyze the expression of FMRP and FMRpolyG in patient´s leukocytes and GCs in parallel with FMR1-gene-expressionanalysis. Firstly, we detected FMRP, both in leukocytes and GCs of women with variable ovarian reserve and different CGG-repeat-length. FMR1/FMRP was detected in a larger amount in GCs than in leukocytes, since it is a major regulator of folliculogenesis. We therefore started a pilot project investigating if variable expression-levels of FMRP and FMRpolyG detected by WB can be related to women with different ovarian reserve parameters and variable CGG repeats including “normal” and premutated ranges. Preliminary data already showed, in a PM patient undergoing oocytes cryopreservation because of her risk of developing FXPOI, a decreased level of FMRP, even though the FMR1 transcription level was within the normal range, fitting to the hypothesis of kind of a perturbed protein production in this patient. We plan to further evaluate if results made in GCs can be translated to leukocytes. This would offer prospectively a predictive tool or marker of progression.

X. Nguyen: None. B. Messmer: None. K. Hinderhofer: None. T. Strowitzki: None. J. Rehnitz: None.


Investigation of single nucleotide variants (SNVs) in a fertile population in association with oocyte and embryo quality

G. Kakourou1, T. Mamas1, C. Vrettou1, E. Tsorva2, S. Zacharia2, C. Oraiopoulou3, E. Chatzi4, G. Kourlaba5, D. Christopikou2, M. Mastrominas2, M. Moisidou3, A. Chatziparasidou3, I. Georgiou4, J. Traeger-Synodinos1

1Department of Medical Genetics, National & Kapodistrian University of Athens, St. Sophia’s Children’s Hospital, Athens, Greece, 2Embryogenesis, Assisted Reproduction Unit, Athens, Greece, 3Embryolab SA, IVF Unit, Thessaloniki, Greece, 4Genetics and IVF Unit, Department of Obstetrics and Gynecology, Faculty of Medicine, University of Ioannina, Ioannina, Greece, 5Center for Clinical Epidemiology and Outcomes Research (CLEO), Athens, Greece

Introduction: Human and animal studies have provided information on a number of genes potentially impacting gamete quality and preimplantation development (with a role on meiosis, mitosis, DNA repair etc.). SNVs in these genes have been investigated in mostly infertile couples undergoing IVF, in search of potential biomarkers of IVF success. Recently, maternal SNVs in infertile women (rs1801133/rs1801131 in MTHFR, rs2305957 on chromosome 4 linked to INTU-SLC25A31-HSPA4L-PLK4-MFSD8-LARP1B and PGRMC2) were associated with embryo quality or chromosomal status. We investigated the potential association of SNVs in 26 selected genes with a possible role in preimplantation development, with oocyte/embryo quality in fertile women undergoing Preimplantation Genetic Testing for Monogenic Disorder (PGT-M).

Materials/Methods: Maternal DNA was genotyped using exonic NGS (QiaseqTM-Targeted Custom Panel, Miseq) for 18 genes (AIRE-AMH-AURKA-AURKB-AURKC-FSHR-HSPA4L-HUWE1-INTU-KHDC3L-LARP1B-MFSD8-MTHFR-PGRMC2-PLK4-SENP7-SLC25A31-WBP1) and 9 selected SNVs in a further 8 genes: rs175080(MLH3), rs1799963(F2), rs6025(F5), rs5918(ITGB3), rs5985(F13A1), rs1805087(MTR), rs1801394(MTRR), rs28756992(MLH3), rs2305957(HSPA4L). The study involved 85 women and 107 PGT-M cycles performed between 2013-2019. Maternal genotyping was investigated in association with number of oocytes collected/fertilized and blastocyst formation.

Results: A 20x coverage was achieved in all regions investigated, identifying 121 variants. Significant associations (p < 0.05) were revealed for 16 SNVs in: AIRE-AMH-AURKB-HSPA4L-KHDC3L-MTHFR-PLK4-SENP7-MLH3. No significant associations were revealed for rs1801131 and rs2305957.

Conclusions: Our understanding of genes that may impact the preimplantation stage and the search for genomic biomarkers predicting IVF-success may benefit by investigating a fertile population, minimizing many confounding variables. This study was supported by the State Scholarships Foundation (IKY), co-funded by national grants and the European union (ESPA 2014-2020).

G. Kakourou: None. T. Mamas: None. C. Vrettou: None. E. Tsorva: None. S. Zacharia: None. C. Oraiopoulou: None. E. Chatzi: None. G. Kourlaba: None. D. Christopikou: None. M. Mastrominas: None. M. Moisidou: None. A. Chatziparasidou: None. I. Georgiou: None. J. Traeger-Synodinos: None.


Further indication of a role for de novo mutations in cases of severe idiopathic male infertility

H. E. Smith1, M. S. Oud2, P. F. de Vries2, B. Alobaidi1, A. Virdi1, B. J. Houston3, R. M. Smits4, L. Batty1, M. K. O’Bryan3, J. Coxhead5, S. Cockell6, J. Greenwood7, G. van der Heijden2,4, K. McEleny7, L. Ramos4, L. E. L. M. Vissers2, M. J. Xavier1, J. A. Veltman1,2

1Biosciences Institute, Newcastle University, Newcastle Upon Tyne, United Kingdom, 2Department of Human Genetics, Donders Institute for Brain, Cognition and Behaviour, Radboudumc, Nijmegen, Netherlands, 3School of Biological Sciences, Monash University, Melbourne, Australia, 4Department of Obstetrics and Gynaecology, Radboudumc, Nijmegen, Netherlands, 5Genomics Core Facility, Newcastle University, Newcastle Upon Tyne, United Kingdom, 6Bioinformatics Support Unit, Newcastle University, Newcastle Upon Tyne, United Kingdom, 7Newcastle Fertility Centre, Newcastle University, Newcastle Upon Tyne, United Kingdom

Infertility affects 1 in 6 couples worldwide and in approximately 50% of these cases the infertility can be attributed to a male factor. Whilst causes such as Klinefelter’s and Y chromosome microdeletions have been well distinguished, the genetic causes behind severe spermatogenic failure are largely unknown, with around 40% of all male infertility cases remaining idiopathic. De novo mutations (DNMs) arise spontaneously in the germline or post-zygotically, and are known to be associated with early onset disorders with reduced reproductive fitness. These mutations, however, are vastly understudied in the field of male infertility due to the difficulty in obtaining parental samples. Here we report on the continuation and expansion of exome sequencing in our cohort of infertile patients in order to identify novel genes involved in male infertility. The role of DNMs was investigated in a total of 194 patients suffering from non-obstructive azoospermia or severe oligozoospermia and their fertile parents by adding 95 additional trios to the previously reported 99 cases. In these additional 95 trios, we have identified and validated 75 protein-altering DNMs, of which 19 are likely to affect normal gene function. Genes of particular interest are PPP1R7, RBM5 and MSH5, all of which are involved in spermatogenesis and linked to infertility in mice. At this moment, none of these genes have been recognised as human male infertility genes. Our data further indicates that DNMs may play an important role in severe male infertility and emphasises the need for further replication studies in larger cohorts.

H.E. Smith: None. M.S. Oud: None. P.F. de Vries: None. B. Alobaidi: None. A. Virdi: None. B.J. Houston: None. R.M. Smits: None. L. Batty: None. M.K. OBryan: None. J. Coxhead: None. S. Cockell: None. J. Greenwood: None. G. van der Heijden: None. K. McEleny: None. L. Ramos: None. L.E.L.M. Vissers: None. M.J. Xavier: None. J.A. Veltman: None.


A case report of a rare nonsense ZP1 variant in a patient with oocyte maturation defect

E. G. Berkay, B. Karaman, G. Toksoy, B. Ozsait Selcuk, Z. O. Uyguner, S. Basaran

Istanbul Medical Faculty, Istanbul University, Istanbul, Turkey

Introduction: Oocyte maturation defect (OOMD) is a rare condition causing female infertility that can be diagnosed during assisted reproduction techniques (ART). OOMD related genes are ZP1, ZP2, ZP3, PANX1, PATL2, TUBB8, WEE2 (OMIM, 2020). We report a case of a 31-year-old woman who had four ART failures diagnosed as empty follicle syndrome and OOMD. She has short stature (-3 SD), bilateral limited extension-flexion on elbows.

Materials and Methods: Chromosome analysis and fluorescence in-situ hybridization (FISH) using X chromosome centromeric and SHOX-probe on interphase nuclei of lymphocytes and mucosal cells was investigated. Whole-exome sequencing (WES) performed via the Illumina platform. Confirmation and familial segregation analysis were performed by Sanger sequencing.

Results: Karyotyping and FISH resulted in normal, possible mosaicism was excluded. WES analysis revealed a known, rare, pathogenic homozygous variant in exon 3 (c.628C>T; p.Q210*) of ZP1 gene, and her parents being first degree cousins were carriers for this variant.

Conclusions: ZP1 with autosomal recessive inheritance is related to OOMD-1 (MIM_615774). Zona pellucida (ZP) is a glycoprotein structure surrounding oocytes and is essential for oocyte development. ZP contains four types of receptor proteins (ZP1-4). Our variant in ZP1 is nonsense, premature stop codon causes to truncate ZP1 receptor proteins. This is the first homozygous occurrence of this variant associated with OOMD. WES findings were also analyzed for known genes related to short stature and no pathogenic variant has been observed. WES is a valuable method to identify the genetic origin in complex, multigenic conditions like in infertility. Istanbul University Project-Number:TSA-2018-32135

E.G. Berkay: None. B. Karaman: None. G. Toksoy: None. B. Ozsait Selcuk: None. Z.O. Uyguner: None. S. Basaran: None.


Prenatal diagnosis and molecular characterization of a jumping translocation leading to 18p monosomy

J. B. Gaillard1, T. Guignard1, J. Puechberty2, A. Schneider1, V. Gatinois1, M. Duport-Percier3, J. M. Faure3, F. Fuchs3, F. Pellestor1, P. Blanchet4, C. Wells4

1Génétique Chromosomique, CHU, Montpellier, France, 2Maladies Génétiques de l’Enfant et de l’Adulte, CHU, Montpellier, France, 3Médecine Maternelle et Foetale, CHU, Montpellier, France, 4Génétique Prénatale et Foetopathologie, CHU, Montpellier, France

Jumping translocations are rare events in cytogenetics, characterised by a donor chromosome and multiple recipient chromosomes. To date, only ten prenatal cases have been described.

Patient: This pregnancy was a second spontaneous pregnancy of a non-consanguinous couple. A first trimester ultrasound showed an increased nuchal translucency persisting at 18 gestational weeks (GW) along with retrognatia, cleft palate and femoral shortening. Choriocentesis was performed at 14 GW.

Results: Direct examination on cytotrophoblast found a der(18)t(18;18) whereas the cultured karyotype on mesenchyme showed a der(18)t(2;18). Due to tissue discordance, an amiocentesis was performed at 18 GW. Surprisingly, we found, neither of the two previously identified cell lines, but a mosaic der(18)t(12;18) with a ring 18. In addition, array CGH provided breakpoints for chromosomes 2 and 12 in REEP1 gene (2p11.2) and TAOK3 gene (12q24.23) respectively. Whole genome sequencing on minION (Nanopore) was conducted to clarify breakpoint in chromosome 18. Using data obtained by array CGH, a long read with hard clipping showed a breakpoint in repeated sequence with about 90% homology with D18Z1 alpha satellite. Finally, all identified cell lines carried a 18p monosomy which could explain ultrasound abnormalities. The pregnancy was terminated. Following fetal autopsy, FISH analysis was performed on blood, thymic, lung and liver tissue and confirmed 18p monosomy in all tissues.

Conclusion: We report here a new case of jumping translocation with precise molecular characterization of breakpoints : D18Z1 as donor region and at least 3 recipient chromosomes : 2p11.2, 12q24.3 and 18q21.

J.B. Gaillard: None. T. Guignard: None. J. Puechberty: None. A. Schneider: None. V. Gatinois: None. M. Duport-Percier: None. J.M. Faure: None. F. Fuchs: None. F. Pellestor: None. P. Blanchet: None. C. Wells: None.


Molecular biomarkers of ageing in men with Klinefelter syndrome

E. Pohl1, S. Muschal2, S. Kliesch3, M. Zitzmann3, J. Rohayem3, J. Gromoll2, S. Laurentino2

1Institute of Human Genetics, University of Münster, Münster, Germany, 2Centre of Reproductive Medicine and Andrology (CeRA), Institute of Reproductive and Regenerative Biology, University Hospital Münster, 48149 Münster, Germany, Münster, Germany, 3Centre of Reproductive Medicine and Andrology (CeRA), Department of Clinical and Surgical Andrology, University Hospital Münster, 48149 Münster, Germany, Münster, Germany

Introduction: Males with Klinefelter syndrome (non-mosaic karyotype 47,XXY), the most common sex chromosome aneuploidy, exhibit increased rates of ageing-related morbidities and mortality. Associated with that, a significantly reduced life span has been observed. The aim of this study was to investigate whether Klinefelter patients show molecular signs of premature ageing.

Patients and Methods: We assessed age-associated DNA methylation patterns (by pyrosequencing) and relative telomere length (TL; by quantitative polymerase chain reaction) as molecular ageing biomarkers in blood from Klinefelter patients (n = 178 and 266 for DNA methylation and TL, respectively) aged 18-71 years. Age-matched healthy males (n = 184 and 196 for DNA methylation and TL, respectively) and females (n = 50) served as controls.

Results: Age-associated DNA methylation patterns did not indicate accelerated ageing/premature ageing in men with Klinefelter syndrome. In young Klinefelter individuals aged 18-24 years, we observed significantly longer telomeres compared to controls (mean = 1.51 vs. 1.09 and 1.26 in female and male controls, respectively). Telomere length in subsequent age groups, however, were not significantly different to controls.

Conclusions: We did not find molecular hints for premature ageing in Klinefelter patients based on DNA methylation patterns. However, we observed higher baseline TL at adolescent age but comparable TL with progressive age in other age groups. Whether this is due to an accelerated TL attrition, putatively related to the observed co-morbidities in Klinefelter syndrome, remains to be addressed in a further longitudinal study.

Funding: German Research Foundation (DFG No. GR1547/19-1; CRU 326 No. GR1547/25-1), IZKF Münster (Grant CRA03/09)

E. Pohl: None. S. Muschal: None. S. Kliesch: None. M. Zitzmann: None. J. Rohayem: None. J. Gromoll: None. S. Laurentino: None.


Maternally inherited copy-number variations may explain cases of severe idiopathic male infertility

F. K. Mastrorosa1, M. J. Xavier1, A. Mikulasova1, M. S. Oud2, R. M. Smith3, G. Astuti2, B. Alobaidi1, J. Greenwood4, S. J. Cockell5, C. Gilissen6, J. Coxhead7, H. Sheth1, G. van der Heijden2,3, K. McEleny4, L. Ramos3, J. A. Veltman1,2

1Biosciences Institute, Newcastle University, Newcastle upon Tyne, United Kingdom, 2Department of Human Genetics, Donders Institute for Brain, Cognition and Behaviour, Radboudumc, Nijmegen, Netherlands, 3Department of Obstetrics and Gynaecology, Radboudumc, Nijmegen, Netherlands, 4Newcastle Fertility Centre, Newcastle University, Newcastle upon Tyne, United Kingdom, 5Bioinformatics Support Unit, Newcastle University, Newcastle upon Tyne, United Kingdom, 6Department of Human Genetics, Radboud Institute for Molecular Life Sciences, Radboudumc, Nijmegen, Netherlands, 7Genomics Core Facility, Newcastle University, Newcastle upon Tyne, United Kingdom

Severe cases of infertility such as azoospermia and oligozoospermia often have genetic causes, with Klinefelter syndrome and chromosome Y microdeletions being the most common. However, of all infertile men, 40% have unknown aetiology after being tested for all known causes. In this study, we explored for the first time the role of maternally inherited copy-number variations (CNVs) on a large scale, sequencing the exome of 160 patient-parents trios, a methodology primarily used to detect single nucleotide variants (SNVs) but also robust for CNV detection. Among all rare maternally inherited CNVs detected, 3 were considered of potential pathogenicity. One is a large duplication (~1.7 Mb) involving, among others, VCX gene, an X-linked multi-copy gene that leads to apoptosis and cell growth inhibition when duplicated in murine germ cells in vitro. A second case carried a ~80 kb duplication involving two genes, one of which was FAS, a known regulator of testicular germ cell apoptosis. The third CNV, instead, was a deletion of ~200 kb affecting, among others, PSME4, a gene required for normal fertility in male mice but dispensable in females. The results of this study, although preliminary, provide a first indication that maternally inherited CNVs may explain cases of severe spermatogenic failure. Currently, additional 35 patient-parents trios and 149 patients are being screened to detect variations affecting the same genes and additional rare variations. Genome sequencing data will soon be available for all the trios, which will improve CNV detection and clarify the contribution of these variations to the disorder.

F.K. Mastrorosa: None. M.J. Xavier: None. A. Mikulasova: None. M.S. Oud: None. R.M. Smith: None. G. Astuti: None. B. Alobaidi: None. J. Greenwood: None. S.J. Cockell: None. C. Gilissen: None. J. Coxhead: None. H. Sheth: None. G. van der Heijden: None. K. McEleny: None. L. Ramos: None. J.A. Veltman: None.


Improved classification of testicular histopathology to promote uniform diagnosis and discovery of genetic causes for male infertility

G. W. van der Heijden1, M. J. Wyrwoll2, L. Ramos1, J. Veltman3, F. Tüttelmann4

11. Division of Reproductive Medicine, Department of Obstetrics and Gynecology, Radboud University Medical Center, Nijmegen, Netherlands, 2Centre of Reproductive Medicine and Andrology, Department of Clinical and Surgical Andrology, University of Münster, Münster, Germany, 3Institute of Genetic Medicine, Newcastle University, Newcastle, Netherlands, 4Institute of Human Genetics, University of Münster, Münster, Germany

With the availability of Next Generation Sequencing the genetic causes of male infertility are increasingly being chartered. A genetic diagnosis combined with a clear unambiguous description of accompanying testis histology will hugely benefit interpretation of the function of identified genes. However, a universal ontology for testis histopathology is lacking. The Human Phenotype Ontology (HPO) is a formal ontology, which describes phenotypes encountered in human diseases. The HPO allows phenotype driven differential diagnostics in clinical routine and research. The current hierarchy of terms regarding azoospermia, is not a systematical one. Moreover, the majority of testicular phenotypes are not annotated yet. Building on what has been published previously and our own experience we propose a framework for testis histopathology that consists of five main categories. Four of these are subdivided to obtain information on the timing of a germ cell arrest or the severity of overall loss of spermatogenesis. To establish a lower threshold for complete spermatogenesis we determined the number of tubules containing elongating spermatids in a collection of samples of men with an obstructive azoospermia due to a previous vasectomy (N=117). This analysis suggested 50% to be a reasonable cut-off to distinguish hypospermatogenesis from complete spermatogenesis. The framework is easy to understand and straightforward in its application. It gathers most relevant information to study the impact of an identified mutation on spermatogenesis. We conclude that once this framework is incorporated in the HPO, the standardized vocabulary will facilitate communication and interpretation in both a clinical routine and research.

G.W. van der Heijden: None. M.J. Wyrwoll: None. L. Ramos: None. J. Veltman: None. F. Tüttelmann: None.


Identification of two homozygous mutations, in the male reproductive tract specific β-defensin 126/128 genes, potentially underlie a severe sperm dysfunction.

S. Kane1, D. Lester1, S. Cameron1, C. Barratt2, S. Martins da Silva2, S. Brown1

1School of Science, Engineering & Technology, Abertay University, Dundee, United Kingdom, 2Reproductive and Developmental Biology, School of Medicine, Ninewells Hospital and Medical School, University of Dundee, Dundee, United Kingdom

Introduction: Idiopathic infertility affects 15% couples globally and recent evidence suggests genetic abnormalities are a causative factor in a significant number of cases. Hence, we conducted whole exome sequencing of men undergoing assisted conception to identify genetic lesions. The β-defensin 126 (DEFB126) and β-defensin 128 (DEFB128), are genes preferentially expressed in the epididymis, where these proteins may help protect the maturing sperm cells and play a vital role during sperm capacitation.

Materials and Methods: DNA extracted from the blood of 26 unrelated, sub-fertile men were sequenced using whole exome sequencing. Pathogenic rare variants were identified using bioinformatics analysis and validated using Sanger sequencing.

Results: A novel and rare homozygous pathogenic 1-bp frame-shift insertion causing a premature stop codon was identified in DEFB128 gene from one patient. Six patients showed a previously reported homozygous 2bp deletion, causing a frame-shift mutation in the DEFB126 gene (p.Pro106ArgfsTer?). All patients ascertained with the DEFB126/DEFB128 mutations were unable to naturally conceive and hence were sub-fertile.

Conclusions: The DEFB128 mutation (rs11396059), is rare and has not been associated with any disease to date. As the patient with the DEFB128 mutation has no other medical condition we predict that it is the cause of infertility. The DEFB126 mutation (rs140685149), is a common variant, and this homozygous variant in sub-fertile men has previously been implicated in facilitating sperm movement in the female reproductive tract. These results suggest that, β-defensin genes play a significant role in male reproduction.

This work was supported by the R-LINCS studentship, Abertay University.

S. Kane: None. D. Lester: None. S. Cameron: None. C. Barratt: None. S. Martins da Silva: None. S. Brown: None.


The sperm epigenome does not display recurrent epimutations in patients with severely impaired spermatogenesis

E. Leitao1, S. Di Persio2, S. Laurentino2, M. Wöste3, M. Dugas3, S. Kliesch4, N. Neuhaus2, B. Horsthemke1,5

1Institute of Human Genetics, University Hospital Essen, Essen, Germany, 2Centre of Reproductive Medicine and Andrology, University Hospital Münster, Münster, Germany, 3Institute of Medical Informatics, University Münster, Münster, Germany, 4Centre of Reproductive Medicine and Andrology, Department of Clinical and Surgical Andrology, University Hospital Münster, Münster, Germany, 5Institute of Human Genetics, University Hospital Münster, Münster, Germany

Introduction: Previous studies have identified aberrant DNA methylation of imprinted genes (e.g. MEST and H19) in the sperm of infertile patients, but the prevalence and genomic extent of these abnormal methylation patterns have remained unknown.

Material and Methods: We collected swim-up sperm samples from 40 normozoospermic and 93 oligoasthenoteratozoospermic patients and determined H19 and MEST methylation levels by deep bisulfite sequencing. Based on this screening, we defined three groups: normal controls (NC), abnormally methylated infertile (AMI; n = 7) and normally methylated infertile (NMI; n = 86), and performed whole genome bisulfite sequencing of five NC, five AMI and six NMI samples.

Results: Whole-genome comparison of NC and AMI samples revealed that each AMI sample had abnormal methylation levels of all 50 imprinting control regions and >2,000 germ cell-specific genes with distinct DNA methylation differences in blood and germ cells. This is consistent with the presence of somatic cell DNA in AMI samples. The comparison of NC and NMI samples revealed 19 differentially methylated regions, none of which could be validated in an independent cohort of 40 men. Analysis of the H19 CTCF-binding region 6 in samples without somatic cell DNA revealed an association between DNA methylation levels and genotype (rs2071094), but not fertility phenotype.

Conclusions: Our results suggest that somatic DNA contamination and genetic variation confound methylation studies in sperm of infertile men and that the prevalence of aberrant methylation in infertile men has been overestimated, which is reassuring for patients undergoing assisted reproduction. Funded by DFG grants HO 949/23-1 and NE 2190/3-1.

E. Leitao: None. S. Di Persio: None. S. Laurentino: None. M. Wöste: None. M. Dugas: None. S. Kliesch: None. N. Neuhaus: None. B. Horsthemke: None.


miR-200 family inhibits trophoblast invasion through regulating epithelial-mesenchymal transition

M. Su1, H. Tsai1, C. Wang2

1Department of Obstetrics and Gynecology, National Cheng-Kung University Hospital, Tainan, Taiwan, 2Department of Cell Biology and Anatomy, National Cheng-Kung University, Tainan, Taiwan

Epithelial-to-mesenchymal transition (EMT) is a biological process that epithelial cells lose cell polarity and cell-cell adhesion, and gain migratory and invasive abilities to become mesenchymal cells. In human placenta, the chorionic villi gradually transform from proliferative cytotrophoblasts and extravillous trophoblasts into a migratory and invasive trophoblast phenotype. Failure in altering the behavior and morphology of trophoblasts in placenta may result in insufficient trophoblast invasion, which leads to placental dysfunction and the diseased placenta. MiR-200 family, including miR-200a and -141, were highly expressed in human placenta and were upregulated in patients of preeclampsia. miR-200a and miR-141 were demonstrated to inhibit the formation of primary clila in trophoblasts and therefore have a less ability for cell invasion. In this study, miR-200a and miR-141 were shown to inhibit trophoblasts (HTR-8/SVneo) invasion and migration by a transfection assay. In western blotting and qRT-PCR, miR-200a and miR-141 stimulated epithelial markers, such as E-cadherin and ZO-1, whereas suppressed mesenchymal and transcriptional factors, such as ZEB1, MMP2, MMP9 and TGF-b1. These results suggest trophoblast migration and invasion mediated by miR-200 family may through the mechanism of EMT, and may therefore predispose to develop preeclampsia in human pregnancy.

M. Su: None. H. Tsai: None. C. Wang: None.


Identification of plasma miRNAs associated with endometriosis in Bulgarian patients

V. Spasova1, V. Karamisheva1,2, O. Antonova1, Z. Hammoudeh1, L. Koleva3, R. Staneva1, M. Ganev1, D. Nesheva1, A. Nachev1,2, D. Toncheva1, S. Hadzhidekova1

1Medical University of Sofia, Sofia, Bulgaria, 2University obstetrics and gynecology hospital “Maichin dom, Sofia, Bulgaria, 3University Hospital “Pirogov”, Sofia, Bulgaria

Introduction: Diagnosis of endometriosis still poses a significant challenge for it usually requires surgery. The need of invasive procedure significantly contributes to late diagnosis and less effective treatment. The aim of this study is to screen for changes in miRNAs expression in peripheral blood that could be further researched as potential non-invasive diagnostic tool for endometriosis.

Materials and methods: 15 patients with surgically confirmed advanced endometriosis and 15 healthy controls were included in the study. Peripheral blood samples were collected after signed informed consent. Plasma RNA was isolated with miRNeasy-Serum/Plasma Kit in conjunction with Plasma Spike-in-Control (Qiagen®). Equimolar amounts of RNA were used to prepare two pools: 1)late stage endometriosis and 2)healthy controls. Reverse transcription of the pool samples was performed by miScript II RT Kit (Qiagen®). Expression spectra of 84 miRNAs (miScript miRNA-PCR-Array-Human Serum & Plasma, Qiagen®) by real-time PCR quantification via miScript-SYBRGreen PCR Kit (Qiagen®) were analyzed on Applied Biosystems™7500 instrument.

Results: The preliminary results revealed 65 upregulated miRNAs and no downregulated miRNAs between cases and controls. The fold change between 5 and 10 was detected in 21 miRNAs and fold change above 10 was detected in 44 miRNAs. The most significantly overexpressed miRNAs were miR-106a and miR-17-3p which are associated with cell proliferation and apoptosis.

Conclusions: MiRNA expression level differences were found in blood between endometriosis patients and healthy controls. This may lay the foundations for future in-depth research on novel non-invasive biomarker for endometriosis. Acknowledgements: Contract Д-212/12.12.2018

V. Spasova: None. V. Karamisheva: None. O. Antonova: None. Z. Hammoudeh: None. L. Koleva: None. R. Staneva: None. M. Ganev: None. D. Nesheva: None. A. Nachev: None. D. Toncheva: None. S. Hadzhidekova: None.


Sequence variants in TBX6 are associated with disorders of the Müllerian ducts - an Update

A. Tewes1, J. Hucke2, T. Römer3, K. Kapczuk4, C. Schippert5, P. Hillemanns5, A. Bohring1, P. Wieacker1, S. Ledig1

1Institute of Human Genetics, Münster, Germany, 2Department of Obstetrics and Gynecology, Wuppertal, Germany, 3Department of Obstetrics and Gynecology, Köln-Weyertal, Germany, 4Division of Gynecology, Poznan, Poland, 5Department of Obstetrics and Gynecology, Hannover, Germany

Müllerian anomalies include formation, fusion or absorption defects of the Müllerian ducts such as unicornuate, didelphys, bicornuate or septate uterus and vaginal septa as well as the Mayer-Rokitansky-Küster-Hauser syndrome (MRKHS) which is characterized by the congenital absence of uterus and vagina. The incidence of MRKHS is estimated to be 1:4.500 and 1:250 in other Müllerian anomalies. Most cases of Müllerian anomalies are sporadic; familial cases seem to be autosomal dominant inherited with reduced penetrance. Until now the knowledge about the etiology of anomalies of the Müllerian ducts is very limited. In approximately 1% of patients with Müllerian anomalies recurrent deletions in 16p11.2 encompassing TBX6 were found as causative in these patients. Moreover, in previous studies, we and others found possibly pathogenic variants in patients with Müllerian anomalies in TBX6. Since TBX6 encodes a conserved transcription factor playing an essential role in developmental processes like mesoderm formation and specification, we suggested TBX6 as strong candidate gene. Thus we performed analysis of the TBX6 gene (NM_004608) in additional 126 patients with Müllerian anomalies. We detected 4 possibly pathogenic variants (c.358A>G p.(Met120Val), c.704G>A p.(Gly235Asp), c.815G>A p.(Arg272Gln), c.1146C>A p.(Tyr382*)) in TBX6 in 5/126 patients and could exclude the previously reported variant c.484G>A p.(Gly162Ser) as being causative for disorders of the Müllerian ducts in the European non-Finnish population. In summary, we present increasing evidence for the association of variants in TBX6 with malformations of the Müllerian ducts.

A. Tewes: None. J. Hucke: None. T. Römer: None. K. Kapczuk: None. C. Schippert: None. P. Hillemanns: None. A. Bohring: None. P. Wieacker: None. S. Ledig: None.


Circulating fetal trophoblastic cell eligible for NIPD: the exception rather than the rule

L. Cayrefourcq1, M. C. Vincent2, S. Pierredon2, C. Moutou3, M. Imbert-Bouteille2, E. Haquet4, J. Puechberty4, M. Willems4, C. Liautard-Haag2, N. Molinari5, C. Zordan6, V. Dorian6, A. Chaussenot7, L. Monteil8, C. Miry9, Y. Petrov10, E. Le Boette11, M. Fradin12, C. Alix-Panabiere1, C. Guissart2

1Laboratoire de cellules circulantes rares humaines_University Medical Center of Montpellier, Montpellier, France, 2Laboratoire de Genetique Moleculaire_University Medical Center of Montpellier, Montpellier cedex 5, France, 3Laboratoire de diagnostic pré-implantatoire_CMCO_University Medical Center of Strasbourg, Schiltigheim, France, 4Departement de Genetique Medicale_University Medical Center of Montpellier, Montpellier, France, 5DIM_University Medical Center of Montpellier, Montpellier, France, 6Service de Genetique Medicale_University Medical Center of Bordeaux, Bordeaux, France, 7Service de Genetique Medicale_University Medical Center of Nice, Nice, France, 8Service de Genetique Medicale_University Medical Center of Toulouse, Toulouse, France, 9Department of Medical Medecinee_University Medical Center of Strasbourg, Schiltigheim, France, 10Laboratoire de cytologie et cytogenetique_University Medical Center of Nimes, Nîmes, France, 11Service de Genetique Medicale_ Medical Center of Saint Brieuc, Saint Brieuc, France, 12Service de Genetique Medicale_Medical Center of Saint Brieuc, Saint Brieuc, France

Introduction: Non-Invasive Prenatal Diagnosis (NIPD), based on the analysis of circulating cell-free fetal DNA (cff-DNA), is successfully implemented for an increasing number of monogenic diseases. However, technical issues related to cff-DNA characteristics remain, and not all mutations can be screened with this method, particularly triplet expansion mutations that frequently concern prenatal diagnosis requests. The objective of this study was to develop an approach to isolate and analyze Circulating Trophoblastic Fetal Cells (CFTCs) for NIPD of monogenic diseases caused by triplet repeat expansion or point mutations.

Materials and Methods: We developed a method for CFTC isolation based on DEPArrayTM sorting and used Huntington’s disease as the clinical model for CFTC-based NIPD. Then, we investigated whether CFTC isolation and Whole Genome Amplification (WGA) could be used for NIPD in couples at risk of transmitting different monogenic diseases.

Results: Our data showed that the allele drop-out rate was 3-fold higher in CFTCs than in maternal cells processed in the same way. Moreover, we gave new insights into CFTCs by compiling data obtained by extensive molecular testing by microsatellite multiplex PCR genotyping and by WGA followed by mini-exome sequencing.

Conclusions: CFTCs appear to be often characterized by a random state of genomic degradation. This might challenge CFTC use for routine NIPD. This emphasizes the importance to have not a single diagnostic cell but multiple diagnostic cells.

This research was funded by French Ministry of Health - DGOS, grant number PHRCI-16-068 (DIACCIMEX) and by Agence de la biomédecine, grant number 18AMP009 (DIAFEXOME)

L. Cayrefourcq: None. M.C. Vincent: None. S. Pierredon: None. C. Moutou: None. M. Imbert-Bouteille: None. E. Haquet: None. J. Puechberty: None. M. Willems: None. C. Liautard-Haag: None. N. Molinari: None. C. Zordan: None. V. Dorian: None. A. Chaussenot: None. L. Monteil: None. C. Miry: None. Y. Petrov: None. E. Le Boette: None. M. Fradin: None. C. Alix-Panabiere: None. C. Guissart: None.


Development of non-invasive prenatal diagnosis for monogenic diseases: the example of Marfan syndrome and GCK diabetes

T. Nouspikel1, A. Bottani2, V. Schwitzgebel1

1University Hospitals of Geneva, Geneva, Switzerland, 2Hôpital du Valais, Sion, Switzerland

Introduction: The presence of foetal DNA in maternal plasma allows for non-invasive prenatal diagnosis of Mendelian diseases, through detection of known parental mutations. While identifying paternal mutations is straightforward, determining whether a maternal mutation is present in the foetus requires relative dosage of maternal haplotypes (RHDO), via heterozygous SNPs on either side of the mutation.

Results: A custom library of 95 SNPs spread over a ~1 Mb region around the FBN1 gene was used for a posteriori analysis of two pregnancies in a couple with a paternal FBN1:c.5143delA mutation causing Marfan syndrome. In both cases, the mutation was detected in maternal plasma and confirmed in amniotic fluid or cord blood. We also verified that the method could determine which maternal haplotype each foetus had inherited.

In another couple with a maternal GCK:p.Gly246Arg mutation causing MODY2, a panel of 99 SNPs over 0.7 Mb around GCK was used to analyse maternal plasma at 16 and 30 weeks gestation. In both samples, we correctly determined the maternal haplotype inherited by the foetus and diagnosed the presence of the mutation, confirmed by cord blood DNA analysis. The same results were obtained by quantifying the number of DNA molecules, obtained through molecular barcodes, rather than sequence reads. Bioinformatic reduction of foetal fractions showed that determination of maternal haplotypes remained possible down to 4.3% foetal DNA.

Conclusions: Although preliminary, our results indicate that a molecular barcode library of ~100 SNPs is adequate to achieve non-invasive prenatal diagnosis of monogenic diseases, even at low foetal fractions.

T. Nouspikel: None. A. Bottani: None. V. Schwitzgebel: None.


NIPT revealed in a pregnant woman 5Mbp-13q duplication, proved as a familial CNV by array CGH

I. Dimova1, I. Bradinova2, V. Peycheva3, R. Bozhilova3, R. Raynova2, K. Kercheva1, A. Savov2, I. Kremensky3, R. Kaneva3

1Medical University Sofia, Sofia, Bulgaria, 2SBALAG "Maichin dom", Medical University Sofia, Sofia, Bulgaria, 3Molecular Medicine Center, Medical University Sofia, Sofia, Bulgaria

Subchromosomal copy number variations (CNVs), known as segmental deletions and duplications, are extensively distributed in human genome. A substantial proportion of these CNVs are associated with severe diseases, even with some newly identified microdeletion and microduplication syndromes. Recently it was showed that NIPT has a good performance in detecting fetal subchromosomal CNVs, especially for CNVs >10 Mb, and can be incorporated into the routine NIPT chromosomal aneuploidies screening with high sensitivity and specificity. The prenatal detection of CNVs with clinical significance would benefit all the pregnant women in genetic counselling and clinical management of the pregnant outcomes. We report here a result from NIPT performed in a first trimester of a pregnant woman with third normal pregnancy. The test revealed 5Mbp-13q duplication and array CGH was indicated for confirmation of the aberration after amniocentesis. Array CGH confirmed the aberration in a fetus with the following size: 13q21.2q21.31 (60,540,987-65,431,854)x3 (4.89 Mb). Such duplications in 13q are described in the literature to be associated with the developmental delay, intellectual deficits, behavioral problems, and facial dysmorphisms. We tested all family members by array CGH for the presence of the aberrations - the parents and two siblings (9 years old girl and 12 years old boy). The same aberration was detected in clinically healthy mother and sister, thus determining the familial character of the aberration. In conclusion, careful evaluation should be done for each submiscroscopic aberration detected by NIPT in order to make proper classification and give an adequate genetic counselling.

I. Dimova: None. I. Bradinova: None. V. Peycheva: None. R. Bozhilova: None. R. Raynova: None. K. Kercheva: None. A. Savov: None. I. Kremensky: None. R. Kaneva: None.


False positive trisomy 21 NIPT-result due to maternal inherited CNV

M. Blankenburg1, A. Weber1, S. Rauschke1, R. Becker2, K. Pötschick3, M. Stumm1

1Medicover Genetics GmbH, Berlin, Germany, 2Zentrum für Pränataldiagnostik Kudamm 199, Berlin, Germany, 3Medicover Humangenetik-Berlin-Mitte, Berlin, Germany

Introduction: Noninvasive prenatal tests (NIPT) are screening methods to detect fetal aneuploidies with high specificity and sensitivity. Nevertheless, maternal factors as copy number variations (CNV) are reasons for discrepancies between NIPT-results and fetal karyotypes. We report a case with a conspicuous trisomy 21 result after NIPT, an unsuspicious fetal ultrasound and a normal karyotype following conventional chromosome analysis (CA) of amniotic fluid cells.

Material and Methods: Next Generation sequencing (NGS) of cfDNA, CA from peripheral blood, amniotic fluid and placental cells, Fluorescence-in-situ-Hybridization (FISH) on metaphases and interphases, array-CGH (aCGH) and quantitative PCR (qPCR).

Results: NIPT indicated a fetal trisomy 21. In contrast, ultrasound examination and amniocentesis showed normal results. Reanalysis of NIPT by WISECONDOR algorithm showed a 10 Mb gain on chromosome 21. No comparable cases were described in literature or databases. The parents declined further diagnostics until birth. Postpartum CA and FISH from blood and placental cells resulted in normal karyotypes. In contrast, aCGH confirmed a 1,44 Mb gain in chromosomal region 21q21.3q22.11 and the aberration was classified as variant of unclear significance (VUS). Additional qPCR analyses confirmed the gain and showed it was maternal inherited. Therefore, we reclassified the aberration as a familial benign CNV.

Conclusions: To best of our knowledge, this is the first report of a small benign maternal chromosome 21 CNV leading to a false positive trisomy 21 NIPT result. That case demonstrates again the importance of a critical reflection of genotype/phenotype correlations after NIPT.

M. Blankenburg: None. A. Weber: None. S. Rauschke: None. R. Becker: None. K. Pötschick: None. M. Stumm: None.


Circulating miRNAs as biomarkers to assess fetal fraction level

G. Santoro1, S. Caporilli1, C. Lapucci1, M. Rusin2, M. Giannoccaro1, A. Della Gatta2, S. Amodeo2, A. Farina2

1Synlab Italia, Castenedolo, Italy, 2Università di Bologna, Dipartimento di Scienze Mediche e Chirurgiche (DIMEC), Bologna, Italy

Non-invasive prenatal testing (NIPT) methods are widely used as screening tool for fetal aneuploidies. They are based on the isolation of cell-free DNA (cfDNA) from maternal plasma which for the majority is maternal, but a small percentage of it comes from the placenta (cell-free fetal DNA or cffDNA). cffDNA percentage or fetal fraction (FF) ranges from < 4% to >30% and it influences NIPT outcome. According to neoBona® NIPT method used in our laboratory, invalidation occurs with a FF <4%. Recently, circulating microRNAs (miRNAs) have been investigated as potential biomarkers for screening in pregnancy. We aimed to investigate them as biomarkers in relation to FF level. Previously tested neoBona® samples were divided into one “control group” (FF >4%) and one “test group” (FF <4%). Samples were analysed in the nanostring nCounter® platform by using a pre-customised panel of 799 miRNAs. Each miRNA expression value was normalised to 1 in the “control group” and “test group” samples were analysed for expression defects. Data filtering was set using 1.5-fold of changes in signal as threshold. Ninety-three per cent of miRNAs showed no significant changes of expression whilst 4 miRNAs (named miR-579, miR-3144, miR-612 and miR-6721) were significantly over- or under-expressed. Bioinformatic analysis showed involvement of these miRNAs and their target genes in several biological pathways. Further investigations of these preliminary data could provide a new insight into the biological roles of the miRNAs in relation to FF level and in their potential use as biomarkers.

G. Santoro: None. S. Caporilli: None. C. Lapucci: None. M. Rusin: None. M. Giannoccaro: None. A. Della Gatta: None. S. Amodeo: None. A. Farina: None.


An Italian public regional healthcare system experience offering non invasive aneuploidy screening test based on cfDNA (NIPT)

C. Pescucci1, F. Gerundino1, C. Giuliani1, B. Minuti1, F. Sbernini1, S. Frusconi1, F. Marin1, L. Giulietti1, C. Berni2, L. Pasquini3, C. Luchi4, F. Severi5, A. Cordisco6, B. Melani7, E. Mainandi8, E. Pelo1

1SOD Diagnostica Genetica, Azienda Ospedaliero Universitaria Careggi, Firenze, Italy, 2P.O. Rete integrata delle malattie rare e dei difetti congeniti e sviluppo della rete dei punti nascita e della pediatria specialistica, Settore Qualità dei servizi e reti cliniche, Regione Toscana, Firenze, Italy, 3Medicina e Diagnosi Fetale, Azienda Ospedaliero Universitaria Careggi, Firenze, Italy, 4U.O. Ostericia e Ginecologia, Azienda Ospedaliero-Universitaria Pisana, Pisa, Italy, 5Dipartimento di Medicina Molecolare dello sviluppo, Azienda Ospedaliero-Universitaria Senese, Siena, Italy, 6Centro Unico di Diagnosi Prenatale del P.O. Piero Palagi, Firenze, Italy, 7UO ginecologia e ostetricia - ASL DI PISTOIA, Pistoia, Italy, 8Consultori e Diagnosi Prenatale USL 4, Prato, Italy

Introduction: Non invasive prenatal testing (NIPT) to investigate the risk for fetal aneuploidies (chromosomes 13, 18 e 21 trisomies and X/Y aneuploidies) has been demonstrated to have high sensitivity and specificity and it is worldwide accepted both as contingent screening as primary test in first trimester.

Materials and Methods: since March 2019, NIPT is offered by public healthcare system in Tuscany to pregnant women with conventional first trimester screening test (FTS) risk (nuchal translucency and biochemical measurements) ranging from 1:301-1:1000 or 1:2-1:300 if they opt-out invasive prenatal diagnosis. Women are counseled in different centers spread out over the regional area. Blood samples are collected in a single regional center authorized to perform the test for chromosomes 13, 18, 21, X and Y by CE-IVD VeriSeq™ NIPT Solution (Illumina Inc.).

Results: Since March to December 2019, 2199 women have been screened of which 429 with a FTS risk ranging from 1:2-1:300 and 940 from 1:301-1:1000. 396 out of 429 NIPT results were at low risk for fetal aneuploidies whereas 7/429 resulted at high risk for T21 (n = 4), T18 (n = 1)and XXX (n = 2). All these results have been confirmed by IPD. In the FTS 1:301-1:1000 risk group, 738 cases resulted at low risk for fetal aneuploidies and 5 at high risk for T21 (n = 1), T18 (n = 1), XXY (n = 2) and XXX (n = 1). Only sex chromosome aneuploidy results have been confirmed by IPD. Conclusions. Preliminary data show that of the Tuscany region protocol improve the pregnancy care pathway by reducing inappropriate invasive tests.

C. Pescucci: None. F. Gerundino: None. C. Giuliani: None. B. Minuti: None. F. Sbernini: None. S. Frusconi: None. F. Marin: None. L. Giulietti: None. C. Berni: None. L. Pasquini: None. C. Luchi: None. F. Severi: None. A. Cordisco: None. B. Melani: None. E. Mainandi: None. E. Pelo: None.




1Genoks Genetic Diseases Diagnostic Center, Ankara, Turkey, 2Department of Obstetrics and Gynecology, Division of Perinatology, Gazi University Faculty of Medicine, Ankara, Turkey

Objective: When non invasive prenatal testing (NIPT) incorporating whole genome sequencing (WGS) is performed, the autosomal aneuploidies other than trisomy 21, 18 and 13 has the potential to be detected. We present the frequency of rare chromosomal trisomies (RATs) that were identified between 1 July 2018 and 31 December 2019

Methods: Standard centrifugation methods were used for plasma isolation. Cell free DNA (cfDNA) was isolated and library construction was prepared according to BGI’s documented protocol. WGS of cfDNA was performed with BGISEQ-500 sequencer. Data was analysed using BGI’s proprietary bioinformatic programme, HALOS.

Results: WGS data was generated from 13,802 NIPT cases. The mean maternal age was 33 years, fetal fraction was 10.7% and gestational age at testing was 14 weeks. Over an 18-month period, there were 30 RATs identified. The overall frequency of RATs was 0.21% in our study. Trisomies 16 and 22 were the most frequently observed RATs while other chromosomes were less frequently involved. Invasive prenatal diagnosis was performed in 37% (11/30) of cases with RATs. Three fetal aneuploidies were confirmed with amniocentesis; one was T16 and the others were T22.

Conclusions: Rare autosomal aneuploidies are not uncommon in clinical practice when performing genome-wide NIPT. The review of the published reports show a significant risk for obstetric pathology and fetal anomalies such as early miscarriage, true fetal mosaicism, intrauterine growth restriction, in utero fetal demise, and uniparental disomy. We advise genetic counseling in all cases of rare chromosomal trisomies to improve pregnancy management.

I.M. Tekin: None. H. Ozdemir: None. H. Ozgur: None. Y. Sahin: None.


Next generation sequencing detects premeiotic errors in human oocytes and provides evidence of genetic influence

H. Ghevaria1, S. SenGupta1, R. Naja2, R. Odia3, P. Serhal4, X. Viñals Gonzalez3, X. Sun1, J. Delhanty1

1Preimplantation Genetics Group, University College London, London, United Kingdom, 2Molecular Genetics Laboratory, Igenomix UK Ltd, London, London, United Kingdom, 3Embryology Department, The Centre For Reproductive and Genetic Health, London, United Kingdom, 4Clinical Department, The Centre For Reproductive and Genetic Health, London, United Kingdom

As women age the risk of an aneuploid oocyte increases with most errors affecting meiosis I/II. Application of next generation sequencing (NGS) has allowed us to confirm our finding, from molecular cytogenetics, that a significant proportion of apparently meiotic aneuploidy is in fact present in the early embryo, leading to a risk of oocyte aneuploidy irrespective of age. It has also provided evidence that genetic factors influence premeiotic oocyte aneuploidy. NGS was performed on oocytes using Ion ReproSeqPGS (ThermoFisher)/VeriSeqPGS (Illumina). Immature oocytes (84) included germinal vesicles & metaphase I oocytes. Mature oocytes (27) were metaphase II - 1st polar body complexes. Totally, 111 oocytes from 33 women, (average maternal age, 35.09 years) were tested. Overall 15/111 (13.5%) oocytes showed premeiotic (PM) errors. Eight of 33 patients had oocytes with PM errors. Aneuploidy analysis was based on the reproductive histories of female partners donating oocytes. Interestingly the first two groups of infertility patients showed almost identical aneuploidy rates in contrast to the social oocyte freezing group. Two young women preserving oocytes prior to breast cancer treatment showed complex aneuploidies. We conclude that the application of NGS has provided accurate information regarding the frequency of aneuploidy that is due to premeiotic errors compared with that caused by errors at MI of oogenesis and that on an individual basis this is influenced by genetic factors. Grant support: CRGH

Classification of female partners donating oocytes
Classification of female partners based on reproductive historiesNo. of patientsTotal oocytes testedOocytes with premeiotic aneuploidy
1. Primary/secondary female factor infertility13325 (15.6%)
2. Females not known to be infertile (probable male factor infertility)11324 (12.5%)
3. Oocyte preservation due to social reasons2211 (4.7%)
4. Oocyte preservation due to breast cancer2105 (50%)
5. Female carriers of structural rearrangements or monogenic disorders (non-cancer related)360
6. Females at increased risk of developing breast/ovarian cancer2100

H. Ghevaria: None. S. SenGupta: None. R. Naja: None. R. Odia: None. P. Serhal: None. X. Viñals Gonzalez: None. X. Sun: None. J. Delhanty: None.


Interrogating the parental and zygotic origin of de novo mutations in severe male infertility using long read sequencing

G. S. Holt1, B. Alobaidi1, L. Batty1, M. Oud2, H. Smith1, P. de Vries2, R. Smits2, L. Vissers2, L. Ramos2, M. Xavier1, J. Veltman1

1Biosciences Institute, Newcastle University, Newcastle Upon Tyne, United Kingdom, 2Department of Human Genetics, Donders Institute for Brain Cognition and Behavior, Radboud University, Nijmegen, Netherlands

Male infertility affects approximately 7% of the male population and while genetic abnormalities are predicted to make up at least 15% of the cases, only ~4% are currently identified. In severe forms of male infertility, patients are found to have high numbers of genetic abnormalities and de novo mutations (DNMs). Interestingly DNMs are known to play an important role in severe genetic diseases with reduced reproductive fitness. Our research investigates the role of DNMs in severe male infertility by studying a unique cohort of azoospermic and severe oligozoospermic patients, and their fertile parents. Employing the Illumina’s Novaseq6000, 100 DNMs were identified in the exomes of 99 patients. While short-read sequencing data was used to collect the location and genotypes of all informative SNPs (iSNPs) surrounding the DNMs, limitations in fragment size prevented further investigation. In-depth examination of DNMs in context with their surrounding iSNPs requires targeted capture approaches for long-read sequencing that span the entire region of interest. Using PCR amplicon sequencing on the Oxford-Nanopore MinION platform we have been able to phase ~90% of the DNMs. Phasing analysis indicated that ~71% of DNMs occurred in the paternal gamete pre-fertilization, which is in concordance with current literature. A link between paternal DNMs and likely causation for disease has been noted, though not all data was available for this association at this time. Investigation of allele frequency identified 91% of the DNMs occurred pre-zygotically, why they failed to impact father spermatogenesis is of particular interest for further study.

G.S. Holt: None. B. Alobaidi: None. L. Batty: None. M. Oud: None. H. Smith: None. P. de Vries: None. R. Smits: None. L. Vissers: None. L. Ramos: None. M. Xavier: None. J. Veltman: None.


Polycystic ovary syndrome: Does it affect the gene expression patterns in human oocytes?

P. Tulay, Z. Al-Omar, B. Ozbakir

Near East University, Nicosia, Cyprus

Polycystic ovary syndrome is a common disorder among women of reproductive age. The aim of this study was to investigate the gene expression levels of a number of genes that is functioning in steroidogenesis and in the maintenance of telomeres.

The spare oocytes were collected from IVF patients with polycystic ovaries and routine IVF patients as controls. DNA and RNA were extracted from each oocyte. Reverse transcription of RNA was performed and real time PCR was conducted to quantify the expression levels of eleven genes, TERF1, TRF2, TINF2, TPP1, POT1, RAP1, CYP11, CYP17, CYP19, HSD17B1 and HSD3B2.

The results of this study showed that the expression levels of TERT, TRF, TINF, TPP, RAP, CYP19 and HSD3B2 were similar between the two groups. However, the expression levels of POT and CYP11A1 genes were shown to be statistically different in the oocytes obtained from control patients (p < 0.05), whereas statistically significant expression levels were observed for CYP17 in the oocytes obtained from the study group (p < 0.05). The expression level of HSD17B1was also shown to be statistically different in the oocytes (p < 0.05).

The results of this study showed that different gene expression levels are observed in the oocytes obtained from females with polycystic ovaries compared to the controls. The extrapolation of the results indicates that the genes essential in maintaining DNA chromosome end integrity and steroidogenesis are crucial for the correct development of the oocytes. Any variations of the expression levels of these genes may result in anomalies of the oocyte maturation.

P. Tulay: None. Z. Al-Omar: None. B. Ozbakir: None.


Regulatory variants upstream of FLT1 as risk factors for preeclampsia

T. Kikas1, R. Inno1, K. Ratnik1,2, K. Rull1,3,4, M. Laan1

1University of Tartu, Institute of Bio- and Translational Medicine, Tartu, Estonia, 2SYNLAB Estonia OÜ, Tartu, Estonia, 3Women’s Clinic, Tartu University Hospital, Tartu, Estonia, 4Department of Obstetrics and Gynecology, University of Tartu, Tartu, Estonia

Introduction: The first GWAS assessing the genetic risk to preeclampsia (PE) in placental samples by McGinnis et al., 2017 identified one statistically significant variant associated with PE (rs4769613 C>T; OR=1.2, P=5.4×10−11). The variant is located near a well-known PE gene FLT1 encoding sFlt-1. The aim of this study was to further investigate the link between two variants highlighted in GWAS near FLT1 gene (rs4769613, rs12050029; 69kb/158kb, respectively) and PE in two independent cohorts.

Materials and Methods: The study utilized placental samples from Estonian REPROMETA and HAPPY PREGNANCY (placentas n=329/1768, respectively) sample sets, including PE and non-PE controls (total n=96/2001). SNPs (rs4769613, rs12050029) were genotyped using Taqman assays. Association analyses and case-control testing were conducted in PLINK (linear or logistic regression, covariates gestational age, sex).

Results: Case-control testing showed an increased risk for PE with rs4769613 C allele in both Estonian sample sets, concordant with the previous study. Meta-analysis across the two cohorts improved the significance (OR=1.75, P = 2.49×10−3). As a novel finding, no associations between rs4769613 and other gestational complications (gestational diabetes, fetal growth, preterm birth) were detected, which suggests the variant is PE-specific. The gene expression of FLT1 was upregulated only in PE placentas carrying CC- and CT genotypes (t-test, P < 0.05), whereas in maternal serum, sFlt-1 was increased in PE cases in all genotype groups.

Conclusions: The study emphasizes the placental rs4769613 as the only known robust and specific risk variant for PE, modulating placental FLT1 expression.

Funding: IUT34-12 (Estonian Research Council); HAPPY PREGNANCY, 3.2.0701.12-0047 (European Regional Development Fund)

T. Kikas: None. R. Inno: None. K. Ratnik: A. Employment (full or part-time); Significant; SYNLAB Estonia OÜ. K. Rull: None. M. Laan: None.


The role of new genetic markers and natural selection in the formation of a hereditary predisposition to preeclampsia

V. Serebrova, E. Trifonova, V. Stepanov

Research Institute of Medical Genetics, Tomsk National Research Medical Center of the Russian Academy of Sciences, Tomsk, Russian Federation

Introduction: Preeclampsia (PE) is a pregnancy-specific complication with the poorly understood etiopathogenesis. There is a hypothesis that the risk to the development of PE may be a consequence of natural selection affecting on placental genes responsible for trophoblast invasion and remodeling of spiral arteries. The aim of this work was to characterize the genetic components of PE by analysis of regulatory single-nucleotide polymorphisms (rSNPs) of differentially expressed genes (DEGs) in placenta and to detect the role of natural selection in its formation.

Materials and Methods: In this report we analyzed 46 rSNPs in 21 DEGs, associated with PE according to the analysis of the transcriptome in 551 patients with PE and 718 women with uncomplicated pregnancies of Buryat, Russian and Yakut populations using MassArray iPLEX (Sequenom). For detection of the signals of natural selection we used new INSIGHT method.

Results: We have detected significant associations of PE with 14 rSNPs in 10 genes: BHLHE40, CORO2A, INHA, LHB, NDRG1, PPP1R12C, PLIN2, RDH13, SASH1, SYDE1. The effect of weak negative selection has being shown for 4 rSNPs in 4 DEGs: rs10985257 in CORO2A gene, rs72959687 in INHA gene, rs34845949 in SASH1 gene, rs2227262 in NDRG1 gene. It should be noted that these results corresponds to number of evolutionary hypotheses of the occurrence of PE.

Conclusions: Our results demonstrate the significant role of studied rSNPs of DEGs and their adaptive changes in the hereditary predisposition to PE. This work was supported by the Russian Foundation for Basic Research (grant №18-29-13045).

V. Serebrova: None. E. Trifonova: None. V. Stepanov: None.


Genetic risk factors for pre-eclampsia in the Finnish population

T. Kaartokallio1, S. Ruotsalainen2, T. Palviainen2, T. Hiekkalinna3,4, K. Auro2,5, M. Perola5,6, FINNPEC, K. Kivinen2, H. Laivuori1,2,7

1Medical and Clinical Genetics, University of Helsinki and Helsinki University Hospital, Helsinki, Finland, 2Institute for Molecular Medicine Finland, Helsinki Institute of Life Science, University of Helsinki, Helsinki, Finland, 3Finnish Institute for Health and Welfare, Helsinki, Finland, 4Clinical and Molecular Metabolism Research Program, University of Helsinki, Helsinki, Finland, 5National Institute for Health and Welfare, Helsinki, Finland, 6Diabetes and Obesity Research Program, University of Helsinki, Helsinki, Finland, 7Department of Obstetrics and Gynecology, Tampere University Hospital and Tampere University, Faculty of Medicine and Health Technology, Tampere, Finland

Introduction: Pre-eclampsia (PE) is a vascular pregnancy disorder with maternal and fetal genetic components. As the disease negatively affects reproductive success, the risk variants are assumed to be under purifying evolutionary selection. The amount of low-frequency pathogenic variants has shown to be increased in Finland due to recent bottlenecks, increasing statistical power in genetic studies. We aim to screen for genetic risk factors for PE in the families mainly originating from a northeastern Finland population isolate, and generalise the results for the whole population.

Methods: The PE family cohort consists of 15 extended families. DNA is available for 174 family members (mothers: 62 PE+32 ctrl; babies: 41 PE+39 ctrl). Using genome-wide imputed data, we have performed linkage and family-based association analyses with PSEUDOMARKER and PLINK to study segregation of loci with both maternal and fetal phenotypes. We will seek replication of the findings by association analysis in two Finnish cohorts with genome-wide imputed data available, the FINRISK population survey (426 PE+8357 ctrl) and the FINNPEC case-control cohort (mothers:1533 PE+995 ctrl; babies:818 PE+925 ctrl).

Results: In the linkage screen, we identified ~35 maternal and 40 fetal candidate loci when using a lenient p-value threshold p < 5x10−5. The FINRISK association analysis showed suggestive replication (p < 5x10−6) of two of the loci, maternal signal in chr9 close to GAS1 and fetal signal in chr16 in CDH13. Next steps include association analysis in FINNPEC and meta-analysis in both cohorts to verify these preliminary findings.

Funding: Academy of Finland, Erkko, Kordelin and Sohlberg Foundations, and VTR funding.

T. Kaartokallio: None. S. Ruotsalainen: None. T. Palviainen: None. T. Hiekkalinna: None. K. Auro: None. M. Perola: None. K. Kivinen: None. H. Laivuori: None.


A Retrospective Study of In Vitro Fertilization and Preimplantation Genetic Testing for chromosomal aberrations (PGT-SR and PGT-A) combined with Clinical Outcome

M. Baetens1, I. De Croo2, A. Dheedene1, K. Tilleman2, B. Menten1

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

PGT for structural rearrangements (PGT-SR) can be offered to patients carrying an inversion, balanced reciprocal or Robertsonian translocation, and PGT for aneuploidy screening (PGT-A) to patients with fertility problems or recurrent miscarriages. Selecting against embryos without a chromosomal rearrangement could improve live birth rates while reducing miscarriage rates or avoiding birth of an affected child. PGT is an extended, stressful trajectory while precise outcome predictions are still hard to grasp. Counseling PGT couples remains delicate due to numerous parameters influencing outcome. Comparing success rates to (inter)national reference data is part of good clinical practice. PGT technologies evolve at high speed, going from different biopsy timepoints to more extensive genetic methodologies, making good comparisons difficult. By detailed evaluation of our in-house data, and comparison to published data from (inter)national clinics, we obtained additional insights and can provide couples with more patient-focused information. We performed a retrospective cohort study at the Department for Reproductive Medicine and the Center for Medical Genetics (Ghent University Hospital), including patients who underwent ICSI-IVF (January 2016-April 2019) followed by PGT-SR or PGT-A after trophectoderm biopsy (68 and 90 oocyte retrieval cycles respectively). Making use of comprehensive chromosome screening (shallow whole genome sequencing or sWGS), we detected chromosomal abnormalities in 65% of PGT-SR embryos. In the PGT-A group, 40% of embryos showed a numerical or structural rearrangement. For translocation carriers, pregnancy rate per transfer was 58% and live birth rate per transfer 44%. For PGT-A patients, pregnancy rate and live birth rate were 39% and 25% respectively.

M. Baetens: None. I. De Croo: None. A. Dheedene: None. K. Tilleman: None. B. Menten: None.


Preimplantation Genetic Testing (PGT) for cancer predisposition: A single center large cohort experience

S. Zuckerman1, O. Freireich1, T. Eldar-Geva2, S. Lieberman1, S. Shaviv1, A. Yadin1, E. Farhi1, E. Hakam-Spector1, M. Ben-Shlomo1, T. Azar1, E. Zivi2, E. Rubinstein2, O. Schonberger2, I. Ben-Ami2, G. Altarescu1

1Medical Genetics Institute, Shaare Zedek Medical Center, Jerusalem, Israel, 2IVF unit, Shaare Zedek Medical Center, Jerusalem, Israel

In the last decade an increasing number of people carriers of cancer predisposition mutations (CCPM) are performing PGT-M. PGT-M for these late-onset and partial penetrant conditions implies that the procedure selects against embryos that might have been cancer-free. Moreover, preventive and therapeutic options for decreasing morbidity and mortality are available and ongoing cancer research might reveal treatments altering the natural history of cancer in the future. Most studies exploring perceptions of PGT-M among CCPM, were based on participants’ theoretical declarative intentions. Actual use has rarely been reported and analyzed. Between 2006-2019 overall 173 CCPM were counseled in our center: One couple between 2006-2008 and gradual increase until 102 CCPM between 2018-2019. Six were single women (3.4%) and 167 couples (96.5%). Among them 145(83.8%) were BRCA1/BRCA2 carriers (92/48 respectively; 5 BRCA1+BRCA2), 10(5.8%) Lynch carriers, 10(5.8%) APC carriers, 4(2.3%) TP53 carriers, 2(1.2%) MEN2 carriers, 1(0.6%) CHEK2 carrier and 1(0.6%) BMPR1A carrier. Overall, 150(86.7%) of the carriers were healthy and 23(13.2%) were diagnosed with cancer or related condition. In 120(69.4%) couples the carrier was the women partner; in 51(29.5%) couples the carrier was the men partner and in 2(1.2%) couples both partners were carriers. Five)2.8%) couples had additional reason for PGT-M/PGT-SR. Overall Live birth rate for embryo transfer was 32%. Reproductive decision-making regarding PGT-M for CCPM is a complex process. While this application for PGT-M becomes acceptable, health professionals involved in the process should be aware and address the specific issues and concerns of this group regarding medical, emotional and moral aspects.

S. Zuckerman: None. O. Freireich: None. T. Eldar-Geva: None. S. Lieberman: None. S. Shaviv: None. A. Yadin: None. E. Farhi: None. E. Hakam-Spector: None. M. Ben-Shlomo: None. T. Azar: None. E. Zivi: None. E. Rubinstein: None. O. Schonberger: None. I. Ben-Ami: None. G. Altarescu: None.


Obstetric risks associated with preimplantation genetic testing for monogenic disorders and structural chromosomal rearrangements (PGT-M/SR) compared to regular IVF/ICSI.

M. Heijligers1, Y. Arens1, R. van Golde1, M. van der Hoeven1, J. Dumoulin1, I. Homminga2, C. van Tilborg3, C. de Die-Smulders1, A. van Montfoort1

1Maastricht University Medical Centre+, Maastricht, Netherlands, 2University Medical Center Groningen, Groningen, Netherlands, 3University Medical Center Utrecht, Utrecht, Netherlands

Introduction: Earlier studies on the obstetric outcome in pregnancies following PGT are inconclusive since both an increased risk and no increased risk for adverse obstetric outcome are reported. Moreover, not all of these studies distinguish between PGT for monogenic disorders or structural chromosomal rearrangements (PGT-M/SR) and PGT for aneuploidies (PGT-A).

Methods: This retrospective cohort study included all pregnancies resulting from PGT-M/SR (n = 389) and IVF/ICSI treatments (n = 1667) conducted between 2004 and 2014. Data were collected from the medical files and from questionnaires. These data included parental characteristics, characteristics of the treatment preceding the pregnancy and obstetric and neonatal characteristics.

Results: Obstetric complications were reported in 18% and 19% of the singleton pregnancies in the PGT and regular IVF/ICSI group, respectively (P = 0.103). The risk for stillbirth was lower in the PGT group (P = 0.044). Birth weights of all singletons were converted into z-scores and showed a comparable birth weight for the two groups (0.09 vs. -0.02; P = 0.084). Within the multiple pregnancies, the risk for obstetric complications (P = 0.535) and the incidence of fetal loss (P = 0.617) was comparable between the PGT and IVF/ICSI group. The mean pregnancy duration was longer (P = 0.037) and the mean birth weight was higher (P = 0.010) in the PGT group. More monozygotic pregnancies were observed in the PGT group (P = 0.009).

Conclusions: The more invasive nature of the PGT-M/SR technique does not lead to an increased risk for obstetric complications as compared to a regular IVF/ICSI treatment.

M. Heijligers: None. Y. Arens: None. R. van Golde: None. M. van der Hoeven: None. J. Dumoulin: None. I. Homminga: None. C. van Tilborg: None. C. de Die-Smulders: None. A. van Montfoort: None.


Results gained from fetal exome sequencing inform clinical management. A report of 100 cases.

E. Dempsey1, L. Ive2, R. Dubis2, E. Serra2, H. Savage2, A. Haworth2, S. Mansour1, T. Homfray1, S. Drury2

1St George’s University of London, London, United Kingdom, 2Congenica Ltd, Cambridge, United Kingdom

Introduction: Exome sequencing in cases of fetal structural malformation has been shown to increase diagnostic yield (over traditional diagnostic methods) by 8-80%, depending on phenotype. This technique is soon to be made available throughout England as part of a centralisation and standardisation of genetic laboratory services.

Materials and Methods: We have undertaken exome sequencing in 100 cases of fetal anomaly as part of a service improvement project at St George’s Hospital. In half of cases sequencing was undertaken with the intention of informing the management of an ongoing pregnancy. In the remaining cases exome sequencing was undertaken post-mortem. Exome data was analysed via the application of patient-specific phenotype panels, reducing the risk of incidental findings.

Results: Our diagnostic rate across all cases is ~ 40 % and higher in cases with skeletal and oedema phenotypes. Exome results have influenced the management of pregnancy and labour. They have also directed early neonatal care, including enabling the treatment of a newborn with enzyme replacement therapy. The results of exome sequencing have been used for accurate assessment of recurrence risk. In addition, results can be used for prenatal testing or preimplantation genetic diagnosis in a future pregnancy.

Conclusions: Using specific case examples we will describe the way in which the results of fetal exome sequencing have influenced clinical management. We will also describe the challenges we have encountered in the implementation of a fetal exome sequencing service, including the analysis of variants of uncertain significance in phenotype-limited situations.

E. Dempsey: None. L. Ive: A. Employment (full or part-time); Significant; Congenica Ltd. R. Dubis: A. Employment (full or part-time); Significant; Congenica Ltd. E. Serra: A. Employment (full or part-time); Significant; Congenica. H. Savage: A. Employment (full or part-time); Significant; Congenica Ltd. A. Haworth: A. Employment (full or part-time); Significant; Congenica Ltd. S. Mansour: None. T. Homfray: None. S. Drury: A. Employment (full or part-time); Significant; Congenica Ltd.


Whole-exome sequencing in the prenatal setting: relating fetal phenotype to genotype

L. Leeuwen, K. Bouman, N. Corsten-Janssen

UMCG, Groningen, Netherlands

Introduction: Whole-exome sequencing (WES) is increasingly being used in the prenatal setting. However, interpretation of WES data is complicated due to various factors including limited knowledge on the prenatal fetal phenotype of rare monogenic disorders. We therefore aimed to describe the prenatal fetal phenotype of genetic variants found by WES.

Methods: In this single-center study, we included fetuses in whom WES was indicated in a prenatal diagnostic setting between March 1, 2018 and January 1, 2020 during pregnancy or after termination of pregnancy. We performed trio WES using a phenotype-dependent filtering strategy. We documented prenatal and postnatal characteristics of fetuses and compared the phenotype with literature using PubMed.

Results: WES was performed in 93 fetuses. A pathogenic or likely pathogenic variant was found in 29 (31.2%) fetuses (21 different genes). For eight genes, it was the first time a causative genetic variant was found in a prenatal setting (C2CD3,CNOT1,GATA6, PLD1,POMK,SAMD9,SMC3,THOC6). In three fetuses, some detected structural anomalies have not previously been described (PEX1-,POMK-,THOC6-variants). In three fetuses, diagnosis was filtered out in the prenatal setting due to various reasons, but could be established using additional postnatal phenotypic characteristics. In general, the prenatal phenotype on ultrasound corresponded well with postnatal phenotype although some features were not detected by the prenatal ultrasound examination.

Conclusions: Increased knowledge about the prenatal fetal phenotype is important for improving the interpretation and classification of fetal genetic variants. This will lead to improvement of parental genetic counseling as well as prenatal and neonatal care.

L. Leeuwen: None. K. Bouman: None. N. Corsten-Janssen: None.


Lessons learned from rapid Exome Sequencing(rES)as a standard diagnostic test in a prenatal setting for fetuses with ultrasound abnormalities.

N. Corsten-Janssen1, K. Bouman1, A. J. Scheper1, M. T. Meems-Veldhuis1, J. B. G. M. Verheij1, R. Suijkerbuijk1, L. K. Duin2, G. T. R. Manten3, I. M. van Langen1, B. Sikkema-Raddatz1, H. Westers1, C. C. van Diemen1

1Department of Genetics, University of Groningen, University Medical Centre Groningen, Groningen, Netherlands, 2Department of Obstetrics, Gynecology and Prenatal Diagnosis, University Medical Centre Groningen, Groningen, Netherlands, 3Department of Obstetrics and Gynecology, Isala, Zwolle, Zwolle, Netherlands

Background: Adding rES to conventional genetic tests for fetuses with ultrasound abnormalities improves the diagnostic yield, but differs from performing postnatal rES for instance by its rapid turnaround time and the impact of a diagnosis. We will present the lessons we learned by performing rES as a diagnostic test in prenatal care from March 2018.

Methods: We performed trio rES analysis using a custom virtual gene panel of ~4,300 OMIM genes during pregnancy. Inclusion criteria were: 1) two or more independent major fetal anomalies, or 2) hydrops fetalis or bilateral polycystic kidneys, or 3) one major fetal anomaly and a first-degree relative with the same anomaly.

Results: We established a genetic rES-based diagnosis in 14 out of 54 (26%) eligible fetuses with a mean turnaround time of 14 days. We learned the following lessons: (1) We made unexpected diagnoses using our broad virtual panel, like Zellweger syndrome in a fetus suspected of a ciliopathy. (2) We missed four diagnoses that were made postnatally using additional phenotypic information and adapted our filter strategy. 3) We were confronted with incidental unsolicited findings and changed our filter strategy to minimize them. (4) There is a lack of knowledge about prenatal phenotype of known genetic conditions, which hinders diagnostics. Further, we experienced the impact of time pressure to generate a diagnosis in limited time and had to deal with changing fetal phenotypes.

Conclusion: rES in the prenatal setting is feasible but challenging, and improves by adapting the process based on insights from previous experiences.

N. Corsten-Janssen: None. K. Bouman: None. A.J. Scheper: None. M.T. Meems-Veldhuis: None. J.B.G.M. Verheij: None. R. Suijkerbuijk: None. L.K. Duin: None. G.T.R. Manten: None. I.M. van Langen: None. B. Sikkema-Raddatz: None. H. Westers: None. C.C. van Diemen: None.


Improved fetal sex determination in twin pregnancies in low coverage whole-genome based non-invasive prenatal testing

T. Sedlackova1, M. Hyblova2,3, J. Budis1,4,5, M. Harsanyova1,6, M. Kucharik1,5, O. Pos1,6, M. Sekelska2,3, B. Nagy7, G. Minarik2,3, T. Szemes1,5,6

1Geneton Ltd., Bratislava, Slovakia, 2Medirex Inc., Bratislava, Slovakia, 3Trisomy Test Ltd., Bratislava, Slovakia, 4Slovak Centre of Scientific and Technical Information, Bratislava, Slovakia, 5Comenius University Science Park, Bratislava, Slovakia, 6Department of Molecular Biology, Faculty of Natural Sciences, Comenius University, Bratislava, Slovakia, 7Semmelweis University, Budapest, Hungary

Introduction: Fetal sex determination is already a common part of the standard non-invasive prenatal testing (NIPT). The current classification methods are almost entirely accurate for fetal sex prediction in singleton pregnancies, but they still show the limitations for multiple pregnancies, with 98% reported accuracy of fetal sex prediction in twins.

Methods: We enrolled 99 maternal samples with twin pregnancies and confirmed fetus sex in this study. Since a precise estimation of the fetal fraction is a key for correct sex determination, we used a previously published improved predictor utilizing two different attributes of analyzed genomic material - length and genomic location of the sequenced DNA fragments. Subsequent fetal sex prediction was performed using an SVM classifier.

Results: Our method has shown perfect prediction accuracy in distinguishing between pregnancies with two female fetuses and pregnancies with at least one male fetus. We observed one incorrect prediction between one and two male fetuses. Overall, the accuracy of fetal sex prediction was 98.99%.

Conclusions: Here we described a novel bioinformatic algorithm to determine the sex of both fetuses in twin pregnancies, by the analysis of cell-free fetal DNA from the maternal plasma. Our algorithm achieved superior accuracy in fetal sex prediction in twins compared to known bioinformatics tools. Moreover, it provides supporting graphs for visual inspection and calculation of prediction confidence.

Keywords: non-invasive prenatal testing, twin pregnancy, whole-genome sequencing, fetal sex prediction

T. Sedlackova: A. Employment (full or part-time); Modest; Geneton Ltd.. M. Hyblova: None. J. Budis: None. M. Harsanyova: None. M. Kucharik: None. O. Pos: None. M. Sekelska: None. B. Nagy: None. G. Minarik: None. T. Szemes: None.


The value of prenatal array to detect submicroscopic chromosomal imbalances in cases with abnormal fetal ultrasonographic findings; a retrospective study

L. OZER, M. Aydin, S. Aktuna, E. Unsal


Chromosomal microarray (CMA) is used in prenatal diagnosis for detecting CNVs which are too small to detect by karyotyping. Here we report our laboratory’s array data of prenatal samples and describe phenotype-genotype correlations. Methods: 180 specimens (149 amniotic fluid, 23 chorionic villi and 8 cord blood samples) were analyzed by array CGH. Detection of genomic CNVs was performed using Agilent, 8x60K CGH Microarray. Interpretation was done according to the ACMG guidelines. Results: %16.1 of patients (29/180), CNVs were detected. 24 of them had segmental duplication or deletion, 5 had trisomy and 1 had monosomy X. 3.3% of patients had aneuploidy, 4.4% of patients had VUS variants and %8.8 of patients had pathogenic segmental deletions/duplications. 61.1% of the segmental pathogenic variants were deletions and most of the VUS variants were duplications (62.5%). 50% of the segmental pathogenic variants were < 7 Mb and the smallest size was 0.5MB (deletion). 22.2% of pathogenic variants were well-defined CNV syndromes. The most prevalent indications were ultrasonographic anomalies, increased triple test risk and advanced maternal age. While 30% of patients with normal results have normal ultrasound, all patients with a pathogenic variant had an ultrasound anomaly. The most defined ultrasonographic findings in cases with pathogenic variants were cardiac anomalies, increased NT and brain anomalies. Conclusion: Detection of submicroscopic imbalances by CMA increases the power of prenatal diagnosis. Our data emphasize the importance of CMA in the prenatal diagnosis of the fetuses with abnormal ultrasonographic findings whereas conventional karyotyping resolution is inadequate to detect submicroscopic changes.

L. Ozer: None. M. Aydin: None. S. Aktuna: None. E. Unsal: None.


Targeted prenatal exome sequencing in a series of fetal structural anomalies detected by ultrasonography: first experience of the University Hospitals of Geneva

L. Quteineh1, J. Pellegrinelli2, M. Billieux2, M. Guipponi1,3, S. Laurent1, E. Hammar1, A. Murphy1, M. Carminho Amaro Rodrigues1, T. Ait-Mouhoub1, F. Sloan-Béna1, S. Unger4, F. Giuliano4, C. Rieubland5, J. Fluss6, P. Extermann7, J. Blouin1,3, M. Abramowicz1, S. Fokstuen1

1Service of Genetic Medicine, Geneva, Switzerland, 2Department of gynecology and obstetrics, Geneva, Switzerland, 3Department of Genetic Medicine and Development, Geneva, Switzerland, 4Service of Genetic Medicine, Lausanne, Switzerland, 5Department of Pediatrics, Division of Human Genetics, Bern, Switzerland, 6Pediatric Neurology Unit, Geneva, Switzerland, 7Dianecho echographic center, Geneva, Switzerland

Introduction: Diagnostic genome-wide sequencing approaches to investigate prenatally detected anomalies are increasingly used in fetal medicine. We aimed to evaluate the value of targeted whole exome sequencing (WES) in fetuses with prenatal sonographic abnormalities and normal microarray and karyotype.

Materials and Methods: After informed consent was obtained, we sequenced the DNA from amniotic fluid cells of 17 fetuses by WES (Illumina NextSEQ500). We then performed bioinformatic analysis of a selected gene panel. All cases were ongoing pregnancies. The genomic results were obtained within 2 weeks.

Results: The 17 fetuses presented with the following anomalies: isolated brain malformations (n = 6), sustained increased nuchal translucency (n = 3), multiple structural anomalies (n = 3), polyhydramnios (n = 1), situs inversus (n = 1), renal cysts (n = 1), hydrops fetalis (n = 1) and skeletal dysplasia (n = 1). Causing variants were identified in 4 cases (24%): two with sustained increased nuchal translucency presented with a de novo variant in RAF1 (p.Ser267Arg) and in BRAF (p.Thr241Arg) genes, compatible with a RASopathy, the fetus with skeletal dysplasia had a de novo variant in COL11A1 gene (p.Gln921_Pro926del) causing Stickler/Marshall syndrome and the fetus with hydrops fetalis had 2 variants in PIEZO1 gene (p.Ser182* and p.Phe1294del) causing autosomal recessive lymphatic dysplasia. Pregnancy was terminated in all 4 cases.

Conclusions: targeted WES is an adequate approach of genome-wide sequencing for ongoing pregnancies with fetal structural abnormalities suspicious for a genetic etiology and normal microarray. It has a diagnostic utility and might facilitates genetic counseling.

L. Quteineh: None. J. Pellegrinelli: None. M. Billieux: None. M. Guipponi: None. S. Laurent: None. E. Hammar: None. A. Murphy: None. M. Carminho Amaro Rodrigues: None. T. Ait-Mouhoub: None. F. Sloan-Béna: None. S. Unger: None. F. Giuliano: None. C. Rieubland: None. J. Fluss: None. P. Extermann: None. J. Blouin: None. M. Abramowicz: None. S. Fokstuen: None.


Prenatal diagnosis of trisomies in CVS: are non-mosaic biallelic QF-PCR results prognostic?

A. R. Pendlebury-Watt1, K. Mann1, C. Ogilvie2

1Viapath analytics, London, United Kingdom, 2Genetics Department, Guys and St. Thomas’ NHS Foundation Trust, London, United Kingdom

Placental mosaicism for chromosome aneuploidy complicates prenatal analysis of chorionic villus (CV) samples and has a reported incidence of up to 2%. Quantitative fluorescence-PCR (QF-PCR) analysis of microsatellite repeats is widely used to establish chromosome copy number of uncultured CV samples; if recommended preparation methods are employed, both cytotrophoblast and mesoderm should be represented. In the case of trisomy results, the presence of three different alleles is observed in ~90% of cases, confirming meiotic non-disjunction as the origin of the abnormal cell line. Where only biallelic 2:1 ratios are observed, the trisomy may have arisen by mitotic non-disjunction following a normal conception, in which case a normal cell line may be present; European and UK best practice guidelines recommend that non-mosaic biallelic (NMB) results should be reported more cautiously, given the increased risk of mosaicism. However, the likelihood of finding a normal cell line following an NMB result is not established. We have therefore audited 10 years of cases and compared NMB results to the follow-up karyotype and/or amniocentesis results. In total, 133 CVS showed NMB results for common trisomies, of which 131 were concordant with follow-up tests. Two trisomy 18 cases showed a normal result on amniocentesis, indicating confined placental mosaicism; neither case had ultrasound abnormalities present at referral. Therefore, in this cohort, ~1.5% of NMB QF-PCR cases subsequently showed evidence of mosaicism, this is reduced to 0% for cases with USS abnormalities. These figures may inform the reporting and pregnancy management of NMB cases.

A.R. Pendlebury-Watt: None. K. Mann: None. C. Ogilvie: None.


Parental experiences of rapid Exome Sequencing in case of serious ultrasound abnormalities during pregnancy

M. Plantinga1, L. Zwienenberg1, E. van Dijk1, H. Breet1, J. Diphoorn1, J. el Mecky2, K. Bouman1, J. Verheij1, A. Ranchor3, N. Corsten-Janssen1, I. van Langen1

1UMCG, Clinical Genetics, Groningen, Netherlands, 2University of Southampton, CELS, Southampton, United Kingdom, 3UMCG, Health Psychology, Groningen, Netherlands

Objective: Adding rapid Exome Sequencing (rES) to conventional genetic tests (QF-PCR and SNP-array) can improve the diagnostic yield of pregnancies showing ultrasound abnormalities but also includes a higher chance of unsolicited findings. rES was offered in the context of a prenatal rES implementation study with a turnaround time of 14 days. We evaluated how this offer was experienced in an explorative mixed-methods parental experience study.

Methods: We investigated reasons for accepting and declining rES, the impact of rES on decision making, experiences of the counseling and test process, and understanding of test information and possible test outcomes. All couples were asked to fill two surveys, one after pre-test and one after post-test counseling. Part of the couples were approached for an interview until data saturation was reached.

Results: 44 couples participated in the survey study, 11 in the interview study. In 22% of pregnancies, a diagnosis was found, in 31% an unsolicited finding. 95% wanted to be informed about unsolicited findings. The most important reason for accepting rES was to find a diagnosis. Most parents already decided to terminate the pregnancy on the basis of the ultrasound abnormalities, although rES results impacted decision in a few cases. The impact for future pregnancies was higher than with conventional testing and the fast turnaround was valued highly. The counseling and test process was experienced positive. Parents suggested to add information of other parents’ experiences and decisional process to the test information.

Conclusion: Parents evaluate rES positively, despite the risk of unsolicited findings.

M. Plantinga: None. L. Zwienenberg: None. E. van Dijk: None. H. Breet: None. J. Diphoorn: None. J. el Mecky: None. K. Bouman: None. J. Verheij: None. A. Ranchor: None. N. Corsten-Janssen: None. I. van Langen: None.


When to test fetuses for RASopathies? Proposition from a systematic analysis of 353 multicenter cases and a postnatal cohort

A. Scott1, N. Di Giosaffatte2, V. Pinna2, P. Daniele2, S. Corno3, V. D’Ambrosio3, E. Andreucci4, A. Marozza5, F. Sirchia6, G. Tortora7, D. Mangiameli8, C. Di Marco9, I. Donati10, A. Zonta11, E. Grosso11, G. Mastromoro12, P. Versacci13, F. Pantaleoni14, F. C. Radio14, T. Mazza15, L. Papi5, T. Mattina8, A. Giancotti3, A. Pizzuti12, A. Laberge1, M. Tartaglia14, M. Delrue1, A. De Luca2

1Pediatrics Department, Medical Genetics Division, CHU Sainte-Justine, University of Montreal, Montreal, QC, Canada, 2Medical Genetics Unit, Fondazione IRCCS-Casa Sollievo della Sofferenza, San Giovanni Rotondo, Italy, 3Department of Maternal and Child Health and Urologic Science, "Sapienza" University, Policlinico Umberto I Hospital, Rome, Italy, 4Medical Genetics Unit, Meyer Children’s University Hospital, Florence, Italy, 5Section of Medical Genetics, Department of Experimental and Clinical Biomedical Sciences, University of Florence, Florence, Italy, 6Institute for Maternal and Child Health, IRCCS "Burlo Garofolo", Trieste, Italy, 7Azienda Ospedaliero-Universitaria Ospedali Riuniti di Ancona, Ancona, Italy, 8Department of Pediatrics, Medical Genetics, University of Catania, Catania, Italy, 9Ospedale Santa Maria Nuova, Florence, Italy, 10Genetic Department, Infermi Hospital, Rimini, Italy, 11Città Della Salute e Della Scienza University Hospital, Medical Genetics Unit, Torino, Italy, 12Department of Experimental Medicine, Università Sapienza, Rome, Italy, 13Department of Pediatrics, Università Sapienza, Rome, Italy, 14Genetics and Rare Diseases Research Division, Ospedale Pediatrico Bambino Gesù, IRCCS, Rome, Italy, 15Laboratory of Bioinformatics, IRCCS Casa Sollievo della Sofferenza, San Giovanni Rotondo, Italy

Introduction: Recent studies have identified some evocative prenatal features of RASopathies e.g. lymphatic dysplasia (cystic hygroma, hydrops, effusions), congenital heart disease, polyhydramnios, and renal anomalies. The aim of this study was to better define the prenatal indications for RASopathy testing and the genotype-phenotype correlations.

Methods: 353 cases sent for prenatal RASopathy testing between 2012-2019, after a normal a-CGH, were collected. For most, 11 RASopathy genes were tested. Detailed ultrasound findings were available for 313 fetuses, whereas 40 cases had only clinical indications for testing collected. Twenty-five RASopathy patients diagnosed postnatally were added to widen the phenotypic spectrum.

Results: See tables below for detailed results. The overall diagnostic yield was 14% (51/353). The largest contributors of mutations were PTPN11 (29%), RIT1 (15%), RAF1 (13%) and HRAS (12%). Severe postnatal phenotypes were in majority diagnosed prenatally and associated with mutations in HRAS, RIT1 and RAF1.

Conclusions: After normal a-CGH, RASopathies should be considered when any ultrasound finding of lymphatic dysplasia or suggestive congenital heart disease is found alone or in association. In the presence of persistent or associated hygroma, NT>6 mm, hypertrophic cardiomyopathy, or any finding combined with polyhydramnios or renal anomaly, the likelihood of RASopathy diagnosis is significantly increased.

Table 1 Prenatal ultrasound findings in 313 patients with a normal array-CGH

Prenatal findingsTotal # of patients (n = 313)Prenatal mutation-positive group (n = 48)Mutation-negative group (n = 265)p-valueaPostnatal mutation-positive group (n = 25)
Increased NT231/313 (74%)30/48 (63%)201/265 (76%)0,031/25 (4%)
NT > 3 < 3,521/231 (9%)0/30 (0%)21/201 (10%)0,048 
NT ≥ 3,5145/231 (63%)18/30 (60%)127/201 (63%) -
NT > 660/231 (26%)12/30 (40%)48/201 (24%)  
unknown5/231 (2%)0/30 (0%)5/201 (2%)  
Increased NT without CH86/313 (27%)7/48 (15%)79/265 (30%)0,021/25 (4%)
Mean NT (mm)b5,01 (0,7-14,9)6,59 (1,25-13,5)4,80 (0,7-14,9)0,00012,58(1,0-4,1)
Cystic hygroma183/313 (58%)31/48 (65%)152/265 (57%)0,31/25 (4%)
1st trimester CH58/183 (32%)4/31 (13%)54/152 (36%)0,02-
Persistent CH125/183 (68%)27/31 (87%)98/152 (64%)  
Isolated*89/183 (49%)5/31 (16%)84/152 (55%)< 0,0001-
in association94/183 (51%)26/31 (84%)68/152 (45%)  
CH without ↑ NT34/183 (19%)9/31 (29%)25/152 (16%)0,11/25 (4%)
CH with ↑ NT149/183 (81%)22/31 (71%)127/152 (84%)  
NT > 3 < 3,517/149 (11%)0/22 (0%)17/127 (13,5%)0,02-
NT ≥ 3,589/149 (60%)11/22 (50%)78/127 (61,5%)  
NT > 643/149 (29%)11/22 (50%)32/127 (25%)  
Increased NF68/313 (22%)18/48 (38%)50/265 (19%)0,0010/25 (0%)
NF > 663/68 (93%)15/18 (83%)48/50 (96%)0,08-
NF > 155/68 (7%)3/18 (17%)2/50 (4%)  
Mean NF (mm)b6,94 (3,0-28)9,39 (4,2-22)6,28 (3,0-28)0,01N/A
Hydrops60/313 (19%)20/48 (42%)40/265 (15%)< 0,00011/25 (4%)
1st trimester hydrops14/60 (10%)2/20 (10%)12/40 (30%)0,08-
Persistent hydrops26/60 (90%)18/20 (90%)28/40 (70%)  
Thoracic effusions or ascites54/313 (17%)22/48 (46%)32/265 (12%)< 0,00014/25 (16%)
Congenital heart disease71/313 (23%)18/48 (38%)53/265 (20%)0,0023/25 (12%)
cardiac defect64/313 (20%)15/48 (31%)49/265 (18%)0,023/25 (12%)
HCM13/313 (4%)9/48 (19%)4/265 (2%)0,0071/25 (4%)
HCM without CD7/13 (54%)3/9 (33%)4/4 (100%)N/A0/25 (0%)
combined HCM + CD6/13 (46%)6/9 (67%)0/4 (0%) 1/25 (4%)
Other associated findings
 Polyhydramnios36/313 (12%)14/48 (29%)22/265 (8%)< 0,00018/25 (32%)
 Renal anomaly46/313 (15%)14/48 (29%)32/265 (12%)0,00035/25 (20%)
 Macrosomia12/313 (4%)6/48 (13%)6/265 (2%)0,0012/25 (8%)
 Mild ventriculomegaly13/313 (4%)7/48 (15%)6/265 (2%)0,00010/25 (0%)
 Short long bones18/313 (6%)8/48 (17%)10/265 (4%)0,00041/25 (4%)
 Only one US finding61/313 (19%)2/48 (4%)59/265 (22%)0,0046/25 (24%)
 Two or more US findings252/313 (81%)46/48 (96%)254/265 (78%) 7/25 (28%)
 No US findingN/AN/AN/A-12/25 (48%)
  1. a : chi-square statistic or Fisher exact test, as appropriate. α;;; error = 0,05, b : t-student test, * without another finding, except increased NT

Table 2 Diagnostic yield of prenatal ultrasound findings

Prenatal findingsDiagnostic yield for NSD according to indications for testing (51 NSD)p-valueaDiagnostic yield for NSD according to prenatal US findings (48 NSD)p-valuea
 n = 353 n = 313 
Global diagnostic yield14% (51/353)-15% (48/313)-
Increased NT11% (29/258)0,413% (30/231)0,7
NT > 3 < 3,50% 0% 
NT ≥ 3,59%0,0212%0,1
NT > 620% 20% 
in association18% 100% 
Increased NT without CH4,5%0,038%0,09
Cystic hygroma16% (30/189)0,617% (31/183)0,9
1st trimester CHN/AN/A7%0,02
Persistent CHN/A 22% 
in association28% 28% 
CH without ↑ NT23%0,226%0,1
CH with ↑ NT14% 15% 
NT > 3 < 3,5-N/A0%0,08
NT ≥ 3,5- 12% 
NT > 6- 26% 
Increased NF26% (13/50)0,0326% (18/68)0,02
NF > 622%0,124%0,1
NF > 1560% 60% 
Hydrops35% (17/48)0,000133% (20/60)0,0004
1st trimester hydropsN/AN/A14%0,1
Persistent hydropsN/A 39% 
Thoracic effusions or ascites30% (12/40)0,0141% (22/54)< 0,0001
Congenital heart disease23% (12/53)0,125% (18/71)0,03
cardiac defect23% (11/48)0,00223% (15/64)< 0,0001
HCM60% (6/10) 69% (9/13) 
combined HCM + CD100% (5/5) 100% (6/6) 
Other associated findings
 Polyhydramnios50% (8/16)N/A39% (14/36)< 0,0001
 Renal anomaly29% (4/14)N/A30% (14/46)0,01
 Macrosomia57% (4/7)N/A50% (6/12)N/A
 Mild ventriculomegaly-N/A54% (7/13)N/A
 Short long bones-N/A44% (8/18)N/A
General findings
 Only one US finding9% (13/143)0,033% (2/61)0,002
 Two or more US findings18% (38/210) 18% (46/252) 

N.B. Diagnostic yield of each major US finding was compared with the global diagnostic yield for p-value’s calculation

A. Scott: None. N. Di Giosaffatte: None. V. Pinna: None. P. Daniele: None. S. Corno: None. V. D’Ambrosio: None. E. Andreucci: None. A. Marozza: None. F. Sirchia: None. G. Tortora: None. D. Mangiameli: None. C. Di Marco: None. I. Donati: None. A. Zonta: None. E. Grosso: None. G. Mastromoro: None. P. Versacci: None. F. Pantaleoni: None. F.C. Radio: None. T. Mazza: None. L. Papi: None. T. Mattina: None. A. Giancotti: None. A. Pizzuti: None. A. Laberge: D. Speakers Bureau/Honoraria (speakers bureau, symposia, and expert witness); Modest; Sobi. M. Tartaglia: None. M. Delrue: None. A. De Luca: None.


Gene ontology enrichment analysis of congenital renal agenesis-associated genes

S. Kalantari1,2,3, I. Filges1,2,3

1Medical Genetics, Institute of Medical Genetics and Pathology, University Hospital Basel, Basel, Switzerland, 2Department of Clinical Research, University Hospital Basel, Basel, Switzerland, 3University of Basel, Basel, Switzerland

Introduction: Uni- or bilateral renal agenesis (RA) is a commonly occurring major congenital anomaly impacting fetal and neonatal outcomes. Although chromosomal abnormalities, CNVs and pathogenic variants in many genes have been associated to RA, the etiology is still poorly understood. Our aim was to better understand the biological pathways involved in RA and to identify interacting candidate pathways useful for further gene identification and molecular diagnosis in clinical care when applying genome-wide sequencing.

Materials and Methods: We used Phenolyzer to obtain all genes known as causative for syndromic and non-syndromic uni- and bilateral RA. Using ClueGO gene enrichment analysis we classified the relationship between these genes and biological processes as defined by gene ontology.

Results: We identified 289 genes described to be involved in the pathogenesis of RA when mutated. We obtained 69 groups of enriched biological processes. About 50% included pathways directly related to the development of urogenital organ tissues (e.g. metanephros, nephric duct, ureter). Furthermore, several ciliary, axis specification, hindgut development and endocrine pathways are enriched, which may relate to different clinical presentations of RA. The results also support the hypothesis that RA is part of the ciliopathy spectrum and developmentally related to dysplastic kidneys.

Conclusions: Our gene ontology analysis shows that genes representing distinct biological pathways are significantly enriched. They may relate to different clinical presentations of RA, which will lead to an improved delineation of candidate genes and molecular diagnosis of this congenital anomaly in clinical practice.

S. Kalantari: None. I. Filges: None.


Is mitochondrial DNA (mtDNA) copy number in human cleavage-stage embryos a marker of embryonic quality and development, or modified by the presence of a mtDNA mutation?

J. Steffann1, K. Chatzovoulou1, N. Gigarel1, A. Mayeur2, F. Jabot-Hanin3, S. Monnot1, L. Hesters2, N. Frydman2, J. Bonnefont1

1Hopital Necker-Enfants Malades, Paris, France, 2Hopital Antoine-Béclère, Clamart, France, 3Université de Paris, Paris, France

Mitochondria play a critical role during the early stages of human embryo development by supplying sufficient levels of energy required for embryo survival. Because mtDNA replication is silenced during early embryonic development, the presence of a large number of mtDNA copies in the mature oocyte is mandatory. In human, several studies on blastocysts day-5 embryos have paradoxically evidenced a positive correlation between high quantities of mtDNA molecules and severe conditions, like aneuploidy, poor embryo quality, and implantation failure. However, data from day-3 embryos are much more limited and contradictory, making this point a highly controversial one. We have quantified the mtDNA copy number (CN) in residual DNA of 1 or 2 cells, sampled from day-3 embryos, in a context of preimplantation genetic analysis for a non-metabolic, non-mitochondrial DNA disorder (n = 165, control group) and for a mtDNA mutation (n = 16, mitochondrial group). A great dispersion of the mtDNA CN was found, among the different cells of a given embryo, and the different embryos from a given patient. A maternal effect regulating the mtDNA CN was observed, but remained of unknown origin. In particular, no correlation was detected between mtDNA CN and maternal age or ovarian reserve. mtDNA CN did not correlate with embryonic quality and ability of an embryo to implant and give a viable pregnancy. Finally, the mtDNA CN was not influenced by the presence of a mtDNA mutation. Based on these findings, mtDNA CN at day 3 of development cannot be used to select the best embryo to transfer.

J. Steffann: None. K. Chatzovoulou: None. N. Gigarel: None. A. Mayeur: None. F. Jabot-Hanin: None. S. Monnot: None. L. Hesters: None. N. Frydman: None. J. Bonnefont: None.



B. Schreiner, E. Lindström, B. Vanherberghen, A. Hedrum

Devyser, Hägersten, Sweden

Background: Hemolytic disease of the fetus and newborn (HDFN) is a serious complication of maternal and fetal blood group incompatibility. This occurs when a Rhesus-D negative woman, carrying a Rhesus-D positive fetus during her first pregnancy, is alloimmunized against RhD antigen and carries another Rhesus-positive fetus during a subsequent pregnancy. Nowadays, HDFN is rare since anti-D prophylaxis is provided to all RhD negative pregnant women. However, 40% of these women carry a Rhesus-negative fetus and are administered unnecessary treatment including all risks connected to blood-based products.

Aims: We set out to develop a fast and reliable diagnostic test assessing the fetal RHD gene status from maternal blood at the end of the first pregnancy trimester to enable targeted anti-D prophylaxis.

Methods: A real-time PCR-based assay was developed to detect cell-free fetal DNA (cffDNA) in maternal blood. The assay contains primer-probe combination specific for the RHD gene and the GAPDH house-keeping gene as internal control. CffDNA from clinical samples was automatically extracted from 1mL of plasma.

Results: Devyser RHD exhibits high experiment repeatability between different reagent lots. The analytical sensitivity is down to DNA concentrations corresponding to less than 7 gene copies per reaction with 95% probability.

Summary/conclusions: Devyser RHD provides a fast, non-invasive procedure to assess the fetal RHD status in Rhesus-D negative mothers. This product is validated together with an automated DNA extraction process and reaction set-up (QIASymphony AS/SP), enabling high-throughput sample analysis with low-risk for sample mix-up. Various kit sizes are available offering flexibility with sample throughput.

B. Schreiner: A. Employment (full or part-time); Significant; Devyser. E. Lindström: A. Employment (full or part-time); Significant; Devyser. B. Vanherberghen: A. Employment (full or part-time); Significant; Devyser. A. Hedrum: A. Employment (full or part-time); Significant; Devyser.


Diagnosis of Rothmund Thomson Syndrome by whole genome sequencing

Q. Zhou, J. Sun, T. Wang, Y. Xing, Z. Wan, X. Yang, Z. Liu, W. Wang

Genomics Institute of Beijing, Shenzhen, China

Introduction: Rothmund-Thomson Syndrome (RTS) is a rare disorder and has been described in all ethnicities, with several identified pathogenic variants. Since there is fewer than hundreds of individuals have been described, the population prevalence is unknown. The genetic features and follow-up data of individuals with RTS are limited.

Methods: We performed a whole genome sequencing (WGS) using cord blood for an infant, who had RTS like clinical features in infancy.

Results: The infant showed a developmental delay in infancy, with a small size in both height and weight. The erythema started appearing on her face at the age of three months and then spread to the entire body. Besides, she had neither eyelashes nor eyebrows, with sparse scalp hair. After analyzing the WGS data, we found a heterozygous nonsense mutation c.2752G>T (p.Glu918*) and a novel frameshift-insertion c.1547_1548insC (p.Ala516fs) in the gene RECQL4, which is a known pathogenic gene of RTS. Both variants had potential effects on protein synthesis or function. Further validation indicated these were compound variants inheriting from parents.

Conclusions: Taking together with the clinical characteristics and the identification of biallelic pathogenic variants in a recessive manner, we report a diagnosed infant with RTS. Our findings provide potential application of WGS for newborn to clinical diagnosis. The follow-up study can help to further understand the clinical features and the genotype-phenotype interactions of RTS.

Grant: The Shenzhen Municipal Government of China (NO.JCYJ20170412152854656)

Q. Zhou: None. J. Sun: None. T. Wang: None. Y. Xing: None. Z. Wan: None. X. Yang: None. Z. Liu: None. W. Wang: None.


Simpson-Golabi-Behmel syndrome familial case series with an emphasis on prenatal diagnosis

N. Krasovskaja1,2, A. Matulevičienė1,2, K. Šiaurytė2,3, K. Grigalionienė1,2, L. Ambrozaitytė1,2, A. Utkus2

1Vilnius University Hospital Santaros Klinikos, Vilnius, Lithuania, 2Department of Human and Medical Genetics, Institute of Biomedical Sciences, Faculty of Medicine, Vilnius University, Vilnius, Lithuania, 3Faculty of Medicine, Vilnius University, Vilnius, Lithuania

Introduction: Simpson-Golabi-Behmel syndrome (SGBS) is a rare recessively inherited X-linked condition, with less than 300 reported cases. Characteristic features of SGBS include pre/postnatal overgrowth, distinctive craniofacial features (macrocephaly, coarse face with macroglossia), visceromegaly and increased risk of neoplasia. This condition is almost always diagnosed postnatally.

Materials and methods: A 23-y/o woman with uninformative anamnesis on the 21 weeks’ gestation was referred due to abnormal fetal ultrasound findings (polyhydramnios, macrocephaly, corpus callosum agenesis, abdominal visceromegaly and abnormal genitalia). The results of fetal DNA qPCR and SNP-CGH tests from amniotic fluid revealed no chromosomal abnormalities. The precise evaluation of mother’s phenotype (tall stature, coarse facial features, macroglossia) and fetal scan were key to directly sequence GPC3 gene and make the correct SGBS diagnosis prenatally.

Results: Sanger sequencing from amniotic fluid revealed hemizygous pathogenic variant NM_004484.3:c.1159C>T, (NP_004475.1:p.(Arg387Ter), rs122453121, CM073110) in exon 4 of the GPC3 gene, which determines SGBS. Mother was also found to be a carrier. Despite severe polyhydramnios progression on 33 weeks’ gestation, condition of the fetus was manageable and, pregnancy was carried to 38th weeks. The newborn’s phenotype presented with classical SGBS features.

Conclusions: An accurate evaluation of maternal phenotype and fetal scan are essential pathognomonic signs for early suspicion of SGBS. Successful prenatal diagnosis enables to make decisions on time for family and physicians about the outcomes of pregnancy and delivery.

N. Krasovskaja: None. A. Matulevičienė: None. K. Šiaurytė: None. K. Grigalionienė: None. L. Ambrozaitytė: None. A. Utkus: None.


Prenatal diagnosis of skeletal dysplasias/anomalies: high diagnostic yield using microarray and sequencing data

I. Mademont-Soler1, A. Maroto1, J. Meléndez2, A. Cherino1, J. Perapoch1, N. Artigas1, B. Campos3, M. Segura3, C. Pérez4, E. Lloveras4, J. Hernando1, X. Queralt1, M. Obón1

1Hospital Universitari de Girona Dr. Josep Trueta, Salt, Spain, 2Hospital Santa Caterina, Salt, Spain, 3Quantitative Genomic Medicine Laboratories, Barcelona, Spain, 4Synlab, Barcelona, Spain

Introduction: Skeletal dysplasias (SDs) are severe congenital disorders, mostly of genetic origin. Etiology remains unknown in ~50% of cases. Materials and Methods: Nine pregnancies with ultrasound findings compatible with SDs were recruited prospectively from 2017 to 2019. A diagnostic algorithm combining microarray, Sanger and Next-Generation Sequencing was used.

Results: Definite diagnosis was achieved in 6 cases, uncertain results in 2 and negative results in 1.

C1Skeletal anomalies, oligohydramnios, increased NTMicroarray: Normal WES: NormalNo
C2Short long bones, polyhydramniosMicroarray: Normal Sanger: Pathogenic variant FGFR3Achondroplasia
C3Short long bones, narrow thoraxMicroarray: Normal Sanger: Pathogenic variant FGFR3Thanatophoric dysplasia
C4Multiple bone fractures, malposition of extremities, skull hypomineralizationMicroarray: NormalNGS virtual panel: Two VOUS IFT140Uncertain
C5Multiple bone fracturesMicroarray: NormalNGS virtual panel: Pathogenic variant COL1A1Osteogenesis imperfecta
C6Bilateral radial aplasiaMicroarray: arr[GRCh37] 1q21.1(145415156_145899418)x1Sanger: Pathogenic variant RBM8ATAR syndrome
C7Bowing of long bones, oligohydramniosSanger: Two pathogenic variants LIFRStüve-Wiedemann syndrome
C8ArthrogryposisMicroarray: Multiple LOH regionsSanger: Two pathogenic variants SLC6A9Glycine encephalopathy with normal serum glycine
C9ArthrogryposisMicroarray: NormalWES: One VOUS PIEZO2Uncertain

Conclusions: A combination of molecular cytogenetics and sequencing offers a high diagnostic yield (6/9), facilitating genetic counselling and clinical management. An individualized approach, multidisciplinary teams and the contribution of new sequencing technologies to diagnosis are recognized.

I. Mademont-Soler: None. A. Maroto: None. J. Meléndez: None. A. Cherino: None. J. Perapoch: None. N. Artigas: None. B. Campos: None. M. Segura: None. C. Pérez: None. E. Lloveras: None. J. Hernando: None. X. Queralt: None. M. Obón: None.


Embryo haplotype phasing based on non-invasive procedures

L. A. Alcaraz1, C. Pérez-Pelegrín1, S. González-Reig1, P. Brígido1, P. Piqueras2, A. Martí2, F. Sánchez-Martín2, V. Penacho1

1Bioarray, SL, Elche, Spain, 2Ginemed, Sevilla, Spain

Introduction: Preimplantation genetic testing (PGT) represents a widely applied genetic procedure for many IVF centers. Nowadays, for these purposes embryo material is needed. The removal of three cells in a day 5 embryo, represents an invasive procedure that could concern the embryo viability and increase the cost of a PGT. In the past few years there have been an increasing interest in the possibility of obtaining reliable DNA in the spent culture media (SCM) where the embryo is growing (1, 2).

Materials and Methods: For optimal PGT-M results, we aim to compare embryo biopsy and SCM. After that, we want to go further: discern whether the SCM is even a better choice to diagnose an embryo in comparison with the trophectoderm biopsy, comparing a complete embryo. All based on next generation sequencing (NGS) with PGDSeqTM (Journey Genomics S.L.U.) technology.

Results: We can confirm a high coincidence of SCM with complete embryo and the embryo biopsy. Tis primary results are promising, even if there are less polymorphisms obtained from this kind of material, we were able to study the haplotypes and the inheritance of the embryos.

Conclusions: A non-invasive perspective on the PGT world is certainly to be the next generation of samples. Bibliography:1. Huang, L. et al. Noninvasive preimplantation genetic testing for aneuploidy in spent medium may be more reliable than trophectoderm biopsy. Proc. Natl. Acad. Sci. 116, 14105-14112 (2019).2. Li, P. et al. Preimplantation Genetic Screening with Spent Culture Medium/Blastocoel Fluid for in Vitro Fertilization. Sci. Rep. 8, 9275 (2018).

L.A. Alcaraz: None. C. Pérez-Pelegrín: None. S. González-Reig: None. P. Brígido: None. P. Piqueras: None. A. Martí: None. F. Sánchez-Martín: None. V. Penacho: None.


Sperm protamine mRNA ratio in relation to DNA fragmentation and semen analysis

M. A. Ishchuk1,2, E. M. Komarova2, O. V. Malysheva2, I. D. Mekina2, E. A. Lesik2, A. M. Gzgzyan2,1, V. S. Baranov2,1, I. Y. Kogan2

1St. Petersburg State University, Saint-Petersburg, Russian Federation, 2The Research Institute of Obstetrics, Gynecology and Reproductology named after D.O.Ott, Saint-Petersburg, Russian Federation

Introduction: Sperm contain a complex population of RNA, and the utility of sperm RNA in fertility research are currently being explored. During mammalian spermatogenesis, the chromatin structure undergoes substantial condensation. The key role in this process is played by protamines 1 and 2 (PRM1, PRM2). Abnormal condensation could lead to DNA breaks, which is linked to pregnancy loss after assisted fertilization, disturbances in embryo development and increased risk of abortions. The aim of this study was to estimate the relations between protamine mRNA ration and semen quality and further DNA fragmentation. Finding sperm-quality markers would help to understand the causes of male infertility and to improve the male reproductive health.

Materials and Methods: Human ejaculates were obtained from 33 patients and semen analyses were assessed by WHO criteria (2010). We evaluated the sperm DNA fragmentation measured by TUNEL assay. Patient’s ejaculates were purified by density-gradient centrifugation, sperm cells were lysed, mRNA extracted, reverse transcribed and subjected to real-time qPCR using specific primer pairs for protamine-1 and protamine-2.

Results: The sperm protamine mRNA ratio in normozoospermic men (n = 19; 2,86 ± 0,67) differed significantly from that of patozoospermic patients (n = 14; 3,43 ± 0,79; p<0,05). A significant correlation was shown between sperm DNA fragmentation and the PRM2/PRM1 mRNA ratio (r=0,33; p <0,05). In the group of patients with an increased sperm DNA fragmentation (n = 14; 3,26 ± 0,19) the PRM2/PRM1 ratio was significantly higher than in the group of patients with normal rates (n = 19; 2,74 ± 0,18; p < 0,05).

Conclusions: Abnormal sperm protamine ratio associated with poor semen quality and DNA fragmentation.

M.A. Ishchuk: None. E.M. Komarova: None. O.V. Malysheva: None. I.D. Mekina: None. E.A. Lesik: None. A.M. Gzgzyan: None. V.S. Baranov: None. I.Y. Kogan: None.


Investigating PHF13 induced infertility through single cell transcriptomics and non-vertebrate model organisms

E. Bosch1, S. Lukassen2, A. Gregor1, A. B. Ekici1, C. Zweier1, A. Winterpacht1

1Institute of Human Genetics, FAU Erlangen-Nürnberg, Erlangen, Germany, 2Digital Health Center, Berlin Institute of Health (BIH) and Charité, Berlin, Germany

Infertility is a common medical problem worldwide. Of all infertility cases, approximately 40 - 50% are due to “male factor” infertility, mostly caused by spermatogenic failure. Despite extensive research, there is still limited knowledge of the genetic causes and underlying molecular mechanisms. We previously showed that deficiency for the epigenetic reader PHF13 in the mouse results in testis hypoplasia due to a spermatogenic defect.

Here, we obtained single-cell RNA expression profiles from Phf13−/− and Phf13+/- testis and compared them with wild type data. The results revealed a loss of meiotic sex chromosome inactivation (MSCI) in pachytene spermatocytes of PHF13 deficient mice. This is remarkable, since canonical PHF13 is only expressed in spermatogonia. In combination with ChIPseq data demonstrating an enrichment of PHF13 binding sites on the X- and Y-chromosome, we hypothesize that PHF13 mediates epigenetic changes on the sex chromosomes in spermatogonia, rendering the chromosomes competent for later inactivation.

PHF13 is evolutionarily conserved down to Bilateria including sea urchin, acorn worm and several insects like the flour beetle (Tribolium), but strikingly not in the evolutionary “young” genus Drosophila. Consequently, we ubiquitously overexpressed the Tribolium Phf13-homolog in D. melanogaster and compared these lines with wild type lines and lines overexpressing neuronal control genes. Phf13-overexpressing strains but not the control strains show a high grade of infertility in seemingly normal flies. This strongly indicates a conserved and very specific function of this protein in germ cell development, making PHF13 an interesting infertility factor and a fundamental player in germ cell differentiation.

E. Bosch: None. S. Lukassen: None. A. Gregor: None. A.B. Ekici: None. C. Zweier: None. A. Winterpacht: None.


Telomere length in karyotypically normal and abnormal chorion in progressing pregnancies and embryonic losses

A. V. Tikhonov1, M. I. Krapivin1, A. V. Petrovskaia-Kaminskaia1, A. A. Smirnova2, Y. M. Sagurova3, O. A. Efimova1, A. A. Pendina1, O. G. Chiryaeva1, L. I. Petrova1, V. S. Dudkina1, V. S. Baranov1,3

1D.O. Ott Research Institute of Obstetrics, Gynecology and Reproductology, St. Petersburg, Russian Federation, 2St. Petersburg State Pediatric Medical University, St. Petersburg, Russian Federation, 3St. Petersburg State University, St. Petersburg, Russian Federation

Telomere length (TL) is considered to be an indicator of biological age. Its correct regulation during pregnancy is crucial for embryonic development. The possible contribution of TL to the developmental capacity of embryos with abnormal karyotype remains unknown so far. Here, we studied TL in karyotypically normal and abnormal chorion in progressing pregnancies and embryonic losses. The chorionic villus samples were obtained by chorion biopsy from 15 patients with progressing first trimester pregnancy referred to invasive prenatal diagnosis and by curettage from 15 patients with missed abortion. The karyotypically abnormal samples included trisomy 21 and trisomy 16 cases in both progressing pregnancies (n = 7) and missed abortion (n = 7). The telomeric regions were detected on direct metaphase preparations by qFISH. Relative TLs were measured by dividing telomeric by subtelomeric fluorescence intensity assessed in ImageJ1.49v. The results were compared by the Mann-Whitney U-test. In progressing pregnancies, the relative TL was higher in karyotypically abnormal samples compared to karyotypically normal ones (p < 0.0001). In missed abortions, we observed the same tendency although the difference did not reach statistical significance (p=0.4652). When comparing the relative TL between the samples with normal karyotype in progressing pregnancies and missed abortions, we revealed no difference (p=0.8890). However, the relative TL was higher in the samples with abnormal karyotype in progressing pregnancies compared to those in missed abortions (p=0.0013). Our results suggest that in the case of karyotype abnormality, longer telomeres in chorionic villi may contribute to pregnancy success. Supported by RSF №19-75-00023.

A.V. Tikhonov: None. M.I. Krapivin: None. A.V. Petrovskaia-Kaminskaia: None. A.A. Smirnova: None. Y.M. Sagurova: None. O.A. Efimova: None. A.A. Pendina: None. O.G. Chiryaeva: None. L.I. Petrova: None. V.S. Dudkina: None. V.S. Baranov: None.


Telomere length of maternal and paternal chromosomes in human triploid zygotes

M. I. Krapivin1, A. A. Pendina1, O. A. Efimova1, I. D. Mekina1, E. M. Komarova1, Y. M. Sagurova2, I. V. Mayorova2, A. M. Gzgzyan1, I. Y. Kogan1, V. S. Baranov1,2

1D.O. Ott Research Institute of Obstetrics, Gynecology and Reproductology, St. Petersburg, Russian Federation, 2Saint Petersburg State University, St. Petersburg, Russian Federation

Telomeres are complex structures of tandem DNA repeats and proteins at chromosome ends. Telomere shortening due to cell divisions, oxidative stress or genetic effects may result in end-to-end chromosome joining, their degradation, and cell death. In contrast to short telomeres in oocytes, sperm telomeres become longer with age. Telomere length (TL) correlates with embryo developmental potential advocating the importance of TL analysis after fertilization. The study was performed on metaphase chromosomes of 23 triploid human zygotes from 20 couples (women aged 32.04 ± 0.8; men aged 34.15 ± 1.2) referred for in vitro fertilization. Paternal (sperm-derived) chromosomes were identified immunocytochemically by weak DNA methylation and strong hydroxymethylation which contrasted them with strongly methylated and poorly hydroxymethylated maternal (oocyte-derived) chromosomes. After qFISH, relative TL was measured by dividing telomeric by subtelomeric fluorescence assessed in ImageJ1.49v. The relative TL was significantly higher in the paternal compared to the maternal chromosomes (Wilcoxon signed-rank test, р < 0.0001). Rare zygotes demonstrated the opposite pattern: higher TL in the maternal chromosomes. However, they seem to be the exception rather than the rule suggesting that some cases of developmental arrest may be caused by altered TL pattern. Neither in sperm- nor in oocyte-derived chromosomes the relative TL correlated with paternal (r=-0.058;р=0.736) or maternal age (r=0.155;р=0.44), respectively. Thus, after fertilization, TL in paternal chromosomes is “reprogrammed”. Being hypomethylated, paternal chromosomes are prone to recombination and, thus, to alternative lengthening of telomeres, which may be part of developmental program and explains the absence of correlation of TL in sperm-derived chromosomes with paternal age. Supported by RSF№18-75-10046.

M.I. Krapivin: None. A.A. Pendina: None. O.A. Efimova: None. I.D. Mekina: None. E.M. Komarova: None. Y.M. Sagurova: None. I.V. Mayorova: None. A.M. Gzgzyan: None. I.Y. Kogan: None. V.S. Baranov: None.


Transcriptomics of placental tissue: identification of potential biomarkers and biological pathways in the great obstetrical syndromes

E. Trifonova1,2, A. Markov1, A. Zarubin1, T. Gabidulina2, V. Stepanov1

1Research Institute of Medical Genetics, Tomsk National Research Medical Center of the Russian Academy of Sciences, Tomsk, Russian Federation, 2Siberian State Medical University, Tomsk, Russian Federation

The great obstetrical syndromes (GOS) are common pregnancy complications that can have detrimental effects on morbidity and mortality of the mother and fetus as well as long-term health outcomes. Although they are distinct conditions, they are often considered together as they share a common etiology of inadequate placental perfusion. The aim of the work was to search for new genetic markers of GOS on the basis of integrative analysis of transcriptomic data. Genome-wide expression profiling was performed on placental tissue from preeclamptic and normal (n = 47) pregnancies. Thirty original datasets from patients with preterm birth, intrauterine growth restriction, preeclampsia (n = 434) were downloaded from Gene Expression Omnibus and were further integrated and analyzed with our data. Subsequently, 5023 differentially expressed genes (DEGs) between GOS patients and healthy women were identified. We found that the transcriptional activity of 64 DEGs changes in at least two GOS diseases. Weighted gene co-expression network analysis identified distinct gene modules associated with preterm birth, intrauterine growth restriction or preeclampsia. Using Gene Set Enrichment Analysis we identified the significant role of disturbance of intercellular interactions and regulation of proteins modification in placental tissue during the development of the GOS. We applied upstream analysis approach implemented in geneXplain platform and identified master regulators (MECOM, CDC34, UBE2E1, UBE2D4, STAT3 and UBE2R2) that are new therapeutic targets. These key genes may be potential biomarkers of diagnosis, therapy and prognosis for GOS. This work was supported by the Russian Foundation for Basic Research (grant No. 18-29-13045, No. 18-44-700007).

E. Trifonova: None. A. Markov: None. A. Zarubin: None. T. Gabidulina: None. V. Stepanov: None.


The importance of trio WES integration into prenatal diagnosis

C. Rodriguez-Solera1, M. Martinez-Garcia1, I. Diez1, I. Sanchez-Navarro1, E. Barroso1, D. Rodriguez1, P. Maietta1, G. Martin-Serrano1, S. Izquierdo2, M. Miramar2, A. Rodriguez2, F. Diaz-Flores3, M. Zeleniuc4, L. Martorell5, J. Bermejo6, C. Toledo7, S. Lopez8, M. Sanchez8, S. Alvarez1

1NIMGenetics S.L, Madrid, Spain, 2Hospital Universitario Miguel Servet, Zaragoza, Spain, 3Hospital Universitario de Canarias, Tenerife, Spain, 4Personal Genetics, Bucharest, Romania, 5Hospital Sant Joan de Déu, Barcelona, Spain, 6Hospital General de Segovia, Segovia, Spain, 7Hospital Virgen de la Salud, Toledo, Spain, 8Hospital Clínico de Santiago, Santiago de Compostela, Spain

Background: Exome sequencing has proved to be a useful diagnostic tool when fetal structural anomalies suggest a genetic etiology, but other standard prenatal genetic tests did not provide a diagnosis. We describe a single laboratory experience with prenatal and fetal samples.

Material and Methods: This study includes 13 samples with ultrasound images suggesting skeletal abnormalities or rasopathies, among others. The samples origin were amniotic fluid (69%) and fetal tissues (31%). In six cases a whole exome sequencing with a trio analysis was performed. The genomic libraries were generated using SureSelectXT and sequenced on a Novaseq 6000. Data alignment, calling and filtering was performed using an in-house developed pipeline. Informed consent was obtained from all participants.

Results: A molecular diagnosis was obtained on 1 amniotic fluid prenatal sample and on 1 fetal specimen (15% global diagnostic yield). All positive cases were obtained after trio analysis. Two pathogenic variants in the ESCO2 gene associated with Roberts syndrome were identified in the prenatal sample and one homozygous variant in the ASPM gene, associated with a primary microcephaly 5, was identified in fetal tissue. Both variants were cosegregated with the disease. Ultrasound studies did not suggest the candidate genes due to the insufficient and nonspecific information about the prenatal clinical presentation of these syndromes.

Conclusion: This study highlights the importance of trio WES integration into prenatal diagnosis. Compared to ultrasound studies alone, prenatal molecular diagnoses led to a refined recurrence risk estimation, altered medical management and allows an informed reproductive planning for families.

C. Rodriguez-Solera: None. M. Martinez-Garcia: None. I. Diez: None. I. Sanchez-Navarro: None. E. Barroso: None. D. Rodriguez: None. P. Maietta: None. G. Martin-Serrano: None. S. Izquierdo: None. M. Miramar: None. A. Rodriguez: None. F. Diaz-Flores: None. M. Zeleniuc: None. L. Martorell: None. J. Bermejo: None. C. Toledo: None. S. Lopez: None. M. Sanchez: None. S. Alvarez: None.


Pregnancy outcome after failure of emergency contraception with ulipristal acetate - an observational study

M. Hoeltzenbein, J. K. Wagner, C. Schaefer, K. Dathe

Pharmakovigilanzzentrum Embryonaltoxikologie, Institut für Klinische Pharmakologie und Toxikologie, Charité-Universitätsmedizin Berlin, Berlin, Germany

Introduction: Ulipristal acetate (UPA) is a selective progesterone receptor modulator used for emergency contraception (EC) after unprotected sexual intercourse. Data on pregnancy outcome after failure of EC is very limited.

Material and Methods: Observational study of prospectively ascertained pregnancies after UPA exposure for EC. In addition, retrospectively reported pregnancy outcomes were evaluated separately.

Results: Among 216 requests to the German Embryotox institute related to EC with UPA, 95 concerned prospectively ascertained pregnancies after failed EC. Follow-up data on pregnancy outcome was obtained in 56 pregnancies: 7 spontaneous abortions, 12 terminations of pregnancy (ETOP) and 37 live births. Major birth defects were not reported. Only 34% of women had taken UPA during the fertile window. Among the 4 retrospectively reported pregnancies there was one child diagnosed with Beckwith-Wiedemann-syndrome (BWS) due to hypomethylation of IC2.

Conclusions: We provide a preliminary basis for reassuring women who wish to carry their pregnancy to term after failed EC with UPA. However, due to the retrospective case report of a child with BWS after maternal UPA exposure possible epigenetic effects could not be excluded and require further evaluation. As ovarian stimulation alone (independent of artificial reproductive techniques) was previously discussed as a predisposing factor for disturbance of imprinting maintenance, UPA might have influenced preimplantation development and disturbed epigenetic processes.

This work was performed with financial support from the German Federal Institute for Drugs and Medical Devices (BfArM).

M. Hoeltzenbein: None. J.K. Wagner: None. C. Schaefer: None. K. Dathe: None.


Chromosomal microarray confirms a rare prenatal case of WAGR syndrome

M. Shetty, P. Venkatesh, S. Menezes, S. Hegde

Manipal Hospital, Bangalore, India

Introduction: WAGR syndrome is a rare genetic disorder with prevalence of 1 in 500,000 to 1 million. It affects many body systems and is named for its main features: wilms tumor, aniridia, genitourinary anomalies and range of developmental delays. Children with WAGR syndrome present with aniridia, genito-urinary defects in the new born period and are at an increased risk for developing malignancies, varying range of intellectual disability and obesity in childhood. The syndrome is a contiguous gene deletion syndrome involving chromosome 11p13 containing WT1 and PAX6 genes.

Materials and Methods: We report a case of 29-year-old Asian (Indian ethnicity) primigravida with non-consanguineous marriage. Her mid trimester anomaly scan at 18 weeks gestation showed bilateral ventriculomegaly, small cavum septum pellucidum and partial agenesis of corpus callosum. Amniocentesis was performed for chromosomal microarray analysis which was performed using an Agilent 8x60K array. The cytogenomic microarray analysis showed 4.9MB deletion in chromosome 11 (11p13-p12).

Discussion: The prenatal diagnosis of WAGR syndrome is associated with borderline ventriculomegaly, absent corpus callosum and absent cavum septum pellucidum. In order to diagnose rare syndrome, microarray should be the first choice of investigation in the presence of any structural abnormality. This would help in understanding the prenatal presentation, potential co-morbid condition, counseling and accordingly decision making.

Conclusions: The clinical association of WAGR syndrome in postnatal period is well established but the antenatal presentation is not well-understood and reported twice in the literature earlier. Microarray study confirms these rare non-classical prenatal manifestations of WAGR syndrome.

M. Shetty: None. P. Venkatesh: None. S. Menezes: None. S. Hegde: None.


Whole Exome Sequencing in 53 fetuses with abnormal ultrasound: 37% of diagnostic yield

D. Trost1, A. Boughalem1, P. Blanchet2, C. Cenni2, R. Dard3, A. Benachi4, V. Ciorna-Monferrato5, R. Touraine6, P. Kleinfinger1, L. Lohmann1, A. Luscan1, M. Valduga1, J. Costa1

1Laboratoire Cerba, Saint Ouen l’Aumone, France, 2Hôpital Arnaud de Villeneuve, Montpellier, France, 3Hôpital Poissy, Poissy, France, 4Hôpital Antoine Béclère, Clamart, France, 5Hôpital Metz-Thionville, Metz, France, 6Hôpital Nord, Saint Etienne, France

Introduction: Fetal structural abnormalities detected by ultrasonography, have a range of genetic causes. Chromosomal aneuploidy and structural abnormalities are explored by karyotype testing and chromosomal microarray (CGH). The utility of prenatal whole exome sequencing (WES) is discussed. Information on the interest of prenatal WES remains limited.

Methods and results: In our cohort of 53 fetuses with normal karyotype and CGH, Trio WES was performed. All fetuses had ultrasound abnormalities of poor prognosis. Most fetuses (26) were analyzed after medical termination of pregnancy, 24 analyzes were performed on ongoing pregnancies and 3 analyzes after intrauterine fetal demise. Pathogenic or likely pathogenic variants were found in 20 cases resulting in a diagnostic yield of 37%. Of these, 55% (11) were autosomal dominant with de novo variants, 45% of the diagnoses followed an autosomal recessive inheritance pattern (5 homozygous variants, 4 compound heterozygous variants). In urgent cases turn-around-time was 2 weeks. Reporting results for an antenatal Trio WES varies between 2-4 weeks.

Conclusions: Whole exome sequencing identified a genetic cause for the fetal anomalies in 20 of the 53 (37%) cases analyzed. This made it possible to carry out genetic counseling for families and to offer targeted prenatal diagnosis for recessive diseases in case of a new pregnancy.

D. Trost: None. A. Boughalem: None. P. Blanchet: None. C. Cenni: None. R. Dard: None. A. Benachi: None. V. Ciorna-Monferrato: None. R. Touraine: None. P. Kleinfinger: None. L. Lohmann: None. A. Luscan: None. M. Valduga: None. J. Costa: None.

P02 Sensory Disorders (Eye, Ear, Pain)


Disruption of PAX6 translation by 5′ UTR variants causes Congenital aniridia

A. Filatova, T. Vasilyeva, A. Marakhonov, R. Zinchenko, M. Skoblov

Research Centre for Medical Genetics, Moscow, Russian Federation

Introduction: Congenital aniridia (AN) is a rare autosomal dominant panocular disorder caused by mutations in the PAX6 gene. Previously, we performed a molecular genetic study of a large cohort of Russian patients with AN and found six PAX6 nucleotide variants in 5ʹ-UTR. It is known that 5′-UTR variants could influence the translation efficiency and cause some diseases, but for AN such a mechanism has not been previously shown. Thus, to investigate the pathogenicity of 5′-UTR PAX6 variants the functional studies are required.

Materials and Methods: To study 5ʹ-UTR variants we generated luciferase constructs with full-length PAX6 5ʹ-UTR (wt and mutants). The translation efficiency was measured by luciferase dual-assay, mRNA structure and expression level were determined by RT-PCR and qPCR respectively.

Results: Six patient-derived 5ʹ-UTR-variants were found to lead to a significant decrease in the translation efficiency, while mRNA expression level was not significantly reduced. Three of these variants also affect splicing. Further bioinformatics analysis allows us to suggest the mechanism of 5ʹ-UTR variants pathogenicity through disruption of an upstream ORF which possibly exists in PAX6 5ʹ-UTR. To validate this hypothesis, we performed additional mutagenesis experiments and confirmed the essential role of uORF in the molecular pathogenesis of the disease. Moreover, we showed that this uORF normally affects the main protein translation.

Conclusions: We have identified uORF in 5′-UTR of the PAX6 gene and found that mutations that disrupt this uORF cause the congenital aniridia. In addition, we hypothesized that this uORF may participate in the normal regulation of PAX6 expression.

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


Evaluating diagnostic challenges with ABCA4 -related retinal disease - experience with a 7500 IRD patient cohort sent for genetic diagnostics

J. Känsäkoski1, K. Kämpjärvi1, S. Tuupanen1, K. Wells1, L. Sarantaus1, P. von Nandelstadh1, K. Merkkiniemi1, H. Västinsalo1, E. Mårtensson1, R. Perez Carro1, E. Sankila2, J. W. Koskenvuo1, S. Myllykangas1, T. Alastalo1

1Blueprint Genetics, Espoo, Finland, 2Helsinki University Eye Hospital, Helsinki, Finland

Introduction: ABCA4 variants cause autosomal recessive Stargardt disease (STGD1), the most prevalent hereditary macular disease. Major challenges in STGD1 molecular diagnostics include (i) non-coding variants typically not targeted by available genetic tests (ii) copy number variants (CNV) missed by standard testing, and (iii) interpretation challenges with common hypomorphic variants. We evaluated the significance of challenging ABCA4 variants and their contribution to STGD1 diagnostic yield in a cohort of inherited retinal disease (IRD) patients.

Materials and Methods: We included 7500 IRD patients tested at a CLIA laboratory between 2017 and 2019. Sequencing analysis, including established non-coding ABCA4 variants and high resolution CNV detection, was done by using in-house developed and validated NGS platform.

Results: Two disease-causing ABCA4 variants were identified in 663 patients, of which 20 (20/663; 3%) had an established non-coding variant and 11 (11/663; 1.5%) a CNV. In 192 patients with clinically diagnosed ABCA4-related disease, only one disease-causing variant was identified. Among those, the hypomorphic allele ABCA4 c.5603A>T, p.(Asn1868Ile) was identified in 109 patients (109/192: 57%), with a significantly increased allele frequency (126/384=0.328) compared to controls in gnomAD (AF 11928/282712=0.042) (OR: 11.087, 95% CI: 8.953-13.73, p < 0.001). The median age at genetic testing among the 109 patients was 56 years, which was significantly higher compared to 37 years among the 663 patients with two disease-causing variants.

Conclusions: Non-coding variants and CNVs contribute significantly to diagnostic yield in ABCA4-related disease. Our large dataset supports the important role of the hypomorphic allele ABCA4 c.5603A>T, p.(Asn1868Ile) in diagnostics of late-onset STGD1.

J. Känsäkoski: A. Employment (full or part-time); Significant; Blueprint Genetics. K. Kämpjärvi: A. Employment (full or part-time); Significant; Blueprint Genetics. S. Tuupanen: A. Employment (full or part-time); Significant; Blueprint Genetics. K. Wells: A. Employment (full or part-time); Significant; Blueprint Genetics. L. Sarantaus: A. Employment (full or part-time); Significant; Blueprint Genetics. P. von Nandelstadh: A. Employment (full or part-time); Significant; Blueprint Genetics. K. Merkkiniemi: A. Employment (full or part-time); Significant; Blueprint Genetics. H. Västinsalo: A. Employment (full or part-time); Significant; Blueprint Genetics. E. Mårtensson: A. Employment (full or part-time); Significant; Blueprint Genetics. R. Perez Carro: A. Employment (full or part-time); Significant; Blueprint Genetics. E. Sankila: A. Employment (full or part-time); Modest; Blueprint Genetics. J.W. Koskenvuo: A. Employment (full or part-time); Significant; Blueprint Genetics. E. Ownership Interest (stock, stock options, patent or other intellectual property); Significant; Blueprint Genetics. S. Myllykangas: A. Employment (full or part-time); Significant; Blueprint Genetics. E. Ownership Interest (stock, stock options, patent or other intellectual property); Significant; Blueprint Genetics. T. Alastalo: A. Employment (full or part-time); Significant; Blueprint Genetics. E. Ownership Interest (stock, stock options, patent or other intellectual property); Significant; Blueprint Genetics.


A novel splicing variant confirms COL11A1 as a cause of autosomal dominant non-syndromic hearing loss in the DFNA37 locus

B. Vona1, A. Tropitzsch1, A. Rad1, F. Schneider1, M. Müller1, T. Schade-Mann1, S. Biskup2, H. Löwenheim1

1Dept of Otolaryngology—Head & Neck Surgery, Tuebingen Hearing Research Centre, University of Tuebingen, Tuebingen, Germany, 2CeGaT GmbH and Praxis für Humangenetik Tuebingen, Tuebingen, Germany

Introduction: Hereditary hearing loss (HL) is a clinically and genetically heterogeneous disorder. Many genes exhibiting either non-syndromic or syndromic HL have also been identified as causing both, owing to extensive phenotypic manifestations in the form of pleiotropy. The gene COL11A1 has long been associated with autosomal dominant Marshall syndrome and Stickler syndrome type II, as well as autosomal recessive fibrochondrogenesis. Each of these syndromes have overlapping phenotypes including skeletal abnormalities, dysmorphic features, variable cleft palate, ocular and auditory phenotypes that can include mild-to-moderate HL, and outer ear malformations. Recently, COL11A1 has been associated with autosomal dominant non-syndromic HL (DFNA37) through the genetic analysis of a large European-American family presenting a novel splice-site altering variant.

Materials and Methods: A proband from a four generation family with non-syndromic HL was ascertained. DNA from the proband was subjected to a custom-designed high-throughput sequencing panel consisting of 160 HL-associated genes. An in vitro splice assay was performed to study the RNA-level effect of the variant.

Results: The patient was diagnosed with moderate sensorineural HL. Additional syndromic features were absent. A heterozygous variant (c.4338+2T>C, p.?, NM_080629.2) was uncovered in COL11A1 that is predicted to abolish the 5′ splice site in exon 58 out of the 68 exons comprising COL11A1. Furthermore, this novel variant affects a highly conserved nucleotide in the alpha chain domain. In vitro splice testing confirmed abnormal splicing.

Conclusions: We report on the second DFNA37-associated splice-altering variant, providing confirmatory evidence of COL11A1 as a bona fide autosomal dominant non-syndromic HL gene.

B. Vona: None. A. Tropitzsch: None. A. Rad: None. F. Schneider: None. M. Müller: None. T. Schade-Mann: None. S. Biskup: None. H. Löwenheim: None.


Non-Mendelian inheritance in Bardet-Biedl syndrome: triallelism

I. Perea-Romero1,2, F. Blanco-Kelly1,2, I. Lorda-Sanchez1, I. Sanchez-Navarro3, O. Zurita1,2, R. Riveiro-Alvarez1,2, M. Trujillo-Tiebas1,2, A. Avila-Fernandez1,2, M. Corton1,2, D. Valverde4,5, C. Ayuso1,2

1Department of Genetics, Instituto de Investigación Sanitaria – Fundación Jiménez Díaz University Hospital – Universidad Autónoma de Madrid (IIS-FJD, UAM), Madrid, Spain, 2Center for Biomedical Network Research on Rare Diseases (CIBERER), ISCIII, Madrid, Spain, 3Medical Department, NIMGenetics, Madrid, Spain, 4Department of Biochemistry, Genetics and Immunology, Biology Faculty, Vigo University, Pontevedra, Spain, 5Center for Biomedical Network Research on Rare Diseases (CIBERER), ISCIII, Pontevedra, Spain

Introduction: Bardet-Biedl syndrome (BBS) is a ciliopathy characterized by retinal dystrophy, obesity, polydactyly, cognitive impairment, renal failure and hypogonadism. Typically, it has an autosomal recessive mode of inheritance, but this heterogeneous disease has variable expressivity between and within families, which might be partially explained by oligogenic inheritance, like triallelism.

Materials and methods: A total of 61 families (78 affected cases) with an “a priori” diagnosis of BBS or BBS-like were studied using different molecular approaches (commercial genotyping microarrays, Sanger sequencing of specific genes and/or NGS technologies).

Results: The characterization rate in our cohort was 79% (48/61 families). In 5 of the families, we found a possible triallelic inheritance, involving 6 out of the 7 affected cases. Three families carried biallelic BBS1 variants together with one allele in MKKS, one family with biallelic MKKS variants and one allele in BBS5 and one additional family carried biallelic C8orf37 variants and one allele in WDPCP. The MKKS (c.724G>T; p.Ala242Ser) variant was found to be part in 3 families accompanying different biallelic variants in BBS1. In one family, the triallelic case showed a more severe phenotype than the biallelic affected sibling. However, the phenotypic modifier effect of the triallelism could not be stablished in 4 of the families, as they were either unique cases or both affected showed triallelism.

Conclusions: Our study presents triallelism as an option in BBS diagnosis, being important to consider it in genetic counselling.

Funding: FIS (PI16/00425 and PI19/00321), University Chair UAM-IIS-FJD Genomic Medicine and FEDER (European Regional Development Fund)

I. Perea-Romero: None. F. Blanco-Kelly: None. I. Lorda-Sanchez: None. I. Sanchez-Navarro: None. O. Zurita: None. R. Riveiro-Alvarez: None. M. Trujillo-Tiebas: None. A. Avila-Fernandez: None. M. Corton: None. D. Valverde: None. C. Ayuso: None.


Molecular genetic cause of non-syndromic congenital and juvenile cataracts in the Czech population

J. Moravikova1, L. Dudakova1, M. Schwarz2, L. Hlavata1, F. Malinka1,3, P. Liskova1,4

1Department of Paediatrics and Adolescent Medicine, First Faculty of Medicine, Charles University and General University Hospital in Prague, Prague, Czech Republic, 2Department of Biology and Medical Genetics, Second Faculty of Medicine, Charles University and University Hospital Motol, Prague, Czech Republic, 3Department of Computer Science, Czech Technical University in Prague, Prague, Czech Republic, 4Department of Ophthalmology, First Faculty of Medicine, Charles University and General University Hospital in Prague, Prague, Czech Republic

Introduction: Congenital and juvenile cataracts are phenotypically and genetically very heterogeneous group. In this study, we aimed to investigate the molecular genetic cause in Czech patients with bilateral congenital or juvenile cataracts not associated with other ocular or systemic clinical findings.

Materials and Methods: Whole exome sequencing was performed in 10 probands of Czech origin. Variants with minor allele frequency less than 0.005 as per GnomAD v.2 were filtered and those located in genes known to be associated with cataract development as per Cat-Map database were given a priority for further evaluation. Conventional sequencing was used to follow segregation of the presumably pathogenic variants in available first degree relatives.

Results: Out of the 10 probands 6 had a family history of congenital or infantile cataracts. In total 5 variants classified as pathogenic or likely pathogenic as per American College of Medical Genetics and Genomics guidelines were identified. Two variants were novel; c.299T>A in GJA3 (NM_021954) and c.2665G>A in EPHA2 (NM_001329090). No pathogenic variants were found in 5 probands.

Conclusions: Herein we report for the first time mutational spectrum of non-syndromic cataracts in Czech patient population. The failure to identify disease-causing variants in half of the analysed cases suggests, that either the underlying mechanisms are not of genetic origin and/or that the pathogenic changes are not located in coding regions of the genome. Only two probands with family history of cataracts remained unsolved. Alternatively, larger structural variants or rearrangement could be also implicated.

Supported by AZV 17-30500A.

J. Moravikova: None. L. Dudakova: None. M. Schwarz: None. L. Hlavata: None. F. Malinka: None. P. Liskova: None.


Insights into the retinal function of ciliopathy proteins gained through zebrafish models

M. Masek, R. Bachmann-Gagescu

University of Zurich- Medical Genetics, Zürich, Switzerland

Ciliopathies are disorders caused by dysfunction of primary cilia, ubiquitous organelles involved in signal transduction. In retinal photoreceptors (PRs), light sensation occurs in the outer segments (OSs), which represent highly specialized ciliary compartments. Consequently, retinal disease is a common manifestation in ciliopathy patients. Our efforts to elucidate the molecular functions of ciliopathy proteins rely on zebrafish mutants in genes associated with Joubert syndrome, an iconic ciliopathy associated with retinal disease in 25% of patients. We focus on genes that encode proteins participating in distinct ciliary complexes: Cc2d2a at the transition zone, representing the gate to the cilium, or Talpid3/KIAA0586 at the basal body (BB), anchoring the cilium in the cell. While both mutants display decreased visual function, we identify distinct ultra-structural phenotypes indicating specific roles for each protein in primary cilium function. We find that Cc2d2a organizes the vesicle fusion machinery at the periciliary margin and provides a docking point for incoming vesicles through a chain of interactions linking CC2D2A to NINL-MICAL3-RAB8. In contrast, Talpid3 is involved in BB docking and ciliogenesis upstream of Rab8a activation. Retina-specific transcriptomics identifies both shared and distinct effects on gene transcription: while transcription of phototransduction genes is decreased in both mutants, likely as a secondary effect, we observe downregulation of the BBSome, a multi-protein complex associated with the ciliopathy Bardet-Biedl syndrome, only in talpid3 mutants, while Nek8 is upregulated only in cc2d2a mutants. In conclusion, our findings demonstrate how mutations in genes causing the same human disorder result in similar phenotypes through distinct mechanisms.

M. Masek: None. R. Bachmann-Gagescu: None.


Analysis of copy number variants in a large cohort of individuals with anophthalmia, microphthalmia and coloboma

F. Ceroni1, D. A. Bax1, R. J. Holt1, Y. Kesim1, A. Cole2, C. Ogilvie3, D. Bunyan4,5, L. Cooper-Charles2, D. McMullan2, M. Griffiths2, N. K. Ragge1,2

1Faculty of Health and Life Sciences, Oxford Brookes University, Oxford, United Kingdom, 2West Midlands Regional Genetics Service, Birmingham Women’s and Children’s NHS Foundation Trust, Birmingham, United Kingdom, 3Guy’s and St Thomas’ NHS Foundation Trust, London, United Kingdom, 4Wessex Regional Genetics Laboratory, Salisbury District Hospital, Salisbury, United Kingdom, 5Faculty of Medicine, University of Southampton, Southampton, United Kingdom

Introduction: Anophthalmia, microphthalmia and coloboma (AMC) are a genetically heterogenous collection of developmental eye disorders, with variants in >100 genes implicated. However, only 25-60% of patients receive genetic diagnoses. Structural variants are an important source of genetic variation, and have led to the identification of numerous eye development genes, including SOX2 and OTX2. However, few studies systematically address their contribution to AMC. Here, we investigate a large cohort of AMC families for known and novel copy number variants (CNVs).

Materials and methods: A cohort of 320 unrelated probands was screened using either diagnostic genome-wide aCGH platforms (n = 156), including the customised OGT CytoSure 4x180K eye array, or the Illumina Infinium Global Screening Array (n = 164). SNP data were analysed using the algorithms QuantiSNP and PennCNV. Variants of interest were validated using qPCR and long-range PCR.

Results: Our analysis identified multiple variants of interest, including likely pathogenic CNVs affecting known AMC genes and rare CNVs highlighting new candidates. We describe a novel homozygous 113kb deletion 19kb upstream of MAB21L2, a developmental gene involved in eye morphogenesis. This deletion removes multiple conserved cis-regulatory elements. We also identified deletions in regions with recurrent CNVs (1q21.1, 6q16, 10q11, 16p11.2, 22q11.2).

Conclusions: Our study supports the importance of CNV screening in routine diagnostic testing. It also highlights how CNVs affecting noncoding elements can provide insights into the role of regulatory regions of eye development genes in AMC. Further investigation of such variants will improve the genetic diagnostic yield and our understanding of the aetiology of these conditions.

F. Ceroni: None. D.A. Bax: None. R.J. Holt: None. Y. Kesim: None. A. Cole: None. C. Ogilvie: None. D. Bunyan: None. L. Cooper-Charles: None. D. McMullan: None. M. Griffiths: None. N.K. Ragge: None.


Cataract and retinal dystrophy inVps13b∆Ex3/∆Ex3mice.

V. R. M. Lhussiez1, Q. Cesar2, E. Dubus2,3, M. Simonutti2, E. Lizé1, S. Nguyen1, A. Geissler3, A. Bouchot3, S. Picaud2, E. F. Nandrot2, N. Acar4, L. Faivre1,5,6, C. Thauvin1,5,7, L. Duplomb1,5, R. Da Costa1,5

1INSERM, UMR1231, Equipe GAD, Université de Bourgogne Franche Comté, Dijon, France, 2Sorbonne Université, INSERM, CNRS, Institut de la Vision, Paris, France, 3Plateforme d’Imagerie Cellulaire CellImaP/DimaCell, Inserm LNC UMR1231, Dijon, France, 4Centre des Sciences du Goût et de l’Alimentation, AgroSup Dijon, CNRS, INRAE, Université Bourgogne Franche-Comté, Dijon, France, 5FHU TRANSLAD, CHU Dijon, Dijon, France, 6Centre de Référence Anomalies du Développement et Syndromes Malformatifs, CHU Dijon, Dijon, France, 7Centre de Référence Déficiences Intellectuelles de Causes Rares, CHU Dijon, Dijon, France

Cohen syndrome (CS) is a rare genetic disorder due to variation in the VPS13B gene. It is characterized by a wide variety of clinical features that includes a typical facial dysmorphism, hypotonia, neutropenia, microcephaly, intellectual disability and severe visual impairments. In their early childhood, CS patients already suffer from myopia and a retinal dystrophy that affects both peripheral and central vision. In addition, long-term outcome studies showed that cataract reaches a high prevalence in adults with CS in their forties. To understand how VPS13B variants lead to these visual impairments and have the possibility to assess therapeutic approaches, we generated the Vps13b∆Ex3/∆Ex3 mouse model. Cataract was almost systematic in 2-month-old animals. Eye fundi appeared normal until cataract development, but OCT, ERG and histological data suggest that rod homeostasis may be affected in the few Vps13b∆Ex3/∆Ex3 mice without cataract after 5 months of age. Immunostaining of the lens revealed that cataract formation was associated with the appearance of large vacuoles in the cortical area, epithelial-mesenchymal transition and fibrosis. In later stages, cataracts became hypermature, lens capsules ruptured, and sclerotic nuclear parts dissociated. Altogether, our results show that Vps13b has a function in lens homeostasis in mice and that the Vps13b∆Ex3/∆Ex3 mouse line is a useful model to study the pathomechanism leading to CS-related cataract. This work was supported with funds from the JED Fondation and from the Fondation Maladies Rares.

V.R.M. Lhussiez: None. Q. Cesar: None. E. Dubus: None. M. Simonutti: None. E. Lizé: None. S. Nguyen: None. A. Geissler: None. A. Bouchot: None. S. Picaud: None. E.F. Nandrot: None. N. Acar: None. L. Faivre: None. C. Thauvin: None. L. Duplomb: None. R. Da Costa: None.


Dominant cone rod dystrophy (CORD7) previously assigned to mutation of RIMS1 , is fully explained by co-inheritance of a dominant allele of PROM1

G. Arno1,2, E. Schiff2, G. Wright2, N. Waseem1, Genomics England Research Consortium, M. Michaelides1,2, A. T. Moore1,2,3, A. R. Webster1,2

1UCL Institute of Ophthalmology, London, United Kingdom, 2Moorfields Eye Hospital, London, United Kingdom, 3University of California, San Francisco, San Francisco, CA, United States

A single missense variant (p.Arg820His) in RIMS1 has been associated with autosomal dominant cone rod dystrophy 7 (CORD-7) and enhanced cognition. This was discovered in 2003 following linkage (LOD 3.61) to a 7cM region on chromosome 6q, in a four generation British family in 1998. The RIMS1 gene is often included in diagnostic panels for retinal dystrophy. However, despite this, no further families with CORD7 have been reported.

Widespread availability of massively parallel sequencing has enabled detection of genomic variation at a scale not possible in the early 2000s. Access to vast datasets in gnomAD and Genomics England 100k genomes project (GE100KGP) now enables better understanding of the validity of previously accepted genetic associations.

Interrogation of the gnomAD datasets reveal 10 carriers of RIMS1 p.Arg820His (Non Finnish European allele frequency 0.0001). This would be unusual given the severity of visual impairment in adulthood and absence of the variant in further patient cohorts. Four members of the original CORD7 family were recruited to the GE100KGP, where WGS showed the two affected members to be heterozygous for a well characterised dominant allele (p.Arg373Cys) of PROM1 located on chromosome 4p. All affected individuals subsequently were heterozygotes for this variant. Cytogenetic analysis excluded a balanced translocation that might explain co-segregation of a region on 6q and 4p.

This finding suggests the rare occurrence of an alpha error despite robust linkage analysis in a large family. So far there is no evidence that perturbation of RIMS1 affects the retina in humans.

G. Arno: None. E. Schiff: None. G. Wright: None. N. Waseem: None. M. Michaelides: None. A.T. Moore: None. A.R. Webster: None.


CRB1-related retinopathy superimposed on a S-adenosylhomocysteine hydrolase deficiency phenotype.

M. Di Scipio1, M. K. Grudzinska Pechhacker1,2, A. Vig1, A. Anupreet1, E. Tavares1, A. Vincent1,2,3, E. Heon1,2,3

1Genetics and Genome Biology, The Hopsital for Sick Children, Toronto, ON, Canada, 2Department of Ophthalmology and Vision Sciences, University of Toronto, Toronto, ON, Canada, 3Department of Ophthalmology and Vision Sciences, The Hospital for Sick Children, Toronto, ON, Canada

Background: S-adenosylhomocysteine deficiency is a rare metabolic disorder for which no eye phenotype has been documented. CRB1 gene is known to cause wide spectrum of autosomal recessive retinal diseases such as Leber’s congenital amaurosis, early onset cone-rod dystrophy, retinitis pigmentosa, isolated maculopathy and familial foveal retinoschisis; or autosomal dominant pigmented paravenous chorioretinal atrophy.

Materials and Methods: The present report describes a non-consanguineous family, where two children were affected with S-adenosylhomocysteine deficiency and one of them presented with reduced vision due to maculopathy. Comprehensive eye examination was performed in available family members together with color vision test, contrast sensitivity, visual fields, fundus images, OCT, full field electroretinogram, pattern electroretinogram and visual evoked potentials. Genetic testing included WES, retinal dystrophy panel and segregation analysis.

Results: Clinical whole exome sequencing revealed homozygous missense mutations in AHCY gene (c.148G>A, p.Ala50Thr) as a cause of S-adenosylhomocysteine deficiency. Because of a maculopathy in the proband, retinal dystrophy gene panel sequencing revealed two heterozygous missense mutations in CRB1 gene (c.1831T>C, p.Ser611Pro) and (c.3955T>C, p.Phe1319Leu) in the proband (III-1) and her mother. Mutations segregated with disease phenotype in family members.

Conclusions: Establishing an ocular genetic diagnosis may be complicated by the co-existence of a rare systemic genetic disease with previously unknown eye involvement. Extensive phenotyping and genotyping of available family members, and genotype-phenotype correlations helps to establish a diagnosis.

Grant References: Henry Brent Research Chair; Canadian Foundation Fighting Blindness; Career Development Award (Foundation fighting Blindness, USA).

M. Di Scipio: None. M.K. Grudzinska Pechhacker: None. A. Vig: None. A. Anupreet: None. E. Tavares: None. A. Vincent: None. E. Heon: None.


Fundus albipunctatus in a pedigree with autosomal-dominant transmission of G116R mutation in RLBP1

K. Mihova1, K. Kamenarova1, S. Cherninkova2, I. Dimova1, F. Shakola1, V. Mitev1, R. Kaneva1

1Department of Medical Chemistry and Biochemistry, Molecular Medicine Center, Medical University of Sofia, Sofia, Bulgaria, 2Department of Neurology, University Hospital Alexandrovska, Medical University of Sofia, Sofia, Bulgaria

Introduction: Fundus albipunctatus (FA) is a rare form of congenital stationary night blindness in which white or yellow dots can be seen scattered through the fundus. Individuals complain of night blindness early in childhood without progression. By next-generation sequencing of clinical exome including all inherited retinal degeneration causative genes, we aimed to identify the disease-causing mutation in a patient with clinical signs of FA.

Materials and Methods: Using Illumina® platform and TruSight One, we identified heterozygous RLBP1-mutation in a young patient, which results in phenotype consistent with FA.

Results: A 5-years-old patient presenting ophthalmoscopic phenotype including delayed dark adaptation, aggregation of yellowish-white lesions of various sizes in the retina, and slight vascular abnormalities, was screened for mutations in all known genes previously associated with inherited retinal degeneration. We identified a heterozygous mutation, c.346G>C (p.G116R), in the gene for cellular retinaldehyde-binding protein 1 (RLBP1), already described in a pedigree affected by autosomal-recessive FA. All other genetic variants detected in the patient’s DNA were excluded after applying commonly used filtering criteria. Pedigree analysis showed the presence of p.G116R mutation in the patient’s mother who was a heterozygous carrier. Мother’s ophthalmological examination subsequently confirmed pre-existing stationary night blindness, prolonged dark adaptation with preserved visual acuity strongly suggesting a phenotype consistent with FA.

Conclusions: Most reported pedigrees are consistent with a recessive pattern of Mendelian inheritance of FA, although autosomal-dominant inheritance has been also reported. Our results suggest that mutation RLBP1-p.G116R may be considered as dominant-acting in pedigrees with autosomal-dominant FA.

Acknowledgements: D01-285; КП-06-Н33/12.

K. Mihova: None. K. Kamenarova: None. S. Cherninkova: None. I. Dimova: None. F. Shakola: None. V. Mitev: None. R. Kaneva: None.


The G-EAR Consortium study: new candidate genes for normal hearing function (NHF) and age-related hearing loss (ARHL) in a large and deeply phenotyped cohort.

M. Brumat1, A. Morgan1,2, A. P. Nagtegaal3, B. C. Oosterloo3, S. Seshadri4,5, N. L. Heard-Costa4,6, G. Van Camp7, E. Fransen8, P. Gasparini1,2, G. Girotto1,2

1Department of Medicine, Surgery and Health Sciences, University of Trieste, Trieste, Italy, 2Institute for Maternal and Child Health – IRCCS Burlo Garofolo, Trieste, Italy, 3Department of Otorhinolaryngology, Erasmus Medical Center, Rotterdam, Netherlands, 4Framingham Heart Study, Framingham, MA, United States, 5Glenn Biggs Institute for Alzheimer’s and Neurodegenerative Diseases, University of Texas Health Sciences Center, San Antonio, TX, United States, 6Department of Neurology, Boston University School of Medicine, Boston, MA, United States, 7Center of Medical Genetics, University of Antwerp and Antwerp University Hospital, Antwerp, Belgium, 8Department of Biomedical Sciences, University of Antwerp, Antwerp, Belgium

Understanding the genetic basis of hearing traits and diseases (e.g. NHF and ARHL) is central to the development of precision medicine for the hearing impaired; to pursue this aim, fully audiometrically characterised cohorts are fundamental.

Through the international G-EAR Consortium, we united data from ten such cohorts from Europe, USA, Caucasus and Central Asia (overall >9000 individuals) and performed a GWAS meta-analysis on NHF and ARHL, analysing in the former case single hearing thresholds or their average value across specific frequencies, and in the latter a case-control phenotype (PMID:29725052). Analyses used linear mixed model/logistic regression; meta-analysis was performed with METAL.

Suggestive association signals were detected on chromosomes 3, 8, 11, 16 and 18. In particular, three genes - CADM2, CALB1 and LRRC4C - result to be potentially associated with NHF and two more - CDH13 and CTIF - with ARHL.

CADM2 encodes a protein of the nectin-like family, members of which are fundamental for the development of the inner ear; CALB1 encodes a calcium-binding protein that regulates the development of murine cochlear hair cells; LRRC4C encodes a transmembrane protein abundantly expressed in mouse inner ear. Finally, CDH13 and CTIF have been associated with NHF in previous studies, suggesting their possible role also in ARHL.

Replication is in progress in an independent cohort of ~1500 individuals, fully phenotyped; updated results will be presented. These findings provide a strong contribution to our understanding of the genetic bases of both NHF and ARHL, laying the foundation of future therapeutic targets.

M. Brumat: None. A. Morgan: None. A.P. Nagtegaal: None. B.C. Oosterloo: None. S. Seshadri: None. N.L. Heard-Costa: None. G. Van Camp: None. E. Fransen: None. P. Gasparini: None. G. Girotto: None.


Further evidence of involvement of CDC14A in autosomal recessive non-syndromic hearing loss

J. Doll1, S. Kolb1, L. Schnapp1, A. Rad2,3, F. Rüschendorf4, I. Khan5, A. Adli2, A. Hasanzadeh2, D. Liedtke1, S. Knaup1, M. A. H. Hofrichter1, T. Müller6, M. Dittrich1,6, I. Kong7, H. Kim8, T. Haaf1, B. Vona1,3

1Institute of Human Genetics, Julius Maximilians University Würzburg, Würzburg, Germany, 2Cellular and Molecular Research Center, Sabzevar University of Medical Sciences, Sabzevar, Iran, Islamic Republic of, 3Tübingen Hearing Research Centre, Department of Otorhinolaryngology, Head and Neck Surgery, Eberhard Karls University, Tübingen, Germany, 4Max Delbrück Center for Molecular Medicine in the Helmholtz Association, Berlin, Germany, 5Department of Chemistry, Bacha Khan University, Charsadda, Pakistan, 6Institute of Bioinformatics, Julius Maximilians University, Würzburg, Germany, 7Department of Animal Science, Division of Applied Life Science (BK21plus), Institute of Agriculture and Life Science, Gyeongsang National University, Jinju, Korea, Republic of, 8Neurological Disorders Research Center, Qatar Biomedical Research Institute, Hamad Bin Khalifa University, Doha, Qatar

Introduction: Hearing loss (HL) is a highly heterogeneous sensory disorder with a prevalence of 1-3 affected in 1000 newborns. Since 2016, the gene CDC14A (Cell Division Cycle 14A) is associated with autosomal recessive non-syndromic HL (DFNB32) and hearing impairment and infertile male syndrome (HIIMS).

Materials and Methods: In order to identify likely pathogenic variants of already known and novel HL genes in Iranian and Pakistani families with sensorineural HL, exome sequencing, gene mapping approaches and bioinformatics analysis was performed. Segregation testing of variants ensued. To investigate functional consequences on the RNA-level of both loss-of-function variants and to evaluate altered expression levels, we used a minigene assay for the splice variant (c.1421+2T>C, p.?) and RT-qPCR for the c.1041dup variant.

Results: We identified a novel homozygous CDC14A splice site variant (c.1421+2T>C, p.?) responsible for the disruption of a splice donor site based on in silico prediction tools. Moreover, we identified a novel homozygous frameshift variant (c.1041dup, p.Ser348Glnfs*2) in the gene CDC14A. We observed that the c.1421+T>C variant is responsible for the activation of a cryptic splice site resulting in a truncated transcript (c.1414_1421del, p.Val472Leufs*20). Significantly reduced CDC14A mRNA levels indicated a defective transcript due to the c.1041dup variant, which is likely targeted by nonsense-mediated mRNA decay (NMD).

Conclusions: In summary, we identified and assessed the pathogenicity of two novel variants on the RNA-level and expand the spectrum of clinically relevant mutations in the gene CDC14A.

J. Doll: None. S. Kolb: None. L. Schnapp: None. A. Rad: None. F. Rüschendorf: None. I. Khan: None. A. Adli: None. A. Hasanzadeh: None. D. Liedtke: None. S. Knaup: None. M.A.H. Hofrichter: None. T. Müller: None. M. Dittrich: None. I. Kong: None. H. Kim: None. T. Haaf: None. B. Vona: None.


Causes of congenital hearing loss and cochlear implant outcome

C. Landgraf1, S. von Hardenberg1, A. Lesinski-Schiedat2, A. Büchner2, B. Schlegelberger1, B. Auber1

1Department of Human Genetics, Hannover Medical School (MHH), Hannover, Germany, 2Department of Otorhinolaryngology, Hannover Medical School (MHH), Hannover, Germany

Aim: Personalize prognosis and therapy through genetic screening of potential cochlear implant patients.

Background: Hearing loss (HL) can be divided into syndromic and non-syndromic hearing loss (NSHL, approximately 70 %). While pathogenic variants affecting the gene GJB2 account for roughly 10-30 % of NSHL, the majority of NSHL is distributed among more than 100 other genes. For many patients who do not benefit sufficiently from hearing aids, cochlear implants (CIs) are the device of choice. Performance of CI recipients is expected to depend on genetic background and a favorable outcome has been associated with pathogenic variants in genes affecting the function of the cochlear sensory organ (CSO); a negative outcome was reported for patients with variants in genes associated with spiral ganglion neuron (SGN) function.

Patients: 129 unrelated hearing impaired children and adults without pathogenic variants in GJB2 and without evidence for acquired HL.

Methods: Whole exome sequencing was performed analyzing 148 genes associated with HL listed in the Deafness Variation Database and additional 681 genes listed in the Human Phenotype Ontology database based on hearing impairment.

Results: In 33 of 129 patients we identified pathogenic or likely pathogenic variants in 30 different genes, confirming the genetic heterogeneity of hereditary HL. Among these was one patient with a (homozygous) pathogenic variant in a gene proven to affect SGN function (DFNB59).

Conclusions: For meaningful results, a significantly higher number of cases is required. In addition, a further functional subdivision of patients with gene alterations influencing CSO function should be considered.

C. Landgraf: None. S. von Hardenberg: None. A. Lesinski-Schiedat: None. A. Büchner: None. B. Schlegelberger: None. B. Auber: None.


In silico and in vivo analyses of novel variants identified by Whole Exome Sequencing in Argentinean deaf patients: to be or not be pathogenic

P. I. Buonfiglio1, C. D. Bruque2, E. Goldschmidt3, V. Lotersztein4, S. Menazzi5, B. Paoli6, P. Plazas7, A. B. Elgoyhen1, V. K. Dalamón1

1Instituto de Investigaciones en Ingeniería Genética y Biología Molecular “Dr. Héctor Torres” - INGEBI/CONICET, Ciudad Autónoma de Buenos Aires, Argentina, 2Centro Nacional de Genética Médica “A.N.L.I.S. – Dr. Carlos G. Malbrán “, Ciudad Autónoma de Buenos Aires, Argentina, 3Servicio de Genética del Hospital General de Agudos “Dr. Juan A. Fernández”, Ciudad Autónoma de Buenos Aires, Argentina, 4Servicio de Genética del Hospital Militar Central Cirujano Mayor “Dr. Cosme Argerich”, Ciudad Autónoma de Buenos Aires, Argentina, 5Servicio de Genética del Hospital de Clínicas “José de San Martín”., Ciudad Autónoma de Buenos Aires, Argentina, 6Servicio de Otorrinolaringología Infantil del Hospital de Clínicas “José de San Martín”., Ciudad Autónoma de Buenos Aires, Argentina, 7Tercera Cátedra de Farmacología, Facultad de Medicina, Universidad de Buenos Aires, Ciudad Autónoma de Buenos Aires, Argentina

Hereditary hearing loss (HHL) is the most common sensory disorder affecting 1 in 500 newborn children. Since HHL is related to more than 150 target genes, we designed a diagnosis strategy in order to identify pathogenic variants. A total of 1250 patients were analyzed for frequent mutations in GJB2 and GJB6 genes by Sanger Sequencing, genotyping 25% of them. From undiagnosed patients, 29 families were selected to perform Whole exome sequencing. After filtering and analysis process, 45% of patients were genotyped, identifying 23 causative mutations (11 novel, 12 reported) classified according to ACMG Standards. Some of the novel variants were further studied in silico by structural and stability studies of the mutated proteins. In addition, datasets from deafness and specific variant databases were correlated with different protein motifs in order to predict the theoretical pathogenicity effect of the aminoacid changes. Furthermore, knock-down phenotype rescue assays in zebrafish are underway to accomplish in vivo validation. In some cases, extensive analysis reinforced the pathogenicity prediction effect of variants and surprisingly, in one case, discouraged the deleterious effect of a genetic variant to the protein. Preliminary results in zebrafish confirmed the pathogenicity of one novel variant in the hair cell function and auditory system. This study shows that our algorithm is successful for the genetic diagnosis of deafness. Comprehensive analysis is crucial to strengthen prediction of variant pathogenicity. These findings highlight the importance of genetic studies followed by in silico and in vivo validation to better understand the genetic basis of HHL.

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


Genetic spectrum of hereditary hearing loss in multigenerational families with autosomal dominant mode of inheritance

M. Oldak1, M. L. Leja1,2, D. Ozieblo1,3, A. Sarosiak1,3, H. Skarzynski4

1Department of Genetics, Institute of Physiology and Pathology of Hearing, Warsaw, Poland, 2Postgraduate School of Molecular Medicine, Medical University of Warsaw, Warsaw, Poland, Warsaw, Poland, 3Postgraduate School of Molecular Medicine, Medical University of Warsaw, Warsaw, Poland, Warsaw, Poland, Warsaw, Poland, 43. Oto-Rhino-Laryngology Surgery Clinic, Institute of Physiology and Pathology of Hearing, Warsaw, Poland, Warsaw, Poland

Introduction: Hearing loss (HL) is the most common disability of human senses and genetic factors play an important role in its development. Autosomal dominant HL (ADHL) is the second most common form of inherited HL, characterized by postlingual age of onset and progression. To date 63 loci with 47 different genes were causally involved in ADHL.

Materials and Methods: Multigenerational families with ADHL were recruited for the study (n = 44). Genomic DNA was isolated from whole blood and buccal swabs samples. A high-throughput sequencing with custom multigene panel was performed on probands’ DNA samples. Family segregation analysis of the identified variants was conducted using Sanger sequencing. All detected variants were analyzed in the context of population databases and available literature. Pathogenicity of the identified variants was predicted by different computational approaches.

Results: Genetic testing revealed probably pathogenic variants in about 52% (23/44) of the analyzed families. The majority of identified variants were novel, previously not reported and hitherto not linked to the disease. The most frequent genes with identified causative variants were MYO6, COCH, DFNA5, KCNQ4, TECTA and POU4F3.

Conclusions: Our study revealed a high involvement of novel probably pathogenic variants in the development of ADHL and confirmed a high heterogeneity of the identified genetic changes. High-throughput sequencing in HL patients generates large amount of data that should be interpreted carefully and confirmed by family studies. There is also a need for functional validation of the detected novel variants. Supported by: NCN Research Grant no. 2016/22/E/NZ5/00470 SONATA BIS6

M. Oldak: None. M.L. Leja: None. D. Ozieblo: None. A. Sarosiak: None. H. Skarzynski: None.


Wide spectrum of genetic hearing loss causes and large number of novel variants in cochlea implanted children

D. Ozieblo1,2, M. L. Leja1,2, H. Skarzynski3, M. Oldak1

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

Introduction: Every year, approximately 1-6/1000 children are born with severe to profound hearing loss (HL). For this group of patients cochlear implantation (CI) is the treatment of choice. In the majority of deaf children HL is determined by DFNB1 locus pathogenic variants. Here, we aim to dissect the genetic background of HL in DFNB1-negative CI patients.

Materials and Methods: Genomic DNA was isolated from blood samples of carefully selected DFNB1-negative patients with profound prelingual HL (n = 50) and their family members. In all probands whole exome sequencing (WES) followed by bioinformatics and expert analysis were performed. Standard Sanger sequencing was used for validation of selected variants, family segregation analysis and detection of the CEVA haplotype. Copy number variants were confirmed with qPCR.

Results: In every tested subject WES revealed at least 5 probably pathogenic variants in HL-related genes. Causative variants were identified in 33 patients and in 39% (13/33) of them they were inherited in an autosomal dominant manner. Among the detected 53 disease-causing alleles, 55% (29/53) contained novel genetic alterations.

Conclusions: Obtained results show a high heterogeneity of genetic HL causes in the population of Polish DFNB1-negative cochlea-implanted patients. In children with no family history of HL, the possibility of identifying causative variants inherited in both autosomal recessive and dominant manner should be taken into account. In patients with an unknown genetic cause of HL, the tested area should be expanded with whole genome analysis. Supported by NCN grant: 2017/27/N/NZ5/02369

D. Ozieblo: None. M.L. Leja: None. H. Skarzynski: None. M. Oldak: None.


Diagnostic Efficiency of Clinical Exome Solution Panel in patients with Hearing loss/Hereditary Deafness by using Next Generation Sequencing

S. G. Temel1,2, A. Alemdar1, M. Yılmaz3, L. Aliyeva2, S. Ozemri Sag2

1Department of Translational Medicine, Bursa Uludag University, Bursa, Turkey, 2Department of Medical Genetics, Bursa Uludag University, Bursa, Turkey, 3Faculty of Medicine, Bursa Uludag University, Bursa, Turkey

Hearing loss (HL)/Hereditary Deafness (HD) is a heterogeneous disease which multiple genes have been found to be implicated in the disease etiopathogenesis. Genetic diagnosis is highly important for HL/HD patients in order to ensure the etiology of the condition and counsel the patients properly. In this study, 41 patients (22M/19F) who admitted to our clinic between February 2018 and November 2019 with HL/HD were screened for 110 genes related with syndromic/non-syndromic HL/HD. NGS is considered to be the best practice for diagnosis. Large gene panels also can evaluate non-prevelant genes and candidate variants specific to populations. All exons and exon-intron boundaries were amplifed by using Clinical Exome (Sophia Genetics) Solution (CES) kit in MiSeq NGS instrument. Variant analyis (SNV, Indel and CNV) and annotations were performed by Sophia DDM platform, classified according to ACMG criteria with in-silico tools and also investigated by HGMD Professional 2019-4 and other available online bioinformatic tools. We identified CNV in three patients, 26 pathogenic and likely pathogenic variants in 19 of 41 HL/HD patients (46 %) in 10 different genes. Similarly, 23 variants of uncertain significance are determined in 18 of 41 HL/HD patients (43 %) in 18 distinct genes. Besides these variations, we found 31 novel variations that are not reported before in literature or databases. Our study affirms that genetic screening of patients with HL by using CES panel not only easily diagnoses but also help counseling the patients and their family for their future risk of developing the disease.

S.G. Temel: None. A. Alemdar: None. M. Yılmaz: None. L. Aliyeva: None. S. Ozemri Sag: None.


NGS approach for molecular diagnosis of hereditary hearing loss

F. Gerundino1, C. Pescucci1, C. Giuliani1, B. Bianchi2, G. Traficante3, S. Bargiacchi3, S. Romano4, A. Cecconi5, C. Di Marco6, A. Orrico7,8, M. Mencarelli9, L. Candita1, C. Deledda1, M. Trafeli1, B. Minuti1, A. Renieri9, E. Pelo1

1SOD Diagnostica Genetica, AOU Careggi, Firenze, Italy, 2Audiologia SOC Otorinolaringoiatria AOU Meyer, Firenze, Italy, 3SOC Genetica Medica AOU Meyer, Firenze, Italy, 4Medicina 1, Genetica Medica AOU Pisana, Pisa, Italy, 5Ambulatorio Integrato Genetica Medica, Usl Toscana Centro, Firenze, Italy, 6Ambulatorio Integrato Genetica Medica, Usl Toscana Centro, Florence, Italy, 7Interdipartimental Program for molecular diagnosis and characterization of pathogenic mechanisms of rare genetic diseases, Siena, Italy, 8Clinical Genetics. ASL Toscana Sudest. Ospedale della Misericordia., Grosseto, Italy, 9Genetica Medica, Azienda Ospedaliera Universitaria Senese, Siena, Italy

Introduction: hearing loss (HL) is one of the most genetically heterogeneous disorders. Although pathogenic variants in GJB2 account for a large proportion of cases, many patients remain undiagnosed after DFNB1 testing. NGS analysis of HL-genes to simultaneously search for nucleotide variants and copy number variants (CNVs) greatly improve the detection rate.

Materials and methods: clinical exome sequencing followed by in silico analysis of HL-gene panel (106 genes) was carried out on 117 patients on which DFNB1 pathogenic variants were previously excluded. Eighty cases show prelingual or childhood deafness while in 37 patients HL was progressive with variable age of onset. Clinical interpretation of genetic variants was performed according to ACMG2015 by InterVar and Varsome tools and revised according to the specific guidelines for HL-genes.

Results: Forty-eight pathogenic or likely pathogenic variants and eight pathogenic CNVs (6 STRC -CATSPER2 and 2 OTOA whole gene deletions) were identified with an overall detection rate of 26.5%. Specific detection rate was 32% (12/37) for adult cases and 24% in children. In particular, NGS identified 12 different pathogenic variants in dominant, 28 in recessive and 8 in dominant/recessive genes. The genes most involved were SLC26A4 and STRC. A single pathogenic variant in SLC26A4 gene was found in 7 cases.

Conclusions: overall detection rate of the test is in line with available data. MLPA analysis in target genes such as SLC26A4 should be performed to exclude the presence of a deletion at exonic level in cases where a single variant as been identified.

F. Gerundino: None. C. Pescucci: None. C. Giuliani: None. B. Bianchi: None. G. Traficante: None. S. Bargiacchi: None. S. Romano: None. A. Cecconi: None. C. Di Marco: None. A. Orrico: None. M. Mencarelli: None. L. Candita: None. C. Deledda: None. M. Trafeli: None. B. Minuti: None. A. Renieri: None. E. Pelo: None.


Complex assessment of pathogenicity of novel variant c.516G>C (p.Trp172Cys) in the GJB2 gene associated with hearing loss in indigenous peoples of Southern Siberia (Russia)

E. A. Maslova1,2, M. V. Zytsar1, V. Y. Danilchenko1, K. E. Orishchenko1,2, O. L. Posukh1,2

1Institute of Cytology and Genetics, Novosibirsk, Russian Federation, 2Novosibirsk State University, Novosibirsk, Russian Federation

Assessment of pathogenicity of novel variants is a primary task for molecular diagnostics of hereditary diseases. Mutations in gene GJB2 encoding transmembrane protein Cx26 are the common cause for hearing loss worldwide. We present a complex assessment of pathogenicity of novel non-synonymous variant c.516G>C (p.Trp172Cys) in GJB2 found with high frequency in deaf patients belonging to indigenous peoples of Southern Siberia (Tuvinians and Altaians).

Materials & Methods: Genetic analysis was performed in group of deaf patients and ethnically matched controls. The GJB2 knockout HeLa cell line (obtained by CRISPR/Cas9 system) was used to establishment of transgenic HeLa cell lines with different pathogenic GJB2-variants and GJB2-wt. Cellular Cx26 localization was investigated by immunocytochemistry (ICC). The Cx26-channels permeability was analyzed by dye (PI) loading assay.

Results: Segregation of variant c.516G>C (p.Trp172Cys) with hearing loss was established in analysis of pedigrees of deaf patients and its frequency was significantly higher in patient’s group than in controls. Most of in silico programs predicted a likely damaging effect of p.Trp172Cys. ICC showed predominantly cytoplasmic localization of the Cx26-p.Trp172Cys protein in contrast to Cx26-wt which represented distinct conglomerates on cell membrane. Dye (PI) loading assay revealed lower PI loading efficiencies in cells expressing mutant variant Cx26-p.Trp172Cys compared to Cx26-wt while the absence of PI accumulation was shown for other mutant Cx26-variants with known pathogenic effects.

Conclusions: Several lines of evidences support pathogenicity of novel variant c.516G>C (p.Trp172Cys) in gene GJB2 and its involving in hearing loss.

Study was supported by Project #0324-2019-0041-C-01 and RFBR grant #17-29-06016_ofi-m.

E.A. Maslova: None. M.V. Zytsar: None. V.Y. Danilchenko: None. K.E. Orishchenko: None. O.L. Posukh: None.


Genetic heterogeneity underlying hearing loss and Usher syndrome in Saudi population

K. Ramzan, F. Imtiaz, M. Al-Owain, S. Alhazzaa

King Faisal Specialist Hospital and research centre, Riyadh, Saudi Arabia

Hearing loss is one of the most common sensory disorders in humans with both genetic and environmental etiologies. Usher syndrome (USH) is the most common cause of combined blindness and deafness. Molecular diagnosis is of great significance in revealing the molecular pathogenesis and aiding the clinical diagnosis of this disease.

Our study aims to comprehensively delineate the genetic basis of hearing loss in the individuals of Saudi Arabian origin. The identification of the causative gene in affected families with hearing loss is difficult due to extreme genetic heterogeneity and lack of phenotypic variability. Consanguineous families are a powerful resource for genetic linkage studies/homozygosity mapping for recessively inherited hearing impairment. Homozygosity mapping, linkage analysis and next generation sequencing Deafness Gene-panel and Whole Exome sequencing were conducted. Using the combined approaches, so far mutations in 32 different deafness genes including novel genes first time identified in this population, have been identified. More than 300 familial/sporadic cases were solved, including novel variants in known HL genes.

Using these innovative molecular approaches, we were able to document the most common forms of hereditary hearing loss, their incidence and distribution in the Saudi population. The overall results of this study are highly suggestive that the underlying molecular basis of hearing loss in Saudi Arabia is very genetically heterogeneous. The benefit of this study will hopefully provide the foundation for knowledge and awareness through screening of carrier status and genetic counselling, thereby having a major impact upon early intervention for and prevention of hereditary hearing loss.

K. Ramzan: None. F. Imtiaz: None. M. Al-Owain: None. S. Alhazzaa: None.


ENT- Genetics Genome Boards: Over 50% diagnostic yield

M. Carminho-Rodrigues1, P. Senn2, M. Abramowicz1, E. Hammar1, L. Quteineh1, A. Murphy1, S. Laurent1, M. Guipponi1, H. Cao-Van2, A. Paoloni-Giacobino1

1Medical Genetics Unit, Geneva University Hospitals, Geneva, Switzerland, 2ENT Unit, Geneva University Hospitals, Geneva, Switzerland

Introduction: Hearing impairment (HI) is the most common sensory impairment. It can be isolated or syndromic. Molecular diagnosis through whole exome sequencing (WES) can allow an improved management and surveillance of HI and possible associated anomalies. Here we show the results of a 3 years’ experience of Ear-nose-and-throat (ENT) Genetics Genome boards.

Materials and Methods: Our cohort includes children with mild to profound, unilateral or bilateral HI referred by ENT clinic from 2017 to 2019. We performed WES with bioinformatics analysis of 189 genes associated with HI. The results are discussed during a genome board where geneticists and ENT specialists confront the genetic variants found by WES with the auditory phenotype of the patients.

Results: We tested 38 children, most often with moderate, bilateral sensorineural hearing loss. The diagnostic rate was 52.6% (20/38). Among the results we found variants associated with: syndromic forms: Usher, Wolfram-like, Branchio-oto-renal, Alport, Perrault, GATA3; non-syndromic forms: Cx26, ACTG1, OTOA, ILDR1, SLC17A8, LOXHD1, TBC1D24 and STRC related HI associated or not with infertility in males.

Conclusions: The diagnostic yield of our study (52.6%) is comparable to recent data in the literature. A genetic assessment of HI in young children makes the identification of the molecular cause possible, and permits to anticipate possible complications particularly in the case of syndromic forms with medical problems involving other organs or systems. Collaboration between ENT specialists and geneticists is essential to make an accurate and precise diagnosis and to personalize the follow-up and treatment of these young patients.

M. Carminho-Rodrigues: None. P. Senn: None. M. Abramowicz: None. E. Hammar: None. L. Quteineh: None. A. Murphy: None. S. Laurent: None. M. Guipponi: None. H. Cao-Van: None. A. Paoloni-Giacobino: None.


The utility of exome sequencing for hereditary eye diseases: single-centre experience

M. Volk1, N. Teran1, A. Maver1, H. Jaklič1, A. Fakin2, M. Hawlina2, B. Peterlin1

1Institute of Genomic Medicine, UMC Ljubljana, Ljubljana, Slovenia, 2Eye hospital, UMC Ljubljana, Ljubljana, Slovenia

Introduction: Over 650 heritable conditions with ocular involvement have been described to date. These include isolated eye diseases (retinal degeneration, cataract, corneal dystrophy, optic atrophy, developmental ocular anomalies), as well as complex genetic syndromes with ocular manifestations. This group of disorders is genetically heterogeneous and diagnostically challenging. The aim of our study was to determine the clinical diagnostic utility of exome sequencing in hereditary eye diseases.

Materials and Methods: 243 Slovene unselected consecutive patients were referred to our institution from November 2013 to October 2019 because of suspected hereditary eye disease. We performed exome sequencing with the mitochondrial sequence analysis based on the off-target exome reads. Filtered variants were analyzed according to population frequency, characterization in the ClinVar database, the putative impact of the variant, and predicted pathogenicity. Variants were classified according to ACMG standards and guidelines for variant interpretation.

Results: We found causative pathogenic or likely pathogenic variants in 102 patients. The diagnostic yield was 50% for retinal degeneration (78/156), 20% for optic atrophy (6/30), 75% for high myopia (3/4), 29% for cataract (2/7), 45% for retinoblastoma (4/9), 20% for developmental ocular anomalies (3/15), 67% for corneal dystrophy (2/3), and 56% for syndromic ocular disease (11/19).

Conclusions: The overall diagnostic yield for ophtalmogenetic referral in a cohort of unselected consecutive patients was 42%. Therefore, the exome sequencing is a cost-effective and efficient comprehensive approach that should be considered as a first-tier investigation for diagnostics of hereditary eye diseases.

M. Volk: None. N. Teran: None. A. Maver: None. H. Jaklič: None. A. Fakin: None. M. Hawlina: None. B. Peterlin: None.


Analysis of recurrent mutations in different Iranian families with hearing loss

M. A. H. Hofrichter1, A. Rad2,3, S. Kolb1, J. Weingart1, J. Doll1, T. Müller4, C. Remmele5,1, M. Dittrich1,4, R. Maroofian6, T. Haaf1, B. Vona1,3

1Institute of Human Genetics, Julius Maximilians University, Würzburg, Germany, 2Cellular and Molecular Research Center, Sabzevar University of Medical Sciences, Sabzevar, Iran, Islamic Republic of, 3Department of Otorhinolaryngology, Head and Neck Surgery, Tübingen Hearing Research Centre (THRC), Eberhard Karls University, Tübingen, Germany, 4Institute of Bioinformatics, Julius Maximilians University, Würzburg, Germany, 5Center for Rare Diseases, University Hospital Würzburg, Würzburg, Germany, 6Genetics and Molecular Cell Sciences Research Centre, St George’s, University of London, Cranmer Terrace, London, United Kingdom

Introduction: Hereditary hearing loss (HL) is one of the most genetically heterogeneous disorders. The genetic landscape of non-syndromic HL includes over 100 genes. In aggregate, these genes harbor several thousand causally implicated variants. The observation of recurrent variants raises the question about whether variants arose from a single ancestral allele or were the result of independent mutational events. In our study that includes 150 Iranian probands, we identified 15 families with recurrent likely pathogenic variants in the genes SMPX, SLC26A4, TECTA and TMIE.

Methods: DNA samples from index patients were exome sequenced following Illumina exome protocols. Libraries were sequenced with a NextSeq500 benchtop sequencer. Variant and copy number variation analysis were performed using GensearchNGS and an in-house exome analysis pipeline. Segregation analysis was performed for all families. Haplotype reconstruction allowed comparison across families.

Results: Haplotype analysis of the c.99del variant in SMPX that was identified in three Iranian families suggested a common descent. Interestingly, this X-linked variant was described as a founder mutation in an isolated Newfoundland population. In addition, more than one recurrent mutation was identified in the genes SLC26A4 and TECTA in families with autosomal recessive HL that was consistent with a founder effect. In three additional families, the c.250C>T variant in TMIE was implicated in autosomal recessive HL without sharing further common variants, suggesting a mutational hotspot.

Conclusions: The genetic origin of recurrent mutations could be interesting for further characterization of HL genes with respect to ethnic background and susceptibility of mutational events.

M.A.H. Hofrichter: None. A. Rad: None. S. Kolb: None. J. Weingart: None. J. Doll: None. T. Müller: None. C. Remmele: None. M. Dittrich: None. R. Maroofian: None. T. Haaf: None. B. Vona: None.


Genomic approaches for the analysis fo more than 700 Italian patients affected by Hereditary Hearing Loss (HHL) reveal new genes/alleles.

A. Morgan1,2, G. Pelliccione1, F. Faletra1, D. Dell’Orco3, M. Brumat2, P. Gasparini4,2, G. Girotto1,2

1Institute for Maternal and Child Health – IRCCS Burlo Garofolo, Trieste, Italy, 2Department of Medicine, Surgery and Health Sciences, University of Trieste, Trieste, Italy, 3Department of Neurosciences, Biomedicine and Movement Sciences, Section of Biological Chemistry, University of Verona, Verona, Italy, 4Institute for Maternal and Child Health – IRCCS, Burlo Garofolo, Trieste, Italy

Discerning the complex genetic basis of HHL is fundamental to facilitate a correct genetic counselling and molecular diagnosis. The use of high-throughput genome technologies has made great strides in the discovery of new alleles/genes identification. Here, 714 deeply clinically characterized patients were screened with a Targeted Re-Sequencing (TRS) panel of 96 deafness genes and, in negative cases, SNP-arrays were applied. Afterwards, families negative to both approaches were analysed by Whole Exome Sequencing (WES) for new candidates identification. TRS and SNP-arrays characterized ~28% of the HL cases (42% familial and 20% sporadic cases) highlighting STRC and TECTA as the second major players in the Italian population after GJB2. Then, WES allowed the identification of five new HHL genes, two of them (SLC12A2 and USP48) just identified. As regards SLC12A2, a heterozygous missense variation (NM_001046.2, exon25, c.3431C>A, p.(T1144N)) has been identified in a three-generation HHL family. The variant is predicted as deleterious by several bioinformatic prediction tools and is not reported in any public database. Interestingly, studies in mouse and zebrafish models demonstrated Slc12a2 expression in the inner ear and its involvement in hearing loss. Moreover, to better evaluate the pathogenic effect of the identified variant, an in silico protein modelling has been performed suggesting that the substitution identified might alter the correct folding of the protein. Thanks to this approach ~35% (60% familial cases and 20% sporadic ones) of cases have been characterized confirming the large mutation’s spectrum of HHL, and the importance of an accurate clinical diagnosis.

A. Morgan: None. G. Pelliccione: None. F. Faletra: None. D. Dell’Orco: None. M. Brumat: None. P. Gasparini: None. G. Girotto: None.


Genetic causes of paediatric hereditary retinal diseases in Polish population

K. H. Chrzanowska1, B. Kocyła-Karczmarewicz2, A. M. Tracewska3, E. Ciara1, M. Rydzanicz4, P. Stawiński4, R. Płoski4

1Department of Medical Genetics, The Children’s Memorial Health Institute, Warsaw, Poland, 2Department of Ophthalmology, The Children’s Memorial Health Institute, Warsaw, Poland, 3DNA Analysis Laboratory, PORT Polish Center for Research Technology, Wrocław, Poland, 4Department of Medical Genetics, Medical University of Warsaw, Warsaw, Poland

Introduction: Every year, Children’s Memorial Health Institute admits approximately 100 children suffering from vision impairment with suspicion of inherited retinal degeneration (IRD), which is confirmed in half of the cases. In many paediatric patients, genetic diagnosis is crucial for prognostics and future planning.

Methods: 76 probands and 22 affected family members were ascertained, and a detailed medical and family history was obtained. Ophthalmological assessment consisted, when feasible, of: best-corrected Snellen visual acuity (BCVA), colour vision, visual field test, dilated ophthalmoscopy, colour fundus photography, autofluorescence imaging, spectral-domain optical coherence tomography (SD-OCT), fluorescein angiography and electrophysiological assessment. DNA was isolated from blood samples, and proband samples underwent targeted next-generation sequencing for 108 known IRD genes.

Results: In the vast majority of affected individuals the genetic analyses confirmed the initial diagnosis; some diagnoses, however, needed to be revised. Causal variants were found in 2/3 of the families. In five cases, monoallelic pathogenic alterations were found in autosomal recessive genes. Most commonly mutated genes in this cohort were ABCA4 (23 individuals), CNGA3 (5) and CEP290 (4).

Conclusions: Genetic analysis is crucial to achieve accurate diagnosis and prognosis, which enables informed choices for the future of the affected children. Many probands were the first individual suffering from IRD in the family. An important role of genetic diagnostics is to inform the parents about their risk for having another child with the disease. Finally, emerging targeted gene therapies indicate a chance of slowing or stopping the disease progression.

Funding: National Science Centre (Poland) grant no. UMO-2015/19/D/NZ2/03193.


K.H. Chrzanowska: None. B. Kocyła-Karczmarewicz: None. A.M. Tracewska: None. E. Ciara: None. M. Rydzanicz: None. P. Stawiński: None. R. Płoski: None.


Diagnostic exome sequencing in 50 patients with high myopia

V. J. M. Verhoeven1,2, A. E. G. Haarman2,3, M. van Tienhoven1, A. A. H. J. Thiadens2, S. E. Loudon2, A. de Klein1, E. Brosens1, J. R. Polling2,4, A. Bouman1, C. C. W. Klaver5,3,2, A. J. A. Kievit1, L. H. Hoefsloot1

1Dept. of Clinical Genetics, Erasmus Medical Center, Rotterdam, the Netherlands, Rotterdam, Netherlands, 2Dept. of Ophthalmology, Erasmus Medical Center, Rotterdam, the Netherlands, Rotterdam, Netherlands, 3Dept. of Epidemiology, Erasmus Medical Center, Rotterdam, the Netherlands, Rotterdam, Netherlands, 4Dept. of Orthoptics, School of Applied Science Utrecht, Utrecht, Netherlands, 5Dept. of Ophthalmology, UMC St Radboud, Nijmegen, the Netherlands, Nijmegen, Netherlands

Introduction: High myopia (HM; ≤-6 diopters) is a heterogeneous condition, and without clear accompanying features it can be difficult to pinpoint a genetic cause. The aim of this study was to evaluate the diagnostic yield of whole exome sequencing (WES) in patients with HM.

Materials and methods: Patients with HM were recruited by ophthalmologists and clinical geneticists. Clinical features were categorized into isolated HM, HM with other ocular features or syndromic HM with systemic involvement. WES was performed using a vision gene panel including 477 genes.

Results: WES was performed in 50 patients with HM; the majority had isolated HM (75%). A genetic cause was identified in 11 patients (22%; mean age at diagnosis 9.4 years (SD 7.5); mean refractive error -12.60 diopters (SD 6.22)). Four patients had mutations in known retinal dystrophy genes (GUCY2D; FAM161A; PDE6H; CACNA1F); 3 female patients had a ARR3 mutation (X-linked female limited high myopia); 2 patients had homozygous or compound heterozygous COL18A1 mutations (Knobloch syndrome); 1 patient had a COL2A1 mutation (Stickler syndrome); 1 patient had a PAX6 mutation. In 14 patients (28%) a variant of unknown significance was found; follow-up examinations are necessary.

Conclusions: The diagnostic yield of our WES vision gene panel for HM was 22%. It enabled us to diagnose various causes of HM, i.e. retinal dystrophies, connective tissue diseases, and non-syndromic HM, allowing appropriate follow up of systemic and ocular features and genetic counselling.

Horizon 2020 (grant 648268); NWO (grants 91815655, 91617076); Oogfonds; ODAS; Uitzicht (grant 2017-28).

V.J.M. Verhoeven: None. A.E.G. Haarman: None. M. van Tienhoven: None. A.A.H.J. Thiadens: None. S.E. Loudon: None. A. de Klein: None. E. Brosens: None. J.R. Polling: None. A. Bouman: None. C.C.W. Klaver: None. A.J.A. Kievit: None. L.H. Hoefsloot: None.


Genetic architecture of inherited retinal degeneration in Germany: A large cohort study from a single diagnostic center over a 9-year period

N. Weisschuh1, C. D. Obermaier2,3, A. Bernd1, L. Kühlewein1,4, F. Nasser5, D. Zobor1, F. Battke3, E. Zrenner1,6, E. Weber1, B. Wissinger1, S. Biskup2,3, K. Stingl5, S. Kohl1

1Institute for Ophthalmic Research, Centre for Ophthalmology, University of Tübingen, Tuebingen, Germany, 2Praxis fuer Humangenetik Tuebingen, Tuebingen, Germany, 3Center for Genomics and Transcriptomics, CeGaT GmbH, Tübingen, Germany, 4University Eye Hospital, Center for Ophthalmology, University of Tübingen, Tübingen, Germany, 5University Eye Hospital, Center for Ophthalmology, University of Tübingen, Tuebingen, Germany, 6Werner Reichardt Centre for Integrative Neuroscience (CIN), University of Tübingen, Tübingen, Germany

Introduction: The aim of this cohort study was to unravel the molecular basis of inherited retinal degeneration (IRD) in a large cohort of 2,156 affected patients diagnosed and followed-up in the largest center for inherited retinal diseases in Germany. Our study cohort accounts for approximately 7% of the estimated 30,000 IRD patients in Germany, thereby providing representative data for the prevalence and the mutation spectrum of IRD genes and mutations for the population in Germany. Presently, more than 260 `disease genes’ are known to cause IRDs. The identification of the underlying disease-causing mutations has been accelerated by the introduction of next-generation-sequencing technologies (NGS). Within the field of NGS platforms, the targeted capture of known ‘disease genes’ with its strong optimization of coverage on relevant targets has been proven superior over whole exome sequencing with respect to read depth and on-target efficiency.

Materials and Methods: DNA samples were analyzed by targeted NGS and, whenever indicated, multiplex ligation-dependent probe amplification (MLPA). Patients with single-gene disorders were analyzed by Sanger sequencing and MLPA.

Results: We achieved an overall molecular diagnostic solving rate of 70.8%. A total of 1,161 distinct variants were identified, including 501 novel variants, reaffirming the known vast genetic heterogeneity of IRD in a mainly outbred European population.

Conclusions: Our study demonstrates the clinical utility of panel-based NGS in a large and highly heterogeneous cohort from an outbred population and for the first time gives a comprehensive representation of the genetic landscape of IRDs in Germany.

N. Weisschuh: None. C.D. Obermaier: None. A. Bernd: None. L. Kühlewein: None. F. Nasser: None. D. Zobor: None. F. Battke: None. E. Zrenner: None. E. Weber: None. B. Wissinger: None. S. Biskup: None. K. Stingl: None. S. Kohl: None.


Retinal disease as the leading clinical manifestation in patients with unexpected inherited systemic disorders detected by whole genome sequencing

A. Liebmann1,2, P. Mazzola1,2, K. Stingl3, T. Heinrich1,2, K. Schäferhoff1,2, S. Ossowski1,2, M. Sturm1,2, S. Kohl4, N. Weisschuh4, B. Wissinger4, O. Riess1,2, T. Haack1,2

1Institute of Medical Genetics and Applied Genomics, University of Tübingen, Tübingen, Germany, 2Centre for Rare Diseases, University of Tübingen, Tübingen, Germany, 3University Eye Hospital, Center for Ophthalmology, University of Tübingen, Tübingen, Germany, 4Institute for Ophthalmic Research, Centre for Ophthalmology, University of Tübingen, Tübingen, Germany

Introduction: Implementation of genome-wide sequencing approaches such as exome and genome sequencing in routine diagnostics implicates the possibility of incidental findings and unexpected diagnoses. Here we present two patients, a 9-year-old girl with cone dystrophy and a 43-year old man with rod-cone dystrophy, initially referred to the Department of Ophthalmology to confirm inherited retinal disease.

Material and Methods: Genomic DNA was processed using the TruSeq DNA PCR-free Kit (Illumina) for library preparation and paired-end whole genome sequencing (WGS) was performed on a NovaSeq6000 System (Illumina). Data analysis was conducted using an in-house bioinformatics pipeline optimized for structural/copy number variant detection.

Results: WGS of the girl identified a clinically relevant homozygous deletion in CLN3. The detected deletion c.461-280_677+382del, p.? is the most frequent pathogenic deletion in CLN3 associated with autosomal recessive neuronal ceroid lipofuscinosis. This lysosomal storage disease is characterized by seizures, psychomotor degeneration and visual failure. Clinical course is progressive and unfavorable. WGS of the man identified a pathogenic homozygous missense variant c.635G>T, p.Gly212Val in TMEM126B. Variants of TMEM126B are associated with autosomal recessive complex 1 deficiency. This mitochondrial disease is characterized by a variable clinical phenotype including neuromuscular, cardiovascular and metabolic symptoms and visual impairment. The detected variant has been described before in another patient with rod-cone dystrophy.

Conclusions: This study highlights the importance to inform patients about the possibility of retinal disease being the leading clinical manifestation of unexpected inherited systemic disorders. Thus, comprehensive genetic counseling and appropriate documentation of informed consent is crucial prior to performing WGS.

A. Liebmann: None. P. Mazzola: None. K. Stingl: None. T. Heinrich: None. K. Schäferhoff: None. S. Ossowski: None. M. Sturm: None. S. Kohl: None. N. Weisschuh: None. B. Wissinger: None. O. Riess: None. T. Haack: None.


Shift of T-cell repertoire in cornea of keratoconus patients

L. Skorodumova1, A. Kanygina1, A. Belodedova2, E. Sharova1, B. Malyugin2

1Federal Research and Clinical Center of Physical-Chemical Medicine of Federal Medical Biological Agency, Moscow, Russian Federation, 2S. Fyodorov Eye Microsurgery Complex Federal State Institution, Ministry of Health of Russian Federation, Moscow, Russian Federation

Introduction: Keratoconus is a pathologic condition of the eye which results in thinning and cone-shaped deformation of cornea. While its precise cause is still unknown, keratoconus is considered to be a multifactorial disease, and immune processes are assumed to contribute to its development, yet little data is available so far on the immune microenvironment of cornea in affected patients. Here we present the assessment of T cell repertoire in cornea and blood samples from 3 patients with keratoconus who underwent cornea transplantation.

Materials and Methods: RNA was extracted from PBMC and cornea samples with RNeasy Micro Kit (Qiagen). RNA libraries of TCR-alpha and TCR-beta transcripts were prepared using SMARTer Human TCR a/b Profiling Kit (Takara Bio USA Inc.) and sequenced using Illumina MiSeq platform. TRA and TRB clonotypes were assembled and quantified with MiXCR package. CDR3 sequences were annotated against VDJdb, PIRD and McPAS-TCR databases.

Results: Cornea samples for all 3 patients demonstrated significantly lower clonotype diversity than corresponding PBMC samples. We observed a shift of T cell repertoire in cornea compared to PBMC samples. The top abundant TCR clones in cornea were poorly represented or absent in corresponding blood samples, which indicates that they represent a tissue-specific population. For most of the CDR3 variants detected in cornea, no specificities against known antigens were found, and further research is needed to identify possible functions of these T cell populations.

This study was supported by RFBR as part of the research project № 17-29-06077.

L. Skorodumova: None. A. Kanygina: None. A. Belodedova: None. E. Sharova: None. B. Malyugin: None.


Genomic landscape in spanish patients with ocular malformations by next generation sequencing

A. Damián1, A. Tamayo1, J. Moya1, C. Villaverde1, A. Avila-Fernández1, A. Arteche1, V. López2, M. Ballesta2, E. Guillen2, S. Tahsin Swafiri1, I. Lorda1, F. Blanco-Kelly1, M. Trujillo1, C. Ayuso1, M. Corton1

1Instituto de Investigación Sanitaria Fundación Jiménez Díaz, Madrid, Spain, 2University Hospital Virgen de la Arrixaca, Murcia, Spain

Human eye development is coordinated by complex developmental processes which involves several hundreds of genes. Any disruption in this regulation during the eye organogenesis can lead to ocular malformations such as Microphthalmia- Anophthalmia-Coloboma (MAC). These diseases are very clinically and genetically heterogeneous conditions with overlapping phenotypes; consequently the mutational load is not well known and the genotype-phenotype correlations can be difficult to define. Our aim was to perform a comprehensive molecular screening in a cohort of Spanish patients with ocular malformations using different approaches of next-generation sequencing (NGS). We have findings in around 52% of the patients. One-third carried likely causal mutations, 8% were partially characterized and 8% had VUS. Different casual mutations were found and the most prevalent in our cohort are transcription factors such as OTX2, SOX2, VSX2 and PAX6. In contrast, others variants have expanded the phenotypic spectrum of some genes classically associated with other phenotypes such as GJA8, KERA, or the lens crystallins family genes suggesting new roles of them in the early development of the eye. This results show that not only NGS is efficient for the genetic analysis of ocular malformations but also for achieving new candidate genes. Therefore, we have update our gene panels resulting in an increase in our diagnostic yield. In contrast, 34% of patients remain uncharacterized, mostly unilateral or non-syndromic cases. So that, our future purposes are to understand the mechanisms involved in this disease by identifying novel genetic causes and exploring their functional role.

A. Damián: None. A. Tamayo: None. J. Moya: None. C. Villaverde: None. A. Avila-Fernández: None. A. Arteche: None. V. López: None. M. Ballesta: None. E. Guillen: None. S. Tahsin Swafiri: None. I. Lorda: None. F. Blanco-Kelly: None. M. Trujillo: None. C. Ayuso: None. M. Corton: None.


Exome-based panel testing as an efficient method to diagnose the highly heterogeneous ocular disorder spectrum Microphthalmia, Anophthalmia, Coloboma and Anterior Segment Dysgenesis (MAC-ASD)

H. Verdin1, T. Rosseel1, S. Vermeer2, I. Balikova3, P. Kestelyn4, C. Meunier5, F. Meire6, J. Van De Velde1, O. Vanakker1, J. Van Den Ende7, M. De Rademaeker7, M. Meuwissen7, M. De Vries8, Y. Sznajer9, M. Desmet1, T. Sante1, F. Coppieters1, B. P. Leroy1,4,10, E. De Baere1

1Center for Medical Genetics, Ghent University Hospital, Ghent, Belgium, 2Center for Human Genetics, University Hospitals Leuven, Leuven, Belgium, 3Department of Ophthalmology, University Hospitals Leuven, Leuven, Belgium, 4Department of Ophthalmology, Ghent University and Ghent University Hospital, Ghent, Belgium, 5Center for Human Genetics, Institute of Pathology and Genetics, Gosselies, Belgium, 6Department of Ophthalmology, Hôpital Universitaire des Enfants Reine Fabiola, Brussels, Belgium, 7Center for Medical Genetics, University of Antwerp and Antwerp University Hospital, Antwerp, Belgium, 8Department of Ophthalmology, Antwerp University Hospital, Antwerp, Belgium, 9Center for Human Genetics, Cliniques Universitaires St-Luc, Universite Catholique de Louvain, Brussels, Belgium, 10Division of Ophthalmology and Center for Cellular & Molecular Therapeutics, The Children’s Hospital of Philadelphia, Philadelphia, PA, United States

Introduction: Microphthalmia and anophthalmia are part of a spectrum with ocular coloboma, a structural malformation resulting from incomplete fusion of the optic fissure, which likely have a shared, although heterogeneous genetic basis. They are collectively named MAC (Microphthalmia-Anophthalmia-Coloboma), characterized by a tremendous clinical heterogeneity and often associated with other ocular abnormalities such as anterior segment dysgenesis (ASD). We aimed to set up an exome-based gene panel test for MAC-ASD.

Materials and Methods: Sample preparation was done with the SureSelectXT Low Input Human All Exon V7 kit using Bravo (Agilent Technologies) and pooled libraries were sequenced on a HiSeq 3000 or NovaSeq 6000 (Illumina) aiming for a minimal coverage of 20x for at least 90% of the investigated regions. Data analysis was executed with a bcbio-based pipeline and variant filtering with in-house Seqplorer software. Copy number variant (CNV) analysis was done using ExomeDepth.

Results: As of May 2018, 26 patients, 12 with MAC (46.2%) and 14 with ASD (53.8%), underwent MAC-ASD panel analysis. The overall diagnostic yield was 9/26 (34.6%), in line with the reported range (11-36%). ASD has the highest yield (50%), compared to MAC (16.7%). In seven out of the nine cases a (likely) pathogenic variant was identified in ALDH1A3, CPAMD8, FOXE3, MFRP, PAX6, PITX2 and PITX3. In two of the nine patients a CNV encompassing FOXC1 was found.

Conclusions: Exome-based MAC-ASD panel analysis revealed a molecular diagnosis in 34.6% of a MAC-ASD cohort, highlighting the efficacy of exome-based panel testing in heterogeneous conditions.

Funding: FWO/12R3419N; FWO/1802220N; BOF15/GOA/011.

H. Verdin: None. T. Rosseel: None. S. Vermeer: None. I. Balikova: None. P. Kestelyn: None. C. Meunier: None. F. Meire: None. J. Van De Velde: None. O. Vanakker: None. J. Van Den Ende: None. M. De Rademaeker: None. M. Meuwissen: None. M. De Vries: None. Y. Sznajer: None. M. Desmet: None. T. Sante: None. F. Coppieters: None. B.P. Leroy: None. E. De Baere: None.


The enigma of Midfacial Toddler Excoriation Syndrome (MiTES), a second PRDM12 poly-alanine tract expansion disorder

N. Sarveswaran1, G. Woods1, C. Moss2

1Cambridge Institute for Medical Research, Cambridge, United Kingdom, 2Birmingham Children’s hospital, Birmingham, United Kingdom

Mid-face toddler excoriation syndrome (MiTES) is a new sensory disease first described in 2017 (PMID: 28028858). We report 12 cases in 7 families. The phenotype is of intense itch in early childhood, which causes mutilating self-injury by scratching. The intense itch is localised only to the midface and no other body parts; is not associated with altered sweating; it spontaneously subsides by 4-6 years; cognition and pain sensing is normal; there is no excess of Staph Aureus infections (on the face or elsewhere). Inheritance appeared recessive, as parents were unaffected, males and females were equally affected, and most cases were consanguineous. Older individuals “grow out” of the condition, facial injuries heal, and have normal whole-body itch and pain sensing. In six of seven families, we found the same mutation in affected individuals - a homozygous poly-Alanine repeat expansion from the normal range (7-13) to 18. Parents were always carriers. This was also found in a non-Asian, Caucasian, non-consanguineous family. In the seventh family, we found only a heterozygous 7 base pair deletion in PRDM12, but no second mutation. What is extraordinary is that a PRDM12 homozygous 19 poly-Alanine repeat expansion causes Congenital Insensitivity to Pain; which is not associated with excess itch, leads to self-inflicted lip, tongue and corneal damage, and always cause excess S. Aureus infections. No other poly-alanine tract expansion disease has two phenotypes. We present our cellular studies to try and understand the disease mechanism whereby a single additional alanine can cause such different phenotypes.

N. Sarveswaran: None. G. Woods: None. C. Moss: None.


Mutational analysis of 256 Russian patients with congenital aniridia and overlapping phenotypes

T. A. Vasilyeva, A. V. Marakhonov, V. V. Kadyshev, R. A. Zinchenko

Research Centre for Medical Genetics, Moscow, Russian Federation

Congenital aniridia (OMIM#106210) is an autosomal dominant eye disorder mainly caused by heterozygous PAX6 mutations or 11p13 chromosome rearrangements. Pathogenic variants in some other genes could cause overlapping phenotypes, also, PAX6 missense mutations could be associated with other than aniridia phenotypes. A total of 208 unrelated families (256 patients) with clinical diagnoses of either congenital aniridia, counting WAGR syndrome, or overlapping ophthalmological diagnoses characterized by iris hypoplasia were included in the study. Familial to sporadic proportion counted for 44/164. A combination of Sanger PAX6 sequencing, whole-exome sequencing, followed by MLPA analysis of 11p13, 4q25, 6p25.3 loci was implemented. 204 out of 208 families (98%) were found to have either heterozygous pathogenic nucleotide sequence variant or chromosome deletion. Disease-causing variants in PAX6 and deletions in 11p13 were found in 92.7%. Four other genes (FOXC1, PITX2, NDP, CRYAA) explained 5.3% of the examined families. 30.3% of the cohort carried pathogenic CNVs (62 in 11p13 and 1 in 6p25.3). A frequent 11p13 deletion of PAX6 3′-cis-regulatory region was determined (16/208, 7.7%). The frequency of this deletion in the cohort was higher than PAX6 hotspot c.718C>T rate (9/208, 4.3%). In total, repeated PAX6 variants were determined in 39/208 (18,8%) families, together with 11p13 downstream deletion they explained 55/208 (26.4%) of the families. PAX6 missense variants counted for 6/208 (2.8%). Mutational spectrum peculiarities in a large cohort of Russian families with congenital aniridia were established. Supported by grant RFBR 19-015-00122 and state task of the Ministry of education and science of Russia.

T.A. Vasilyeva: None. A.V. Marakhonov: None. V.V. Kadyshev: None. R.A. Zinchenko: None.


Revealing the role of enhancers in myopia

J. Swierkowska1,2,3, M. S. Tedja1,2, A. E. G. Haarman1,2, M. Ghanbari1, S. J. Erkeland4, M. Meester-Smoor1,2, C. C. W. Klaver5,6,7, V. J. M. Verhoeven1,2,8

1Department of Epidemiology, Erasmus Medical Center, Rotterdam, Netherlands, 2Department of Ophthalmology, Erasmus Medical Center, Rotterdam, Netherlands, 3Institute of Human Genetics, Polish Academy of Sciences, Poznan, Poland, 4Department of Immunology, Erasmus Medical Center, Rotterdam, Netherlands, 5Epidemiology and Ophthalmology Departments, Erasmus Medical Center, Rotterdam, Netherlands, 6Department of Ophthalmology, Radboud University Medical Center, Nijmegen, Netherlands, 7Institute of Molecular and Clinical Ophthalmology, Basel, Switzerland, 8Department of Clinical Genetics, Erasmus Medical Center, Rotterdam, Netherlands

Introduction: Previous studies have indicated a crucial role of the non-coding genome in myopia (nearsightedness), a complex genetic trait. We aimed to identify enhancers, cis-regulatory elements regulating gene expression, associated with myopia leveraging summary statistics of previous genome-wide association studies (GWAS).

Materials and Methods: GWAS on myopia related phenotypes (148,485 European, 11,935 Asian individuals) from CREAM and 23andMe were used to examine the association between enhancer-related SNPs and myopia. We obtained enhancer regions (Ntotal=12,244) active in eye-related human tissues from the FANTOM5 database and performed a look-up of these regions in the GWAS. We prioritized enhancers and ANNOVAR determined target genes by ranking system based on LD, eQTLs, enhancer histone marks (HaploReg v4.1) and the association with myopia/ocular phenotype (GWAS catalog).

Results: We selected enhancers expressed in ≥3 eye-related tissue types (N=7,944), of which 4,610 enhancers (NSNPs=9,253) were available in the GWAS for analysis. The look-up of these regions yielded 15 significant SNPs clustering in 12 enhancers regions exceeding the Bonferroni significance threshold (P = 0.05/4,610=1.08x10−5). The highest ranked rs9535268 (P = 1.04x10−9) and rs4415919 (P = 3.76x10−8) were annotated to an intergenic enhancer region between RCBTB1 (~16 kb) and ARL11 (~27 kb) genes. Interestingly, SNPs within RCBTB1 were associated with myopia in the GWAS.

Conclusions: We selected enhancers with putative target genes, as RCBTB1 (RCC1 and BTB domain containing protein 1), that could explain significant loci identified in GWAS and play a role in myopia pathogenesis. Support: Horizon 2020 (648268); NWO (91815655, 91617076); Uitzicht (2017-28; Oogfonds; Landelijke Stichting voor Blinden en Slechtzienden; MaculaFonds), EMBO (8529).

J. Swierkowska: None. M.S. Tedja: None. A.E.G. Haarman: None. M. Ghanbari: None. S.J. Erkeland: None. M. Meester-Smoor: None. C.C.W. Klaver: None. V.J.M. Verhoeven: None.


Functional analysis of a novel Myo6 variant in hereditary hearing loss family from Qatar

M. K. Al-Kowari1,2, S. Da’as1, M. Guarch1, D. Abdurahman1, W. Hasan1, N. Krishnamoorthy1, H. Horn2, X. Estivill1,2

1Sidra Medicine, Doha, Qatar, 2Biological and Biomedical Program, Hamad Bin Khalifa University, Doha, Qatar

Introduction: Hereditary Hearing Loss (HHL) is a common genetic disorder accounting for at least 60% of pre-lingual deafness in children. The long tradition of consanguinity among Qatari population increases the prevalence of HHL. A novel missense variant in Myo6 was detected in two siblinges diagnosed with severe to profound HL (Alkowari et al. 2017). Prediction tools revealed that this variant is pathogenic and conserved.

Materials and methods: We functionally validate the pathogenicity of the novel variant using both cellular and animal models. HeLa cells were transiently transfected with plasmids carrying wildtype and mutated human Myo6. A zebrafish model was generated using morpholino knock down and co-injection with synthetic RNA (Myo6WT and Myo6p.E60Q) to further characterize the phenotype.

Results: HeLa cells transfected with Myo6WT and Myo6p.E60Q, overexpressed the same amount of protein. Cells with Myo6WT showed localization of the protein in both cytoplasm and the plasma membrane. In contrast, Myo6p.E60Q is not expressed in the plasma membrane but it’s restricted to the cytoplasm. Zebrafish model resulted in severe otic defects. Myo6p.E60Q RNA injection resulted in alterations in the saccule that is responsible for hearing in zebrafish ear. Additionally, we found that zebrafish with Myo6p.E60Q displayed shorter, less abundant and disordered hair bundles in otic hair cells when compared to control and Myo6WT injected zebrafish.

Conclusions: In this study we functionally characterized a novel genetic variant associated with hearing loss; in vitro studies demonstrated that c.G178C variant affects Myo6 protein trafficking. A zebrafish model confirmed the pathogenicity and identified significant otic defects.

M.K. Al-Kowari: None. S. Da’as: None. M. Guarch: None. D. Abdurahman: None. W. Hasan: None. N. Krishnamoorthy: None. H. Horn: None. X. Estivill: None.


Characterizing a large Iranian cohort of non-syndromic hearing loss families using molecular inversion probe and exome sequencing

A. Rad1, R. Maroofian2, N. Mazaheri3, J. Oostrik4, S. Loum1, H. Löwenheim1, T. Haaf5, H. Galehdari6, H. Kremer4, B. Vona1

1Department of Otorhinolaryngology, Head and Neck Surgery, Tübingen, Germany, 2Department of Neuromuscular Disorders, UCL Queen Square Institute of Neurology,, London, United Kingdom, 3Department of Genetics, Faculty of Science, Shahid Chamran University of Ahvaz, Ahvaz, Iran, Islamic Republic of, 4Department of Otorhinolaryngology, Radboud University Medical Center,, Nijmegen, Netherlands, 5Institute of Human Genetics, Julius Maximilians University, Würzburg, Germany, 6Department of Genetics, Faculty of Science, Shahid Chamran University of Ahvaz,, Ahvaz, Iran, Islamic Republic of

Introduction: Hearing loss (HL) is the most prevalent sensory deficit. With about 120 genes implicated, non-syndromic hearing loss (NSHL) is extremely genetically heterogeneous. In this study, we investigated extended Iranian families with hereditary NSHL to uncover the genetic landscape and characterize its genetic heterogeneity.

Materials and Methods: Using molecular inversion probes (MIPs), we targeted 89 genes involved in NSHL in 20 patients. Exome sequencing (ES) was performed in 102 GJB2-negative and unresolved MIP-screened NSHL probands. Sanger sequencing, homozygosity mapping, and in vitro functional assays were used to confirm selected variants.

Results: Our analysis resolved 50% (10/20) of the MIP-screened patients and about 76% (78/102) of patients subjected to ES. The overall diagnostic rate is about 78.5% (88/112). Nearly half of the diagnostic yield was attributed to the genes SLC26A4 (13.4%), MYO15A (8.0%), LOXHD1 (7.0%), MYO7A (6.2%), CDH23 (5.3%) and TMC1 (4.5%). Our data also identified CLRN2 as a novel NSHL gene in a family. A novel variant in the SPATA5 gene, known to cause epilepsy, hearing loss, and mental retardation syndrome, was implicated in a family with HL as the sole clinical feature.

Conclusions: Using two approaches, we could resolve roughly 78.5% of the patients undergoing genetic screening for NSHL. Our cohort also uncovered LOXHD1 as a highly prevalent gene in NSHL diagnoses that was not similarly observed in large cohort screenings of Iranian HL patients. Furthermore, we introduce CLRN2 and SPATA5 as novel autosomal recessive NSHL genes. Select unresolved families from this cohort have been followed-up with genome sequencing.

A. Rad: None. R. Maroofian: None. N. Mazaheri: None. J. Oostrik: None. S. Loum: None. H. Löwenheim: None. T. Haaf: None. H. Galehdari: None. H. Kremer: None. B. Vona: None.


Multitopographical effect of mutations in the gene PAX6 for pediatric ophthalmology

V. V. Kadyshev, T. A. Vasilyeva, A. V. Marakhonov, S. I. Kutsev, R. A. Zinchenko

FSBSI Research Centre for Medical Genetics, Moscow, Russian Federation

The expression of the PAX6 gene is realized in the formation of almost all structures of the eye - the cornea, iris, lens, optic nerve and central part of the retina. Accounting this fact, the clinical spectrum expands when there is a single pathogenetic process. Materials and methods. The sample of patients with hereditary ophthalmological pathology was 31 children (0-16 years old). The examination included clinical (ophthalmoscopy, biomicroscopy, gonyoscopy, visometry, tonometry), instrumental (electrophysiological, ultrasound, etc.), genealogical and molecular genetic (NGS, direct Sanger sequencing, MLPA) research methods. Results. Accounting the topographical principle, mutations in the PAX6 gene led to lesions in both the anterior and posterior segments of the eyeball. Structures such as the optic disc and/or macular zone (posterior segment) are affected in 100% of cases. The lesion of the anterior segment of the eye is characterized by the involvement of different structures in the process: 6 patients had corneal changes of different grades (ex.; c. 1184-2_1184delAGGinsGGA PAX6 het; c. 1184-2_1184delAGGinsGGA PAX6 het); in 5 cases - pathology of the lens (NM_000280. 4(PAX6_v001):c. 765G>A het synchronous splicing); 7 patients had different grades of iris pathology. One patient with mutation NM_000280.4(PAX6_v001):c.109dupG had a congenital malformation-colobomatous microphthalmos. Two patients had anterior segment dysgenesis (corneal, iris, anterior chamber angle, lens) (c.142-139T>C PAX6 IVS5a-3T>C het; c.718C>T, p.(Arg240*)). Mutations in the PAX6 gene have a multitopographic effect on the eye, with the development of pathology in the anterior and posterior segments of the eyeball. Supported by RFBR grant 18-015-00090. All authors haven’t conflict of interest.

V.V. Kadyshev: None. T.A. Vasilyeva: None. A.V. Marakhonov: None. S.I. Kutsev: None. R.A. Zinchenko: None.


Nine Tunisian patients with typicalPeters plus syndrome

M. Guirat, S. Guidara, H. Kanoun, I. Boujelbene, N. Gharbi, I. B. Ayed, F. Abdelhedi, H. Kamoun, N. Belguith

Service de génétique médicale, CHU Hedi Chaker, Sfax, Tunisia

Peters plus syndrome is an autosomal recessive rare disorder comprising ocular anterior segment dysgenesis, short stature, hand abnormalities, distinctive facial features. It was related only to mutations in the B3GALTL gene (13q12.3), leading to the inactivation of the B1, 3-glucosyltransferase. In this study, we report 9 patients with typical Peters plus syndrome. Ocular abnormality, facial dysmorphism, and rhizomelic dwarfism were present in all cases. We noted the presence of 2 new anomalies not previously described in Peters Plus syndrome, namely renal malformation and sexual ambiguity. Total sequencing of the B3GALTL gene showed the presence of the splicing mutation (c.597-2A> G) in the B3GALTL gene in 7 patients. This mutation harbours a novel splice site mutation in the B3GALTL gene that modulates the mRNA secondary structure. These data confirm a key role of the genetic study that provides diagnosis confirmation and improves genetic counseling for the families

M. Guirat: None. S. Guidara: None. H. Kanoun: None. I. Boujelbene: None. N. Gharbi: None. I.B. Ayed: None. F. Abdelhedi: None. H. Kamoun: None. N. Belguith: None.


Severe forms of presbycusis are caused by very rare variants in genes underlying early-onset forms of deafness

S. boucher1, F. Wong Jun Tai2, A. Lelli3, S. Delmaghani3, A. Singh-Estivalet3, T. Dupont3, V. Michel3, N. Wolff4, A. Bahloul3, M. Niasme-Grare5, A. Bouaita3, D. Oficjalska-Pham3, S. Chardenoux3, Y. Bouyacoub6, D. Bouccara7, O. Deguine8, B. Fraysse8, L. Collet9, H. Thai-Van10, E. Ionescu9, J. Kemeny11, F. Giraudet12, J. Lavieille13, A. Devèze13, A. Roudevitch-Pujol14, V. Christophe15, C. Renard16, V. Franco-Vidal17, C. Thibult-Apt17, V. Darrouzet17, E. Bizaguet18, A. Coez19, A. Aubois14, G. Lefèvre3, N. Michalski3, P. Avan20, C. Bonnet3, C. Petit21

1INSERM UMRS1120, Institut de l’Audition, Pasteur Institute and University Hospital of Angers, Paris, France, 2INSERM UMRS1120 and Institut de l’Audition, Pasteur Institute, Paris, France, 3INSERM UMRS1120, Institut de l’Audition, Pasteur Institute, Paris, France, 4Institut Pasteur, Unité de RMN des Biomolécules, UMR 3528, CNRS, Paris, France, 5Service de Biochimie et Biologie Moléculaire, Hôpital d’Enfants Armand-Trousseau, AP-HP, Paris, France, 6INSERM UMRS1120, Pasteur Institute, Paris, France, 7Hôpital Beaujon, Hôpitaux Universitaires Paris Nord val-de-Seine, APHP, Paris, France, 8Centre Hospitalier Universitaire de Toulouse, Hôpital Larrey, Service d’Oto-Rhino-Laryngologie et Chirurgie Cervico-Faciale, Toulouse, France, 9Centre Hospitalier Universitaire, Hôpital Lyon-Sud, Audiologie et Explorations Orofaciales, Lyon, France, 10Centre Hospitalier Universitaire, Hôpital Lyon-Sud, Audiologie et Explorations Orofaciales and Institut de l’Audition, Lyon and Paris, France, 11Centre Hospitalier Universitaire de Clermont Ferrand, Hôpital Gabriel Montpied, Service d’Anatomo-Pathologie, Clermont Ferrand, France, 12Centre Hospitalier Universitaire de Clermont Ferrand, Hôpital Gabriel Montpied, Service d’Anatomo-Pathologie, UMR 1107, INSERM and Centre Jean Perrin, Clermont Ferrand, France, 13Centre Hospitalier Universitaire de Marseille, Hôpital Nord, Service d’Oto-Rhino-Laryngologie, Marseille, France, 14Centre Hospitalier National d’Ophtalmologie des Quinze-Vingts, Centre d’Investigation Clinique, Paris, France, 15Centre Hospitalier Universitaire de Lille, Hôpital Roger Salengro, Service d’Otologie et Otoneurologie, Lille, France, 16Laboratoire d’Audiologie Renard, Lille, France, 17Centre Hospitalier Universitaire de Bordeaux, Hôpital Pellegrin, Service d’Oto-Rhino-Laryngologie et Chirurgie Cervico-Faciale, Bordeaux, France, 18Laboratoire de Correction Auditive, Paris, France, 19Laboratoire de Correction Auditive and CEA-Inserm U1000 Neuroimaging and Psychiatry and DRM, DSV, Service Hospitalier Frédéric-Joliot, Paris, France, 20INSERM UMR 1107, Laboratoire de Biophysique Neurosensorielle, Université d’Auvergne, Facultés de Médecine et de Pharmacie, Clermont Ferrand, France, 21INSERM UMRS1120, Institut de l’Audition, Pasteur Institute and Collège de France, Paris, France

Introduction: Presbycusis is a major public health issue world wide with half its variance due to genetic predisposition. The aim of this study was to identify presbycusis causative genes by whole exome sequencing (WES).

Patients and methods: We analysed 105 families, 122 simplex cases and 120 normal-hearing controls. After an in silico bioinformatic analysis to select predicted pathogenic variants, and according to the autosomal dominant mode of inheritance in 95% of the families, we studied very rare heterozygous variants (allele frequency : AF≤0.0001).

Results: We found a significative higher ratio of pathogenic variants in autosomal dominant deafness genes carried by 24.7% of mARHL and 21,8% of sARHL (aside from three individuals carrying GJB2 biallelic variants) than in the controls (6,7%) (p < 0,001). By studing a mouse model Tmc1N321I, we confirmed the pathogenicity of the mutation leading to congenital moderate hearing loss in homozygous mice due to a reduced mechanotransduction current amplitude, and to a progressive hearing loss beginning at 7 months and affecting the high frequencies in heterozygous carriers, validating also the mutation as responsible for a monogenic form of presbycusis.

Conclusions: WES unraveled that a large proportion of severe presbycusis is due to new mutations in autosomal dominant deafness genes arguing for a genetic continuum between early-onset deafness and presbycusis. We demonstrated also the existence of monogenic forms of presbycusis that could be targetted by new personnalized therapies like gene therapy. This study was supported by a grant from “Fondation pour l’Audition” and also by the LabEx Lifesenses (ANR-10-LABX-65).

S. boucher: None. F. Wong Jun Tai: None. A. Lelli: None. S. Delmaghani: None. A. Singh-Estivalet: None. T. Dupont: None. V. Michel: None. N. Wolff: None. A. Bahloul: None. M. Niasme-Grare: None. A. Bouaita: None. D. Oficjalska-Pham: None. S. Chardenoux: None. Y. Bouyacoub: None. D. Bouccara: None. O. Deguine: None. B. Fraysse: None. L. Collet: None. H. Thai-Van: None. E. Ionescu: None. J. Kemeny: None. F. Giraudet: None. J. Lavieille: None. A. Devèze: None. A. Roudevitch-Pujol: None. V. Christophe: None. C. Renard: None. V. Franco-Vidal: None. C. Thibult-Apt: None. V. Darrouzet: None. E. Bizaguet: None. A. Coez: None. A. Aubois: None. G. Lefèvre: None. N. Michalski: None. P. Avan: None. C. Bonnet: None. C. Petit: None.


Genetic analysis of a Dutch discovery cohort suggests an association between primary open-angle glaucoma and the mitochondrial variant m.11915G/A in the MT-ND4 gene

V. Lo Faro1, I. Nolte1, J. ten Brink2, H. Snieder1, N. Jansonius1, A. Bergen2

1UMCG, Groningen, Netherlands, 2AMC, Amsterdam, Netherlands

Introduction: To determine a possible association between mitochondrial variations and primary open-angle glaucoma (POAG) in a Dutch discovery cohort.

Materials and Methods: 140 mtDNA single nucleotide polymorphisms (SNPs) were genotyped using the Illumina Infinium Global Screening Array-24 (GSA) 700K array set. Genetic analysis was performed in a case-control discovery cohort of 734 patients with POAG and 1,417 controls. The variants were classified and analyzed both as single SNPs and as haplogroups. SNP association was performed using logistic regression. The bioinformatics software HaploGrep was used to determine the haplogroup. The Chi-square test and Fisher’s two-sided exact test were used for haplogroups association.

Results: For discovery, a nominal association with POAG was found for the allele A of m.11915G/A (rs2853496, p=0.016), within the MT-ND4 gene. The odds ratio was 0.80 (95% confidence interval 0.67-0.95) per copy of the rare allele and adjusted for gender. No haplogroup was associated with POAG in our cohort.

Conclusions: The present study reinforces the concept that mitochondrial variation contributes to POAG susceptibility. Further genetic and functional studies are required to highlight the pathophysiological mechanisms underlying primary open-angle glaucoma.

V. Lo Faro: None. I. Nolte: None. J. ten Brink: None. H. Snieder: None. N. Jansonius: None. A. Bergen: None.


Whole Exome analysis versus a targeted panel for Polish patients with retinal dystrophies

E. Matczyńska1,2, P. Łyszkiewicz1, A. Wasowska1,2, R. Szymańczak1, K. Stradomska1, E. Suchecka1, M. Jędrzejowska1, S. Teper2, M. Wiącek3, A. Machalińska3, A. Boguszewska-Chachulska1

1Genomed SA, Warsaw, Poland, 2Chair and Clinical Dept. of Ophthalmology, Medical University of Silesia, Katowice, Poland, 3Pomeranian Medical University, Szczecin, Poland

A Whole Exome Sequencing (WES) approach was adopted, as a part of the recently accomplished NeuStemGen project, for identifying genetic causes of inherited retinal dystrophies (IRD) in a cohort of Polish patients. Project outcomes prompted the development of a targeted retinal panel, covering most pathogenic variants occurring in the Polish population.

WES was performed for 105 patients with clinical symptoms of retinal dystrophies. A targeted panel, consisting of 278 IRD genes (including deep intronic regions for ABCA4), was used to sequence 29 IRD samples, including 6 samples not resolved by WES. Bioinformatic analysis was performed using standard BWA-GATK best practice pipeline and several databases, including POLGENOM, for variant frequency filtration. Copy number variants (CNVs) were analysed using the CoNVaDING program.

Over 50 pathogenic/potentially pathogenic variants were identified using the WES analysis, involving genes already known to cause IRD. The diagnostic yield of this approach, however, did not exceed 60%. The pilot sequencing of the targeted retinal panel resulted in a significantly higher coverage (389x vs 78x), 98-99% concordance of identified variants and allowed to develop a CNV analysis pipeline.

Data obtained using these two approaches were compared and support the decision of applying the large targeted panel for routine diagnostics of retinal dystrophies. This approach, focused on the complete set of retinal genes, assuring a high quality coverage and allowing for reliable analysis of CNVs and deep intronic variants, should be most efficient and cost-effective for a large cohort of Polish patients.

Partially supported by STRATEGMED1/234261/2/NCBR/2014.

E. Matczyńska: None. P. Łyszkiewicz: None. A. Wasowska: None. R. Szymańczak: None. K. Stradomska: None. E. Suchecka: None. M. Jędrzejowska: None. S. Teper: None. M. Wiącek: None. A. Machalińska: None. A. Boguszewska-Chachulska: None.


A genetic and clinical study of families with non-syndromic retinopathy consequent upon sequence variants in HGSNAT , the gene causing Sanfilippo C mucopolysaccharidosis

E. R. Schiff1,2, G. Arno1,2,3, K. Pierpoint1, R. Ba-Abbad1,2, S. Nutan4, O. A. Mahroo1,2,5, M. Michaelides1,2, K. Harvey6, A. R. Webster1,2

1Genetics Service, Moorfields Eye Hospital, London, United Kingdom, 2UCL Institute of Ophthalmology, London, United Kingdom, 3North East Thames Regional Genetics Service, Great Ormond Street Institute of Child Health, London, United Kingdom, 4London North Genomic Laboratory Hub, Great Ormond Street NHS Foundation Trust, London, United Kingdom, 5Section of Ophthalmology, King’s College London, London, United Kingdom, 6Enzyme Unit, Chemical Pathology, Paediatric Laboratory Medicine, Great Ormond Street Hospital for Children NHS Foundation Trust, London, United Kingdom

Distinct genotypes in several genes (e.g. USH2A, FLVCR1, CEP290, CLN3, MFSD8) have been shown to be associated with either syndromic or non-syndromic forms of retinitis pigmentosa (RP), a progressive degenerative condition initially affecting rod photoreceptors with subsequent cone phototreceptor loss; RP is the most prevalent form of inherited retinal disease (IRD), which is the commonest cause of blindness in the working-age population of England and Wales. HGSNAT (heparan-α-glucosaminide N-acetyltransferase) sequence variants cause mucopolysaccharidosis type IIIC (MPS-IIIC) or Sanfilippo C syndrome - a severe childhood-onset lysosomal storage disorder. Specific genotypes have, however, been associated with isolated retinal disease in a few reported individuals, thereby further extending the genes that can result in both syndromic and non-syndromic IRD. We have identified likely pathogenic variants in 13 individuals (11 families) with late onset (30s to 60s) and slowly progressing RP. None showed any other symptoms of MPS-IIIC. 7/11 families were compound heterozygotes with the allele c.1843G>A, p.(Ala615Thr). We detected 6 novel sequence variants (p.(Ser296Leu); p.(Phe428Cys); p.(Gly248Ala); p.(Gly418Arg), c.1543-2A>C; c.1708delA), and 5 variants that were previously described in MPS-IIIC patients. In one family, two siblings, genetically identical for the HGSNAT locus, were discordant for retinal disease. A mild but significant reduction of HGSNAT enzyme activity in leukocytes was observed in affected individuals but urinary glucosaminoglycans were not elevated. This case series expands the phenotypic and genotypic spectrum of non-syndromic HGSNAT-associated retinopathy, and in one family suggests the influence of trans-acting modifying factors. Funding: NIHR BRC, Moorfields Eye Hospital and UCL Institute of Ophthalmology.

E.R. Schiff: None. G. Arno: None. K. Pierpoint: None. R. Ba-Abbad: None. S. Nutan: None. O.A. Mahroo: None. M. Michaelides: None. K. Harvey: None. A.R. Webster: None.


First systematic molecular genetic analysis using NGS analysis of 100 Greek patients with retinal dystrophy

S. Kamakari1, S. Koukoula2, V. Kokkinou1, L. Haer-Wigman3, I. Datseris4, M. Tsilimbaris5

1Ophthalmic Genetics Unit, Athens, Greece, 2Ophthalmica Institute of Ophthalmology and Microsurgery, Thessaloniki, Greece, 3Department of Human Genetics, Donders Centre for Neuroscience, Radboud University Nijmegen Medical Centre, Nijmegen, Netherlands, 4Department of Retinal Disorders, OMMA Ophthalmological Institute of Athens, Athens, Greece, 5Department of Ophthalmology, University of Crete School of Medicine, Heraklion, Greece

Purpose: Inherited Retinal Dystrophies (IRDs) are characterized by clinical variability and genetic heterogeneity. The aim of this study was to molecularly diagnose 100 Greek patients with different forms of IRDs.

Materials and Methods: 100 unrelated Greek patients were analyzed by Next Generation Sequencing (NGS), 13 and 87 of them using a 105 retinal and a 287 ophthalmic gene panel, respectively as described (Ellingford JM et al. J Med Genet 2016, Haer-Wigman L et al. Eur J Hum Genet. 2017). Additional analysis methods were used (Sanger, MLPA, array-CGH) in 6 cases.

Results: Potentially pathogenic mutations were detected in 45 retinal dystrophy genes including ABCA4, PRPF31, SPATA7, MERTK, FAM161A, CDHR1, USH2A, CNGB1, PROM1 and RP2 genes. The detection mutation rates were 46.15% (6/13) and 81.6% (71/87) for the 105 and 287 gene panels, respectively. These mutation rates were achieved using complementary methods in 6 cases. Final diagnoses included retinitis pigmentosa, Usher syndrome, cone-rod dystrophy and Leber congenital amaurosis and two rare cases of Knobloch and Oliver-McFarlane syndromes due to mutations in the COL18A1 and PNPLA6 genes, respectively.

Conclusions: This is the first systematic investigation of the molecular identity of 100 Greek patients with various subforms of IRDs by NGS and complementary methods leading to an overall mutation rate of 77%. A plethora of novel mutations was documented further expanding the genetic heterogeneity. The molecular identification established the complete diagnosis of the patients thus contributing to family making decision, prognosis and candidacy to current and future treatments.

S. Kamakari: None. S. Koukoula: None. V. Kokkinou: None. L. Haer-Wigman: None. I. Datseris: None. M. Tsilimbaris: None.


A possible epistatic interaction of two variants in autosomal dominant retinitis pigmentosa

R. A. Zinchenko, T. A. Vasilyeva, V. V. Kadyshev, A. V. Marakhonov

Research Center for Medical Genetics, Moscow, Russian Federation

Retinitis pigmentosa (RP) refers to a heterogeneous group of inherited ocular diseases that result in a progressive retinal degeneration affecting 1 in 3,000 to 5,000 people. To date, more than 80 genetic forms of RP are described. Here we perform genetic analysis of the two-generation family with 5 affected members with the terminal severe stage of RP. Whole exome sequencing (WES) of one of the patients revealed two candidate variants. The first one is pathogenic variant in the exon 16 of SNRNP200 gene (chr2:96958829G>A), leading to the missense substitution (p.Arg681Cys, NM_014014.4) in the heterozygous state. Mutations in the SNRNP200 gene in heterozygous state were described in patients with RP33 (OMIM 610359). The second variant is a novel single nucleotide variant in exon 1 of the PRPH2 gene (chr6:42689991C>T) resulting in the missense substitution (p.Val28Met, NM_000322.4) in a heterozygous state. Mutations in the PRPH2 gene in heterozygous, compound heterozygous and homozygous states were described in patients with Leber congenital amaurosis 18 (OMIM:608133), RP7 (OMIM:608133), retinitis punctata albescens (OMIM:136880), in heterozygous state - in patients with choroidal dystrophy, central areolar 2 (OMIM:613105), macular dystrophy, patterned, 1 (OMIM:169150), macular dystrophy, vitelliform, 3 (OMIM:608161). Segregation analysis revealed SNRNP200 to be co-segregated with the disease while PRPH2 variant was not found in 2 out of 5 patients. Nevertheless, it could make an impact on the clinical presentation by aggravation of clinical picture in patients bearing two variants. Supported by grant RFBR № 18-015-00090 and state task of the Ministry of education and science of Russia.

R.A. Zinchenko: None. T.A. Vasilyeva: None. V.V. Kadyshev: None. A.V. Marakhonov: None.


Loss of function of RIMS2 , a synaptic membrane exocytosis gene, causes a new phenotype characterized by congenital cone-rod synaptic disease associated with neurodevelopmental and pancreatic involvement

S. Mechaussier1, B. Almoallem2, C. Zeitz3, K. Van Schil2, L. Jeddawi4, J. Van Dorpe5, C. Condroyer3, O. Pelle6, M. Polak7, N. Boddaert8, N. Bahi-Buisson9, M. Cavallin10, J. Bacquet11, A. Mouallem-Bézière11, O. Zambrowski11,12, J. Sahel3,13,14, I. Audo3,14,15, J. Kaplan1,11, J. Rozet1, E. De Baere2, I. Perrault1

1Laboratory of Genetics in Ophthalmology (LGO), INSERM UMR1163, Institute of Genetic Diseases, Imagine and Paris University, Paris, France, 2Center for Medical Genetics, Ghent University and Ghent University Hospital, Ghent, Belgium, 3Sorbonne Université, INSERM, CNRS, Institut de la Vision, Paris, France, 4Pediatric Ophthalmology Division, Dhahran Eye Specialist Hospital, Dhahran, Saudi Arabia, 5Department of Pathology, Ghent University and Ghent University Hospital, Ghent, Belgium, 6Cell Sorting Facility, INSERM UMR1163, Institute of Genetic Diseases, Imagine and Paris University, Paris, France, 7Endocrinology, Gynecology and Pediatric Diabetology Department, University Hospital Necker-Enfants Malades, Paris, France, 8Department of Pediatric Radiology, Hôpital Necker-Enfants Malades, Paris University, Paris, France, 9Pediatric Neurology Department, University Hospital Necker-Enfants Malades, Paris, France, 10Laboratory of Embryology and genetics of human malformation, INSERM UMR1163, Institute of Genetic Diseases, Imagine and Paris University, Paris, France, 11Service d’Ophtalmologie, Centre Hospitalier Intercommunal de Créteil (CHIC), Créteil, France, 12Ophthalmology Department, University Hospital Necker-Enfants Malades, Paris, France, 13Department of Ophthalmology, The University of Pittsburgh School of Medicine, Pittsburgh, PA, United States, 14Centre Hospitalier National d’Ophtalmologie des Quinze-Vingts, INSERM-DHOS CIC 1423, Paris, France, 15Institute of Ophthalmology, University College of London, London, United Kingdom

Introduction: Anomalies in pre- and post-synaptic transmission of visual information from photoreceptors to bipolar cells are responsible for stationary congenital night blindness (CSNB). Pre-synaptic disorders, characterized by congenital nystagmus and photophobia, night blindness, are known as cone-rod synaptic disorder (CRSD) can be mistaken for Leber congenital amaurosis (LCA). Full-field electroretinogram is crucial for the differential diagnosis, but it can be difficult to perform in young children. Here, we report (i) the identification of biallelic mutations in the RIMS2 gene encoding a pre-synaptic protein not yet associated with human diseases in 3/4 families initially referred for LCA and (ii) the diagnostic adjustment to CRSD with variable associated alteration including autistic behavior and insulinopathy.

Methods: Exome sequencing combined with targeted RIMS2 testing were performed in 46 prescreened patients with CRSD and 94 with LCA. Biallelic RIMS2-mutated cases underwent ophthalmologic, neurologic and metabolic assessment. RIMS2 immunostaining was performed on human retina, brain and pancreas. Nonsense variants were functionally investigated in mammalian cells.

Results: Biallelic RIMS2 sequence variants were found in four unrelated families. RIMS2, a protein regulating synaptic membrane exocytosis localizes in the human retinal outer plexiform layer, Purkinje cells and pancreatic islets. Apart from CRSD, neurodevelopmental disease was observed in all cases, and abnormal glucose homeostasis in the eldest patient. Nonsense variants lead to truncated RIMS2 and decreased insulin secretion in mammalian cells.

Conclusions: This is the first report of syndromic CRSD with neurodevelopmental or pancreatic involvement due to RIMS2 loss-of-function. The disease presentation is consistent with the localization and function of RIMS2.

S. Mechaussier: None. B. Almoallem: None. C. Zeitz: None. K. Van Schil: None. L. Jeddawi: None. J. Van Dorpe: None. C. Condroyer: None. O. Pelle: None. M. Polak: None. N. Boddaert: None. N. Bahi-Buisson: None. M. Cavallin: None. J. Bacquet: None. A. Mouallem-Bézière: None. O. Zambrowski: None. J. Sahel: None. I. Audo: None. J. Kaplan: None. J. Rozet: None. E. De Baere: None. I. Perrault: None.


Whole exome sequencing in Slovak patients with bilateral sensorineural hearing impairment

Z. Slobodova1,2, L. Varga1,2, S. Borecka2, I. Masindova1, D. Danis1, L. Langova3, M. Skopkova1, M. Profant2, D. Gasperikova1

1Biomedical Research Center, Slovak Academy of Sciences, Bratislava, Slovakia, 2Department of Otorhinolaryngology–Head and Neck Surgery, Faculty of Medicine, Bratislava, Slovakia, 3Pediatric ENT Department of Medical Faculty and National Institute of Child Diseases, Bratislava, Slovakia

Background: The genetic heterogeneity of sensorineural hearing loss (SNHL) is a major hurdle to the discovery of disease-causing variants. Whole exome sequencing (WES) is a method that allows an efficient analysis of all known genes associated with hereditary hearing loss.

Aim: Using WES to identify new genetic causes of hearing impairment in the Caucasian and Roma populations in Slovakia, where standard genetic tests performed so far have not elucidated the etiology of deafness.

Patients and methods: We collected 1,369 individuals from 870 families with bilateral SNHL. The previous analysis revealed that 30% of the families have mutations in the DFNB1 locus. From the DFNB1 negative patients we selected 52 families, 42 of Caucasian and 10 of Roma ethnicity, for WES.

Results: Using WES, we identified the genetic etiology in 22 probands of Caucasian ethnicity and in 1 proband of Roma ethnicity. The detected variants were found in 18 genes (COCH, COL4A5, CREBBP, EDNRB, EYA4, ILDR1, LOXHD1, MYO6, MYO15A, OTOA, OTOG, P2RX2, SIX1, TECTA, TMC1, TMPRSS3, DIAPH1, USH1C) harboring 17 pathogenic variants and 11 likely pathogenic variants responsible for hearing impairment. 16 variants have not yet been described in the literature. In one Roma family the causal variants were found in two genes (PTPRQ and MYO15A) in one individual.

Conclusion: Using WES, we detected the genetic etiology of hearing impairment in 52% probands of Caucasian ethnicity and 10% of Roma probands. WES dramatically increases the detection of pathogenic variants in known genes associated with HL. Supported by: VEGA 1/0214/16, APVV 15-067

Z. Slobodova: None. L. Varga: None. S. Borecka: None. I. Masindova: None. D. Danis: None. L. Langova: None. M. Skopkova: None. M. Profant: None. D. Gasperikova: None.


Genotype-phenotype correlations in a Spanish cohort of 506 families with bi-allelic ABCA4 mutations

M. del Pozo-Valero1, R. Riveiro-Álvarez1, F. Blanco-Kelly1, J. Aguirre-Lamban1, I. Martín-Mérida1, I. Iancu1, S. Swafiri1, I. Lorda-Sanchez1, E. Rodriguez-Pinilla1, M. Trujillo-Tiebas1, B. Jiménez-Rolando2, E. Carreño2, I. Mahillo-Fernandez3, C. Rivolta4, M. Corton1, A. Avila-Fernandez1, B. García-Sandoval2, C. Ayuso1

1Department of Genetics, Instituto de Investigación Sanitaria–Fundación Jiménez Díaz University Hospi, Madrid, Spain, 2Department of Ophthalmology, Instituto de Investigación Sanitaria–Fundación Jiménez Díaz University Hospi, Madrid, Spain, 3Department of Epidemiology, Instituto de Investigación Sanitaria–Fundación Jiménez Díaz University Hospi, Madrid, Spain, 4Institute of Molecular and Clinical Ophthalmology Basel (IOB), Basel, Switzerland, Basel, Switzerland

Introduction: ABCA4 is the most mutated gene in inherited retinal dystrophies. A genotype-phenotype correlation model was described in 1999 with few cases reported. Here, we define genotype-phenotype correlations in the largest cohort study worldwide of patients: 434 with Stargardt disease (STGD1) and 72 with cone-rod dystrophy (CRD), all carrying biallelic ABCA4 mutations.

Methods: Conventional genetic tools and Next Generation Sequencing technologies. Medical history and ophthalmological data were obtained for 372 patients. Genotype-phenotype correlation studies were carried out for the following variables: variant type, age at onset of symptoms (AO), and clinical phenotype (STGD1 or CRD).

Results: Genotype-phenotype correlations showed that patients with biallelic truncating variants represented 41% of CRD and 6.5% of STGD1 patients in our cohort; these cases had a significantly earlier AO than patients carrying two mutations, one of which is a missense variant. CRD patients presented earlier AO than STGD1 patients.

Conclusions: Our study, conducted in the largest ABCA4-associated disease cohort reported to date, supports the genotype-phenotype model established for ABCA4 variants, and broadens the mutational spectrum of the gene. ABCA4 truncating variants were mostly associated with earlier AO and a CRD phenotype. According to our observations, ABCA4 patients presenting with two truncating variants may first present features of STGD1 but eventually develop rod dysfunction, underscoring the importance of an accurate genetic diagnosis. Grants: Instituto de Salud Carlos III (ISCIII), CIBERER (06/07/0036), IIS-FJD Biobank PT13/0010/0012, and FIS (PI16/00425) RAREGenomics-CM (CAM, B2017/BMD-3721), Conchita Rábago Foundation, Autonomous Community of Madrid (CAM, PEJ-2017-AI/BMD7256), Miguel Servet Program (CP12/03256) from ISCIII

M. del Pozo-Valero: None. R. Riveiro-Álvarez: None. F. Blanco-Kelly: None. J. Aguirre-Lamban: None. I. Martín-Mérida: None. I. Iancu: None. S. Swafiri: None. I. Lorda-Sanchez: None. E. Rodriguez-Pinilla: None. M. Trujillo-Tiebas: None. B. Jiménez-Rolando: None. E. Carreño: None. I. Mahillo-Fernandez: None. C. Rivolta: None. M. Corton: None. A. Avila-Fernandez: None. B. García-Sandoval: None. C. Ayuso: None.


Functional characterization of a Xenopus tropicalis knockout and a human cellular model of RCBTB1 -associated inherited retinal disease shows involvement of RCBTB1 in the cellular response to oxidative stress

M. Carron1,2, T. Naert1, G. Ascari2, S. Demuynck1, T. Van Nieuwenhuyzen1, T. Rosseel2, D. Priem3,4, A. Kremer4, H. Van Landeghem1, S. Hoogstoel2, F. Coppieters2, C. Guérin5, K. Vleminckx1, E. De Baere6

1Ghent University, Biomedical Molecular Biology, Ghent, Belgium, 2Ghent University, Center for Medical Genetics and Dept of Biomolecular Medicine, Ghent, Belgium, 3Ghent University, Ghent, Belgium, 4VIB-UGent Center for Inflammation Research, Ghent, Belgium, 5VIB Bioimaging Core, Ghent, Belgium, 6Ghent University and Ghent University Hospital, Center for Medical Genetics and Dept of Biomolecular Medicine, Ghent, Belgium

Introduction: The function of RCBTB1, recently implicated in syndromic and non-syndromic inherited retinal disease (IRD), remains unknown so far. Patients with biallelic missense variants in RCBTB1 display diverse IRD phenotypes such as retinitis pigmentosa. Here, we tested the hypothesis that RCBTB1 is involved in NRF2-regulated protection against reactive oxygen species in the eye, more specifically in the retinal pigment epithelium (RPE).

Materials and Methods: A Xenopus tropicalis rcbtb1−/− knockout (KO) was generated using CRISPR/Cas9 editing. Histology and three-dimensional electron microscopy was performed on retinas of rcbtb1−/− frogs. RNA-seq analysis was performed on RCBTB1-mutated patients’ lymphocytes, treated with H2O2, as well as on embryos from the rcbtb1−/− KOs treated with CdCl2. An RCBTB1 knockdown cell line was generated in ARPE-19 cells and functional assays (flow cytometry, MTT-assay, cell death kinetics) assessed the consequences of RCBTB1 loss-of-function.

Results: Rcbtb1−/− animals showed changes in the RPE, similar to observations in human cases, including loss of apical-basal cell polarity, cuboidal cell morphology, spreading of the pigment granules and vacuolisation. NRF2 downstream targets and several metallothioneins were found to be differentially expressed, both in the KO and cellular models. Functional assays in ARPE-19 cells revealed that RCBTB1 depletion affects cellular responses to external insults of oxidative stress.

Conclusions: We showed that the Xenopus tropicalis rcbtb1−/− KO recapitulates the human IRD phenotype. Both in vivo and in vitro functional data show involvement of RCBTB1 in the cellular response to oxidative stress. This provides insight into the mechanism underlying RCBTB1-associated IRD and uncovers potential therapeutic opportunities.

Funding: FWO/1802220N; FWO/1S52818N; BOF15/GOA/011; BOF20/GOA/023.

M. Carron: None. T. Naert: None. G. Ascari: None. S. Demuynck: None. T. Van Nieuwenhuyzen: None. T. Rosseel: None. D. Priem: None. A. Kremer: None. H. Van Landeghem: None. S. Hoogstoel: None. F. Coppieters: None. C. Guérin: None. K. Vleminckx: None. E. De Baere: None.

P03 Internal Organs & Endocrinology (Lung, Kidney, Liver, Gastrointestinal)


A cost effective experience in diagnosis of autosomal-dominant polycystic disease by custom-target NGS panel in a reference center.

J. NEVADO1,2, R. Mena1, R. Peces3, M. Melgosa4, A. Alonso4, R. Selgas3, L. Espinosa4, P. Barruz1, P. D. Lapunzina1,2

1INGEMM-IdiPaz, Madrid, Spain, 2CIBERER, Madrid, Spain, 3Sº Nefrología Adultos, HULP, Madrid, Spain, 4Sº Nefrología Infantil, HULP, Madrid, Spain

ADPKD is the most common inherited kidney disease that is caused by mutations in the PKD1/PKD2. Most mutations take place in PKD1. Its molecular diagnosis is a challenge by the high allelic heterogeneity and overlap of pseudogenes between 1-32 exons. So far, there are few data on PKD1/PKD2 variants in the Spanish population. The main objective of this study has been to establish and know the mutations in ADPKD in our cohort. Mutational analysis of both PKD1 and PKD2 was performed on 158 unrelated families, by NGS using a custom exon-target panel from inherited nephropathies, Nephroseq®. To confirm SNVs, Long-Range-PCR and Sanger sequencing were performed on PKD1 or PCR, and Sanger sequencing for PKD2. In addition, MLPA was used to rule out CNVs. SNVs were identified in 122 families (77.23%): 96 families in PKD1 (78.7%) and 26 families in PKD2 (22.3%). Globally: 64 frameshift and nonsense; 11 splicing, 36 missense, 8 in frame ins/del, and 4 large-deletions). Overall, 15% of variants were de novo and 50% were not previously described. This study demonstrates that NGS is a cost-effective experimental approach for simultaneous analysis of the PKD1 and PKD2. The analysis of mutations in the ADPKD-Spanish population expands the understanding of the genetic diversity of the different ethnic groups, enriches the databases with the new mutations, and contributes to their genetic counselling. As well as, reproductive (six pregnancies were currently successful) and/or therapeutic decision-making (a significant number of patients with highly pathogenic variants are currently under treatment with Tolvaptan).

J. Nevado: None. R. Mena: None. R. Peces: None. M. Melgosa: None. A. Alonso: None. R. Selgas: None. L. Espinosa: None. P. Barruz: None. P.D. Lapunzina: None.


Clinical and molecular characteristics of young Russian patients with homo- and heterozygous AIRE mutations

E. N. Suspitsin1,2, L. V. Ditkovskaya1, T. V. Gabrusskaya1, N. B. Ulanova1, D. L. Strekalov1, M. N. Guseva1

1St.-Petersburg State Pediatric Medical University, St.-Petersburg, Russian Federation, 2N.N. Petrov Institute of Oncology, St.-Petersburg, Russian Federation

Introduction: Hereditary defects of the AIRE gene are associated with a rare autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy syndrome (APECED). The disorder has been found in many populations worldwide. The clinical manifestations are quite variable even within families, but major features include hypoparathyroidism, Addison disease, and mucocutaneous candidiasis. Although the condition usually follows autosomal-recessive inheritance there is an increasing body of evidence that some cases may be caused by monoallelic gain-of-function (GOF) mutations.

Materials and methods: 10 Russian patients (age 3-18 years) were subjected to the AIRE gene analysis. Of those, 5 had clinical signs of APECED syndrome suspected by immunologist, gastroenterologist or endocrinologist. In these patients Sanger sequencing of AIRE was performed. Another 5 patients underwent targeted sequencing of primary immunodeficiency (PID) genes including AIRE because of suspected PID condition.

Results: 6 patients had biallelic AIRE mutations with the common c.769C>T (p.R257*) allele being detected in 4 individuals. One patient was homozygous for c.173C>T (p.A58V) variant recently described in Russian patients (Orlova et al, 2017). Rare c.32T>C (p.L11P) mutation was reported in Russian population for the first time. All monoallelic mutations (n = 4) were represented by the c.901G>A (p.V301M) allele. This variant altering functional properties of the protein was once reported in a Russian female with late-onset classical APECED (Oftedal et al, 2015).

Conclusions: Our study indicates that some GOF variants of AIRE are common in young Russian patients with manifestations of autoimmune disease.

This work was supported by RFBR grant 17-29-06069

E.N. Suspitsin: None. L.V. Ditkovskaya: None. T.V. Gabrusskaya: None. N.B. Ulanova: None. D.L. Strekalov: None. M.N. Guseva: None.


Genetic and molecular analysis of urinary magnesium concentration in Scottish and Croatian populations

C. B. Joseph1, C. M. Stanton1, C. Drake1, T. S. Boutin1, J. F. Wilson2, O. Polasek3, O. Devuyst4, T. Hurd1, C. Hayward1

1MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, United Kingdom, 2Centre for Global Health Research, Usher Institute for Population Health Sciences and Informatics, University of Edinburgh, Edinburgh, United Kingdom, 3Split Medical School, Split, Croatia, 4Institute of Physiology, University of Zurich, Zurich, Switzerland

Magnesium is the second most abundant bivalent cation in the body and is essential for many cellular processes. Renal magnesium handling plays an important role in maintaining magnesium homeostasis, however the exact biological mechanisms remain unclear. A recent genome-wide association study (GWAS) identified an association between urinary magnesium concentration (uMg) and variants in the ARL15 gene on chromosome 5. ARL15 encodes a GTP-binding protein that regulates the magnesium transporter channel TRPM6, and other proteins involved in magnesium homeostasis in physiologically relevant cell lines. We conducted meta-analyses for urinary magnesium in 11, 617 individuals from Scottish and Croatian populations to determine possible genes involved in these traits. Mass spectrometry analysis was conducted to determine interactors of Arl15 and in-vitro knockout models of Arl15 was established using CRISPR-Cas9. The top SNP associated with uMg in our meta-analyses lies within a transcription factor binding site in an enhancer region of ARL15. Individuals homozygous for the alternate allele of rs35931 has lower urinary magnesium levels compared to individuals with the homozygous reference allele. Arl15 interacts with magnesium transporters within the distal convoluted tubule segment of the kidneys. Furthermore, we find that homozygous deletion of Arl15 is lethal at the organismal level. We hypothesise that the variant found affects the expression of ARL15, which, in turn modulates magnesium transport by regulating magnesium transporters. We are performing further functional studies to elucidate the role ARL15 plays in magnesium homeostasis both in vitro and in vivo.

C.B. Joseph: None. C.M. Stanton: None. C. Drake: None. T.S. Boutin: None. J.F. Wilson: None. O. Polasek: None. O. Devuyst: None. T. Hurd: None. C. Hayward: None.


Blood chimerism in monochorionic dizygotic twins (MCDZT) identified at molecular genetic testing of permanent neonatal diabetes mellitus

B. Lorenz-Depiereux1, U. Ahting2, G. Eckstein1, K. Warncke3, T. Meitinger1,2, S. Andres2

1Helmholtz Zentrum München German Research Center for Environmental Health, Institute of Human Genetics, Neuherberg, Germany, 2Technical University of Munich, Institute of Human Genetics, Munich, Germany, 3Technical University of Munich School of Medicine, Kinderklinik München Schwabing, Department of Pediatrics, Munich, Germany

In most cases dizygotic (DZ) twins have a dichorionic placenta, but in some cases - especially in the context of assisted reproduction - blood chimerism in DZ twins due to placental sharing has been shown, identifying them as monochorionic dizygotic twins (MCDZT). Here we report the first case of blood chimerism in MCDZT detected as an additional finding at molecular genetic testing. Dizygotic twin brothers were born to a female couple after assisted reproduction (IVF, in vitro fertilization). One presented with permanent neonatal diabetes mellitus. Molecular genetic analysis identified a heterozygous pathogenic variant within the insulin gene (INS, dominant inheritance). Interestingly, the donor of the oocytes for IVF had been diagnosed with type 1 diabetes as an infant. Screening for the INS variant in her blood DNA was positive and altered the diagnosis to permanent neonatal diabetes mellitus. Testing of the unaffected brother for the INS variant surprisingly showed that he also carried the INS variant in blood DNA but not in DNA from buccal membrane cells. Additional microsatellite studies confirmed blood chimerism in both twins but ruled out chimerism in buccal membrane cells. Whether our findings should be classified as confined blood chimerism which is relatively common, or as an exceedingly rare reported tissue chimerism has to be elucidated by studies of other tissues.

B. Lorenz-Depiereux: None. U. Ahting: None. G. Eckstein: None. K. Warncke: None. T. Meitinger: None. S. Andres: None.


Ultra-rare variants in the TRPV6 gene cause autosomal dominant chronic pancreatitis

E. Masson1,2, J. M. Chen1, V. Rebours3, M. Le Rhun4, H. Audin5, A. Lachaux6, B. Caumont7, D. Lorenzo3, M. Fron8, K. Billiemaz9, R. Besnard10, L. Buscail11, GREPAN (Genetic REsearch on PANcreatitis) Study Group, C. Férec1

1Univ Brest, Inserm, EFS, UMR 1078, GGB, Brest, France, 2CHRU Brest, Service de Génétique Médicale et de Biologie de la Reproduction, Brest, France, 3Department of Gastroenterology and Pancreatology, Beaujon Hospital, APHP, Clichy, and Paris-Diderot University, Paris, France, 4Service d’Hépato-Gastro-Entérologie, Cancérologie Digestive et Assistance Nutritionnelle, CHU Nantes, Nantes, France, 5Médecine ‘Chauvet’ à Orientation Gastro-Entérologique, CH Gabriel Martin, Saint Paul, France, 6Service de Gastroentérologie, Hépatologie et Nutrition Pédiatriques, HCL, Bron, France, 7Service de Médecine à Orientation Hépato-–Gastro- Entérologique, CH Sud Gironde, Langon, France, 8Service d’Hépato-Gastro-Entérologie, CHD Vendée, La Roche-Sur-Yon, France, 9Service de Pédiatrie, CHU Saint-Etienne, Saint-Etienne, France, 10Service d’Hépato-Gastro-Entérologie et Oncologie Digestive, CHR Orléans, Orléans, France, 11Service de Gastroentérologie et Pancréatologie, CHU Toulouse, Toulouse, France

Until very recently, variants in the so far reported chronic pancreatitis (CP) genes such as PRSS1 have been found to cause/predispose to the disease through increasing trypsin activity or eliciting endoplasmic reticulum stress. In January 2020, an international collaboration reported association of functionally deficient variants in the TRPV6 gene with CP, identifying a novel mechanism of pancreatitis due to Ca2+ dysregulation. This discovery was made essentially by analyzing young patients with idiopathic CP. Whether TRPV6 variants are responsible for autosomal dominantly inherited CP remains unknown. Herein, we analyzed the TRPV6 gene in probands of 235 such families by NGS. All families had at least two affected members and had remained genetically unexplained prior to the current analysis. We identified 11 ultra-rare variants (all were absent in 570 French controls and had an allele frequency of 0-0.000008 in gnomAD) in 11 families. Of the 11 variants, 5 were previously reported to be pathogenic. All the remaining six variants were novel, comprising a clear loss-of-function variant, a single amino acid deletion variant, and four missense variants. All these four missense variants were predicted to be of pathogenic relevance by Polyphen and/or SIFT and had a CADD score comparable to known pathogenic missense variants. In summary, we identified ultra-rare TRPV6 variants in ~5% of the studied families. This establishes TRPV6 as a relatively frequent autosomal dominant CP gene for which genetic testing is warranted. Our results also suggest that haploinsufficiency of the TRPV6 gene is sufficient to cause CP. (Work supported by INSERM).

E. Masson: None. J.M. Chen: None. V. Rebours: None. M. Le Rhun: None. H. Audin: None. A. Lachaux: None. B. Caumont: None. D. Lorenzo: None. M. Fron: None. K. Billiemaz: None. R. Besnard: None. L. Buscail: None. C. Férec: None.


Truncating LBX1 mutation in Congenital Central Hypoventilation Syndrome

M. Elbracht, M. Begemann, I. Kurth

Institute of Human Genetics, Aachen, Germany

Undine syndrome, a congenital central hypoventilation syndrome (CCHS), derives its name from the legend of the water nymph Ondine/Undine who punished her mortal husband for his unfaithfulness with a curse of stopping breathing and dying when he fell asleep. Normally, increasing CO2 blood levels induce the hypercapnic reflex which accelerates breathing via specialized neurons of the pre-Bötzinger complex in the ventral hindbrain. Polyalanine expansions in PHOX2B and rarely other genes have been described to cause genetic forms of CCHS by disturbing the breathing reflex. Recently, a homozygous mutation in the homeodomain transcription factor LBX1 was reported in two brothers with neonatal hypoventilation and complex autonomic symptoms. The mutation affects the C-terminus of the protein and impairs cooperativity with PHOX2B, without disrupting the DNA-binding domain. We here report a female patient with severe hypoventilation who required continuous mechanical ventilation immediately after birth. During her first 11 years of life she was supplied with a tracheostomy and mask ventilation at night because of episodes of apnea. In childhood, she had severe constipation and, developed a paralytic ileus like Hirschsprung disease. Since the age of 11 she used a respiratory mask for sleeping and showed normocapnia. With consequent breathing control starting immediately after birth the now 23 years old woman developed excellently and completes a university education. Trio exome sequencing identified a homozygous frameshift-mutation in LBX1 (hg19: chr10:102987166del, NM_006562.4:c.707del, p.(Val236Alafs*59)). Our patient corroborates the role of C-terminal LBX1 mutations in CCHS and emphasizes the favorable clinical outcome in case of consequent treatment.

M. Elbracht: None. M. Begemann: None. I. Kurth: None.


Deletion and duplication: unusual case of diagnostics for patient with cystic fibrosis

S. Deryabina1,2, O. Lagutina1, N. Nikitina1, E. Nikolaeva1

1Medical Center “Health Care of Mother and Child”, Yekaterinburg, Russian Federation, 2Federal State Autonomous Educational Institution of Higher Education «Ural Federal University named after the first President of Russia B.N. Yeltsin, Yekaterinburg, Russian Federation

Background: Cystic fibrosis (CF) is an autosomal recessive disease caused by a mutation in the CFTR (cystic fibrosis transmembrane conductance regulator) gene. Newborn screening for CF was introduced in the Russian Federation in 2006. The incidence of CF varies among different regions, in Sverdlovsk region it is 1: 9,500. The vast majority of recurrent CFTR variants are point mutations, but several populations have larger deletions and duplications, involving one or more complete exons. Here we want to report a case of CF with simultaneously existing deletion and duplication of different regions of 11 exon CFTR.

Case presentation: The patient (a 1-month-old girl) was born at term to non-consanguineous parents. For newborn’s diagnostics CF we use a three-step IRT/IRT1↑/DNA algorithm. Our patient had elevated levels of immunoreactive trypsinogen both times (328.6 ng/ml and 299.8 ng/ml respectively). Moreover, the girl had pathological sweat chlorides - 110 mmol/L.

Results: The trio genetic sequencing of the CFTR gene showed heterozygous delF508 mutation in exon 11 for the child and her father. For searching another variant we were done Multiplex Ligation-mediated Probe Amplification (MLPA) of the CFTR gene. We have found heterozygous deletion of exon 9 for the girl and her mother and have proved the deletion F508 for father, but this analysis has shown also duplication of exon 11 and 2 copies F508 for proband.

Conclusions: We assume the proband has 3 copies of exon 11: two normal and one with del508F, and it is the defect copy, that is crucial for protein synthesis.

S. Deryabina: None. O. Lagutina: None. N. Nikitina: None. E. Nikolaeva: None.


Genotypic spectrum of cystic fibrosis in Cyprus with emphasis on the endemic L346P and the novel CFTRdup2 cases

A. Matthaiou1, P. Anagnostopoulou1,2, P. Kouis1, V. Neocleous3, T. Adamidi4, P. Ioannou5, P. Fanis3, C. Costi3, A. Georgiou4, L. A. Phylactou3, P. Yiallouros1,5

1Medical School, University of Cyprus, Nicosia, Cyprus, 2Institute of Anatomy, University of Bern, Bern, Switzerland, 3Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus, 4Department of Pulmonology, Nicosia General Hospital, Nicosia, Cyprus, 5Pediatric Pulmonology Unit, Archbishop Makarios III Hospital, Nicosia, Cyprus

Introduction: The aim of this study was to describe the epidemiology of CFTR mutations in the cystic fibrosis population in Cyprus with particular emphasis on the endemic L346P (p.Leu346Pro) cases and a novel case which bears the poorly described CFTRdup2 in homozygosity.

Materials and Methods: The national cystic fibrosis patients’ registry in Cyprus was searched for the demographic, diagnostic, and genotypic data of all cases whose diagnosis was genetically confirmed via the identification of two mutant CFTR alleles. In all cases, genotyping was done by Sanger sequencing for the detection of common mutations and was followed by next-generation sequencing for the identification of uncommon mutations.

Results: From an overall of 47 cases, the most common mutation was p.Phe508del with an allelic frequency of 46.9%, which is compatible with its northwest-to-southeast decreasing frequency gradient in Europe. Most mutations were classified as CFTR protein processing (class II, 50%) or conductance (class IV, 12.3%) defects. Multiple mutations were detected in 3 cases. Seven patients with mild clinical manifestations, mostly presenting with dehydration and/or electrolyte imbalance, carried p.Leu346Pro (7.1%), a class IV mutation detected solely in individuals of Cypriot descent. A novel case of a patient homozygous for the CFTRdup2 mutation was associated with a severe phenotype with hepatic involvement, biliary cirrhosis and eventually hepatic failure leading to death.

Conclusions: The small population of cystic fibrosis patients in Cyprus follows the regional genotypic trends, although several cases bear unique mutational profiles, which contribute to the wide genotypic and phenotypic spectrum of the disease.

A. Matthaiou: None. P. Anagnostopoulou: None. P. Kouis: None. V. Neocleous: None. T. Adamidi: None. P. Ioannou: None. P. Fanis: None. C. Costi: None. A. Georgiou: None. L.A. Phylactou: None. P. Yiallouros: None.


Distinct regulatory elements enhance SLC6A14 expression modifying cystic fibrosis pancreas and lung phenotypes

M. Esmaeili1, N. Panjwani1, G. He1,2, F. Lin1, J. M. Rommens1,3, L. J. Strug1,2

1Program in Genetics and Genome Biology, The Hospital for Sick Children, Toronto, ON, Canada, 2Division of Biostatistics, Dalla Lana School of Public Health, University of Toronto, Toronto, ON, Canada, 3Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada

Introduction: Cystic Fibrosis (CF) individuals with the same CFTR mutations have variable lung and pancreatic disease severity. Genome wide association studies (GWAS) have indicated that SNP clusters in the immediate promoter region and far upstream of SLC6A14 modify meconium ileus (MI) occurrence at birth and progressive lung disease, respectively. SLC6A14 is a sodium- and chloride-dependent neutral and cationic amino acid transporter that is expressed in several tissues including lung and pancreatic duct.

Materials and Methods: We are using public data and in vitro transcriptional reporter studies to understand how the SLC6A14 promoter and upstream SNPs contribute to modification of CF.

Results: Colocalizing SLC6A14 promoter SNPs with expression quantitative trait loci (eQTL) of multiple organs from Genotype-Tissue Expression (GTEx) suggests that the pancreas drives MI susceptibility and indicates that the eQTL effect is driven primarily by males. Reporter assays in the CFPAC-1 pancreas cell line with a 2.4kb segment containing MI-associated SNPs display increased gene expression with the risk haplotypes, and exhibit transrepression with a steroid hormone receptor. The core promoter activity and haplotype effects were also evident in the lung cell lines Calu-3 and HBE. Analyses of ReMap and JASPAR databases pinpoint a series of transcription binding elements in the upstream region for ongoing investigations to understand how these segments crosstalk with the core promoter of SLC6A14 in the lung.

Conclusions: Our studies implicate differences in gene regulation of SLC6A14 in the lung versus the pancreas, with increased expression level being detrimental to CF phenotypes in both organs.

M. Esmaeili: None. N. Panjwani: None. G. He: None. F. Lin: None. J.M. Rommens: None. L.J. Strug: None.


Impact of next-generation sequencing on diagnosis of cystic kidney diseases

L. Obeidová1, T. Seeman2, V. Elisakova1, K. Hirschfeldova1, I. Zednikova1, B. Janosikova1, M. Urbanova1, F. Liska1, O. Seda1, B. Chylikova1, J. Stekrova1

1Institute of Biology and Medical Genetics of the First Faculty of Medicine, Charles University and General University Hospital in Prague, Prague, Czech Republic, Prague, Czech Republic, 2Department of Pediatrics, 2nd Faculty of Medicine, Charles University in Prague and Motol University Hospital, Prague, Czech Republic, Prague, Czech Republic

Introduction: Cystic kidney diseases are a very heterogeneous group of chronic kidney diseases with sometimes ambiguous phenotype. Thus, the clinical diagnosis is often difficult to be made. The aim of our study was to test the ability of next-generation sequencing (NGS) to improve the diagnosis precision in a heterogeneous group of children with clinically suspected cystic kidney diseases.

Materials and Methods: Next-generation sequencing of genes responsible for the formation of cystic kidneys was performed in 32 unrelated patients with various clinically diagnosed cystic kidney diseases without a definitive genetic diagnosis.

Results: The underlying pathogenic variants were detected in 69% of patients (n = 22). The result of NGS correlated with the clinical diagnosis made before the NGS in 53% of patients (n = 17), in the remaining 15 children (47%) the result of NGS revealed another type of cystic kidney disease than clinically suspected or did not find any causal mutation. The most common unexpected findings were variants in nephronophthisis (NPHP) genes in children with clinically suspected autosomal recessive polycystic kidney disease (n = 4). Overall, we found 25 (probably) pathogenic variants in the PKHD1 gene, 8 variants in the TMEM67 gene, 4 variants in the PKD1 gene, 2 variants in the HNF1B gene and 2 variants in BBS1 and NPHP1 genes, respectively.

Conclusions: NGS is a valuable tool in the diagnostics of various forms of cystic kidney diseases. Its results changed the clinically based diagnoses in 15% of the children.

Supported by the ESIF - Operational programme Prague CZ.07.1.02/0.0/0.0/17_049/0000828, GAUK 1015 and PROGRES- Q25/LF1

L. Obeidová: None. T. Seeman: None. V. Elisakova: None. K. Hirschfeldova: None. I. Zednikova: None. B. Janosikova: None. M. Urbanova: None. F. Liska: None. O. Seda: None. B. Chylikova: None. J. Stekrova: None.


Circulating miRNA as a predictive biomarker of type 2 diabetes in prediabetic patients-outcomes of 5-year prospective observational study

I. Sidorkiewicz, M. Niemira, K. Maliszewska, A. Bielska, A. Szałkowska, J. Raczkowska, G. Sokołowska, M. Górska, A. Krętowski

Medical University of Bialystok, Bialystok, Poland

Introduction: Type 2 Diabetes Mellitus (T2DM) remains the most prevalent form of diabetes. The detection of current T2DM diagnostic markers is generally late and occurs when displaying metabolic imbalance thus, novel biomarkers that could be useful as a new tool for predicting the progression from prediabetes to T2DM are needed. The goal of this study was to conduct a baseline comparison of serum-circulating miRNA in prediabetic individuals with distinction between those who later progressed to T2DM and those who did not.

Materials and methods: The study group was divided – patients with newly diagnosed T2DM over 5 year observational period (n = 18) and control group (n = 12) with no known history of dysglycemia. The expression level of 798 miRNAs in serum samples using the NanoString technology was examined.

Results: The study revealed that 18 miRNAs were deregulated in serum samples of patients who developed T2DM after 5 years versus non T2DM patients. Pathway analysis showed that miRNA target genes were mainly significantly enriched in homeostasis, neuronal signaling, protein complex oligomerization and core matrisome. ROC analysis demonstrated that miR-491-5p, miR-1307-3p, miR-298 and miR-1290 can be introduced as a diagnostic tool for the prediction of T2DM (AUC=0.940; 0.894; 0.877; 0.722, respectively).

Conclusions: The results suggest that circulating miRNAs could potentially be used as predictive biomarkers of T2DM in prediabetic patients.

I. Sidorkiewicz: None. M. Niemira: None. K. Maliszewska: None. A. Bielska: None. A. Szałkowska: None. J. Raczkowska: None. G. Sokołowska: None. M. Górska: None. A. Krętowski: None.


Identification of a germline interstitial deletion in14q32.13 in a child and mother with DICER1 syndrome

I. Wieland1, I. Schanze1, A. Gnekow2, M. Kuhlen2, F. Maier3, D. Wahl3, M. Frühwald2, M. Zenker1

1Institute of Human Genetics, University Hospital, Otto-von-Guericke University, Magdeburg, Germany, 2Swabian Children’s Cancer Center, University Children’s Hospital, Augsburg, Germany, 3Praxis für Humangenetik, Augsburg, Germany

Introduction: DICER1 syndrome (MIM 601200) is a rare tumor predisposition syndrome with autosomal dominant inheritance usually manifesting in childhood and adolescence. Tumor development follows Knudson’s two-hit model involving the gene DICER1 located on chromosome 14q32.13. Predisposing germline pathogenic variants (first hit) are mainly small loss-of-function mutations. Tumor-related driver mutations are specific hot-spot missense mutations in trans (second hit) located in the RNAse IIIb domain of the DICER1 protein.

Patients: We report on a female child with cystic nephroma of the right kidney and developmental delay at age 1 year. Facial features included frontal bossing, flat nasal bridge, hypertelorism and small mouth with thin lips. Body measurements were unremarkable. Her mother was reported with Sertoli-Leydig cell tumor (SLCT) and multinodular goiter at age 12 to 16 years. Clinical presentation of mother and child suggested familial inheritance of DICER1 syndrome.

Results: No typical DICER1 loss-of-function germline mutation was detected in the child by standard Sanger sequencing. The child’s tumor tissue of the right kidney revealed DICER1 hot-spot mutation c.5437G>A p.Glu1813Lys. Subsequent molecular karyotyping of DNA from blood of the child using Affymetrix CytoScan HD SNP-Array showed a 2.31MB interstitial contiguous gene deletion in 14q32 (arr[hg19] 14q32.12q32.13(93835222_96155830)x1). The interval harbors 23 OMIM-listed genes including DICER1. The array result was finally confirmed in the blood of child and mother by MLPA.

Conclusions: Patients with DICER1 syndrome and germline 14q32 contiguous gene deletions infrequently have been detected. In the few patients reported to date various deletion intervals were identified suggesting no consistent chromosomal breakpoints.

I. Wieland: None. I. Schanze: None. A. Gnekow: None. M. Kuhlen: None. F. Maier: None. D. Wahl: None. M. Frühwald: None. M. Zenker: None.


Two cases with central precocious puberty caused by paternally inherited novel variants in DLK1gene

V. Karaman1, E. Karakılıç Özturan2, F. Baş2, S. Başaran1, Z. Uyguner1

1Department of Medical Genetics, Istanbul Faculty of Medicine, Istanbul University, Istanbul, Turkey, 2Department of Pediatric Endocrinology, Istanbul Faculty of Medicine, Istanbul University, Istanbul, Turkey

Introduction: Gonadotropin-dependent or central precocious puberty (CPP) is caused by premature activation of the hypothalamic-pituitary-gonadal (HPG) axis. There are several genes playing roles in this pathway and two of them are maternally imprinted; 1) MKRN3 gene, located at 15q11.2, upstream suppressor of HPG axis, 2) DLK1 gene, located at 14q32, affecting kisspeptin signaling pathway hence important role for regulating pubertal timing.

Materials and Methods: Coding regions of the MKRN3 (NM_005664.3) and DLK1 (NM_003836.6) genes were Sanger sequenced in 17 patients (15 female, 2 male). In presence of pathogenic variant, segregation analysis were performed in families.

Results: We identified two novel heterozygous variants in two unrelated cases in DLK1 gene. The first variant was in exon 4 (c.357C>G (p.Tyr119Ter)) and the second was in intron 1 (c.67+78C>T), both were classified under probably pathogenic by in silico analysis. Segregation analysis presented the paternal inheritance of both, complying with the inheritance model of the genes. No pathogenic variant identified in MKRN3 gene in our cases.

Conclusions: Previously, three small and one gross deletions of DLK1 were associated with CPP. Nonsense and splicing variant have first time presented in our study. The alteration identified in intron one needs further investigation on transcript level to show whether it causes splicing defect. Nevertheless, inheritance pattern complies with imprinted gene model of DLK1. These results suggest that investigation of DLK1 gene may contribute to the elucidation of the genetic assosiation of familial CPP. This study was supported by Scientific Research Projects Coordination Unit of Istanbul University (TDK-2018-32682)

V. Karaman: None. E. Karakılıç Özturan: None. F. Baş: None. S. Başaran: None. Z. Uyguner: None.


Elucidating the molecular mechanism underlying laterality defects

D. Antony1,2,3, A. Maver4, M. A. Deardorff5,6, L. Slagter2, K. Wu2, Z. Bakey1,2, E. Y. Gulec7, A. Gezdirici7, H. Brunner2,3, M. Schmidts1,2,3

1Center for Pediatrics and Adolescent Medicine, University Hospital Freiburg, Freiburg, Germany, 2Genome Research Division, Department of Human Genetics, Radboud UMC, Nijmegen, Netherlands, 3Radboud Institute of Molecular Life Sciences, Nijmegen, Netherlands, 4Centre for Mendelian Genomics, Clinical Institute of Medical Genetics, UMC Ljubljana, Ljubljana, Slovenia, 5Department of Pediatrics and Pathology, The Perelman School of Medicine, University of Pennsylvania, Pennsylvania, PA, United States, 6Laboratory Medicine, The Perelman School of Medicine, University of Pennsylvania, Pennsylvania, PA, United States, 7University of Health Sciences, Kanuni Sultan Suleyman training and research Hospital, Department of Medical Genetics, Istanbul, Turkey

Introduction: Laterality defects are rare developmental disorders, occurring isolated or as part of complex syndromes. Underlying cause is frequently dysfunction of motile cilia causing Primary Ciliary Dyskinesia (PCD) and rarely, non-motile cilia defects or non-ciliary causes. In addition to randomization of the left right body axis, PCD is characterized by frequent respiratory infections and infertility.

Methods: We used Whole Exome Sequencing (WES) to delineate the underlying molecular cause in 37 mainly consanguineous families with laterality defects.

Results: We identified causative variants in 32% with majority of mutations detected in genes previously associated with PCD, including 2 small homozygous CNVs. Variants in non PCD genes, PKD1l1 and GJA1 were also detected. We further identified homozygous null mutations in two genes not previously associated with human disease, MNS1 and DNAH9, both in cases with situs inversus but little or no respiratory symptoms. Gene matcher database revealed two additional families with biallelic DNAH9 mutation and immunofluorescence analysis revealed complete absence of DNAH9 from respiratory cilia of one affected while DNAH9 localises to the distal half of cilia in controls. Further, we found direct interaction of DNAH9 with the ODA docking complex protein CCDC114 using Y2H screening also interaction with DNAH5 and DNAI2 using co-Immunoprecipitation.

Conclusions: We could only solve a third of the laterality cases genetically using WES, pointing towards non coding variations, genetic mosaicism or epigenetic effects while we were able to identify two novel human disease causing genes, MNS1 and DNAH9, both causing laterality defects with no or little respiratory disease.ERC: TreatCilia

D. Antony: None. A. Maver: None. M.A. Deardorff: None. L. Slagter: None. K. Wu: None. Z. Bakey: None. E.Y. Gulec: None. A. Gezdirici: None. H. Brunner: None. M. Schmidts: None.


Insights into the proteomics of renal function: a trans-ethnic meta-analysis and Mendelian randomization study

P. R. Matias-Garcia1,2,3, R. Wilson1,2, Q. Guo4, S. B. Zaghlool5, S. Sharma1,2, P. Schlosser6, A. Köttgen6,7, A. Peters2,8,9, D. O. Mook-Kanamori10, J. Graumann11,12, W. König8,13,14, K. Hveem15,16, C. Jonasson15,16, A. Butterworth4, K. Suhre5, C. Gieger1,2,8, A. Teumer17,18, M. Waldenberger1,2,8

1Research Unit of Molecular Epidemiology, Helmholtz Zentrum München, German Research Center for Environmental Health, Neuherberg, Germany, 2Institute of Epidemiology, Helmholtz Zentrum München, German Research Center for Environmental Health, Neuherberg, Germany, 3School of Medicine, Technical University of Munich, Munich, Germany, 4Cardiovascular Epidemiology Unit, Department of Public Health and Primary Care, University of Cambridge, Strangeways Research Laboratory, Wort’s Causeway, Cambridge, United Kingdom, 5Department of Physiology and Biophysics, Weill Cornell Medicine-Qatar, Doha, Qatar, 6Institute of Genetic Epidemiology, Faculty of Medicine and Medical Center-University of Freiburg, Freiburg, Germany, 7Department of Epidemiology, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, United States, 8German Research Center for Cardiovascular Research (DZHK), partner site Munich Heart Alliance, Munich, Germany, 9German Center for Diabetes Research (DZD), München-Neuherberg, Neuherberg, Germany, 10Department of Clinical Epidemiology, Leiden University Medical Centre, Leiden, Netherlands, 11Scientific Service Group Biomolecular Mass Spectrometry, Max Planck Institute for Heart and Lung Research, W.G. Kerckhoff Institute, Bad Nauheim, Germany, 12German Centre for Cardiovascular Research (DZHK), partner site Rhine-Main, Max Planck Institute of Heart and Lung Research, Bad Nauheim, Germany, 13Deutsches Herzzentrum München, Technical University of Munich, Munich, Germany, 14Institute of Epidemiology and Medical Biometry, University of Ulm, Ulm, Germany, 15K.G. Jebsen Center for Genetic Epidemiology, Department of Public Health, Norwegian University of Science and Technology, Trondheim, Norway, 16HUNT Research Center, Department of Public Health, Norwegian University of Science and Technology, Levanger, Norway, 17Institute for Community Medicine, University Medicine Greifswald, Greifswald, Germany, 18German Center for Cardiovascular Research (DZHK), partner site Greifswald, Greifswald, Germany

Introduction: Renal health is reflected in the kidneys’ ability to filter blood, clinically measured as the estimated glomerular filtration rate (eGFR). A number of plasma proteins have been found to be associated with renal phenotypes, but prior studies have been limited by smaller sample sizes and fewer proteins. Moreover, the causal mechanisms underlying these associations have not been investigated.

Methods: We conducted the largest observational study of plasma proteins and renal function in terms of sample size (up to N=5,285 across four studies: KORA, INTERVAL, HUNT, QMDiab) and number of proteins (k=1,100). Associations with eGFR and chronic kidney disease (CKD) were tested using regression models in a trans-ethnic discovery-replication approach. We further examined the causal nature of these associations using two-sample bidirectional Mendelian randomization.

Results: 57 proteins were found to be consistently associated with eGFR in population samples of European and admixed ancestry; of these, 18 were associated with CKD. Many well-known biomarkers of renal function (e.g. cystatin-C) were identified, as were JAM-B and contactin-4 as novel biomarkers. The strongest evidence of causality was observed in the positive effect of eGFR on testican-2 (p=2.84E-04), a finding consistent with the biological role of testican-2 in glomeruli remodeling and kidney injury repair.

Conclusions: Our study uncovers novel plasma proteomic biomarkers for renal function and presents evidence for selected causal relationships. Plasma levels of testican-2 are reflective of mechanisms underlying progressive renal function loss, thus suggesting its applicability as a biomarker of early renal dysfunction and disease progression in the general population.

P.R. Matias-Garcia: None. R. Wilson: None. Q. Guo: None. S.B. Zaghlool: None. S. Sharma: None. P. Schlosser: None. A. Köttgen: None. A. Peters: None. D.O. Mook-Kanamori: None. J. Graumann: None. W. König: None. K. Hveem: None. C. Jonasson: None. A. Butterworth: None. K. Suhre: None. C. Gieger: None. A. Teumer: None. M. Waldenberger: None.


Gain-of-function variant of the calcium-sensing receptor gene is associated with longer survival on renal replacement therapy in Caucasian patients

H. M. Rothe1,2, S. Komolka2, C. Wanner3, G. Mayer4

1Centre for Nephrology and Metabolic Disorders, Weisswasser, Germany, 2Coburg University for Applied Sciences, Coburg, Germany, 3University of Würzburg, Division of Nephrology, University Hospital, Würzburg, Germany, 4Medical University Innsbruck, Department of Internal Medicine IV, Nephrology and Hypertension, Innsbruck, Austria

Introduction: Patients with end-stage kidney disease require renal replacement therapy, i.e. dialysis or kidney transplantation, for survival. Here we report for the first time a genetic variant which is associated with longer survival on renal replacement therapy in Caucasian patients.

Materials and Methods: We conducted a survival analysis of three European end-stage kidney disease patient populations in Austria and Bavaria. 198 patients (118 male, 80 female) were genotyped for three variants of the calcium-sensing receptor gene after signing the consent form. The study started in 2005 and survival analysis was performed in 2018. During these years all patients received care according to the standards of the Austrian and German societies of nephrology.

Results: Survival on renal replacement therapy was significantly different (age and sex adjusted Cox regression p=0.04) between carriers of the glycine and arginine alleles at position 990 of the calcium-sensing receptor molecule (rs1042636.) The two rs1042636 cohorts AA (two arginine alleles) and GA/GG (at least one glycine allele) were in Hardy Weinberg equilibrium with a minor allele frequency of 7%.

Conclusions: The glycine variant of rs1042636 calcium-sensing receptor molecules has been shown to result in a gain-of-function, as compared with the arginine variant, in cell culture studies with transfected human embryonic kidney cells. It was also associated with lower baseline parathyroid hormone levels in the Caucasian patient cohort of the Evaluation of Cinacalcet Hydrochloride Therapy to Lower Cardiovascular Events (EVOLVE) trial. Our study shows that it is associated with longer survival on renal replacement therapy in Caucasian patients.

H.M. Rothe: None. S. Komolka: None. C. Wanner: None. G. Mayer: None.


Genes regulating enteric nervous system development are impacted by Copy Number loss and modify penetrance in epistasis with RET

L. Kuil1, C. S. Tang2, J. D. Windster1,3, T. Le4,5, A. Karim2,6, B. M. de Graaf1, R. van der Helm1, Y. van Bever1, C. E. J. Sloots3, C. Meeussen3, D. Tibboel3, A. de Klein1, R. M. H. Wijnen1, J. Amiel4,7,5, S. Lyonnet4,7,5, M. Garcia-Barcelo2, P. K. H. Tam2, M. M. Alves1, A. S. Brooks1, R. M. W. Hofstra1,8, E. Brosens1

1Clinical Genetics department, Erasmus Medical Centre – Sophia Children’s Hospital, Rotterdam, Netherlands, 2Department of Surgery, Li Ka Shing Faculty of Medicine, University of Hong Kong, Hong Kong, China, 3Paediatric Surgery department, Erasmus Medical Centre – Sophia Children’s Hospital, Rotterdam, Netherlands, 4Paris Descartes - Sorbonne Paris Cité University, Imagine Institute, Paris, France, 5Laboratory of embryology and genetics of congenital malformations, INSERM UMR1163, Paris, France, 6Chittagong Research Institute for Children Surgery (CRICS), Chittagong, Bangladesh, 7Service de Génétique, Necker Enfants Malades University Hospital, APHP, Paris, France, 8Stem Cells and Regenerative Medicine, UCL Great Ormond Street Institute of Child Health, London, United Kingdom

Hirschsprung disease (HSCR) is characterized by absence of enteric ganglia, primarily in the distal colon. Approximately 18% of patients have additional anatomical malformations or associated neurodevelopmental disorders (HSCR-complex). We hypothesized that genes affected by rare Copy Number Variants contribute to disease development in “HSCR-complex” patients. Indeed, we could confirm that Copy Number (CN) losses (n = 11) are enriched in these patients (n = 23), if no other causal variant was identified (p=3.64E−7). At least five patients had a large de novo CNV (3 losses, 2 gains). The identified rare CN losses are enriched for variant intolerant genes, overexpressed in the developing mouse enteric nervous system (p=1.760E-10): SLC8A1, GNL1, GABBR1, MAPK8, UFD1L, AKT3, TBX2, USP32 and TUBB. Additionally, a loss of function variant in TUBB was identified in a HSCR patient without a deleterious CNV, and AKT3 as well as UFD1L are impacted by CN losses in other patients. The contribution of the known HSCR predisposing risk haplotypes was comparable between patients with a pathogenic coding variant and controls (P=0.826). All other HSCR subgroups differed significantly with controls and between each subgroup (P=0.02 or lower), patients with isolated HSCR and without a RET coding variant had the highest burden, unaffected controls the lowest. Our zebrafish model reveals that disruption of six of these 9 genes - alone or via epistasis with ret- results in a HSCR phenotype. This confirms the epistatic effect of losing a copy of a gene regulating enteric nervous system development with the predisposed downregulation of RET expression.

L. Kuil: None. C.S. Tang: None. J.D. Windster: None. T. Le: None. A. Karim: None. B.M. de Graaf: None. R. van der Helm: None. Y. van Bever: None. C.E.J. Sloots: None. C. Meeussen: None. D. Tibboel: None. A. de Klein: None. R.M.H. Wijnen: None. J. Amiel: None. S. Lyonnet: None. M. Garcia-Barcelo: None. P.K.H. Tam: None. M.M. Alves: None. A.S. Brooks: None. R.M.W. Hofstra: None. E. Brosens: None.


Identification of disease-causing variants in children and adolescents with focal segmental glomerulosclerosis

S. Draut1, M. C. Braunisch1,2, K. M. Riedhammer1,2, P. M. Herr1, R. Günthner1,2, M. Wagner1,3,4, B. Alhaddad1, L. Renders2, U. Heemann2, C. Schmaderer2, J. Hoefele1

1Institute of Human Genetics, Klinikum rechts der Isar, Technical University of Munich, School of Medicine, Munich, Germany, 2Department of Nephrology, Klinikum rechts der Isar, Technical University of Munich, School of Medicine, Munich, Germany, 3Institute of Human Genetics, Helmholtz Zentrum München, Neuherberg, Germany, 4Institute of Neurogenomics, Helmholtz Zentrum München, Neuherberg, Germany

Introduction: A monogenic cause can be identified in approximately 30% of young patients (< 25 years of age) with steroid-resistant nephrotic syndrome (SRNS). SRNS in children and adolescents is mostly inherited in an autosomal recessive pattern. The histological correlate of SRNS is often focal segmental glomerulosclerosis (FSGS). The aim of this study was to identify genetic causes in a cohort of children and adolescents with FSGS.

Material and Methods: 35 unrelated individuals with disease onset <18 years of age and suspected hereditary FSGS were analyzed using exome sequencing. Suspected hereditary FSGS was defined if at least one of the following criteria was present: absence of a secondary cause, <18 years of age, kidney biopsy with suspicion of a hereditary cause, extrarenal manifestations, and/or positive familial history/consanguinity. Variants were rated according to the ACMG classification. The clinical phenotype was assessed using a standardized questionnaire.

Results: In seven patients, (likely) pathogenic variants according to ACMG were identified in known disease-causing genes (diagnostic yield: 20%). Three variants in two different FSGS genes (INF2, TRPC6) had to be classified as VUS (Table 1).

Conclusions: The diagnostic yield of 20% highlights the importance of genetic testing in patients <18 years of age with nephrotic syndrome. Early genetic diagnosis could allow a personalized treatment approach with weaning of unnecessary immunosuppressive treatment, avoidance of renal biopsies and provision of well-informed genetic counselling.

Table 1. Results of exome sequencing

Study-IDGen (transcript)Nucleotide changeAmino acid changeSegregationZygositySolved/VUS
c.598G>Tp.(Gly170Cys)de novoheterozygoussolved
c.515_517delp.(Thr172del)p, mhomozygoussolved

m, maternal; n.d., not determined; p, paternal; VUS, variant of unknown significance

S. Draut: None. M.C. Braunisch: None. K.M. Riedhammer: None. P.M. Herr: None. R. Günthner: None. M. Wagner: None. B. Alhaddad: None. L. Renders: None. U. Heemann: None. C. Schmaderer: None. J. Hoefele: None.


Genetic linkage analysis identifies a genomic region in 2q24.3 as a candidate modulator of reduced penetrance in heritable pulmonary arterial hypertension.

I. Madrigal1,2, I. Blanco3,4, J. Barberà3,4, C. Badenas1,2, D. Geiger5, P. Puigdevall6, L. Piccari3, R. Castelo6,7

1Biochemistry and Molecular Genetics, IDIBAPS, Hospital Clinic, Barcelona, Spain, 2Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Barcelona, Spain, 3Hospital Clínic de Barcelona, Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain, 4Centro de Investigación Biomédica en Red de Enfermedades Respiratorias (CIBERES), Madrid, Spain, 5Faculty of Computer Science, Technion Israel Institute of Technology, Haifa, Israel, 6Department of Experimental and Health Sciences, Universitat Pompeu Fabra, Barcelona, Spain, 7nstitut Hospital del Mar d’Investigacions Mèdiques (IMIM), Barcelona, Spain

Hereditary pulmonary arterial hypertension (hPAH) is an autosomal dominant disease caused, in most cases, by mutations in the BMPR2 gene. However, not all BMPR2 mutation carriers develop the disease, highlighting the presence of reduced penetrance. In order to identify genomic regions that could be contributing to incomplete penetrance, high-throughput genotyping arrays and next generation sequencing were conducted in an hPAH family. The family comprises 65 Spanish individuals spanning five different generations. One BMPR2 mutation (c.1472G> A; p.Arg491Gln) was identified in 22 members, but only 9 of them developed the disease. The SNP arrays analysis was performed in 33 family members and we identified a genomic region in 2q24.3, located at 38 Mb from the BMPR2 gene, which could contribute to increase the susceptibility of pulmonary hypertension in mutation carriers (Puigdevall et al., J Med Genet, 2019). Several genetic association studies have identified variants in this region associated with phenotypes involved in cardiovascular pathology, such as hypertension or ischemic cardiomyopathy, which could be related to some of the symptoms of pulmonary hypertension. In order to identify the specific variant/s that may act as genetic modifiers of hPAH within the candidate region, we have performed whole-genome sequencing in 16 BMPR2 mutation carriers: 6 affected individuals and 7 asymptomatic carriers. The identification of a possible genetic modifier in this family will allow us to advance in our understanding of reduced penetrance in hPAH.

Acknowledgments: ISCIIII (PI15/00483), ‘fondos FEDER’, Agència de Gestió d’Ajuts Universitaris i de Recerca (2017SGR1134), “CERCA Programme / Generalitat de Catalunya”

I. Madrigal: None. I. Blanco: None. J. Barberà: None. C. Badenas: None. D. Geiger: None. P. Puigdevall: None. L. Piccari: None. R. Castelo: None.


HNF1B-related syndrome: genotype-phenotype correlation

C. P. Cristalli1,2, A. Mattiaccio2, C. Graziano3, I. Capelli1, O. Baraldi1, G. Comai1, M. Pariali2,3, L. Gargano2,3, M. Seri3, G. La Manna1, V. Mantovani2,3

1Nephrology Dialysis and Transplantation Unit, DIMES, Bologna University, Bologna, Italy, 2Center for Applied Biomedical Reasearch (CRBA), Bologna University, Bologna, Italy, 3Medical Genetics Unit, S.Orsola-Malpighi University Hospital, Bologna, Italy

Molecular defects of HNF1B are associated with RCAD syndrome, a clinically heterogeneous disorder characterized by diabetes (MODY5), kidney abnormalities (cysts), cholestasis and uro-genital malformations. We performed targeted NGS of 15 genes and MLPA on 118 patients recruited by nephrology or diabetology units. 19 index cases showed molecular defects in HNF1B gene (10 pediatric and 9 adult). Six patients carried a likely pathogenic novel variant (p.G83D p.R295C, c.-67C>T, c.1207-1G>A, p.S379*, p.Q484Sfs*22) and 4 a VUS (p.G76C, p.E105K, p.A453T, p.M532V). A functional analysis for c.-67C>T indicated reduced activity in Caco2 and Mcf7 cells. Nine cases showed the whole HNF1B gene deletion (de novo in five patients). The median age of diagnosis was 19 (SNVs) and 6 (deletion) years old, respectively. Clinical presentation included earlier onset in deleted patients (median 6 vs 19 years) and different spatial cysts localization (cortical for SNVs and medullary in at least 3 deleted cases). Diabetes was present in six patients only, possibly due to the young age of many cases involved. Two patients with urogenital malformation and 3 with cholestasis were identified. A 21-year-old woman had mild developmental delay: additional array-CGH was performed and a large (1.3 Mb) deletion was found. Contrary, a 29-years-old man homozygous for p.G83D did not show intellectual disability. In conclusion, the patients with SNVs developed a milder phenotype than those deleted, and the molecular defects were frequently of de novo origin. We confirm the wide phenotypic heterogeneity of HNF1B-related disorders and we point out that liver disease can be a major complication.

C.P. Cristalli: None. A. Mattiaccio: None. C. Graziano: None. I. Capelli: None. O. Baraldi: None. G. Comai: None. M. Pariali: None. L. Gargano: None. M. Seri: None. G. La Manna: None. V. Mantovani: None.


Impact of common genetic risk variants on familial aggregation of inflammatory bowel disease

H. Lee1,2, L. Hannes1, M. Vancamelbeke1, V. Ballet3, M. Ferrante3,4, S. Vermeire3,4, I. Cleynen1

1KU Leuven, Leuven, Belgium, 2University of Ulsan College of Medicine, Seoul, Korea, Republic of, 3University Hospitals Leuven, Leuven, Belgium, 4KU leuven, Leuven, Belgium

Family history is the strongest risk factor for inflammatory bowel disease (IBD). The contribution of common genetic variants to familial aggregation in multiple-affected IBD families, however, remains unclear. We included 54 multiple-affected families (≥ 3 first-degree relatives affected), including 189 affected (IBD), and 133 unaffected first-degree relatives; as well as 1768 sporadic cases and 868 unrelated non-IBD controls. For all individuals, Immunochip genotypes were available. Weighted polygenic risk scores (PRS) were calculated using PRSice-2.0 across different p-value thresholds (pTs), and compared between groups. The association between PRS and familial IBD was further evaluated in age- and sex-adjusted generalized mixed regression models including family id as random effect. Using pT=0.05, the affected relatives had a higher PRS than unaffected relatives (P=1.0x10−02), sporadic cases (P=4.6x10−02), and non-IBD controls (P < 2.2x10−16). The risk of IBD in families increased by 1.23-fold for every incremental standard deviation in PRS. However, the proportion of the explained variance between affected and unaffected family members was smaller than that between sporadic IBD and non-IBD controls (5% versus 17%, respectively). Of note, in familial IBD, variance explained by the PRS dropped dramatically for pT>0.01. A plausible hypothesis is that while familial cases differ from sporadic cases in low-risk (high p-value) variants, familial cases differ from their unaffected relatives mostly in high-risk (low p-value) variants. Higher PRS increases the risk for familial IBD as it does for sporadic IBD. The proportion of the explained variance in families however is limited, because of shared genetic background between family members. BIRD Grant

H. Lee: None. L. Hannes: None. M. Vancamelbeke: None. V. Ballet: None. M. Ferrante: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Modest; Amgen, Biogen, Janssen, Pfizer, Takeda. D. Speakers Bureau/Honoraria (speakers bureau, symposia, and expert witness); Modest; Abbvie, Amgen, Biogen, Boehringer-Ingelheim, Falk, Ferring, Janssen, Lamepro, MSD, Mylan, Pfizer, Takeda. F. Consultant/Advisory Board; Modest; Abbvie, Boehringer-Ingelheim, Janssen, MSD, Pfizer, Sandoz, Takeda. S. Vermeire: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Modest; MSD, Abbvie, Takeda, Janssen, Pfizer. D. Speakers Bureau/Honoraria (speakers bureau, symposia, and expert witness); Modest; AbbVie, MSD, Takeda, Ferring, Hospira, Pfizer, Janssen, and Tillots. F. Consultant/Advisory Board; Modest; AbbVie, MSD, Takeda, Ferring, Genentech/Roche, Shire, Pfizer Inc, Galapagos, Mundipharma, Hospira, Celgene, Second Genome, Progenity, Lilly, Arena, Gilead and Janssen. I. Cleynen: None.


Combined exome/genome sequencing with in-house variant data repository mining confirm USP53 as a causal gene for intrahepatic cholestasis

S. Alawbathani1, M. Rocha1, H. Arshad Cheema2, R. Boustany3, S. Nampoothiri4, A. Alswaid5, N. El Koofy6, V. Karageorgou1, I. Hövel1, S. Khan1, C. Beetz1, A. Rolfs1,7, P. Bauer1, A. M. Bertoli-Avella1

1Centogene AG, Rostock, Germany, 2Children’s Hospital and Institute of Child Health, Lahore, Pakistan, 3AUBMC American University of Beirut Medical Center, Beirut, Lebanon, 4Amrita Institute of Medical Sciences, Kerala, India, 5King Abdulaziz Medical City, Riyadh, Saudi Arabia, 6Abu El Reesh Children’s Hospital, Cairo, Egypt, 7University of Rostock, Rostock, Germany

Cholestatic liver disease is a significant cause of morbidity and mortality in the paediatric population, starting in the early postnatal life. Severity is variable, from benign idiopathic neonatal hepatitis to intrahepatic cholestasis leading to liver failure and transplant. There are several genes described for different genetic diseases that lead to cholestasis. USP53 has been recently suggested as a candidate gene for paediatric intrahepatic cholestasis based on one family with two affected siblings. Interestingly, USP53 interacts with tight junction protein 2 (TJP2) for which pathogenic variants are known to cause progressive familial intrahepatic cholestasis. To date, the USP53 gene has not been linked to an OMIM phenotype. By combining exome/genome sequencing and mining our in-house variant database we identified five patients from unrelated families. All affected individuals display a similar phenotype, mainly including early onset intrahepatic cholestasis. Moreover, one family showed hearing loss as an additional phenotype. All patients were homozygotes for different loss-of-function variants in the USP53 gene. USP53 (ubiquitin specific peptidase 53) is a tight junction-associated protein that is known to involve in the survival of auditory hair cells and hearing. Our results confirm USP53 as a novel gene for autosomal recessive intrahepatic cholestasis. Additionally, we demonstrate that combined exome/genome sequencing and variant database mining is an effective approach to discover and confirm novel genes related to human diseases. This accelerate the process of gene discovery and ultimately shortens the diagnostic odyssey in rare genetic disorders.

S. Alawbathani: A. Employment (full or part-time); Significant; Centogene AG, Rostock, Germany. M. Rocha: A. Employment (full or part-time); Significant; Centogene AG, Rostock, Germany. H. Arshad Cheema: None. R. Boustany: None. S. Nampoothiri: None. A. Alswaid: None. N. El Koofy: None. V. Karageorgou: A. Employment (full or part-time); Significant; Centogene AG, Rostock, Germany. I. Hövel: A. Employment (full or part-time); Significant; Centogene AG, Rostock, Germany. S. Khan: A. Employment (full or part-time); Significant; Centogene AG, Rostock, Germany. C. Beetz: A. Employment (full or part-time); Significant; Centogene AG, Rostock, Germany. A. Rolfs: A. Employment (full or part-time); Significant; Centogene AG, Rostock, Germany. P. Bauer: A. Employment (full or part-time); Significant; Centogene AG, Rostock, Germany. A. M. Bertoli-Avella: A. Employment (full or part-time); Significant; Centogene AG, Rostock, Germany.


A common haplotype of the alternative serotonin transporter gene SLC6A4 promoter P2 impacts gene expression and shows disease association in women with Irritable Bowel Syndrome

S. Mohr1, N. Hattensperger1, C. Hammer2, C. Martinez3, S. Berens4, S. Schmitteckert1, L. A. Houghton5,6, M. Goebel-Stengel7, I. Vulic8, M. D’Amato9,10, T. Zheng9,10, H. Mönnikes11, F. Engel4, A. Gauss12, J. Tesarz4, V. Andresen13, T. Frieling14, J. Keller13, C. Pehl15, G. Clarke16, R. Spiller17, T. G. Dinan16, E. M. Quigley16, C. Beltrán18, E. A. Mayer19, G. Sayuk20, M. Gazouli21, L. Kapur-Pojskic8, M. Bustamante22, K. Rabionet22, X. Estivil22, G. Boeckxstaens23, M. M. Wouters23, M. Simrén24, G. A. Rappold1, M. Vicario25, J. Santos25, R. Schäfert4,26, J. Lorenzo Bermejo27, B. Niesler1

1Department of Human Molecular Genetics, Heidelberg University, Heidelberg, Germany, 2Department of Cancer Immunology, Genentech, South San Francisco, CA, United States, 3Institut de Recerca Biomèdica de Lleida, Lleida, Spain, 4Department of General Internal Medicine and Psychosomatics, University Hospital Heidelberg, Heidelberg, Germany, 5Division of Gastroenterology & Surgical Sciences, Leeds, United Kingdom, 6Mayo Clinic, Jacksonville, FL, United States, 7Helios Klinikum Rottweil, Rottweil, Germany, 8Institute for Genetic Engineering and Biotechnology, Sarajevo, Bosnia and Herzegovina, 9School of BioSciences, Monash University, Melbourne, Australia, 10Karolinska Institute, Solna, Sweden, 11Martin-Luther-Krankenhaus, Berlin, Germany, 12Department of Gastroenterology, University Hospital Heidelberg, Heidelberg, Germany, 13Israelitisches Krankenhaus, Hamburg, Germany, 14Helios Klinik Krefeld, Krefeld, Germany, 15Krankenhaus Vilsbiburg, Vilsbiburg, Germany, 16Department of Psychiatry and Neurobehavioral Science, University College Cork, Cork, Ireland, 17Nottingham Digestive Diseases Centre, University of Nottingham, Nottingham, United Kingdom, 18Laboratory of Immuno-Gastroenterology, Universidad de Chile, Santiago de Chile, Chile, 19Oppenheimer Centre for Neurobiology of Stress, University of California, Los Angeles, CA, United States, 20Washington University School of Medicine, St. Louis, MO, United States, 21Biology National and Kapodistrian University of Athens, Medical School Athens, Athens, Greece, 22Centre for Genomic Regulation, Barcelona, Spain, 23TARGID, University Hospital Leuven, Leuven, Belgium, 24Institute of Medicine, University of Gothenburg, Gothenburg, Sweden, 25Institut de Recerca Vall d’Hebron, Hospital Vall d’Hebron, Barcelona, Spain, 26Universitätsspital Basel, Basel, Switzerland, 27Institute of Medical Biometry and Informatics, Heidelberg University, Heidelberg, Germany

Background: Irritable bowel syndrome (IBS) is a gut-brain disorder in which imbalances within the central and peripheral serotonergic systems contribute to disease pathophysiology and thereby shape the phenotype. The serotonin transporter gene SLC6A4 has earlier been implicated in IBS.

Material and Methods: We sequenced the alternative promoter P2 that primarily drives SLC6A4 expression in the gut and analyzed single nucleotide polymorphisms (SNP) for association with IBS. We also assessed their functional relevance by gene reporter assays, in silico analysis and comparative expression studies. In addition, we correlated detected SNPs with expression data and IBS symptoms.

Results: Detected SNPs were linked in different haplotypes and the tagging SNP rs2020938 associated with female IBS with constipation (IBS-C) in a discovery sample comprising 287 individuals. Subsequent validation and meta-analysis of rs2020938 in 2175 IBS patients and 6128 controls from eight countries confirmed the association with female IBS-C. Expression data obtained from different intestinal regions showed that the alternative promoter drives SERT expression primarily in the small intestine. Additionally, gene reporter assays pointed to a functional impact of the SNPs within this region. In silico analyses indicated differential expression regulation by transcription factors that have previously been implicated in GI disorders. Further follow up in tissue samples and clinical data from IBS patients and controls revealed that rs2020938 correlates with differential expression in the jejunum and stool consistency.

Conclusions: Our results indicate differential expression regulation driven by a functionally relevant polymorphic promoter in female IBS-C and consolidates the importance of SLC6A4 in IBS pathogenesis.

S. Mohr: None. N. Hattensperger: None. C. Hammer: None. C. Martinez: None. S. Berens: None. S. Schmitteckert: None. L.A. Houghton: None. M. Goebel-Stengel: None. I. Vulic: None. M. D’Amato: None. T. Zheng: None. H. Mönnikes: None. F. Engel: None. A. Gauss: None. J. Tesarz: None. V. Andresen: None. T. Frieling: None. J. Keller: None. C. Pehl: None. G. Clarke: None. R. Spiller: None. T.G. Dinan: None. E.M. Quigley: None. C. Beltrán: None. E.A. Mayer: None. G. Sayuk: None. M. Gazouli: None. L. Kapur-Pojskic: None. M. Bustamante: None. K. Rabionet: None. X. Estivil: None. G. Boeckxstaens: None. M.M. Wouters: None. M. Simrén: None. G.A. Rappold: None. M. Vicario: None. J. Santos: None. R. Schäfert: None. J. Lorenzo Bermejo: None. B. Niesler: None.


Application of NGS sequencing for improved diagnosis in the pediatric nephrology setting

O. Beltcheva1, K. Kamenarova1, K. Mihova1, G. Zlatanova2, F. Shakola1, D. Roussinov2, M. Gaydarova2, V. Mitev1, R. Kaneva1

1Molecular Medicine Center, Dept. of Medical Chemistry and Biochemistry, Medical University-Sofia, Sofia, Bulgaria, 2SBAL Pediatric Diseases, Nephrology and Hemodialysis Clinic, Department of Pediatrics, Medical University - Sofia, Sofia, Bulgaria

Introduction: Renal disease first diagnosed in childhood often progresses towards chronic or end-stage kidney failure in adults. Identifying the molecular cause of the condition in each case may allow a more personalized approach to therapy and consequently increase the likelihood of preserving the renal function of the patients in adulthood.

Materials and Methods: TruSight One Sequencing Panel (Illumina) on MiSeq platform was used for mutation screening in 10 children with hematuria, proteinuria and/or compromised renal function, who did not respond well to standard treatment or presented with unusual course of the disease. Sanger sequencing was applied for confirmation and establishing the origin of the mutations. The pathogenicity of each novel variant was evaluated according to the established ACMG criteria.

Results: The NGS yielded both known and novel mutations in the sample set. In addition to well-known genes, such as WT1, GATA3, SALL1, AGXT and COL4A4/5, the analysis of the data revealed that pathogenic variants in CD96, HNF1? and TREX1 may also contribute to the etiology of renal disease in children.

Conclusions: The application of massive parallel sequencing of a large gene panel allowed us to identify the molecular cause of renal disease in a number of pediatric patients where diagnoses were hindered by atypical presentation. In several of the cases the findings allowed us to expand the clinical spectrum of the phenotypic presentation of the mutations in certain genes.

Grant references: D-73/2019; D01-285/17.12.2019

O. Beltcheva: None. K. Kamenarova: None. K. Mihova: None. G. Zlatanova: None. F. Shakola: None. D. Roussinov: None. M. Gaydarova: None. V. Mitev: None. R. Kaneva: None.


New LUTO-candidate genes identified by systematic analysis of copy number variations in 152 patients

L. M. Schierbaum1,2, J. Fabian2, S. Herms3, S. Sivalingam2,3, S. Schneider2, S. Weber4, W. Rösch5, W. M. Merz6, M. Feldkötter7, H. Reutter8,2, B. Hoppe1, G. Schalk1, M. Zaniew9, A. Hilger1,2

1Division of Pediatric Nephrology, Department of Pediatrics, University Hospital Bonn, Bonn, Germany, 2Institute of Human Genetics, Children’s Hospital, University of Bonn, Bonn, Germany, 3Department of Genomics, Life & Brain Centre, Bonn, Germany, 4Department of Pediatrics, University Hospital Marburg, Marburg, Germany, 5Deparment of Pediatric Urology, St. Hedwig Krankenhaus Barmherzige Brüder, Regensburg, Germany, 6Department of Obstetrics and Prenatal Medicine, University of Bonn, Bonn, Germany, 7Division of Pediatric Nephrology, Children’s Hospital, University of Zürich, Zürich, Switzerland, 8Department of Neonatology and Pediatric Intensive Care, Children’s Hospital, University of Bonn, Bonn, Germany, 9Department of Pediatrics, University of Zielona Góra, Zielona Góra, Poland

Introduction: Lower urinary tract obstruction (LUTO) is caused by anatomical blockage of the bladder outflow tract or by functional impairment of urinary voiding. For anatomical blockages so far only variants in BNC2 have been described as causal. Genomic copy number variations (CNVs) have been previously described as a cause of congenital anomalies of the kidneys and the urinary tract.

Used Methods: Array-based molecular karyotyping was performed in 152 LUTO patients. Raw intensity data were collected for all persons and CNVs were called with CNVPartition, QuantiSNP and PennCNV. Using PLINK version 1.07 and Excel the CNVs, identified in the patients, were compared to CNVs of 4168 controls. Overlapping CNVs between both groups were discarded. Further filtering steps were used.

Results: We identified 30 potential disease causing CNVs: 8 deletions and 22 duplications. Based on expression of the comprised genes in developing mouse urethra we prioritized 6 CNVs. One of those CNVs comprised the gene GPC5 (MIM 602446), located on chromosome 13q31.3. Interestingly, of 8 patients reported in the literature with 13q duplications that comprise GPC5, four showed urogenital malformations. So far, GPC5 was not yet correlated with a specific clinical phenotype.

Conclusions: Systematic analysis of copy number variations in patients with LUTO is suitable for the identification of new candidate genes. We prioritized GPC5 as candidate gene. For further verification of this candidate gene, re-sequencing of LUTO patients to identify further mutation carriers and functional characterization in zebrafish is warranted.

Acknowledgment: A.C.H. is supported by BONFOR grant O-149.0123.

L.M. Schierbaum: None. J. Fabian: None. S. Herms: None. S. Sivalingam: None. S. Schneider: None. S. Weber: None. W. Rösch: None. W.M. Merz: None. M. Feldkötter: None. H. Reutter: None. B. Hoppe: None. G. Schalk: None. M. Zaniew: None. A. Hilger: None.


ZSCAN12 , a candidate gene for congenital lower urinary tract obstruction (LUTO)

L. Waffenschmidt1, S. Schneider1, J. Altmüller2,3, H. Thiele4, S. Weber5, W. Rösch6, W. M. Merz7, M. Feldkötter8, B. Hoppe9, G. Schalk9, M. Zaniew10, H. Reutter1,11, M. F. Schreuder12,13, G. M. A. Beckers14, J. A. E. van Wijk12, A. C. Hilger1,9

1Institute of Human Genetics, Children’s Hospital, University of Bonn, Bonn, Germany, 2Center for Molecular Medicine Cologne (CMMC), University of Cologne, Cologne, Germany, 3Cologne Center for Genomics, University of Cologne, Cologne, Germany, 4University of Leipzig-Heart Center, Department of Internal Medicine/ Cardiology, Leipzig, Germany, 5Department of Pediatrics, University Hospital Marburg, Marburg, Germany, 6Department of Pediatric Urology, St. Hedwig Krankenhaus Barmherzige Brüder, Regensburg, Germany, 7Department of Obstetrics and Prenatal Medicine, University of Bonn, Bonn, Germany, 8Division of Pediatric Nephrology, Children’s Hospital, University of Zürich, Zürich, Switzerland, 9Division of Pediatric Nephrology, Department of Pediatrics, University Hospital Bonn, Bonn, Germany, 10Department of Pediatrics, University of Zielona Góra, Zielona Góra, Poland, 11Department of Neonatology and Pediatric Intensive Care, Children’s Hospital, University of Bonn, Bonn, Germany, 12Department of Pediatric Nephrology, VU University Medical Center, Pediatric Nephro-urology Center, Amsterdam, Netherlands, 13Department of Pediatric Nephrology, Erasmus MC – Sophia Children’s Hospital, University Medical Center, Rotterdam, Netherlands, 14Department of Pediatric Urology, VU University Medical Center, Amsterdam, Netherlands

Congenital lower urinary tract obstruction (LUTO) is usually characterised by urinary bladder outflow obstruction, it can be functional, based on a congenital dysfunction in voiding or on anatomical blockage of the urethra. LUTO has a birth prevalence of about three per 10.000 pregnancies.

Whole-Exome-Sequencing (WES) in eight families revealed two rare variants in the ZSCAN12 gene. The first variant (c.1695-2A>C, minor allel frequency (MAF): 0,00005814) was identified in three affected brothers, the second variant (c.62T>C; p.Ile21Thr, MAF: 0,00002595) was identified in an affected father and his affected son. Re-sequencing of ZSCAN12 in 263 LUTO patients identified three further patients who all carried the same variant c.637T>C, p.Phe213Leu (MAF: 0.0002046). It has not been reported homozygous. Currently no genetic material of the parents is available for testing.

ZSCAN12 is expressed in the developing mouse urethra at embryonic day 15.5 (GUDMAP: 5824; GUDMAP Consortium) and has been found to be differentially methylated in prostate carcinoma.

Based on these preliminary data we suggest ZSCAN12 as a novel LUTO candidate gene. Further functional studies are warranted.

A.C.H. is supported by BONFOR grants O-149.0123.

L. Waffenschmidt: None. S. Schneider: None. J. Altmüller: None. H. Thiele: None. S. Weber: None. W. Rösch: None. W.M. Merz: None. M. Feldkötter: None. B. Hoppe: None. G. Schalk: None. M. Zaniew: None. H. Reutter: None. M.F. Schreuder: None. G.M.A. Beckers: None. J.A.E. van Wijk: None. A.C. Hilger: None.


Biallelic mutations in the LSR gene cause a novel type of infantile intrahepatic cholestasis

T. Uehara1, M. Yamada1, S. Umetsu2, H. Nittono3, H. Suzuki1, T. Fujisawa2, T. Takenouchi4, A. Inui2, K. Kosaki1

1Center for Medical Genetics, Keio University Hospital, Tokyo, Japan, 2Department of Pediatric Hepatology and Gastroenterology, Saiseikai Yokohama-shi Tobu Hospital, Kanagawa, Japan, 3Junshin Clinic BA Institute, Tokyo, Japan, 4Department of Pediatrics, Keio University Hospital, Tokyo, Japan

LSR is expressed in the liver and other organs, and represents a component protein of tricellular tight junctions. The roles of LSR in human health and disease have not been established. Only one patient with intrahepatic cholestasis with a rare variant of LSR has been reported. Herein, we report a patient who fulfilled this criterion establishing LSR deficiency as a novel cause of human liver disease. The proband was a 5-year-old female patient. At 5 months of age, the infant developed severe and uncontrollable itching. The results of blood examination at 9 months of age showed elevated serum AST, ALT and TBA. Her verbal milestones were slightly delayed. A needle biopsy of the liver at 5 years of age showed paucity of interlobular bile duct. At 6 years of age, whole-exome analysis of the samples from the patient and her parents was performed. The de novo non-synonymous variant (c.602C>T) and the maternally derived heterozygous frameshift variant (c.1396dup) were detected in the LSR gene. Immunohistochemical staining using anti-LSR antibodies showed absent expression of LSR at the tricellular tight junctions in the patient. Documentation of biallelic pathogenic variants of the LSR gene and lack of LSR protein expression in the liver in our patient with infantile intrahepatic cholestasis presented in this report establishes LSR deficiency as a novel cause of infantile intrahepatic cholestasis.

T. Uehara: None. M. Yamada: None. S. Umetsu: None. H. Nittono: None. H. Suzuki: None. T. Fujisawa: None. T. Takenouchi: None. A. Inui: None. K. Kosaki: None.


Neo-TAD formation on 7q36.3 causes lung agenesis in three affected fetuses

U. Melo1, J. Piard2, M. Klever3, M. Mensa4, R. Schöpflin3, M. Holtgrewe3, F. Arbez-Gindre2, V. Guigue2, D. Gaillard2, V. Kremer2, R. Ramanah2, C. Cabrol2, D. Beule3, F. Harms3, K. Kutsche3, M. Spielmann3, B. Fischer-Zirnsak3, L. Van Maldergem3, S. Mundlos3

1MPI, Berlin, Germany, 23Centre de Génétique Humaine, Université de Franche-Comté, Besançon, France, 31Max Planck Institute for Molecular Genetics, RG Development & Disease, Berlin, Germany, Berlin, Germany, 41Max Planck Institute for Molecular Genetics, RG Development & Disease, Berlin, Germany, Berlin, Dominican Republic

Lung development is a tightly regulated process under the control of a large number of signaling molecules. The sonic hedgehog (SHH) signaling pathway is one of the main regulators of lung development during embryogenesis and mutations in SHH-related genes can lead to lung malformation. Here we report on three fetuses from one family with severe malformation of the respiratory system. By ultrasound screening and autopsy after termination, a complete bilateral lung agenesis and near-complete agenesis of the tracheae was observed in all fetuses. Using a combination of array-CGH, Illumina whole-genome sequencing and Hi-C we identified a complex rearrangement at 7q36.3, ~580 kb downstream the SHH gene. The rearrangement consisted of a 449 kb triplication, a 232 kb duplication and a 61 kb deletion, involving parts of the SHH and NOM1/MNX1/UBE3C topologically associated domains (TADs). Due to incorporation of a boundary between two TADs in the triplicated CNV, the rearrangements lead to the formation of two neo-TADs each containing the known SHH enhancer MACS1 which is now placed next to MNX1, a transcription factor involved in caudal development. This situation can be expected to result in an ectopic activation of MNX1 in the lung at a time and place where SHH is normally expressed; a misexpression that likely explains the lung aplasia. The rearrangement was present in mosaic state in the clinically unaffected father, explaining the recurrence of the phenotype in his offspring’s. Thus, whole-genome sequencing in combination with Hi-C is a powerful tool to resolve and functionally interpret complex rearrangements.

U. Melo: None. J. Piard: None. M. Klever: None. M. Mensa: None. R. Schöpflin: None. M. Holtgrewe: None. F. Arbez-Gindre: None. V. Guigue: None. D. Gaillard: None. V. Kremer: None. R. Ramanah: None. C. Cabrol: None. D. Beule: None. F. Harms: None. K. Kutsche: None. M. Spielmann: None. B. Fischer-Zirnsak: None. L. Van Maldergem: None. S. Mundlos: None.


Recessive and dominant patterns in Noonan Syndrome associated with LZTR1 variants

A. Cambra1, M. López-Blázquez2, J. Sánchez del Pozo3, J. Cruz-Rojo3, M. Güemes4, A. Carcavilla5, V. Seidel6, C. Medrano2, B. Ezquieta1

1Molecular Diagnostic Laboratory, Department of Laboratory Medicine, Hospital General Universitario Gregorio Marañón, Instituto de Investigación Sanitaria Gregorio Marañón (IiSGM), Madrid, Spain, 2Pediatric Cardiology, Department of Pediatrics, Hospital General Universitario Gregorio Marañón, Madrid, Spain, 3Pediatric Endocrinology, Department of Pediatrics, Hospital Universitario 12 de Octubre, Madrid, Spain, 4Pediatric Endocrinology, Department of Pediatrics, Hospital Universitario Niño Jesús, Madrid, Spain, 5Pediatric Endocrinology, Department of Pediatrics, Hospital Universitario La Paz, Madrid, Spain, 6Clinical Genetics, Department of Pediatrics, Hospital General Universitario Gregorio Marañón, Madrid, Spain

Noonan syndrome (NS) is an autosomal dominant disease caused by germline mutations in genes involved in the Ras/MAPK pathway. Nevertheless, a recessive form related to biallelic variants in LZTR1 gene was described in 2018. Following publications discussed phenotype and inheritance patterns associated with this gene. Here we report seven unrelated patients with clinically suspected NS and LZTR1 variants. Clinical features are provided in table1. Three novel variants and four previously reported variants were identified. Two patients, NS1564 and NS960, presented a recessive form of NS. In the first patient, c.2074T>C;p.(F692L) and c.2070-2A>G were identified and the second one was homozygous for c.2074T>C;p.(F692L), both affect the BTB/POZ domain. In the rest of patients only one affected allele was identified: NS1414 had a stop-gain variant, c.1084C>T;p.(R362*), located on kelch-5 domain; NS895 presented c.742G>A;p.(G248R), which lies in kelch-4 domain; NS883 presented c.851G>A;p.(R284H), located on kelch-5 domain; NS1199 presented a splicing variant, c.791+1G>T; and the patient 99 was heterozygous for c.905C>T;p.(Ala302Glu), located on kelch-5 domain. Overall, these cases provide more evidence on the clinical phenotype of NS associated with LZTR1 variants. These results are in accordance with the model that specifies that the location throughout the gene determines the inheritance form of NS. Our experience indicates that LZTR1 is a not infrequent cause of NS, so this gene should be included in next-sequencing panels for molecular diagnosis of NS.

Face dismorphologyTypicalSuggestiveSuggestiveTypicalSuggestiveSuggestiveTypical
Short/webbed neckYesYesNoNoYesNoNo
Chest wall deformityBroad thoraxNoNoPectus excavatumPectus carinatumNoNo
Short statureYesYesNoNoNoNoYes
CryptorchidismYesYes Yes   
Developmental delayYesNoYesNoNoYesNo
Prenatal findingsYesNoNoNoNoNoNo

A. Cambra: None. M. López-Blázquez: None. J. Sánchez del Pozo: None. J. Cruz-Rojo: None. M. Güemes: None. A. Carcavilla: None. V. Seidel: None. C. Medrano: None. B. Ezquieta: None.


Profiling of microRNA isoforms in ulcerative colitis

D. Nikitina1, S. Juzėnas1,2, V. Šaltenienė1, A. Franke2, G. Kiudelis3, J. Kupčinskas1,3, L. Kupčinskas1,3, J. Skiecevičienė1

1Lithuanian University of Health Sciences, Medical Academy, Institute for Digestive Research, Laboratory of Clinical and Molecular Gastroenterology, Kaunas, Lithuania, 2Kiel University, Institute of Clinical Molecular Biology, Kiel, Germany, 3Lithuanian University of Health Sciences, Medical Academy, Department of Gastroenterology, Kaunas, Lithuania

Introduction: Recent research has shown that so far widely studied reference miRNA sequences in miRNA databases have a large number of isoforms that can be involved in other physiological pathways compared to the reference miRNA. Ulcerative colitis (UC) is a chronic disease of the digestive system of the autoimmune origin and is attributed to the group of inflammatory bowel diseases. The aim of this study was to perform isomiRNA analysis in ulcerative colitis tissue.

Materials and Methods: In total, 76 individuals were included in this study (32 controls, 24 patients with active UC and 20 patients with UC in remission phase). Small RNA sequencing was performed using the TruSeq protocol and the HiSeq2500 platform (Illumina, USA). Bioinformatic microRNA analysis was performed using the following software tools: MiRDeep2, mirAligner, Cutadapt, FASTX, RNAfold, Bowtie, BioPerl, BLASTn and R.

Results: The analysis revealed that the reference microRNA sequences make up 2.1% of all microRNA sequences and 97.9% are microRNA isoforms. Altered microRNAs and their isoforms (padj <0.01, log2FC> | 0.9 |) were detected in three groups: (i) active UC group compared to the HC (healthy control group) - 874; (ii) UC in the remission group compared to the HC - 93; (iii) active UC group compared to UC remission group - 632. The expression of 9 isomiRNA was strongly correlated with the intensity of the inflammation (rho> 0.7).

Conclusions: An altered profile of microRNAs and their isoforms was found in UC colon tissue, which also correlated with inflammation intensity.

D. Nikitina: None. S. Juzėnas: None. V. Šaltenienė: None. A. Franke: None. G. Kiudelis: None. J. Kupčinskas: None. L. Kupčinskas: None. J. Skiecevičienė: None.


Screening for genetic polymorphisms in Bulgarian children with non-alcoholic fatty liver disease

D. Kofinova1, I. Yordanova2, P. Hadzhiyski1, R. Shentova-Eneva1, M. Baycheva1, P. Yaneva1, M. Panayotova3, A. Todorova2, D. Avdjieva-Tzavella1

1University Children’s Hospital "Prof. Ivan Mitev", Sofia, Bulgaria, 2Genetic Medico-Diagnostic Laboratory Genica, Sofia, Bulgaria, 3Trakia Hospital, Stara Zagora, Bulgaria

Introduction: Non-alcoholic fatty liver disease (NAFLD) is a highly prevalent liver disease in children. NAFLD is associated with insulin resistance, obesity, and dyslipidemia. NAFLD is a multifactorial disease. Growing evidence suggest the leading role of certain gene variants, associated with rapid progression of the liver disease and dyslipidemia. The aim of this study was to investigate whether various single-nucleotide polymorphisms play any role in the development of NAFLD in Bulgarian children.

Materials and Methods: We prospectively included 22 patients with NAFLD and 10 healthy controls. Ten of the children with NAFLD underwent liver biopsy. PCR amplification with specific primers pairs for variants in the genes PNPLA3 (c.444C>G, p.I148 M), GCKR (c.1337C>T, p.P446L) and TM6SF2 (c.499C>T, p.E167K) was performed followed by direct sequencing using BigDye Terminator v.3.1 Cycle Sequencing kit.

Results: The GCKR P446L gene polymorphism was found in 75.0% (24/32) of the study participants (homo- or heterozygous carriers). It was significantly higher in homozygous patients with NAFLD than in healthy controls (54.5% vs. 10.0%, p=0.025). PNPLA3 I148M was detected in 9 of the patients and 7 of the controls. TM6SF2 E167K was the rarest genetic variant, only 3 children (2 patients and 1 healthy child) were carriers. Two of the patients, who underwent liver biopsy had significant fibrosis F2. Patient N1 was homozygous for PNPLA3 I148M and heterozygous for GCKR P446L. Patient N2 was GCKR P446L heterozygous carrier.

Conclusions: GCKR P446L polymorphism is associated with development of NAFLD in Bulgarian children. Medical University Sofia, Grant "Young Investigator" Contract № D-142/02.05.2017

D. Kofinova: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Modest; Medical University Sofia, Grant "Young Investigator" Contract № D-142/02.05.2017. I. Yordanova: None. P. Hadzhiyski: None. R. Shentova-Eneva: None. M. Baycheva: None. P. Yaneva: None. M. Panayotova: None. A. Todorova: None. D. Avdjieva-Tzavella: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Modest; Medical University Sofia, Grant "Young Investigator" Contract № D-142/02.05.2017.


Molecular characterization of familial intrahepatic cholestasis by multi-gene panel analysis

A. Mattiaccio1,2, G. Vitale3, C. P. Cristalli1, M. Pariali1,4, L. Gargano1,4, M. Seri4, P. Andreone5,2, V. Mantovani1,4

1Center for Applied Biomedical Research (CRBA), Bologna University, Bologna, Italy, 2DIMEC, Bologna University, Bologna, Italy, 3Internal Medicine for the Treatment of Severe Organ Failure, S. Orsola-Malpighi Hospital, Bologna, Italy, 4Medical Genetics Unit, S.Orsola-Malpighi University Hospital, Bologna, Italy, 5Department of Medical and Surgical Sciences, Maternal, Child and Adult Sciences, University Hospital, Modena, Italy

Introduction: Progressive Familial Intrahepatic Cholestasis (PFIC) is a group of autosomal recessive diseases that affects especially newborns and children, with progression to liver failure in the first decades of life. PFIC is classified into five types (PFIC1-5) based on the genetic defect involved in bile transport. Other benign late-onset phenotypes and non-progressive forms (BRIC, LPAC, DIC and ICP) are caused by heterozygous mutations in the same gene pattern.

Materials and Methods: DNA analysis of 15 candidate genes involved in PFIC and other related benign phenotypes was carried out. Amplicon-based NGS on 80 patients was performed. Bioinformatic and statistic pipelines were applied.

Results: A total of 184 different variants has been identified in our cohort: 18 pathogenic, 46 VUS, 44 likely benign and 76 benign. P/LP mutations were found in 12% of patients: 2 in ATP8B1, 3 in ABCB11, ABCB4 and TJP2 each, one in ABCC2, JAG1, NOTCH2. Many patients had multiple variants in several genes. Patients had from 7 to 35 variants each and some SNPs were significantly associated with biochemical parameters and phenotypic features that could better explain clinics and accelerate the progression to liver failure.

Conclusions: Our detection rate is according to other studies proposing multi-gene panels. Our analysis may be useful for the molecular diagnostics of PFIC and a better characterization and understanding of the linking between molecular defects and different subtypes of the disease. The high SNPs prevalence let us to hypothesize a synergistic haplotype effect in determining different multifactorial cholestasis phenotypes and overlapping features.

A. Mattiaccio: None. G. Vitale: None. C.P. Cristalli: None. M. Pariali: None. L. Gargano: None. M. Seri: None. P. Andreone: None. V. Mantovani: None.


Telomere length (TL) and oxidative stress in C57BL/6J mice

E. Kidd1, J. Pender1, M. J. Gatt1, J. Williams2, A. A. I. F. Blakemore3, E. Meimaridou1, A. J. Walley4, U. L. Fairbrother1

1London Metropolitan University, London, United Kingdom, 2Queen Mary, London, United Kingdom, 3Brunel University, London, United Kingdom, 4St George’s University of London, London, United Kingdom

High levels of oxidative stress may lead to an increased rate of telomere shortening and contribute to loss of telomere integrity. Most in vivo studies have looked for correlations between biomarkers of oxidative stress and TL, and have used leucocytes. We have used a murine model to investigate the potential impact of oxidative stress on TL in a tissue-specific manner and to assess telomere oxidation directly. Our model is a C57BL/6J mouse strain with a naturally occurring nicotinamide nucleotide transhydrogenase (NNT) deficiency. The absence of Nnt results in high levels of reactive oxygen species (ROS) in cells. DNA was extracted from healthy control mice, Nnt-deficient mice and Nnt-rescued mice (Nnt reinserted at the blastocyst stage) and their telomeres analysed. Two qPCR methods were used: mmQPCR to assess relative telomere length (RTL), and a formamidopyrimidine DNA glycosylase (FPG) enzyme-based qPCR method to directly determine the extent of telomere oxidation. Analysis of RTL from kidney revealed no significant difference (p>0.05) between the three groups. This is predictable since rapidly dividing leucocytes probably best represent early life replicative responses and tissues such as kidney, a slowly dividing tissue, represents the heritable load of TL. QPCR analysis of the DNA extracts, before and after FPG digestion showed there was no significant difference in the ΔCT values between the three groups (p>0.05), implying there was no discernible difference in telomere oxidation levels. Further analysis will investigate tissues with variable metabolic and cellular turnover rates such as the brain, spleen, liver, heart and adipose tissue.

E. Kidd: None. J. Pender: None. M.J. Gatt: None. J. Williams: None. A.A.I.F. Blakemore: None. E. Meimaridou: None. A.J. Walley: None. U.L. Fairbrother: None.


Molecular markers of severity of nonalcoholic fatty liver disease

D. Sidorenko, V. Nazarov, S. Lapin, V. Emanuel

Pavlov First Saint Petersburg State Medical University, Saint-Petersburg, Russian Federation

Introduction: Nonalcoholic fatty liver disease (NAFLD) is one of the most common parenchymal liver disorders. There are no reliable markers for assessment of progression of NAFLD from steatosis to steatohepatitis that leads to liver fibrosis and cirrhosis. Polymorphism I148M in PNPLA3 gene is associated with inhibition of lipid hydrolysis. Pathological variants of SERPINA1 gene promote accumulation of mutant alpha-1-antitrypsin in endoplasmic reticulum. Polymorphisms in HFE gene leads to intracellular iron accumulation and fatty degeneration of liver.

Materials and Methods: Group of 59 patients with NAFLD was selected. The assessment of structural changes in liver was carried out by transient elastometry with controlled attenuation parameter. Polymorphisms in PNPLA3, SERPINA1 and HFE genes were determined by real-time PCR.

Results: Association between homozygous carriage of I148M polymorphism and increase of ALT was found (p=0.011). A connection was established between the presence of the polymorphism and more severe steatosis (p=0.045). AST activity was higher in PiZ carriers than in patients without changes in this gene (p=0.049). H63D and C282Y polymorphisms of the HFE gene are associated with higher ALT (p=0.020 and p=0.030). A correlation between changes in mentioned genes and more severe fibrosis was not demonstrated.

Conclusions: Polymorphisms in the PNPLA3, SERPINA1, and HFE genes are associated with an increase in ALT and AST in patients with NAFLD. The homozygous variant of the I148M polymorphism in PNPLA3 gene and the heterozygous C282Y in HFE gene are associated with more severe steatosis. However, conflicting data regarding liver fibrosis rates need further study.

D. Sidorenko: None. V. Nazarov: None. S. Lapin: None. V. Emanuel: None.


Gene expression differences in non-obese, obese and previously obese subjects.

M. Tessarech1, E. Mileti2, K. Kwok2, P. Arner3, D. Bonneau1, E. Colin1, M. Rydén3, C. Daub2

1CHU Angers, Angers, France, 2Karolinska Institutet, Stockholm, Sweden, 3Karolinska University Hospital Huddinge, Stockholm, Sweden

Obesity is a worldwide public health issue associated with many comorbidities. Understanding its underlying causes and mechanisms is one of the keys to prevent and treat this condition. Besides obvious environmental causes, as physical inactivity or poor nutrition, genetics is one of the main component of obesity. Here, we perform a transcriptomic study by 5′Cap-mRNA sequencing of subcutaneous white adipose tissue of non-obese (NO), obese (OB) and previously obese (POB) subjects, at fasting and hyperinsulinemia states. Differential gene expression analysis showed few difference in the analysis of NO compared to POB subjects, while the comparison of OB subjects versus NO and versus POB displayed many transcriptomic changes, with most of the genes in common in both comparison. Moreover, many genes were specifically involved in expression changes between OB and POB subjects. Gene ontology analysis revealed that those genes were involved in metabolic and cellular process pathways and in cardiovascular diseases. Fasting and hyperinsulinemia metabolic states had little influence on the results. This study demonstrates that a major gene expression difference is observed between obese and lean subjects, whether they were never obese or got rid of their fat mass after a weight loss surgery, and that after this kind of surgery, many changes in gene expression appear. Further studies are needed to identify precisely which genes are involved and if they can be a target for personalized medicine in the treatment of obesity by weight loss surgery.

M. Tessarech: None. E. Mileti: None. K. Kwok: None. P. Arner: None. D. Bonneau: None. E. Colin: None. M. Rydén: None. C. Daub: None.


Autosomal Dominant Polycystic Kidney Disease in Israel: Genetic diagnosis reveals recurrent pathogenic variants

T. Paperna1, A. Mory1, A. Ofir1, N. Damouny1, D. Magen2,3, I. Maya4,5, K. Weiss1, H. Baris Feldman6,5

1The Genetics Institute, Rambam Health Care Campus, Haifa, Israel, 2Pediatric Nephrology Institute, Rambam Health Care Campus, Haifa, Israel, 3Faculty of Medicine, Technion, Haifa, Israel, 4The Raphael Recanati Genetic Institute, Rabin Medical Center, Beilinson Hospital, Petah Tikva, Israel, 5Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel, 6The Genetics Institute, Tel Aviv Sourasky Medical Center, Haifa, Israel

Introduction: The genetic diagnosis of Autosomal Dominant Polycystic Kidney disease (ADPKD) is complicated by the genomic structure of the main causative gene PKD1, due to the presence of overlapping pseudogenes. Classification of the missense variants is challenging as allelic heterogeneity is rife, and few recurrent variants are known. Data on ADPKD in Israeli populations is yet unavailable.

Materials and methods: Patient samples (n = 112) From Israel were referred to Rambam Genetics Institute for ADPKD gene panel testing (PKD1, PKD1, HNF1B). Next generation sequencing (NGS) was performed using either Trusight One (Illumina, n = 77), or exome sequencing (ES, n = 35); analysis employed the Genoox platform. NGS was supplemented by Sanger sequencing to achieve full coverage of panel. GANAB, DNAJB11 were included in ES testing.

Results: A clinically significant variant (CSV) was identified in 82% of the patients (PKD1-88%, PKD2-7%, HNF1B-5%). Three PKD1 CSVs were identified only following Sanger sequencing. Truncating CSVs comprised ~55% of pathogenic variants. Most CSVs (72%) were unique; however, eight PKD1 CSVs recurred in two or more patients, notably, in Jews of Persian and Ethiopian descent. HNF1B-related kidney disease was diagnosed in four individuals. Positive family history was linked with higher CSV detection rates, 81%, compared with 60% when negative.

Conclusions: This first report on ADPKD genetics in Israel highlights recurrent CSVs in Israeli sub-ethnicities. HNF1B CSVs can account for an ADPKD-like phenotype. Family history was a strong predictor for detection of a CSV. Our experience underscores the importance of utilizing ES as well as Sanger sequencing for variant detection.

T. Paperna: None. A. Mory: None. A. Ofir: None. N. Damouny: None. D. Magen: None. I. Maya: None. K. Weiss: None. H. Baris Feldman: None.


Genome-wide association study identifies CDH12 as candidate gene for renal injury in patients with posterior urethral valves

L. F. M. van der Zanden1, I. A. L. M. van Rooij1, J. S. L. T. Quaedackers2, R. Nijman2, M. Steffens3, L. L. L. de Wall1, E. M. H. F. Bongers1, F. Schaefer4, M. Kirchner5, R. Behnisch5, A. K. Bayazit6, S. Caliskan7, L. Obrycki8, G. Montini9,10, A. Duzova11, M. Wuttke12, R. Jennings13,14, N. A. Hanley13,14, N. J. Milmoe15, P. J. D. Winyard15, K. Y. Renkema16, M. F. Schreuder1, N. Roeleveld1, W. F. J. Feitz1

1Radboud university medical center, Nijmegen, Netherlands, 2University Medical Center Groningen, Groningen, Netherlands, 3Isala, Zwolle, Netherlands, 4University Hospital Heidelberg, Heidelberg, Germany, 5University of Heidelberg, Heidelberg, Germany, 6Cukurova University, Adana, Turkey, 7Istanbul University-Cerrahpasa, Istanbul, Turkey, 8Children`s Memorial Health Institute, Warsaw, Poland, 9Fondazione IRCCS Ca’ Granda – Ospedale Maggiore Policlinico di Milano, Milan, Italy, 10University of Milan, Milan, Italy, 11Hacettepe University Faculty of Medicine, Ankara, Turkey, 12University of Freiburg, Freiburg, Germany, 13University of Manchester, Manchester, United Kingdom, 14Manchester University NHS Foundation Trust, Manchester, United Kingdom, 15UCL Great Ormond Street Institute of Child Health, London, United Kingdom, 16University Medical Center Utrecht, Utrecht, Netherlands

Introduction: Posterior urethral valves (PUV) and ureteropelvic junction obstruction (UPJO) are congenital obstructive uropathies that may impair renal development. We performed the first GWAS for renal injury in obstructive uropathy patients.

Methods: We included patients with pyeloplasty or PUV resection before age 18. Signs of renal injury were defined as dialysis, nephrectomy, kidney transplantation, estimated glomerular filtration rate<60 ml/min/1.73m2, high blood pressure, antihypertensive medication use, proteinuria, and/or one kidney functioning<45%. In the GWAS, we compared 141 and 216 obstructive uropathy patients with and without signs of renal injury 5 years post-surgery, respectively. For the associated SNPs, we performed stratified analyses for PUV and UPJO, and Kaplan-Meier and Cox regression analyses in the discovery and two replication samples of 102 PUV patients each. For the associated gene, we performed RNA and protein expression analyses in fetal tissues.

Results: We observed suggestive associations with six SNPs in three loci, of which rs6874819 in CDH12 was the strongest (P = 7.5×10−7). This SNP also seemed to be associated with time to renal injury in the PUV discovery and replication samples. RNA expression analyses showed clear expression in fetal kidneys, which was confirmed by protein immunolocalization.

Conclusions: This study identified CDH12 as candidate gene for renal injury in PUV. CDH12 is a target of the proinflammatory MCP-1-MCPIP pathway. We hypothesize that in PUV, MCP1 pathway upregulation due to increased urinary tract pressure is augmented in carriers of the CDH12 variant, exacerbating renal injury.

Grant support: Dutch Kidney Foundation (13OKJ36), Netherlands Organisation for Scientific Research (91618036).

L.F.M. van der Zanden: None. I.A.L.M. van Rooij: None. J.S.L.T. Quaedackers: None. R. Nijman: D. Speakers Bureau/Honoraria (speakers bureau, symposia, and expert witness); Modest; Astra Zeneca. M. Steffens: None. L.L.L. de Wall: None. E.M.H.F. Bongers: None. F. Schaefer: None. M. Kirchner: None. R. Behnisch: None. A.K. Bayazit: None. S. Caliskan: None. L. Obrycki: None. G. Montini: F. Consultant/Advisory Board; Modest; Merck. A. Duzova: None. M. Wuttke: None. R. Jennings: None. N.A. Hanley: F. Consultant/Advisory Board; Modest; APIS Assay Technologies. N.J. Milmoe: None. P.J.D. Winyard: None. K.Y. Renkema: None. M.F. Schreuder: None. N. Roeleveld: None. W.F.J. Feitz: None.


RNA-seq approach to detect splicing alterations in primary ciliary dyskinesia and non-CF bronchiectasis

J. Legebeke1, G. Wheway1, H. Wai1, W. Walker1,2, J. S. Lucas1,2, D. Baralle1,2

1University of Southampton, Southampton, United Kingdom, 2University Hospital Southampton, Southampton, United Kingdom

Mutations that cause PCD have been reported in >40 genes. Most cases are recessive, caused by biallelic changes in these genes. These account for around 70% of cases, suggesting that additional genes, and non-coding or synonymous changes in known genes, remain to be identified. Non-CF bronchiectasis is a heterogeneous group of disorders and adults may need to be investigated for PCD. Here we present an optimised pipeline for RNA analysis from nasal brushings taken from the inferior turbinate of patients with suspected PCD or non-CF bronchiectasis. We present analysis pipelines to investigate differences in gene expression, alternative splicing and the identification of deleterious splicing events. We developed gene-neutral and gene-specific analysis protocols, from both nasal brushings stored in RNA-later and cells from nasal brushings cultured at the air-liquid interface (ALI). We present this RNA-seq approach as a particularly useful diagnostic tool to investigate patients in whom a single pathogenic variant has been identified in a known PCD gene and where we suspect an alteration in splicing of the same gene due to a second intronic or synonymous / non-synonymous exonic variant. This represents a step towards using RNA analysis for increasing diagnostic yield in rare recessive respiratory disease. JL, DB and HT are supported by a NIHR Research Professorship to DB (RP- 2016-07-011). JL is further supported by Southampton NIHR BRC. GW is funded by a Wellcome Trust Seed Award in Science (Grant No. 204378/Z/16/Z) and a University of Southampton Faculty of Medicine Research Management Committee research grant.

J. Legebeke: None. G. Wheway: None. H. Wai: None. W. Walker: None. J.S. Lucas: None. D. Baralle: None.


Genetic testing for hereditary hyperparathyroidism in a large UK cohort

S. Mariathasan1, K. Andrews2, E. Thompson3, B. Challis1,4, S. Wilcox2, H. Pierce2, J. Hale1, S. Spiden3, G. Fuller5, H. Simpson6, B. Fish7, P. Jani7, I. Seetho8, R. Armstrong2, L. Izatt9, M. Joshi10, A. Velusami10, R. Casey1, S. Park2

1Department of Endocrinology, Cambridge University Hospitals NHS Foundation Trust, Cambridge, United Kingdom, 2Department of Clinical Genetics, Cambridge University Hospitals NHS Foundation Trust, Cambridge, United Kingdom, 39.10. East Midlands and East of England NHS Genomic Laboratory Hub, Cambridge University Hospitals NHS Foundation Trust, Cambridge, United Kingdom, 4Translational Science & Experimental Medicine, Research and Early Development, Cardiovascular, Renal and Metabolism, BioPharmaceuticals R&D, AstraZeneca,, Cambridge, United Kingdom, 59.10. East Midlands and East of England NHS Genomic Laboratory Hub, Cambridge University Hospitals NHS Foundation Trust, Cambridge, United Kingdom, 64. Department of Endocrinology, University College Hospital, London, United Kingdom, 7Department of Head and Neck Surgery, Cambridge University Hospitals NHS Foundation Trust, Cambridge, United Kingdom, 8Department of Endocrinology, Northwick Park Hospital, London North West University Hospital NHS Trust, London, United Kingdom, 9Department of Clinical Genetics, Guy’s and St Thomas’ NHS Foundation Trust, London, United Kingdom, 10Department of Endocrinology, Guy’s and St Thomas’ NHS Foundation Trust, London, United Kingdom

Primary hyperparathyroidism (PHPT) is common with a prevalence of 0.86%. 10% are familial. Syndromic PHPT includes multiple endocrine neoplasia (MEN)1, MEN4, MEN2A and CDC73-related disorders. Non-syndromic causes include familial hypocalciuric hypercalcaemia (FHH), familial isolated HPT and neonatal severe PHPT. Establishing the underlying genetic cause allows for targeted, cost effective management. The 2019 National Genomic Test Directory PHPT testing criteria are PHPT and calcium creatinine clearance ratio (CCCR) >0.02 presenting <35y or <45y with one of a) multi-glandular disease, or b) hyperplasia, or c) ossifying fibromas of the jaw, or d) a family history (FH) of PHPT. The testing criteria for FHH is CCCR <0.02.

We reviewed 121 PHPT patients from the Cambridge and Guy’s Endocrine Genetics clinic over a 4y period referred with multi-glandular disease, relevant FH, CCCR <0.01, age <50y, or >1 risk factor. We offered NGS panel gene testing of all known PHPT genes. Of the 121 patients, (92F, mean age 41y), a pathogenic variant was identified in 16% (11 CASR, 6 MEN1, 1 CDC73, 1 AP2S1) and we detected 4 VUS’s. The mean CCCR was 0.0073 in those with a CASR pathogenic variant vs 0.016 in those without (p 0.003). A CCCR <0.01 had a sensitivity of 82% and specificity of 81% for FHH. A positive FH was a strong predictor of a pathogenic variant whereas multi-glandular disease was not.

In summary, genetic testing is recommended in PHPT at any age with i) syndromic PHPT, ii) relevant FH iii) a CCCR<0.01 and iv) multiple risk factors.

S. Mariathasan: None. K. Andrews: None. E. Thompson: None. B. Challis: None. S. Wilcox: None. H. Pierce: None. J. Hale: None. S. Spiden: None. G. Fuller: None. H. Simpson: None. B. Fish: None. P. Jani: None. I. Seetho: None. R. Armstrong: None. L. Izatt: None. M. Joshi: None. A. Velusami: None. R. Casey: None. S. Park: None.


Biallelic loss of function GFRA1 variants cause bilateral renal agenesis

S. Khan1, I. Verma2, V. Arora2, R. Puri2, A. El-Hattab3, M. Rocha1, R. Merdzanic1, O. Paknia1, C. Beetz1, A. Rolfs1,4, A. Bertoli-Avella1, P. Bauer1

1Centogene AG,, Rostock, Germany, 2Sir Ganga Ram Hospital, Institute of Genetics and Genomics, Rajinder Nagar, New Delhi, India, 3Department of Clinical Sciences, College of Medicine, University of Sharjah, Sharjah, United Arab Emirates, 4University of Rostock, Rostock, Germany

Congenital anomalies of the kidney and urinary tract comprise a broad spectrum of renal and urinary tract malformations with renal agenesis being at the most severe end of the spectrum. It usually results in death in utero or in the perinatal period. Only three genes have been identified so far as causing renal agenesis. By exome/genome sequencing within a diagnostic setting, with combined database analyses, we identified two unrelated patients presenting with congenital bilateral renal agenesis and homozygous loss of function variants (LoF) in the GFRA1 gene. Both patients had parental consanguinity and similarly affected, deceased siblings, suggesting an autosomal recessive inheritance. Patient 1 was homozygote for the nonsense variant NM_001348097.1:c.676C>T, p.(Arg226*). The second patient was homozygote for a frameshift variant (NM_001348097.1:c.1294delA, p.(Thr432Profs*13)). Both variants are novel, identified for first time in our database, and presumed to lead to LoF. The GFRA1 gene is not reported to be associated to any human phenotype in OMIM, but GFRA1 missense variants have been reported as associated to Hirschsprung disease in several patients, based on an oligogenic model. Gfra1-deficient mice demonstrate absence of enteric neurons and agenesis of the kidney, characteristics that are reminiscent of both Gdnf-and Ret-deficient mice. The phenotype described in our patients is identical to the renal phenotype reported in Gfra1 null mice, however, no abnormalities of the enteric nervous system was reported in these two cases. In conclusion, these findings strongly support the causal role of GFRA1 inactivating variants for an autosomal recessive form of bilateral renal agenesis.

S. Khan: A. Employment (full or part-time); Significant; Centogene AG, Rostock, Germany. I. Verma: None. V. Arora: None. R. Puri: None. A. El-Hattab: None. M. Rocha: A. Employment (full or part-time); Significant; Centogene AG, Rostock, Germany. R. Merdzanic: A. Employment (full or part-time); Significant; Centogene AG, Rostock, Germany. O. Paknia: A. Employment (full or part-time); Significant; Centogene AG, Rostock, Germany. C. Beetz: A. Employment (full or part-time); Significant; Centogene AG, Rostock, Germany. A. Rolfs: A. Employment (full or part-time); Significant; Centogene AG, Rostock, Germany. A. Bertoli-Avella: A. Employment (full or part-time); Significant; Centogene AG, Rostock, Germany. P. Bauer: A. Employment (full or part-time); Significant; Centogene AG, Rostock, Germany.


WES expands the mutational spectrum in patients with Silver-Russell syndrome features

R. Meyer1, M. Begemann1, C. Hübner1, D. Dey1, A. Küchler2, M. Elgizouli2, P. Kroisel3, G. Fekete4, L. Ambrozaityte5, A. Matuleviciene5, M. Elbracht1, T. Eggermann1

1Institute of Human Genetics, Medical Faculty, RWTH Aachen University, Aachen, Germany, 2Institute of Human Genetics, University Hospital Essen, Essen, Germany, 3Institute of Human Genetics, Medical University of Graz, Graz, Austria, 4II. Department of Pediatrics, Semmelweis University, Budapest, Hungary, 5Department of Human and Medical Genetics, Institute of Biomedical Sciences, Faculty of Medicine, Vilnius University, Vilnius, Lithuania

Silver-Russell syndrome (SRS) is one of the most prominent growth retardation disorders. However, both, the phenotype and underlying molecular alterations of SRS show a marked heterogeneity. SRS itself is an imprinting disorder, mainly associated with specific molecular changes at specific imprinted chromosomal regions. Disease causing point mutations are less common and vary widely in means of the affected gene. The suitability of panel-based NGS assays to identify monogenetic causes of SRS-like phenotypes has already been proven. The reasonableness of whole exome sequencing to identify point mutations in patients with Silver-Russel syndrome features however, is still the subject of ongoing discussions. We examined samples of 75 patients referred for molecular testing for SRS using different NGS-based approaches. The typical SRS-associated molecular alterations had been excluded before. The results of multi gene panel, whole exome sequencing, and trio-based whole exome sequencing approaches were compared. Taking together the results of all different approaches, in 19/75 patients a disease-causing variant could be identified. Most of these variants are associated with monogenetic differential diagnoses of Silver-Russell syndrome. The diagnostic yield raised from panel to whole exome sequencing to trio-based whole exome sequencing. Broadening the molecular sequencing approach increases the mutation detection rate in patients referred for molecular testing of SRS without one of the typical molecular alterations. Some of the monogenetic disorders detected in our cohort have an important impact on the clinical management and genetic counseling of affected families mainly in terms of recurrence risks, tumor development, and mental development. Funding: Deutsche Forschungsgemeinschaft (EG110/15-1).

R. Meyer: None. M. Begemann: None. C. Hübner: None. D. Dey: None. A. Küchler: None. M. Elgizouli: None. P. Kroisel: None. G. Fekete: None. L. Ambrozaityte: None. A. Matuleviciene: None. M. Elbracht: None. T. Eggermann: None.


‘The pathogenicity analysis of mutations identified in AR gene in patients with AIS from Ukraine’

L. A. Livshits1, D. A. Sirokha1, O. V. Gorodna1, D. M. Lozhko1, G. B. Livshyts1, N. B. Zelinska2

1IMBG NANU, Kyiv, Ukraine, 2Ukrainian Scientific and Practical Center for Endocrine Surgery, Transplantation of Endocrine Organs and Tissues of the Ministry of Health of Ukraine, Kyiv, Ukraine

Androgen Insensitivity Syndrome (AIS) is a Disorder of Sex Development (DSD) that occurs at a frequency of 1 in 20,000 live births, is the most common DSD in people with karyotype 46, XY, caused by mutations in the Androgen Receptor (AR) gene. More than 1000 AR gene mutations have been described in patients with AIS and prostate cancer. Almost 400 of them are located in ligand binding domain (LBD). The pathogenicity of mutations detected in AIS patients from Ukraine was analyzed. Directional sequencing of the PCR products of LBD-coding exons 7 and 8 was performed on DNA samples of AIS patients after informed consent. The substitution X:67722905 T>C (rs9332970), exon 7, missense mutation Ile843Thr was identified in PAIS-patient. In another PAIS-patient the substitution X: 67723745 C>T (rs137852594) samesence variant was identified. It was predicted the creation of a new enhancer motif for SRp55 splisosome protein, and as a consequence a new donor splice site at the end of the last exon 8. The substitution X:67722943 C> T (rs886041132), exon 7 - missense mutation Arg856Cys, was identified in CAIS-patient. Mutation X: 67722884 T> G, exon 7, a missense mutation Ile836Ser, was identified in CAIS-patient was not annotated in Varsome, gnomAD and ARDB and identified as novel. The effect of listed missense mutations was predicted according to SIFT and PolyPhen values and classified as damaging. Analysis the substitution Ile836Ser on 3D model showed that the mutant protein does not get the correct conformation and binds dihydrotestosterone ~ 460 times worse than wild-type AR.

L.A. Livshits: None. D.A. Sirokha: None. O.V. Gorodna: None. D.M. Lozhko: None. G.B. Livshyts: None. N.B. Zelinska: None.


Cell free DNA as potential marker of inflammatory diseases

D. Smolak1,2, G. Roman3, J. Budis2,4,5, M. Maronek3, B. Gromova3, R. Liptak3, M. Harsanyova2,1, J. Radvanszky6,2, T. Szemes2,1,4

1Department of Molecular Biology, Faculty of Natural Sciences, Comenius University, Bratislava, Slovakia, 2Geneton Ltd., Bratislava, Slovakia, 3Institute of Molecular Biomedicine, Faculty of Medicine, Comenius University, Bratislava, Slovakia, 4Comenius University Science Park, Bratislava, Slovakia, 5Slovak Center of Scientific and Technical Information, Bratislava, Slovakia, 6Institute for Clinical and Translational Research, Biomedical Research Center, Slovak Academy of Sciences, Bratislava, Slovakia

Introduction: Ulcerative colitis is an inflammatory bowel disease (IBD) characterised by chronic inflammation of the human digestive tract. Despite efforts, the availability of effective non-invasive diagnostics assays is still limited. Cell free DNA (cfDNA) represents extracellular genetic information that already showed promises for various fields of genetic testing, including, non-invasive prenatal testing (NIPT) and cancer testing.

Materials and methods: Using a murine dextran sulfate sodium (DSS) induced colitis model we collected blood from 46 laboratory mice, grouped by stage of ulcerative colitis modeled by the duration of DSS administration. cfDNA was isolated and sequenced using the MiSeq Illumina System. Preprocessed sequenced data were mapped against mouse genome (GRCm38.p6). Statistical analyses were performed by in-house Python and R scripts.

Results: Besides the observed different fragment length distributions between control and colitis groups, we observed also a significantly increasing amount of mouse genomic material with the advancing stage of the disease, measured by the proportion of sequenced reads mappable to the mouse genome. In addition, to further investigate the pathophysiological processes behind the observed changes, we performed a sequence based differentiation of reads according to their mappability to the mouse nuclear and mitochondrial genomes, as well as of the detected bacterial communities.

Conclusions: We observed significant differences between the control group and various disease stages in sequenced data. Our results suggest the utility of cfDNA as a potential screening marker for IBD and even its specific stages and pathophysiological processes behind them.

Funded by: Slovak Research and Development Agency, grant APVV-17-0526.

D. Smolak: A. Employment (full or part-time); Significant; Geneton Ltd.. G. Roman: None. J. Budis: A. Employment (full or part-time); Significant; Geneton Ltd.. M. Maronek: None. B. Gromova: None. R. Liptak: None. M. Harsanyova: A. Employment (full or part-time); Significant; Geneton Ltd. J. Radvanszky: A. Employment (full or part-time); Modest; Geneton Ltd. T. Szemes: A. Employment (full or part-time); Significant; Geneton Ltd..


Functional characterization of variants of unknown significance in the Wilson disease-associated ATP7B gene

A. Stalke, F. Hennig, E. Pfister, U. Baumann, M. Eilers, V. Schäffer, B. Vajen, N. Huge, T. Reinkens, B. Schlegelberger, T. Illig, B. Skawran

Hannover Medical School, Hannover, Germany

Introduction: Wilson disease (WD) is an autosomal recessive disorder of copper excess due to pathogenic ATP7B variants. ATP7B codes for a copper-transporting ATPase. Sequencing of ATP7B is an important tool to diagnose the disease. However, variants of unknown significance (VUS) are detected frequently, impeding a clear diagnosis. Functional analyses can help to unambiguously classify VUS. We aimed to functionally characterize four ATP7B VUS, detected in patients with clinically distinct or suspected WD.

Methods: The ATP7B open reading frame was cloned into an expression vector and the VUS were generated by site-directed mutagenesis. After HEK293T cell transfection, ATP7B expression of the mutants was quantified at mRNA and protein level by qRT-PCR and western blot. To assess the mutant’s ability to export copper a copper-sensitive luciferase-based assay was used.

Results: For all VUS ATP7B mRNA expression was significantly reduced compared to wild-type and a benign polymorphism. Three VUS also led to decreased protein expression and copper transport capacity. These VUS had been detected in clinically distinct WD patients. One VUS led to protein expression and copper transport capacity at wild-type level. It had been detected in a patient with suspected WD.

Conclusions: Our results strongly suggest a pathogenic effect for three of the VUS. For one VUS our results hint at a benign effect. On-going immunofluorescence assays will further address a possible protein mislocalization. Besides clarifying the clinical relevance of variants, functional characterization will help to understand the variant’s pathomechanism contributing to personalized treatment with fewer side effects.

Supported by: DFG

A. Stalke: None. F. Hennig: None. E. Pfister: None. U. Baumann: None. M. Eilers: None. V. Schäffer: None. B. Vajen: None. N. Huge: None. T. Reinkens: None. B. Schlegelberger: None. T. Illig: None. B. Skawran: None.

P04 Skeletal, Connective Tissue, Ectodermal and Skin Disorders


Geleophysic and acromicric dysplasias: clinical and molecular review of 38 patients

P. Marzin1,2, B. Thierry3,4, A. Dancasius1,2, C. Michot1,2, S. Rondeau1,2, G. Baujat1,2, G. Phan5, M. Le Bourgeois6, D. Khraiche7, C. Delacourt6, V. Cormier-Daire1,2

1Fédération de Génétique et Institut Imagine, AP-HP, Hôpital Necker-Enfants Malades,, F-75015, France, 2Université de Paris, UMR1163, INSERM, F-75 015, Paris, France, 3Service d’ORL pédiatrique, AP-HP, Hôpital Necker-Enfants Malades, F-75015, France, 4Université de Paris, UMR976, INSERM, F-75 010,, Paris, France, 5Laboratoire de Cristallographie et RMN Biologiques, UMR 8015, CNRS, Université Paris Descartes, Faculté de Pharmacie, 4 Avenue de l'Observatoire, 75006, paris, France, 6Service de Pneumologie et Allergologie Pédiatriques, AP-HP, Hôpital Necker-Enfants Malades, F-75 015, Paris, France, 7Unité Médico-Chirurgicale de Cardiologie Congénital et Pédiatrique, AP-HP, Hôpital Necker-Enfants Malades, F-75 015,, Paris, France

Introduction: Geleophysic dysplasia (GD) and acromicric dysplasia (AD) are skeletal dysplasia characterized by short stature, brachydactyly and progressive joint limitation. Cardiorespiratory involvement may be responsible for a severe prognosis in GD. Dominant mutations in the FBN1 and LTBP3 genes are associated with AD and GD, whereas recessive mutations in the ADAMTSL2 gene only with GD. These three genes encode for proteins of microfibrillar network. The aim of this study is to improve understanding of natural history and genotype-phenotype correlations.

Materials and methods: This retrospective study included patients with AD and GD followed in our center, with identified mutation(s). Data from the medical records were abstracted, recorded in an Excel spreadsheet and analyzed using simple frequency analysis.

Results: We described clinical and molecular data from 38 patients (20GD, 18 AD) with a mean age of 17 years. Mutations in the FBN1, ADAMTSL2 and LTBP3 genes were identified in 23, 12 and 3 patients, respectively. Early death occurred in nine cases. 45% of patients presented with valvulopathy, progressive in half of cases. Respiratory disorders were asthma (50%), non-cardiogenic restrictive syndrome (30%), laryngo-tracheo-bronchial involvement (18%) and acute non-cardiogenic pulmonary hypertension (four cases). Patients with a variant involving a cysteine in the FBN1 gene or mutations in the ADAMTSL2 gene presented a higher proportion of severe cardiorespiratory complications compared to a more benign course in patients with other mutations.

Conclusions: Genetic findings could have importance not only in the diagnosis, but also in risk stratification and clinical management of patients with suspected acromelic dysplasia.

P. Marzin: None. B. Thierry: None. A. Dancasius: None. C. Michot: None. S. Rondeau: None. G. Baujat: None. G. Phan: None. M. Le Bourgeois: None. D. Khraiche: None. C. Delacourt: None. V. Cormier-Daire: None.


Acromesomelic dysplasia Maroteaux type, nine patients with seven novel NPR2 mutations

E. Kilic1, B. Cavdarli2, G. Buyukyilmaz3, M. Kilic4

1Division of Pediatric Genetics, Department of Pediatrics, University of Health Sciences, Ankara City Hospital, Ankara, Turkey, 2Department of Medical Genetics, Ankara City Hospital, Ankara, Turkey, 3Division of Pediatric Endocrinology, Department of Pediatrics, Ankara City Hospital, Ankara, Turkey, 4Division of Metabolism, Department of Pediatrics, Sami Ulus Children Hospital, Ankara, Turkey

Background: Acromesomelic Dysplasia, type Maroteaux (AMDM), is an autosomal recessive skeletal dysplasia caused by loss of function mutations of NPR2 gene which encodes C-type natriuretic peptid reseptor B. This rare type of dwarfizm characterized with shortening of the middle and distal segments of the limbs. Herein we reported nine patients from eight families with seven novel NPR2 mutations.

Methods: Nine patients were diagnosed AMDM in a single centre from 2015 to 2020 were enrolled in this study. Their clinical, radiological and molecular genetic findings examined retrospectively. The mutations in NPR2 gene were confirmed with direct sequencing.

Results: All nine patiens in this group were admitted to hospital with the complaint of short stature. All had short limb type dwarfism with marked shortening middle and acral segment of the limbs. All had typical dysmorphic signs, brachydactyly, significant greater toes and normal intelligence. We identified one previously reported (c.2729 C>T), five novel missense (c.3029 A>G, c.1738-1739 del ins TT, c.1163 G>A, c.3058 C>T, c.329 G>A) and two novel nonsense (c.1213 delT fs*, c.14 C>A fs*) mutations. All new mutations identified in this group are likely pathogenic and meaningfull with the patients typical clinical presentations.

Conclusions: Reporting new patients with novel mutations would contribute to better clinical dealination of this rare dysplasia and would help to established pathogenic mutations.

References:1. Maroteaux P, Martinelli B, Campailla E.[Acromesomelic dwarfism]. Presse Med. 1971; 79:1839-42.2. Khan S, Basit S, Khan MA, et al. Genetics of human isolated acromesomelic dysplasia. Eur J Med Genet. 2016;59:198-203.

E. Kilic: None. B. Cavdarli: None. G. Buyukyilmaz: None. M. Kilic: None.


Severe congenital cutis laxa with generalized muscle hypotonia due to ATP6V1A mutations

G. Vogt1,2, N. El Choubassi1,2, M. Holtgrewe3, S. Krause4, A. Lekaj1, R. Horvath5, M. Schülke6, T. Gardeitchik7, S. Mundlos1,2, Á. Herczegfalvi8, H. Lochmüller9,10, V. Karcagi11, U. Kornak1,2,12, B. Fischer-Zirnsak1,2

1Institute of Medical Genetics and Human Genetics, Charité - Universitätsmedizin, Berlin, Germany, 2RG Development & Disease, Max Planck Institute for Molecular Genetics, Berlin, Germany, 3Core Unit Bioinformatics-CUBI, Berlin Institute of Health, Berlin, Germany, 4Department of Neurology, Friedrich-Baur-Institute, Munich, Germany, 5Department of Clinical Neurosciences, University of Cambridge School of Clinical Medicine, Cambridge, United Kingdom, 6Department of Neuropediatrics, Charité Universitätsmedizin, Berlin, Germany, 7Department of Human Genetics, Radboud University Medical Centre, Nijmegen, Netherlands, 8II. Department of Paediatric Neurology, Semmelweis Medical University, Budapest, Hungary, 9Division of Neurology, Children’s Hospital of Eastern Ontario Research Institute, Ottawa, IL, Canada, 10Brain and Mind Research Institute, Ottawa, IL, Canada, 11Department of Molecular Genetics and Diagnostics, National Institute of Environmental Health, Budapest, Hungary, 12Institute of Human Genetics, University Medical Center, Göttingen, Germany

Introduction: Autosomal recessive cutis laxa type 2D is a severe multisystem disease due to pathogenic variants affecting ATP6V1A, encoding a component of the V-Type H+ATPase. Affected individuals show a strong cutis laxa phenotype with large skin folds at birth, hypotonia and cardiac abnormalities.

Material and Methods: We performed single whole exome sequencing (WES) to identify the causative molecular defect in our index patient. Furthermore, Sanger sequencing, qRT-PCR and Immunoblot analyses were performed to further investigate causality of the variants identified.

Results: Here we report on two affected individuals from a non-consanguineous family from Hungary suffering from a multisystem condition with severe, generalized muscle hypotonia and and strong juvenile cutis laxa. One child died in infancy, while the other significantly improved. Using WES and co-segregation analyses we found our proband to be compound heterozygous for a novel missense and the first so far described nonsense pathogenic variant affecting ATP6V1A. Expression studies revealed a strong degradation of the allele carrying the nonsense alteration. Additionally, patients’ dermal fibroblasts showed strong alteration of Cathepsin D processing indicating alterations of lysosomal function.

Conclusions: This is the second report on pathogenic variants in ATP6V1A as the cause for a severe condition with cutis laxa. Our data expand the clinical spectrum of ATP6V1A-related phenotypes and show that beside missense, also nonsense pathogenic variants are a possible cause of ATP6V1A-ARCL. Funding: The study was supported by the Deutsche Forschungsgemeinschaft (FI 2240/1-1) to BF-Z and by EURO-CDG-2 to UK.

G. Vogt: None. N. El Choubassi: None. M. Holtgrewe: None. S. Krause: None. A. Lekaj: None. R. Horvath: None. M. Schülke: None. T. Gardeitchik: None. S. Mundlos: None. Á. Herczegfalvi: None. H. Lochmüller: None. V. Karcagi: None. U. Kornak: None. B. Fischer-Zirnsak: None.


New duplication downstream of the BMP2 gene in a French family with Brachydactyly type A2 (BDA2)


1Department of Biochemistry and Genetics, Angers University Hospital, ANGERS, France, 2Department of Gynecology and Obstetrics, Angers University Hospital, ANGERS, France, 3Pole of Biology Genetic Pathology, Lille University Hospital - Center of Biology Genetic Pathology, LILLE, France

Brachydactyly type A2 (BDA2, MIM 112600) is an autosomal dominant pathology characterized by deviation and shortening of the middle phalanx of the index and / or second toe. It may be due to mutations in the BMPR1B gene (MIM 603248) or the GDF5 gene (MIM 601146) but may also depend on structural changes in 20p12.3 involving the BMP2 gene. Indeed, microduplications downstream of the BMP2 gene have been described in 5 families (2 German and 3 Chinese). Using an SNP-array technique, we identified a new duplication of 45kb downstream of the BMP2 gene in a French family. We describe the phenotypic and genotypic characteristics of this family and review the literature.

E. Colin: None. A. Guichet: None. F. Boussion: None. M. Tessarech: None. A. Ziegler: None. M. Gorce: None. C. Houdayer: None. S. Manouvrier: None. D. Bonneau: None.


Loss-of-function variants in C3ORF52 result in localized autosomal recessive hypotrichosis

N. Cesarato1, L. Malki2,3, O. Sarig2,3, J. Mohamad2,3, T. Canter4, S. Assaf2,3, M. Pavlovsky2, D. Vodo2,3, Y. Anis5, O. Bihari2, K. Malovitski2,3, A. Gat2, H. Thiele6, B. E. Perez White4, L. Samuelov2, A. Nanda7, A. S. Paller4, R. C. Betz1, E. Sprecher2,3

1Institute of Human Genetics, University of Bonn, Bonn, Germany, 2Division of Dermatology, Tel Aviv Sourasky Medical Center, Tel Aviv, Israel, 3Department of Human Molecular Genetics & Biochemistry, Sackler Faculty of Medicine, Tel Aviv University, Ramat Aviv, Israel, 4Department of Dermatology, Northwestern University Feinberg School of Medicine, Chicago, IL, United States, 5Institute of Endocrinology, Tel Aviv Sourasky Medical Center, Tel Aviv, Israel, 6Cologne Center for Genomics, University of Cologne, Cologne, Germany, 7As’ad Al-Hamad Dermatology Center, Kuwait, Kuwait

Introduction: Localized autosomal recessive hypotrichosis has been associated with pathogenic variants in DSG4, encoding a desmosomal protein as well as in LIPH and LPAR6, encoding respectively lipase H, which catalyzes the formation of 2-acyl lysophosphatidic acid (LPA), and lysophosphatidic acid receptor 6, a receptor for LPA. LPA promotes hair growth and differentiation. In this study, we aimed at delineating the genetic basis of LAH in patients without pathogenic variants in these three genes.

Materials and Methods: Variant analysis was conducted using exome and Sanger sequencing. We then performed RT-qPCR, immunofluorescence staining, immunoblotting, enzymatic and co-immunoprecipitation assays in cell cultures and hair follicles to evaluate the consequences of the identified variants.

Results: We identified homozygous variants in C3ORF52 in four individuals from two families with LAH. C3ORF52 was found to be co-expressed with lipase H in the inner root sheath of the hair follicle and the two proteins were found to directly interact. The LAH-causing variants were associated with decreased C3ORF52 expression and resulted in markedly reduced lipase H-mediated LPA biosynthesis.

Conclusions: We could connect C3ORF52 to the LPA-mediated signaling pathway which, together with LPAR6 and LIPH, plays a crucial role in hair growth. LAH can be caused by abnormal function of any of these three proteins.

N. Cesarato: None. L. Malki: None. O. Sarig: None. J. Mohamad: None. T. Canter: None. S. Assaf: None. M. Pavlovsky: None. D. Vodo: None. Y. Anis: None. O. Bihari: None. K. Malovitski: None. A. Gat: None. H. Thiele: None. B.E. Perez White: None. L. Samuelov: None. A. Nanda: None. A.S. Paller: None. R.C. Betz: None. E. Sprecher: None.


Evidence for a role of fibrillin-2 in de pathogenesis of carpal tunnel syndrome

S. Peeters1, A. Decramer2, S. Cain3, P. Houpt4, F. Verstreken5, J. Noyez2, C. Hermans6, W. Jacobs7, M. Lammens8, E. Fransen1, A. Kumar9, G. Vandeweyer1, B. Loeys1, W. Van Hul1, C. Baldock3, E. Boudin1, G. Mortier1

1Department of Medical Genetics, University of Antwerp and Antwerp University Hospital, Antwerp, Belgium, 2Department of Orthopaedics and Traumatology, AZ Delta, Roeselare, Belgium, 3Division of Cell-Matrix Biology and Regenerative Medicine, Wellcome Centre for Cell-Matrix Research, the University of Manchester, Manchester, United Kingdom, 4Department of Plastic Surgery, Isala clinic Zwolle, Zwolle, Netherlands, 5Department of Orthopaedic surgery, AZ Monica, Deurne, Belgium, 6Center for Oncological Research Antwerp (CORE), University of Antwerp, Edegem (Belgium), Antwerp, Belgium, 7Department of Forensic Medicine and Pathology, Antwerp University Hospital and University of Antwerp, Antwerp, Belgium, 8Department of Pathological Anatomy, Antwerp University Hospital, Antwerp, Belgium, 9Department of Paediatrics, Wellcome - MRC Cambridge Stem Cell Institute Cambridge, Cambridge, United Kingdom

Introduction: Carpal tunnel syndrome (CTS) is the most common form of peripheral entrapment neuropathy. Although CTS is quite common, its pathogenesis remains largely unknown. An estimated heritability index of 0.46 and a familial occurrence in 27-39% of the cases indicate that genetic factors must play a role in the pathogenesis. We report on a peculiar family in which CTS occurred in subsequent generations at an usually young age. Additional clinical features included brachydactyly and short Achilles tendons resulting in toe walking in childhood.

Material and methods: We performed exome sequencing and genome-wide linkage analysis to identify the pathogenic variant in this family. Several functional assays were performed to further study the causality of the variant. In addition, we sequenced a cohort of 216 patients to assess the role of this new candidate gene in the pathogenesis of CTS in a larger population.

Results: We identified a heterozygous variant (c.5009T>G; p.Phe1670Cys) in the fibrillin-2 (FBN2) gene that co-segregated with the phenotype in the family. Functional assays showed that the FBN2 variant impaired integrin-mediated cell adhesion and migration. Moreover, we observed an increased TGF-β signaling and fibrosis in the carpal tissues of affected individuals. A variant burden test in a large cohort of CTS patients revealed a significantly increased frequency of rare FBN2 variants in patient alleles compared to controls.

Conclusions: Our data strongly suggest a role of FBN2 in the pathogenesis of CTS.

S. Peeters: None. A. Decramer: None. S. Cain: None. P. Houpt: None. F. Verstreken: None. J. Noyez: None. C. Hermans: None. W. Jacobs: None. M. Lammens: None. E. Fransen: None. A. Kumar: None. G. Vandeweyer: None. B. Loeys: None. W. Van Hul: None. C. Baldock: None. E. Boudin: None. G. Mortier: None.


New pathways and variants in genes causing collagenopathies identified in patients with isolated cervical insufficiency

L. Volozonoka, D. Rots, I. Kempa, A. Kornete, D. Rezeberga, L. Gailīte, A. Miskova

Riga Stradins University, Riga, Latvia

Introduction: Cervical insufficiency is phenotypically distinguishable condition causing pregnancy loss or preterm delivery. A strong genetic component and few syndromic forms of cervical insufficiency are described. Knowledge about the genetics of human cervix remodelling during pregnancy is scarce - only 12 maternal genes and their variants are implicated in cervical insufficiency so far as identified by our systematic gene analysis before.

Material and methods: We sequenced DNA from 21 patients with clinical presentation of isolated cervical insufficiency using Illumina’s TruSight One NGS kit. Genes having rare deleterious variants identified in our patients (n = 694) were annotated for the pathway enrichments using ConsensusPathDB. Variants in genes from enriched phenotype-relevant pathways were manually curated based on variant’s pathogenicity, gene’s expression pattern, mode of inheritance and disease mechanism, to assign likelihood for contribution to the patients’ phenotype.

Results: Pathway-enrichment analysis revealed increased variant burden in 15 pathways (max p-value 2.47E-03) playing roles in tissue mechanical and biomechanical properties, i.e. collagen biosynthesis, cell-extracellular matrix communications and previously unnoticed pathways of hemidesmosome assembly, laminins, integrins and focal adhesion. Fourteen variants of unknown significance and (likely) pathogenic variants in 11 genes - TGM2, ITGA6, COMP, COL11A1, including COL1A1, COL1A2, COL6A3, P3H1, PLOD1, PPIB and TNXB causing collagenopathies - in 11 patients were classified as potential to increase susceptibility to the development of non-syndromic cervical insufficiency.

Conclusions: Cervical insufficiency being an expression point in a phenotypic continuum of collagenopathies should be investigated further using multiple approaches. This might open up new opportunities to improve patient evaluation and management in the future.

L. Volozonoka: None. D. Rots: None. I. Kempa: None. A. Kornete: None. D. Rezeberga: None. L. Gailīte: None. A. Miskova: None.


The phenotypic spectrum of Chitayat syndrome: Six additional affected individuals

A. Suter1, A. Abad Perez1, F. Santos-Simarro2, P. Toerring3, R. Ramos-Mejia4, K. Heath2, V. Huckstadt5, M. Parrón-Pajares6, M. Mensah1,7, W. Hülsemann8, M. Holtgrewe1,9, S. Mundlos1,10, U. Kornak1,11, O. Bartsch12, N. Ehmke1,10

1Institute of Medical and Human Genetics, Charité - Universitätsmedizin Berlin, Berlin, Germany, 2Institute of Medical and Molecular Genetics and Skeletal dysplasia multidisciplinary Unit (UMDE), Hospital Universitario La Paz and CIBERER, ISCIII, Madrid, Spain, 3Department of Clinical Genetics, Odense University Hospital, Odense, Denmark, 4Growth and Development Department, Garrahan Hospital, Buenos Aires, Argentina, 5Genetics Department, Garrahan Hospital, Buenos Aires, Argentina, 6Department of Radiology and Skeletal dysplasia multidisciplinary Unit (UMDE), Hospital Universitario la Paz, Madrid, Spain, 7Berlin Institute of Health (BIH), Anna-Louisa-Karsch-Str. 2, 10178, Berlin, Germany, 8Handchirurgie Kinderkrankenhaus Wilhelmstift, Hamburg, Germany, 9Core Unit Bioinformatics – CUBI, Berlin Institute of Health, Chariteplatz 1, Berlin, Germany, 10RG Development & Disease, Max Planck Institute for Molecular Genetics, Berlin, Germany, 11Institut für Humangenetik, Universität Göttingen, Göttingen, Germany, 12Institute of Human Genetics, University Medical Centre of the Johannes Gutenberg University, Mainz, Germany

Introduction: Chitayat Syndrome is characterized by hand hyperphalangism with shortened index fingers and ulnar deviation, hallux valgus, severe respiratory distress requiring assisted ventilation and mild facial dysmorphism. It is caused by the recurrent heterozygous missense variant NM_006494.2:c.266A>G; p.Tyr89Cys in ERF, which encodes the ETS2 repressor factor (ERF) protein. The ETS family of transcription factors are involved in cellular proliferation and differentiation, embryological development, hematopoiesis, lymphocyte function and apoptosis. The exact pathomechanism of Chitayat syndrome remains unknown. So far, only seven individuals with molecularly confirmed Chitayat syndrome have been documented in the literature.

Methods: We performed a systematic phenotype analysis of six unpublished individuals with Chitayat syndrome.

Results: We found unreported phenotypic features and a great variability of pulmonary manifestations. Only one individual presented with a history of assisted ventilation, and one of the remaining presented with an atypical phenotype lacking any obvious pulmonary compromise.

Conclusions: Our findings extend the phenotype spectrum associated with the recurrent ERF variant c.266A>G and contribute to improved diagnostic strategies in individuals with hand hyperphalangism.

A. Suter: None. A. Abad Perez: None. F. Santos-Simarro: None. P. Toerring: None. R. Ramos-Mejia: None. K. Heath: None. V. Huckstadt: None. M. Parrón-Pajares: None. M. Mensah: None. W. Hülsemann: None. M. Holtgrewe: None. S. Mundlos: None. U. Kornak: None. O. Bartsch: None. N. Ehmke: None.


First family from Germany with early-onset chondrocalcinosis and osteoarthritis (CCAL1, OMIM %600668) caused by TNFRSF11B gene mutation

A. C. Blumberg1, M. Wingendorf2, I. Spier3, V. Riehmer4, O. Bartsch1

1Institute of Human Genetics, University Medical Centre of the Johannes Gutenberg University Mainz, Mainz, Germany, 2Practice for Orthopaedics and Trauma Surgery, Kirchen/Sieg, Germany, 3Institute of Human Genetics, University Medical Centre Bonn, Bonn, Germany, 4Institute of Human Genetics, University Medical Centre Cologne, Cologne, Germany

Chondrocalcinosis (CCAL) is a common condition caused by calcium pyrophosphate accumulation and resulting in destruction of articular cartilage, affecting 4-7% of adults. CCAL is usually seen in polygenic disorders (hypercalcemia, gout, arthritis), but in rare cases runs in families as a dominant trait (usually CCAL2, MIM #118600, ANKH gene). CCAL1 is extremely rare with only four families known to date: an Ashkenazy-Jewish kindred (PMIDs 2383078 and 29578045), a family from Maine (PMID 7887424), a family from the Netherlands and France (PMID 24743232), and a Long Island NY family (PMID 29578045). These families all demonstrated a specific heterozygous TNFRSF11B variant, c.1205A>T, modifying the stop codon (AAT>ATT) and predicting an elongated protein (p.Ter402Leuext*19). We report another 4-generation CCAL1 kindred from Rhineland-Palatinate (comprising 8 females and 3 males) also demonstrating the TNFRSF11B c.1205A>T variant. All patients (11/11, 100%) had early-onset osteoarthritis and chondrocalcinosis with an onset of symptoms between 16 and 30 years, and all had swelling of joints, motion restriction, and intense pain. Three probands (3/10, 30%; no data in a deceased family member) also had digital polyarthritis. First, knee, elbow, ankle and wrist joints were affected, followed by spinal destruction. Remarkably, ball-and-socket joints (e.g. hip joints) were only minimally affected (only an affected 30-year-old male). The early onset and familial clustering led to the molecular diagnosis. This is the first CCAL1 family from Germany, providing further understanding of the clinical signs and treatment (including physical therapy, orthoses, antirheumatic drugs, experimental treatment with interleukin inhibitors, and multiple joint surgery).

A.C. Blumberg: None. M. Wingendorf: None. I. Spier: None. V. Riehmer: None. O. Bartsch: None.


Three families with suspected Ehlers-Danlos syndrome and glycine substitution in COL1A1 and COL1A2 genes

S. Döhnert1, H. Reichenbach2, S. Demuth3, S. Weidensee3, K. Wilhelm4, I. Schreyer4, B. Eichhorn1

1MVZ Mitteldeutscher Praxisverbund Humangenetik MVZ, Dresden, Germany, 2MVZ Mitteldeutscher Praxisverbund Humangenetik MVZ, Leipzig, Germany, 3MVZ Mitteldeutscher Praxisverbund Humangenetik MVZ, Erfurt, Germany, 4Zentrum für ambulante Medizin, Uniklinikum Jena gGmbH, Jena, Germany

Introduction: Glycine substitutions in the Gly-X-Y-triplet disrupt the type I collagen triple helix structure and folding, and typically result in osteogenesis imperfecta (OI) and have not yet been associated with Ehlers-Danlos syndrome (EDS). OI is characterized by bone fragility, whereas EDS affects the connective tissue resulting in joint hypermobility, skin hyperextensibility, and tissue fragility. Although, an OI/EDS overlap” syndrome characterized by clinical hallmarks of EDS and mild or absent OI-phenotype is also described. The underlying mutation spectrum is wide, but includes glycine substitutions.

Material and Methods: Here, we report three patients mainly showing joint hypermobility and unusual skin. Panel diagnostic via sequencing by synthesis was conducted including candidate genes contributing to connective tissue disorders. Sanger sequencing was used for analyses of segregation patterns in parents.

Results: All index patients carried each an undescribed, heterozygous glycine substitution. In two cases the variant (c.1270G>A/p.(Gly424Ser) in COL1A1 gene; c.434G>A/p.(Gly145Arg) in COL1A2 gene) was maternal inherited and segregated with the symptoms in both families. In the other case the substitution occurred de novo (c.179G>A/p.(Gly60Asp) in COL1A2 gene). Comparable to our patients, glycine substitutions in the more N-terminal region of both genes are already described with “OI/EDS overlap” syndrome. In detail, one of our families showed an EDS-phenotype with additional minimal OI-symptoms (short stature, blue sclera), whereas the others were clinical classified as EDS-phenotype.

Conclusions: Despite the detection of glycine substitution, the patients could manifest a more typical EDS phenotype rather than OI. Certainly, subclinical symptoms for OI can additionally occur and should be considered.

S. Döhnert: None. H. Reichenbach: None. S. Demuth: None. S. Weidensee: None. K. Wilhelm: None. I. Schreyer: None. B. Eichhorn: None.


Morphological and molecular characterization of ectopic cartilage in Crouzon Syndrome

E. Candelo

Fundacion Valle del Lili, Cali, Colombia

Background: Craniosynostosis is the premature closure of the skull sutures. Patients with these condition undergo several surgical procedures, which are not able to fully restore appearance and functionality due to insufficient bone growth and suture refusion. Most mutations disrupt genes that encode for members of the fibroblast growth factor receptor (FGFR) family.

Materials and Methods: A knock out mouse with Fgfr2c342Y/C342Y was generated for phenotypic analysis by histological staining of sections of WT, Fgfrc342y/+ heterozygous (HET) and Fgfr2c342Y/C342Y homozygous (HOM) mouse embryos from embryonic day (E) 14.5 and E 16.5 stages. Morphological characterisation of the ectopic cartilages in the mutant specimens was done against the HET and WT by calculating the surface area, perimeter, width on the parietal cartilage in each genotype and cellular characterisation by obtaining the cellular density and shape descriptors. Molecular characterisation was determined by mmunohistochemistry of Collagen I and II.

Results: We identified a significant parietal cartilage expansion in HOM embryos since E14.5 with higher perimeter, surface area, and high cellular density. Additionally, characteristic cell morphology comprised; high cellular area and perimeter and circular or rounded shape. These findings were associated with abnormal intensity and localization pattern of Collagen type I and II.

Conclusions: A possible switch off on the intramembranous ossification of the parietal and frontal primordial bone and switch on of endochondral ossification process supported by the cellular, morphological and molecular findings of the abnormal cartilage expansion lead to a novel hypothesis of coronal suture fusion that can future drug target on this condition.

E. Candelo: None.


The first case of Dyssegmental Dysplasia Rolland-Desbuquois type with a variant in HSPG2

T. Kalayci1, N. Balanda2, C. R. Ferreira3, U. Altunoglu1,4

1Istanbul University, Istanbu Faculty of Medicine, Medical Genetics Department, Istanbul, Turkey, 2Undiagnosed Diseases Program, Common Fund, National Institutes of Health, Bethesda, MD, United States, 3Medical Genomics and Metabolic Genetics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD, United States, 4Koc University, School of Medicine, Medical Genetics Department, Istanbul, Turkey

Introduction: Dyssegmental dysplasia (DD) is a rare, autosomal recessive disorder characterized by micromelia and anisospondyly. Two distinct forms are described; Silverman-Handmaker (DDSH) characterized by severe shortening of limbs and perinatal lethality, and Rolland-Desbuquois (DDRD) with survival beyond infancy and milder skeletal findings. DDSH is caused by biallelic variants in HSPG2 but the aetiology of DDRD has not been explained. Here, we present the first DDRD patient reported to have an HSPG2 variant.

Materials and Methods: Detailed phenotyping and next-generation sequencing were performed.

Results: The patient is a 15-month-old female with short bones, flared metaphyses, camptodactyly, anisospondyly, pes equinovarus, flat face, micrognathia, cleft palate and the history of respiratory distress in infancy. She was diagnosed as DDRD given milder skeletal findings and a prolonged survival in comparison to DDSH. Whole exome analysis revealed a homozygous NM_005529.7:c.9970G>A(p.Gly3324Arg) variant on HSPG2 which was predicted to be pathogenic by in silico analyses.

Conclusions: The phenotypic heterogeneity and clinical overlap between DDSH and DDRD raise the question of whether HSPG2 variants lead to distinct DD forms or represent a single disorder with variable phenotypic spectrum. In a review of the literature, we recognised three patients reported as DDSH whereas they had milder skeletal findings and survival age between 13 months and 7 years, compatible with DDRD. Strikingly, they harboured novel HSPG2 variants, one of which was detected in our case. In the light of all these findings, we conclude that DDSH and DDRD are allelic and represent a single disorder with a spectrum of severity.

T. Kalayci: None. N. Balanda: None. C.R. Ferreira: None. U. Altunoglu: None.



P. Marin Reina1, A. Zuñiga Cabrera1, A. Parra Llorca1, P. Molina Aguilar2, J. Blasco2, A. Monzo Blasco2, M. Evole Buselli1

1Hospital UyP La Fe, Valencia, Spain, 2Forensic Department, Valencia, Spain

Introduction: The misdiagnosis of Ehler Danlos syndrome (EDS) and child abuse is well known, usually in relationship to skin lesions or bone fractures. At our case, the first manifestation was an aborted lethal episode in an infant due to a cerebral hemorrhage.

Clinical Case: A 4-month-old boy was founded unconscious after sleep. He was admitted to the pediatric intensive care unit with a subdural and subarachnoid hemorrhage and severe cerebral edema. Child abuse was suspected. Familiar antecedents: Mother, 34 years, first gravida. She was followed up during infancy due to small size. She has joint hypermobility. Her brother died at sixth month due to supposed sudden infant death. One 50 years old aunt presented a cerebral hemorrhage. Healthy father, 30 years old. No consanguinity. The child presented hypermovility and skin hyperextensibility. EDS vascular type was suspected. NGS analysis was performed using Illumina (NextSeq 500) technology with a ClearSeq Inherited Disease panel (Agilent Thecnologies) and consisted in the analysis of the coding and intronic regions of collagen deficiency related gene. The study did not show any pathogenic variant and was completed by analysis of gene dosage by MLPA (P331; P332), resulting in patient HETEROZYGOUS for deletion of exons 5-7 of the COL5A1 gene. Presence of deletion was confirmed in affected familial members.

Conclusions: Vascular complications in nonvascular subtypes of the EDS could be life-threatening. - Misdiagnosis of child abuse should be considered in EDS - The relevance of study microdeletions in cases not diagnosed by gene sequencing.

P. Marin Reina: None. A. Zuñiga Cabrera: None. A. Parra Llorca: None. P. Molina Aguilar: None. J. Blasco: None. A. Monzo Blasco: None. M. Evole Buselli: None.


Follow-up findings and genotype-phenotype correlation in 18 Turkish patients with biallelic mutation in the FKBP10 gene

A. Yüksel Ülker1, D. Uludag Alkaya1, G. Yeşil2, G. Toksoy3, Z. Uyguner3, B. Tüysüz1

1Istanbul University, Cerrahpasa Medical Faculty, Istanbul, Turkey, 2Bezmialem Vakif University, Medical Faculty, Department of Medical Genetics, Istanbul, Turkey, 3Istanbul University, Medical Faculty, Department of Medical Genetics, Istanbul, Turkey

Introduction: Osteogenesis Imperfecta (OI)-typeXI and Bruck syndrome type-I are two very rare disorders caused by biallelic FKBP10 gene mutations. While both disorders are characterized by early onset bone fractures, progressive bone deformity and scoliosis, Bruck syndrome type 1 also has congenital contractures and pterygium. FKBP10/KRT14 mutations have been reported to be present in patients with OI-typeXI and Epidermolysis bullosa simplex (OIEBS). The aim of this study is to investigate the clinical findings and genotype-phenotype correlation in the Turkish cohort with FKBP10 mutation.

Materials and Methods: Eighteen patients from fifteen families were enrolled. FKBP10 mutations studied via sanger sequencing or next generation sequencing. The parents of all patients had consanguineous marriages. 16 patients were followed up for 6.2 years (7 months-15.5 years).

Results: We identified p.Gly300Ter in eleven patients and p.Gly278Argfs*95 in one patient who live same region. Ten of these patients were compatible with OI-XI, two patients had Bruck1 phenotype. One patient with Bruck1 who lives different region had a novel (c.1256+1G>A) mutation. We also found same mutation (c.321_353del) in five patient with OIEBS who lives same city. Pamidronate treatment was given to 16 follow-up patients.

Conclusions: The clinical course of the patients was progressive; bone fractures were started in the infantile period (15/18) and continued as more than 1 fracture (9/16) per year. During the follow-up period, severe limb deformity (7/16) and scoliosis (11/16) developed and no patient could walk (16/16). Although most families are not related, they had the same mutation which is attributed to the founder effect.

A. Yüksel Ülker: None. D. Uludag Alkaya: None. G. Yeşil: None. G. Toksoy: None. Z. Uyguner: None. B. Tüysüz: None.


Wide sutures and fontanels, delayed speech development and a hoarse voice useful signs in diagnosing KBG syndrome, clinical, molecular and cytogenetic description of 12 patients.

A. Kutkowska-Kazmierczak1, M. Gos1, J. Castaneda1, M. Boczar1, A. Barczyk1, A. Pietrzyk1, O. Kordowska1, T. Gambin1, P. Własienko1, B. Wiśniowiecka-Kowalnik1, K. Kowalczyk1, M. Bartnik-Głaska1, M. Smyk1, M. Paczkowska1, I. Plaskota1, K. Niepokój1, J. Kelly2, B. Nowakowska1, E. Obersztyn1, J. Bal1

1Institute of the Mother and Child, Warsaw, Poland, 2Quest Diagnostics Nichols Institute, Chantilly, VA, United States

Introduction: KBG syndrome is a neurodevelopmental disorder characterized by short stature, macrodontia, developmental delay, behavioral problems as hyperactivity, velopharyngeal insufficiency causing feeding problems and speech delay and by delayed closing of sutures and fontanels. Most of the patients with KBG syndrome have a mutation of ANKRD11 gene or micro-rearrangement encompassing this gene.

Patients and methods: We present clinical evaluations of 12 patients with the clinical features of KBG syndrome at the age from 4 months to 17 years. The mutation analysis in these patients was performed using panel next generation sequencing (6pts), whole exome sequencing (WES) (2pts) and array CGH (4pts).

Results: Besides typical dysmorphic features for KBG syndrome found in all patients, the majority of patients had wide sutures and fontanels (7/12), delayed speech (9/11), feeding problems (9/12). Hoarse voice was noticed in 4 patients and short stature, below 2 SD in two patients, between -1-2 SD in 7 patients. Abnormal behavior was noted in 4 patients, macrodontia in 3 patients.

Conclusions: The fact that the delayed closing of sutures and fontanels was observed in more than half of our patients with KBG syndrome caused by mutations or rearrangements of ANKRD11 gene confirms the role of this gene in skull and suture formation. Speech delay is found in majority of patients with this syndrome. Rarely described sign - hoarse voice could reinforce clinical knowledge in diagnosing KBG syndrome. The studies on craniosynostoses were supported from Institute of Mother and Child in Warsaw intramural grant no. OPK-510-18-63

A. Kutkowska-Kazmierczak: None. M. Gos: None. J. Castaneda: None. M. Boczar: None. A. Barczyk: None. A. Pietrzyk: None. O. Kordowska: None. T. Gambin: None. P. Własienko: None. B. Wiśniowiecka-Kowalnik: None. K. Kowalczyk: None. M. Bartnik-Głaska: None. M. Smyk: None. M. Paczkowska: None. I. Plaskota: None. K. Niepokój: None. J. Kelly: None. B. Nowakowska: None. E. Obersztyn: None. J. Bal: None.


Patients with suspected Erythrodermia variabilis of a large family carry variants in GJB3 and KRT10

A. Hotz1, S. Redler2, J. Kopp1, A. Zimmer1, J. Fischer1

1Institute of Human Genetics, University Medical Center Freiburg, Faculty of Medicine, Freiburg, Germany, 2Institute of Human Genetics, Heinrich-Heine-University, Medical Faculty, Düsseldorf, Germany

Introduction: Erythrokeratoderma variabilis (EKV) is a rare skin disease characterized by two features: red skin areas with sharp borderlines, which tend to shift positions; and hyperkeratosis, which can either be generalized or localized as thickened plaques. EKV is mostly inherited in an autosomal dominant manner and is mainly caused by pathogenic variants in GJB3, GJB4 or GJA1.

Results: We describe a large family with twelve patients of five generations affected by a skin disorder. The index patient shows typical features of EKV. Mutation analysis in seven affected members from three generations using Next Generation and Sanger sequencing methods revealed that all of them carry both the heterozygous unclear variant c.783G>C, p.(Gln261His) in GJB3 and the novel heterozygous pathogenic variant c.1346A>C, p.(Tyr449Ser) in KRT10. Pathogenic variants in KRT10 lead to Epidermolytic ichthyosis, which is characterized by diffuse erythroderma and blistering at birth and progressive hyperkeratosis later in life and is usually inherited in an autosomal dominant manner. Furthermore, two unaffected members were analyzed, one of them carries none of the variants, the other only the GJB3 variant.

Conclusions: It is remarkable, that all seven tested affected family members from three generations carry both variants in GJB3 and KRT10. Although the KRT10 variant is pathogenic, it cannot be excluded that the GJB3 variant has an impact on the phenotype, since the initial suspected diagnosis was EKV. However, it cannot be excluded that the GJB3 variant is neutral. In this case, analysis of patients with suspected EKV should include pathogenic variants in KRT10.

A. Hotz: None. S. Redler: None. J. Kopp: None. A. Zimmer: None. J. Fischer: None.


When textbooks fall short - earlier detection of Hypochondroplasia - a large UK cohort experience and systematic review.

A. Sabir1, E. Morley2, J. Sheikh2, A. Singh2, A. Cocca1, M. Cheung1, M. Irving1

1Guy’s and St Thomas’ Hospital, London, United Kingdom, 2University of Birmingham, Birmingham, United Kingdom

Introduction: Hypochondroplasia (HCH) is a rare autosomal dominant skeletal dysplasia caused by FGFR3 mutations leading to disproportionate short stature. Classically HCH presents in toddlers or school-age children, as limb-to-trunk disproportion is often mild and easily overlooked during infancy. We report experiences from our UK HCH cohort of ~30 patients and review recent literature to explore the antenatal detection of HCH.

Methods: We reviewed the rate of antenatal HCH detection in our 29 patient cohort and describe relevant case-date. We then conducted a systemic, retrospective literature review using PUBMED and MEDLINE, identifying antenatally suspected / detected HCH cases. Paper inclusion criteria: Antenatally suspected HCH. Molecular confirmation of denovo HCH. Exclusion criteria: unclear antenatal scanning history.

Results: Antenatally suspected (and concurrent or deferred molecularly confirmation) HCH has been reported 10 times in the literature (between 2004-2019) and in 6 of our 29 patient cohort. Key sonographic markers included reduced; femur length, biparietal diameter and humeral length.

Conclusions: HCH is increasingly detected both antenatally and in infancy, contrary to previous descriptions. This is likely due to greater HCH awareness, improved imaging and easier molecular testing. Thus one should consider HCH outside the classical presenting period. Studying the natural history of earlier cases is important with the advent of several targeted FGFR3 therapies currently in trials for Achondroplasia (Biomarin’s Vosorotide, Ascendis’ Transchon, QED’s Infagratinib and Pfizer’s TA-46) as such therapies may soon be trialled in HCH and are likely most efficacious during earliest skeletal maturation.

A. Sabir: None. E. Morley: None. J. Sheikh: None. A. Singh: None. A. Cocca: None. M. Cheung: None. M. Irving: None.


Mono-allelic deleterious variants in autosomal recessive skeletal dysplasia genes are strongly associated with idiopathic short stature

C. T. Thiel1, N. Hauer1, C. Vogl1, S. Uebe1, H. Sticht2, A. B. Ekici1, C. Kraus1, H. Dörr3, A. Reis1

1Institute of Human Genetics, Friedrich-Alexander-Universität Erlangen-Nürnberg FAU, Erlangen, Germany, 2Institute of Biochemistry, Friedrich-Alexander-Universität Erlangen-Nürnberg FAU, Erlangen, Germany, 3Department of Pediatrics and Adolescent Medicine, Friedrich-Alexander-Universität Erlangen-Nürnberg FAU, Erlangen, Germany

Short stature is a common concern in childhood with an incidence of 3%. After exome analysis about 60% remain without diagnosis (idiopathic short stature, ISS). Recent identification of heterozygous mutations in ACAN in 1.4% of ISS individuals confirmed that single, large-effect variants strongly affect height (appr. 15cm final adult height). We therefore hypothesized that heterozygous variants in other autosomal recessive skeletal dysplasia genes also contribute to ISS. We selected exome data from 368 ISS families where previous analysis showed no mutations in a known growth-associated gene and selected 114 genes known to be involved in autosomal recessive inherited skeletal dysplasia, extracted from the “Nosology and Classification of Genetic Skeletal Disorders” (Bonafe et al. 2015). From a total of 391 heterozygous variants we selected 33 variants in 26 genes which were classified as ACMG class 4/5 and compatible with segregation in the families and were thus considered strong candidates for ISS. This accounted for 31 patients (8.4%). No class 4/5 variant in any of the 26 genes was detected in 343 controls (p<1e-85). Frequently affected genes were ACAN (6 individuals) and NPR2 (3 individuals) representing 2.5% of the study group. In addition to missense variants in 17 genes, a nonsense variant was observed in ADAMTS10, CHST3, CYP27B1, TCIRG1, TRIP11, WDR35 and CUL7. Our findings suggest a strong association between rare mono-allelic deleterious variants in genes for recessive skeletal dysplasia and idiopathic short stature and provide further evidence for the contribution of rare, large-effect variants to the etiology of this common trait.

C.T. Thiel: None. N. Hauer: None. C. Vogl: None. S. Uebe: None. H. Sticht: None. A.B. Ekici: None. C. Kraus: None. H. Dörr: None. A. Reis: None.


IRAK1 gene SNPs rs1059703 and rs3027898 polymorphisms is associated with psoriasis

L. Kucinskas1, A. Bruzaite1, J. Cepaite1, V. Kucinskiene2, R. Ugenskiene1, S. Valiukeviciene2

1Institute of Biological System and Genetic Research LUHS, Kaunas, Lithuania, 2Department of Skin and Venereal Diseases, Lithuanian University of Health Sciences (LUHS), Hospital of LUHS Kauno Klinikos, Kaunas, Lithuania

Psoriasis vulgaris and psoriatic arthritis are multifactorial diseases with an important genetic component. The clinical significance of IRAK1 genes SNPs rs1059703 ir rs3027898 are unknown for psoriasis pathogenesis. The aim of present study was to determinate IRAK1 gene SNPs rs1059703 and rs3027898 clinical importance for psoriatic arthritis and psoriasis development.Methods. 106 patients with psoriasis from Skin and Veneral disease department of Lithuanian University of Helath Sciences hospital and 52 healthy control group subjects were genotyped. The IRAK1 gene SNPs rs1059703 and rs3027898 genotyping was performed using Mva1269I (BSMI) and Eco57I (AcuI) restriction enzymes. Data and statistical analysis was performed using, Microsoft Office Excel“ and, IBM SPSS Statistics 20.0“ programs.Results. The number of patients of IRAK1 gene rs3027898 with C/A, C/C and C genotypes outnumbered control group (32 persons (30 %) vs 5 persons (10 %). The number of patients of IRAK1 rs1059703 with C/T, C/C and C genotype was higher compare with control group (30 persons (30 %) vs 6 persons (11 %). The statistical analysis of patients genotypes and psoriatic arthritis corelation showed no significant data of association (p < 0,05). Odds of ratio calculation (OR) showed IRAK1 rs3027898 C/A, C/C, C and IRAK1 rs1059703 C/T, C/C and C genotypes association with psoriasis (OR 4,06) and (OR 3,39) respectively.Conlusions. Statistically significant association of IRAK1 rs3027898 and IRAK1 rs3027898 SNPs with psoriatic arthritis was not determinated. IRAK1 rs3027898 C/A, C/C, C genotypes and IRAK1 rs1059703 C/T, C/C and C genotypes are associated with psoriasis development.

L. Kucinskas: None. A. Bruzaite: None. J. Cepaite: None. V. Kucinskiene: None. R. Ugenskiene: None. S. Valiukeviciene: None.


Skin cancer screening is indicated in IWC patients due to the identification of heterozygous KRT10 variants in patients with MAUIE syndrome

B. Burger1, A. Ghosh1,2, C. K. Y. Ng3, S. Piscuoglio1, I. Spoerri1, P. H. Itin1, K. Greer4, D. Elbaum5

1University of Basel and University Hospital Basel, Basel, Switzerland, 2University of Zurich and ETH Zurich, Zurich, Switzerland, 3University of Bern, Bern, Switzerland, 4University of Virginia, Charlottesville, VA, United States, 5Mercy Hospitals of Bakersfield, Bakersfield, CA, United States

Ichthyosis with confetti (IWC) is an autosomal dominant skin disorder characterized by a congenital erythema and the development of numerous pale spots. About 20% of the patients are reported to develop skin cancer at young adulthood. All IWC patients are carriers of a heterozygous keratin (KRT)10 or KRT1 variant leading to a shifted reading frame and the translation of an aberrant carboxyl (C)-terminus. This results in the nuclear localization of the KRT10/KRT1 protein instead of cytoplasmic. About 20 years ago, two patients were described as MAUIE patients. They showed the typical IWC-phenotype including the development of pale spots. Both patients developed aggressive and fatal cutaneous squamous cell carcinoma (cSCC). A genetic analysis is not yet done. Our study aimed to identify an IWC-correlated frameshift variant in both patients and to clarify whether they suffered from IWC. We performed whole exome sequencing on genomic DNA from FFPE cSCC tissue of both deceased patients and screened for variants similar to IWC-associated variants. A heterozygous KRT10 variant, encoding for the aberrantly shifted C-terminus, was identified in each patient and the nuclear localization of KRT10 was confirmed by immunofluorescence staining. Our study reclassified both MAUIE patients as patients with a specific form of IWC. The fatal course of the disease is not typical for the other IWC forms, but it emphasizes the importance of skin cancer screening in all IWC-patients. We recommend the regular intensive examination of their skin for cancers and a good education of the patients with reference to their disease.

B. Burger: None. A. Ghosh: None. C.K.Y. Ng: None. S. Piscuoglio: None. I. Spoerri: None. P.H. Itin: None. K. Greer: None. D. Elbaum: None.


Kinesin family member 13A, KIF13A, is a novel gene for hypomelanosis of Ito


The University of Hong Kong, Hong Kong, China

Introduction: Hypomelanosis of Ito is a neurocutaneous syndrome, characterized by distinctive skin manifestations, with or without multisystem involvements. While the exact cause of hypomelanosis of Ito is unknown, chromosomal mosaicism as a result of sporadic gene mutation has been suggested in literature.

Materials and Methods: In our Clinics for Rare diseases Referral (CRareDr), we encountered a 3-year-old girl presenting with characteristic skin pigmentation and developmental delay, suggesting hypomelanosis of Ito This case was enrolled in our undiagnosed diseases program (UDP) for ending diagnostic odyssey. We initiated genetic analysis for neurodevelopmental impairment using whole-genome (WGS) and whole exome sequencing (WGS).

Results: A heterozygous de novo variant NM_022113.6: c.2357dupA; p.Asn786Lysfs*18 in the KIF13A gene was identified in the patient. The variant is a single nucleotide duplication causing a shift in the translational reading frame and premature termination of the protein. The variant is absent in control population in gnomAD exomes and genomes.

Conclusions: KIF13A is a relatively new gene with a limited number of studies on its function and has not been reported to cause any hereditary conditions. To date, KIF13A is known to interact with adaptor complex AP-1, forming a complex that is responsible for the transportation of a number of proteins and receptors to plasma membrane and endosomal organelles. The role of KIF13A in melanosome biogenesis is well-demonstrated in in-vitro studies and may explain the underlying pathogenesis of skin hypopigmentation in hypomelanosis of Ito.

C. Lam: None.


Functional evidence supporting the hypothesis that Neanderthals and Denisovans had a genetically specified high bone mass negatively selected in anatomically modern humans

N. Roca-Ayats1, I. Maceda2, C. Bruque3, M. Cozar1, N. Garcia-Giralt4, L. Mellibovsky4, W. van Hul5, O. Lao2, D. Grinberg1, S. Balcells1

1University of Barcelona, CIBERER, IBUB, IRSJD, Barcelona, Spain, 2Centre Nacional d’Anàlisi Genòmica CNAG-CRG, Barcelona, Spain, 3Centro Nacional de Genética Médica, ANLIS, Instituto de Biología y Medicina Experimental, CONICET, Buenos Aires, Argentina, 4Musculoskeletal Research Group, IMIM (Hospital del Mar Medical Research Institute), Centro de Investigación Biomédica en Red de Fragilidad y Envejecimiento Saludable (CIBERFES), ISCIII, Barcelona, Spain, 5Department of Medical Genetics, University of Antwerp, Antwerp, Belgium

Introduction: Bone density underwent gracilization through hominin evolution; while Neanderthals and Denisovans had robust and dense bones, anatomically modern humans (AMH) show a more gracile skeleton. In this context, mutations in the first β-propeller of LRP5 result in high bone mass (HBM) in AMH, suggesting this gene as a suitable candidate for studying the hominin evolution of bone mass.

Material and Methods: Variants were looked for in the publically available Neanderthal and Denisovan genomes and functionally tested by a luciferase assay in Saos-2 cells. A molecular homology model of LRP5 was generated using MODELLER (v.9.22). Introgression was inferred in the Simons Genome Diversity Project using the CRF method. Evidences of selective pressures at LRP5 in AMH were sought using PopHuman browser.

Results: We identified five Neanderthal or Denisovan missense mutations in LRP5. Four displayed significantly greater Wnt pathway stimulation, and two also showed reduced inhibition by DKK1. Modelling suggested that these mutations either affected the 3D structure of the first β-propeller, or impinged on the interaction of LRP5 with DKK1. We observed that the LRP5 genomic region in AMH is depleted of introgression from Neanderthals and/or Denisovans and that the functional Neanderthal and Denisovan mutations are at a very low frequency in current human populations.

Conclusions: The variants identified in Neanderthal and Denisovan genomes, together with the evolutionary analyses in the LRP5 region suggest that this gene may have been involved in HBM determination in archaic hominins.

Funding: Spanish MINECO (SAF2016-75948-R; PGC2018-098574-B-I00); CIBERER (U720); AGAUR (2017 SGR 937).

N. Roca-Ayats: None. I. Maceda: None. C. Bruque: None. M. Cozar: None. N. Garcia-Giralt: None. L. Mellibovsky: None. W. van Hul: None. O. Lao: None. D. Grinberg: None. S. Balcells: None.


Looking for genetic modifiers in Marfan syndrome

E. Ramos-Luis1,2, G. Teixidó3, J. Limieres3, R. Cruz4,2, C. Granato3, B. Sobrino5,2, J. Amigo5,2, A. Blanco-Verea1,2, A. Carracedo5,2,4, A. Evangelista3, M. Brion1,2

1Xenética Cardiovascular, Instituto de Investigación Sanitaria de Santiago, Santiago de Compostela, Spain, 2Grupo de Medicina Xenómica, Universidade de Santiago de Compostela, Santiago de Compostela, Spain, 3Hospital Vall d’Hebron, Servicio de Cardiología, Barcelona, Spain, 4CIBER enfermedades Raras, Santiago de Compostela, Spain, 5Fundación Pública Galega de Medicina Xenómica, Santiago de Compostela, Spain

Introduction: Marfan syndrome is an autosomal dominant disorder of connective tissue manifested in the ocular, skeletal and cardiovascular systems. It is most frequently caused by mutations in FBN1 with high clinical penetrance, but showing a high inter and intra-familial variability, that cannot be explained by the current knowledge of genotype-phenotype correlation. In order to improve knowledge of the whole genetic background of this disease, this study aims to identify genetic modifiers of the phenotypic expression of Marfan syndrome, looking for the intra-familial variability with known causal mutation in FBN1.

Material and Methods: A total of 22 Marfan syndrome patients with different severity from 8 families with known FBN1 mutation, were exome sequenced. For exome wide gene-set analyses F-SKAT (familial sequence kernell association test) was performed. At the intra-family level, non-common rare protein changing variants among first-degree relatives with severe and mild phenotypes were selected for discriminant analysis of principal components (DAPC).

Results: F-SKAT test showed no gene with significant differences after Bonferroni test between severe and mild phenotype group of patients. Nevertheless, DAPC showed two genes of retinal homeostasis, NXNL1 gene and RP1L1 gene, as one of the principal contributors to the discrimination analysis between mild and severe phenotype.

Conclusions: The genetic architecture of Marfan syndrome is very complex, however, as it was previously described in the literature, this study point to the genes related to retinal homeostasis as possible genetic modifiers of the disease. Founding: Instituto de Salud Carlos III and Fondo Europeo de Desarrollo Regional (PI14/01062, PI16/00903)

E. Ramos-Luis: None. G. Teixidó: None. J. Limieres: None. R. Cruz: None. C. Granato: None. B. Sobrino: None. J. Amigo: None. A. Blanco-Verea: None. A. Carracedo: None. A. Evangelista: None. M. Brion: None.


Reciprocal translocation as a possible cause of familial connective tissue disorder

B. Herr, K. Komlosi, A. Tzschach, J. Fischer, B. Gläser

Department of Human Genetics, Freiburg, Germany

Introduction: Monogenic disorders due to apparently balanced reciprocal translocations are rarely reported. There are few reports of interstitial deletions involving FBN1-gene and only one case of a patient with Marfan syndrome and a complex chromosome rearrangement. However, no disruption of FBN1 due to a translocation has been reported so far.

Clinical report: We report on a mother and daughter with clinical symptoms of connective tissue disorder. The 39-year-old mother presented with congenital strabismus, muscle weakness, muscle hypotrophy of forearms and calves, reduced exercise tolerance and muscle and joint pain. Skeletal manifestations included arachnodactyly, joint laxity and foot deformity. Her father and a paternal uncle had skeletal abnormalities and died unexpectedly in young adulthood. Her 18-year-old daughter fulfilled the Ghent criteria for Marfan syndrome.

Methods and Results: Connective tissue panel diagnostics did not reveal pathogenic variants in the mother. R-banded karyotyping of lymphocyte cultures revealed the same reciprocal translocation in mother and daughter: 46,XX,t(2;15)(q22;q21.1). Chromosome microarray analysis revealed no imbalances. Subsequent FisH analysis with region-specific BAC probes for 2q and 15q narrowed down the translocation break point to a region of approximately 110 kb from g.43663778 proximal the 3`end of FBN1 to g.48772449 in intron 32-33 of FBN1.

Conclusions: In order to prove a possible disruption of FBN1 we plan to perform long range PCR, next generation mapping and RNA sequencing. We emphasize the importance of a combination of cytogenetic and molecular diagnostic techniques to reveal the cause of monogenic diseases typically caused by point mutations.

B. Herr: None. K. Komlosi: None. A. Tzschach: None. J. Fischer: None. B. Gläser: None.


Variant classification in Marfan syndrome: illustrating an underestimated issue in DNA-diagnostics

G. Pals1,2,3, S. A. Stolk1, A. Maugeri1, D. Micha2, M. A. M. M. Mannens1

1Amsterdam UMC, Genome Diagnostics laboratory, Amsterdam, Netherlands, 2Centre for Connective tissue disorders, Amsterdam, Netherlands, 3Prodia laboratories, Jakarta, Indonesia

Introduction: Correct classification is crucial for reliable diagnostic DNA-testing of patients. The classification of variants ranges from benign (class 1) to pathogenic (class 5). General recommendations for classification have been developed, like the American College of Medical Genetics (ACMG). Variant classification differs per method used. We compared three different guidelines, by reanalyzing patients’ data of the FBN1 gene.

Materials and Methods: Variants in the FBN1 gene, found in our lab, were used to compare the outcome of three different variant classifications: the ACMG guidelines, the methods of our accredited laboratory (ISO15189-RvA-M174) and recommendations from the Revised Ghent nosology of Marfan syndrome (Loeys et al. 2010).

Results: We classified, by using our guidelines, 869 variants as pathogenic. 77 of these are classified as benign according to Loeys et al. or VUS according to ACMG. A large number of variants that we classified as benign would be classified as likely benign or VUS according to the other guidelines.

Conclusions: Comparison of various variant interpretations shows that pathogenic variants were classified as VUS or even benign by using the other guidelines. This was mainly caused by using an incorrect consensus sequence for the amino acid motif of the 47 EGF domains in fibrillin-1 (Loeys et al.) or not using specific properties of the protein. Different use of frequency data causes a large difference in interpretation of benign variants. These data emphasize that knowledge of the disease and the protein involved are indispensable in correct interpretation of variants.

G. Pals: None. S.A. Stolk: None. A. Maugeri: None. D. Micha: None. M.A.M.M. Mannens: None.


Identifying the molecular cause in Marfan and Marfan like phenotype in a cohort of patients from Romania - 5-year single center experience

M. Puiu1, A. Chirita-Emandi1, N. Andreescu1, G. Doros2, A. Popoiu2, A. Dumitrescu3, A. Lacatusu4, C. Zimbru5

1Genetics Discipline, Center of Genomic Medicine, “Victor Babes,” University of Medicine and Pharmacy, Timisoara, Romania, 2Pediatric Department University of Medicine and Pharmacy “Victor Babes”, Timisoara, Romania, 3Clinical Emergency Hospital for Children “Louis Turcanu”, Timisoara, Romania, 4“Victor Babes,” University of Medicine and Pharmacy, Timisoara, Romania, 5Department of Automation and Applied Informatics, Politehnica University, Timisoara, Romania

Background: Connective tissue disorder represent a group of life-threatening disorders. The prevalence of these diseases is relatively low, but Marfan, Ehlers-Danlos, and Loeys-Dietz syndromes are ubiquitous, affect both genders and involve several organs, particularly the cardiovascular system, eyes, and skeleton. Mortality generally results from cardiovascular complications, mainly aortic dissection. Identifying the molecular cause can lead to a different therapeutic management and the possibility for prevention of reoccurrence in the family.

Aim: We aimed to retrospectively evaluate the diagnostic yield in these group o patients and present the variants identified.

Methods: Using targeted Next Generation Sequencing (NGS) with a panel for 174 genes (TruSightCardio Illumina) related to cardiologic diseases we screened 52 patients (37 males/15 females) evaluated for a Marfan/Marfan like phenotype using Ghent nosology, between 2015-2020 at the Center for Genomic Medicine University of Medicine and Pharmacy Timisoara, Romania.

Results: Diagnosis was identified in 55.7% (29/52) patients, most(25) pathogenic/likely pathogenic variants were found in FBN1 gene, 3 splicesite, 5 frameshift and 17 missense variants, including two variants associated with neonatal Marfan Syndrome (NM_000138.4:c.3143T>C and c.3217G>A). Of these, 10 are previously unreported variants. Nine out of 52 patients originated from 3 families. Diagnosis in 3 other patients involved COL3A1, TGFBR1 and SLC2A10 genes.

Conclusions: This study broadens the spectrum of pathogenic variants in FBN1 gene, providing an update of the molecular basis of Marfan Syndrome in Romania.

M. Puiu: None. A. Chirita-Emandi: None. N. Andreescu: None. G. Doros: None. A. Popoiu: None. A. Dumitrescu: None. A. Lacatusu: None. C. Zimbru: None.


Characterization of the pathogenetic role of POLD1 gene in Mandibular hypoplasia, Deafness, Progeroid features and Lipodystrophy (MDPL) Syndrome

C. De Masi1, P. Spitalieri1, M. Murdocca1, V. Ferradini1, M. D’Adamo2, P. Sbraccia2, M. Sanchez3, I. Udroiu4, J. Marinaccio4, G. Novelli1, A. Sgura4, M. D’Apice5, F. Sangiuolo1

1Dep. of Biomedicine and Prevention, Tor Vergata University, Rome, Italy, 2Dep. of Systems Medicine, Tor Vergata University, Rome, Italy, 3Core Facilities Center - Section of Cytometry, Istituto Superiore di Sanità, Rome, Italy, 4Dep. of Science. "Roma Tre" University, Rome, Italy, 5Laboratory of Medical Genetics, Tor Vergata Hospital, Rome, Italy

MDPL defines a multisystem disorder identified in 22 patients worldwide and associated to mutations inPOLD1 gene, encoding the DNA polymerase δ, involved in DNA replication and repair. We identified an in-frame deletion (p.Ser605del), segregating for the first time as an autosomal dominantmutation, in a MDPL patient from whom dermal fibroblasts (HDFs) have been derived. Cells displayed in vitro abnormalities of nuclear envelope morphology, presence of micronuclei, a large number of autophagosomesand a significant accumulation of prelamin A, strongly linked to altered cellular proliferation and genomicinstability. DNA damage-induced treatment in MDPL-HDFs revealed a poor capacity of DNA repair, an arrestin G0/G1 phase transition, and a lower number of cells in the S phase. Moreover, Western Blot analysesshowed a nuclear reduction of POLD1 in MDPL-HDFs compared to WT in basal condition but a statisticallysignificant and persistent increase after cisplatin/1-Gy X-ray damage when compared to WT. Analysis of telomere length exhibited an increased rate of telomere shortening, accompanied by anaccumulation of unrepaired telomeric DNA damage. Furthermore, the evaluation of H2AX and TelomereInduced Foci after 1-Gy X-ray treatment enabled us to figure out that POLD1 repairs less efficiently DNAdamage and its activity is mainly associated with the homologous recombination phase. Finally, we also generated human induced pluripotent stem cells from patient’s fibroblasts showing thepeculiar presence of micronuclei. Knowing the mechanistic basis for the association of DNA damage and failed DNA repair with aging wouldopen up new therapeutic possibilities for this rare disease.

C. De Masi: None. P. Spitalieri: None. M. Murdocca: None. V. Ferradini: None. M. D’Adamo: None. P. Sbraccia: None. M. Sanchez: None. I. Udroiu: None. J. Marinaccio: None. G. Novelli: None. A. Sgura: None. M. D’Apice: None. F. Sangiuolo: None.


Molecular analysis of Polish patients with Mendelian Disorders of Cornification

K. Wertheim-Tysarowska1, K. Osipowicz2, A. Kutkowska-Kaźmierczak1, J. Sawicka1, M. Jackiewicz1, O. Kordowska1, M. Gos1, A. M. Rygiel1, K. Niepokój1, K. Woźniak2, C. Kowalewski2, J. Bal1

1Medical Genetic Department, Institute of Mother and Child, Warsaw, Poland, 2Department of Dermatology and Immunodermatology, Warsaw Medical University, Warsaw, Poland

Introduction: Mendelian Disorders of Cornification (MeDOC) comprise syndromic or non-syndromic heterogeneous disorders manifesting by skin hyperkeratosis and/or scaling. Mutations in approximately 40 different genes can cause MeDOCs, that can be inherited in autosomal recessive/dominant or X-linked patterns. Identification of molecular defect is essential for prognosis, proper medical interventions and genetic counseling. Nonetheless, genetic diversity, low incidence of majority of MeDOCs and overlapping phenotypes make diagnosis challenging. The aim of the study was to characterize molecularly Polish MeDOC patients.

Materials and methods: A group of 161 patients with clinical symptoms of MeDOC was enrolled to the study. Genetic analysis was performed using self-designed customized NGS panel. Sanger sequencing or MLPA were used for mutations verification.

Results: We identified full genotype in 125 (78%) patients. In 17 (11%) patients we didn’t detect any mutation, while in 19 (12%) we found mutation in one allele only, despite expected autosomal recessive inheritance. Overall, we detected mutations in 27 distinct genes. The 33 patients had mutations in more than one gene involved in skin barrier functioning: 13 of them had pathogenic mutations in two genes, while in remaining 20 cases variant of unknown significance was detected in at least one of the genes.

Conclusions: Our diagnostic strategy proved to be an efficient and sensitive diagnostic tool for MeDOC patients. Our data provide further information regarding molecular epidemiology of skin barrier defects and also focus on the presence of secondary mutations, which have important impact on genetic counseling and, presumably, may affect therapy. Supported by NCN:2014/13/D/NZ5/03304

K. Wertheim-Tysarowska: None. K. Osipowicz: None. A. Kutkowska-Kaźmierczak: None. J. Sawicka: None. M. Jackiewicz: None. O. Kordowska: None. M. Gos: None. A.M. Rygiel: None. K. Niepokój: None. K. Woźniak: None. C. Kowalewski: None. J. Bal: None.


CRISPR/Cas9-based analysis of a missense variant in Paxip1 suggests subtle effects on palatal process distance

N. Ishorst1,2, E. Z. Kvon2, Y. Zhu2, S. Tran2, I. Plajzer-Frick2, C. S. Pickle2, S. Hölzel1, A. Harrington2, J. Godoy2, J. A. Akiyama2, L. Selleri3,4,5,6,7, I. C. Welsh3, M. M. Nöthen1, K. U. Ludwig1, E. Mangold1, D. E. Dickel2, L. A. Pennacchio2,8,9, A. Visel2,8,10

1Institute of Human Genetics, University of Bonn, School of Medicine & University Hospital Bonn, Bonn, Germany, 2Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, Berkeley, CA, United States, 3Program in Craniofacial Biology, University of California at San Francisco, San Francisco, CA, United States, 4Institute of Human Genetics, University of California at San Francisco, San Francisco, CA, United States, 5Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California at San Francisco, San Francisco, CA, United States, 6Department of Orofacial Sciences, University of California at San Francisco, San Francisco, CA, United States, 7Department of Anatomy, University of California at San Francisco, San Francisco, CA, United States, 8U.S. Department of Energy Joint Genome Institute, Berkeley, CA, United States, 9Comparative Biochemistry Program, University of California Berkeley, Berkeley, CA, United States, 10School of Natural Sciences, University of California Merced, Merced, CA, United States

Nonsyndromic cleft lip with/without cleft palate (nsCL/P) is among the most common congenital malformations and has a multifactorial etiology. Common risk loci explain parts of the trait heritability, but a substantial fraction needs to be elucidated. Parts of the missing heritability could be explained by rare, penetrant mutations that might occur de novo in genes potentially involved in craniofacial development. A previous study by our group identified three nsCL/P susceptibility genes with rare recurrent de novo mutations (PAXIP1, MDN1, ANK1). The present study aims at assessing a contribution of the rare mutations found in these nsCL/P susceptibility genes to cleft development by using CRISPR/Cas9 generated mouse models. We created five knock-in mouse strains that carried human missense mutations in Paxip1 (1 mutation), Ank1 (1 mutation), and Mdn1 (3 different mutations). Embryos at embryonic day E18.5 were dissected and assessed for craniofacial anomalies using skeletal preparations and microscopic assessment. No craniofacial anomalies were observed for E18.5 embryos in the F0 generation in any of the five knock-in strains. In the F1 generation, 2/31 E18.5 pups carrying the knocked-in Paxip1 missense mutation in a heterozygous state presented with abnormal body posture, a broader snout and a subtle increase in distance between the palatal processes, suggesting a role of the missense variant in Paxip1 in craniofacial development. Further follow-up studies in mice homozygous for the variants and more precise phenotyping methods are ongoing.

N. Ishorst: None. E.Z. Kvon: None. Y. Zhu: None. S. Tran: None. I. Plajzer-Frick: None. C.S. Pickle: None. S. Hölzel: None. A. Harrington: None. J. Godoy: None. J.A. Akiyama: None. L. Selleri: None. I.C. Welsh: None. M.M. Nöthen: None. K.U. Ludwig: None. E. Mangold: None. D.E. Dickel: None. L.A. Pennacchio: None. A. Visel: None.


High-throughput screen for rare variants in candidate genes for orofacial clefting

B. Zametica1, F. Thieme1, J. Fazaal1, N. Ishorst1, E. Ongkosuwito2, C. Carels3, I. A. L. M. van Rooij4, E. Mangold5, K. U. Ludwig1

1Institute of Human Genetics, University of Bonn, School of Medicine & University Hospital Bonn, Bonn, Germany, Bonn, Germany, 2Department of Orthodontics and Craniofacial Biology, Radboud Institute for Molecular Life Sciences, Radboudumc, Nijmegen, Netherlands, 3Department of Human Genetics and Department of Oral Health Sciences, KU Leuven and University Hospitals KU Leuven, Leuven, Belgium, 4Department for Health Evidence, Radboud Institute for Health Sciences, Radboud university medical center (Radboudumc), Nijmegen, Netherlands, 5Institute of Human Genetics, University Hospital of Bonn, Bonn, Germany

Nonsyndromic cleft lip with/without cleft palate (nsCL/P) is a common birth defect with multifactorial etiology. Genome-wide association studies (GWAS) have revealed >40 risk loci that contribute to nsCL/P risk, and it has been suggested that ~30% of phenotypic variance is explained by common genetic variation. In the absence of surgery, nsCL/P is a detrimental phenotype, which implies a considerable effect on reproductive fitness. Thus, rare variants are likely to also contribute to nsCL/P etiology. First studies have suggested nsCL/P candidate genes that harbor causative rare variants, or a burden thereof. These studies included the analysis of established “syndromic” genes (n = 6 genes), genes with established role in craniofacial development (n = 5), and the identification of mutations in genes of the epithelial cadherin-p120-catenin complex segregating within multiply affected families (n = 5). However, independent replication is lacking. Here, we aimed at providing further genetic evidence for an involvement of these genes in nsCL/P etiology, through the identification of additional evidence for rare variants from a large Central-European cohort. Using single molecule molecular inversion probes (smMIPs), we obtained high-confident sequencing results for 14 of the 16 genes in 711 nsCL/P cases, 761 population-based controls, and 234 case-parent trios. At individual level, we identified four novel loss-of-function (LoF) variants (3 cases / 1 control). The variants observed in cases were LoF-mutations in CDH1, CTNND1 and MTRR. Further statistical analyses, including analyses of missense variants, burden and pedigree analyses, are currently ongoing and will help to further understand the etiology of nsCL/P.

B. Zametica: None. F. Thieme: None. J. Fazaal: None. N. Ishorst: None. E. Ongkosuwito: None. C. Carels: None. I.A.L.M. van Rooij: None. E. Mangold: None. K.U. Ludwig: None.


Phenotypic spectrum of NPR2 mutations: study of 12 patients with acromesomelic dysplasia, Maroteaux type and 8 patients with short stature.

C. Michot1, S. Prévost1, C. Haudry1, A. Tourré1, A. Dieux-Coeslier2, M. Mathieu-Dramard3, M. Rossi4, A. Toutain5, G. Baujat1, S. Rondeau1, V. Cormier-Daire1

1APHP - Hôpital Necker-Enfants Malades, Paris, France, 2CHU de Lille - Centre de Biologie Pathologie Génétique, Lille, France, 3CHU Amiens-Picardie - Site Sud, Amiens, France, 4CHU de Lyon HCL - GH Est-Hôpital Femme Mère Enfant, Bron, France, 5CHRU de Tours - Hôpital Bretonneau, Tours, France

Introduction: Acromesomelic dysplasia, Maroteaux type (AMDM, MIM 602875) associates disproportionate short stature, short limbs and extremities with cone-shaped epiphyses. AMDM is caused by biallelic mutations of NPR2 (natriuretic peptide receptor B). Heterozygous NPR2 mutations are involved in idiopathic or dyschondrosteosis-like short stature.

Materials and methods: Our study reviewed the clinical and molecular findings of AMDM and short stature patients with NPR2 mutations. 12 AMDM DNAs and more than 200 short stature DNAs were tested, after SHOX MLPA testing, in the molecular biology laboratory of Necker Hospital.

Results: NPR2 mutations were identified in all AMDM cases and in 8 short stature patients. 23 different mutations were identified: nonsense, frameshift, splice and missense ones in AMDM and mostly missense variants in short stature. Heterozygous parents of AMDM patients were relatively small. All AMDM patients were of short birth and final heights (125-130 cm). Apart small extremities, 6 had large hallux and 7 had curved radius with limited pronation. Out of 12 cases, 8 had hyperlaxity, 7 scoliosis and 8 dental misalignment and arched palate. All heterozygous patients were of normal birth size and grew between -2,5 to -1,5 SD. They had normal envergure/height ratio, even if X-rays showed thick radius. 2/8 had IV and V brachymetacarpia.

Conclusions: our study supports AMDM genetic homogeneity. We underline the importance of specific assessments, including laxity, scoliosis and orthodontics. We confirm that NPR2 heterozygous mutations should be considered in short stature with minor skeletal changes. NPR2 function in C-natriuretic peptide signaling might lead to therapeutics options.

C. Michot: None. S. Prévost: None. C. Haudry: None. A. Tourré: None. A. Dieux-Coeslier: None. M. Mathieu-Dramard: None. M. Rossi: None. A. Toutain: None. G. Baujat: None. S. Rondeau: None. V. Cormier-Daire: None.


Non-syndromic cleft lip with/without cleft palate: Genome-wide association study in patients from The Netherlands and Belgium identifies a suggestive risk locus at 16p12.1 and supports SH3PXD2A as a clefting susceptibility gene.

I. van Rooij1, K. U. Ludwig2, J. Welzenbach2, N. Ishorst2, M. Thonissen3, T. E. Galesloot1, E. Ongkosuwito3, S. J. Bergé4, K. Aldhorae5, A. Rojas-Martinez6, L. Kiemeney7, J. Vermeersch8, H. Brunner9, N. Roeleveld1, K. Devriendt10, T. Dormaar11, G. Hens12, M. Kanpp13, C. Carels14,8, E. Mangold2

1Radboud university medical center, Radboud Institute for Health Sciences, Department for Health Evidence, Nijmegen, Netherlands, 2Institute of Human Genetics, University of Bonn, School of Medicine & University Hospital Bonn, Bonn, Bonn, Germany, 3Radboud Institute for Health Sciences, Department of Dentistry, Section of Orthodontics and Craniofacial Biology, Radboud university medical center, Nijmegen, Netherlands, 4Radboud university medical center, Department of Oral and Maxillofacial Surgery, Nijmegen, Netherlands, 5Orthodontic Department, College of Dentistry, Thamar University, Thamar, Zambia, 6Tecnologico de Monterrey, School of Medicine, and Universidad Autonoma de Nuevo Leon, Centro de Investigación y Desarrollo en Ciencias de la Salud, Monterrey, Mexico, 7Radboud university medical center, Radboud Institute for Health Sciences, Department for Health Evidence and Department of Urology, Nijmegen, Netherlands, 8Department of Human Genetics, KU Leuven, Leuven, Belgium, 9Radboud university medical center, Department of Human Genetics, and Donders Institute for Brain, Cognition and Behaviour, Nijmegen, Netherlands, 10Center for Human Genetics, University Hospitals Leuven, KU Leuven, Leuven, Belgium, 11Oral and Maxillofacial Surgery, University Hospitals Leuven, Leuven, Belgium, 12Department of Neurosciences, Experimental Otorhinolaryngology, KU Leuven, Leuven, Belgium, 13Institute of Medical Biometry, Informatics and Epidemiology, University of Bonn, Bonn, Germany, 14Orthodontics, University Hospitals Leuven, Leuven, Belgium

Nonsyndromic cleft lip with or without cleft palate (nsCL/P) ranks among the most common human congenital malformations. The etiology of nsCL/P is multifactorial, whereby genetic risk factors, environmental exposures, and potential gene-environment interactions all contribute to susceptibility. The present project is a genome-wide association study (GWAS) in a medium-sized nsCL/P cohort encompassing a total of 285 nsCL/P patients and 1,212 controls from The Netherlands and Belgium. Twenty of 40 previously reported nsCL/P susceptibility loci were replicated, and at the well-established nsCL/P susceptibility locus at chromosome 8q24.21, the analyses identified 62 genome-wide significant SNVs. These findings underline the validity of this sample. SNV-based analysis of the data identified a yet unreported suggestive locus at chromosome 16p12.1 (p-value of the lead SNV: 4.17×10-07). This association was replicated in two of three patient/control replication series (Central European and Yemeni). The lead SNV rs56383345 maps to a 930 kb non-coding region at 16p12.1, and is not located in any currently known regulatory element. Gene-based analysis of the GWAS data prioritized SH3PXD2A at chromosome 10q24.33 as a candidate gene for nsCL/P. To date, support for this gene as a cleft gene has been restricted to data from zebrafish and a knockout mouse model. The present GWAS is the first to implicate SH3PXD2A in non-syndromic cleft formation in humans. In summary, although performed in a relatively small sample, the present GWAS generated novel insights into nsCL/P etiology, and demonstrates how medium-sized, clinically well characterized GWAS samples can improve knowledge of the genetic basis of nsCL/P.

I. van Rooij: None. K.U. Ludwig: None. J. Welzenbach: None. N. Ishorst: None. M. Thonissen: None. T.E. Galesloot: None. E. Ongkosuwito: None. S.J. Bergé: None. K. Aldhorae: None. A. Rojas-Martinez: None. L. Kiemeney: None. J. Vermeersch: None. H. Brunner: None. N. Roeleveld: None. K. Devriendt: None. T. Dormaar: None. G. Hens: None. M. Kanpp: None. C. Carels: None. E. Mangold: None.


GWAS candidate genes for cleft lip with or without cleft palate: Analysis of expression data during embryonic craniofacial development.

A. Siewert1, B. Reiz2, J. Welzenbach1, H. Dickten2, K. U. Ludwig1

1Institute of Human Genetics, University of Bonn, School of Medicine & University Hospital Bonn, Bonn, Germany, 2FASTGenomics, Comma Soft AG, Bonn, Germany

Molecular defects during craniofacial embryonic development may result in nonsyndromic cleft lip with/without cleft palate (nsCL/P), one of the most common birth defects. Despite the possibility of surgical correction, follow-up treatment is long-term and requires an interdisciplinary approach. Also, epidemiological data indicate that individuals with nsCL/P show an increased risk for morbidities and suffer from a considerable psycho-social burden. The molecular pathways underlying these observations are currently unknown. We here used nsCL/P-GWAS data and array-based expression data from embryonic mice (gestational days E10.5-E12.5, Hooper et. al 2017), to identify pathways in which nsCL/P candidate genes are enriched. The 45 nsCL/P risk loci comprise ~400 genes within their topologically associated domains. Using gene-based analysis, we identified 86 genes with enrichment of common variants, suggesting their involvement in nsCL/P etiology. For each of these genes, we analyzed the expression pattern in ectodermal or mesodermal tissues of mandibular, maxillary and fronto-nasal prominences, and performed hierarchical clustering. One of the three clusters we identified comprised 33 genes, including established nsCL/P-genes like IRF6 and TFAP2a. A pathway analysis of genes within this cluster revealed a significant overrepresentation of cancer-related pathways and processes related to molecular signaling. Interestingly, affecteds with nsCL/P have been reported to be at increased risk of cancer. We will next extend our approach at single-cell level, using systematic data as recently provided (Cao et. al 2019) and the analytical ecosystem FASTGenomics (Scholz et. al 2018). This will generate novel insights into gene regulatory networks across development, and their contribution to nsCL/P.

A. Siewert: None. B. Reiz: A. Employment (full or part-time); Significant; Comma Soft AG. J. Welzenbach: None. H. Dickten: A. Employment (full or part-time); Significant; Comma Soft AG. K.U. Ludwig: None.


Severe Osteogenesis imperfecta with oligodontia: think of MESD

S. Moosa1,2,3, G. L. Yamamoto4,5,3, L. Garbes6, K. Keupp6, A. Beleza-Meireles7,8, C. A. Moreno9, E. R. Valadares10, S. B. de Sousa7,11, S. Maia7,12, J. Saraiva7,13, R. S. Honjo14, C. A. Kim15, H. Cabral de Menezes16, E. Lausch17, P. V. Lorini18,19, A. Lamounier Jr10, T. C. B. Carniero10, C. Giunta20, M. Rohrbach20, M. Janner21, O. Semler22, F. Beleggia23, Y. Li24, G. Yigit24, N. Reintjes25, J. Altmüller26, P. Nürnberg26, D. P. Cavalcanti9, B. Zabel17,27, M. L. Warman28, D. R. Bertola14,5, B. Wollnik24,29, C. Netzer23,30

1Stellenbosch University, Tygerberg, South Africa, 2University Medical Center Göttingen, Göttingen, Germany, 3Boston Children’s Hospital and Harvard Medical School, Boston, MA, United States, 4Unidade de Genética, Instituto da Criança do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo, São Paulo, 05403-000, Brazil;, Brazil, 5Instituto de Biociências da Universidade de São Paulo, São Paulo, Brazil, 6Institute of Human Genetics, University Hospital of Cologne and Faculty of Medicine, University of Cologne, Cologne, Germany, 7Medical Genetics Unit, Hospital Pediátrico de Coimbra, Centro Hospitalar e Universitário de Coimbra, Coimbra, Portugal, 8Department of Clinical Genetics, St Michael’s Hospital, University Hospitals Bristol, Bristol, United Kingdom, 9Skeletal Dysplasia Group, Department of Medical Genetics, Faculty of Medical Sciences, University of Campinas, Campinas, Brazil, 10Hospital das Clínicas da Faculdade de Medicina da Universidade Federal de Minas Gerais, Belo Horizonte, Belo Horizonte, Brazil, 11University Clinic of Genetics, Faculty of Medicine, University of Coimbra, Coimbra, Portugal, 12University Clinic of Genetics, Faculty of Medicine, University of Coimbra,, Coimbra, Portugal, 13University Clinic of Pediatrics, Faculty of Medicine, University of Coimbra, Coimbra, Portugal, 14Unidade de Genética, Instituto da Criança do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo, São Paulo, Brazil, 15Instituto de Biociências da Universidade de São Paulo, São Paulo, Portugal, 16Unidade de Endocrinologia, Instituto da Criança do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo, São Paulo, Portugal, 17Division of Genetics, Children’s Hospital, University of Freiburg, Freiburg, Germany, 18Stellenbosch UniversityDivision of Genetics, Children’s Hospital, University of Freiburg, Freiburg, Germany, 19Institute of Human Genetics, University Hospital Halle, Martin Luther University Halle-Wittenberg, Halle, Germany, 20Connective Tissue Unit, Division of Metabolism, University Children’s Hospital Zurich, Zurich, Switzerland, 21Division of Pediatric Endocrinology, Diabetology and Metabolism, Department of Pediatrics, Inselspital, Bern University Hospital, Bern, Switzerland, 22Faculty of Medicine, University of Cologne, 50931 Cologne, Germany; Department of Pediatrics, University Hospital Cologne, Cologne, Germany, 23Institute of Human Genetics, University Hospital of Cologne, Cologne, Germany, 24Institute of Human Genetics, University Medical Center Göttingen, Göttingen, Germany, 25Institute of Human Genetics, University Hospital of Cologne, Cologne, Ghana, 26Cologne Center for Genomics, University of Cologne, Cologne, Germany, 27Medical Faculty of the University of Magdeburg, Magdeburg, Germany, 28Orthopaedic Research Laboratories, Department of Orthopaedic Surgery, Boston Children’s Hospital and Harvard Medical School, Boston, MA, United States, 29Cluster of Excellence, Multiscale Bioimaging: from Molecular Machines to Networks of Excitable Cells (MBExC), University of Göttingen, Göttingen, Germany, 30Faculty of Medicine, University of Cologne, Cologne, Germany

Osteogenesis imperfecta (OI) comprises a genetically heterogeneous group of skeletal fragility diseases. It is often clinically impossible to predict the underlying causative gene, thus necessitating broad multi-gene panel testing or exome sequencing. We report on five independent families with a progressively-deforming type of OI with the distinguishing feature of oligodontia. We identified four homozygous truncation or frameshift mutations in a novel gene for OI, namely MESD. MESD encodes an endoplasmic reticulum (ER) chaperone protein for the canonical Wingless-related integration site (WNT) signaling receptors LRP5 and LRP6. Because complete absence of MESD causes embryonic lethality in mice, we hypothesized that the OI-associated mutations are hypomorphic alleles since these mutations occur downstream of the chaperone activity domain but upstream of ER-retention domain. This would be consistent with the clinical phenotypes of skeletal fragility and oligodontia in persons deficient for LRP5 and LRP6, respectively. Further functional analysis showed that OI-associated MESD mutations produce hypomorphic alleles whose failure to remain within the ER significantly reduces but does not completely eliminate LRP5 and LRP6 trafficking. Since these individuals have no eye abnormalities (which occur in individuals completely lacking LRP5) and have neither limb nor brain patterning defects (both of which occur in mice completely lacking LRP6), we infer that bone mass accrual and dental patterning are more sensitive to reduced canonical WNT signaling than are other developmental processes. Multigene panels do not yet include MESD. Therefore, if an undiagnosed patient with severe OI and oligodontia presents, it may be prudent to think first of MESD.

S. Moosa: None. G.L. Yamamoto: None. L. Garbes: None. K. Keupp: None. A. Beleza-Meireles: None. C.A. Moreno: None. E.R. Valadares: None. S.B. de Sousa: None. S. Maia: None. J. Saraiva: None. R.S. Honjo: None. C.A. Kim: None. H. Cabral de Menezes: None. E. Lausch: None. P.V. Lorini: None. A. Lamounier Jr: None. T.C.B. Carniero: None. C. Giunta: None. M. Rohrbach: None. M. Janner: None. O. Semler: None. F. Beleggia: None. Y. Li: None. G. Yigit: None. N. Reintjes: None. J. Altmüller: None. P. Nürnberg: None. D.P. Cavalcanti: None. B. Zabel: None. M.L. Warman: None. D.R. Bertola: None. B. Wollnik: None. C. Netzer: None.


Adult osteopetrosis with osteonecrosis of the jaws and abnormal osteoclast resorption due to a LRRK1 splice site mutation

A. Howaldt1, A. Hennig1, T. Rolvien2, U. Roessler1, A. Knaus3, S. Boettger4, S. Geißler1, R. Oheim2, M. Amling2, H. Howaldt4, U. Kornak5

1Charité-Universitaetsmedizin Berlin, Berlin, Germany, 2University Medical Center Hamburg-Eppendorf, Hamburg, Germany, 3School of Medicine & University Hospital Bonn, Bonn, Germany, 4Justus Liebig Universität Gießen, Gießen, Germany, 5Universitaetsmedizin Göttingen, Göttingen, Germany

We report on a 34 year-old patient with sandwich vertebrae, platyspondyly, osteosclerosis of long bones, pathologic fractures, and anemia. In the third decade, he developed osteonecrosis of the jaws, which was progressive in spite of repeated surgical treatment. An iliac crest bone biopsy revealed an elevated trabecular density (BV/TV [%] 32.2 vs. 21.5 + 14.8) and the presence of hypermineralized cartilage remnants leading to an overall increase in mineralization (CaMean [wt%] 25.65 vs. 21.8). We also found large multinucleated osteoclasts with abnormal morphology and inadequate bone resorption. Although these findings were reminiscent of autosomal dominant osteopetrosis type 2, no mutations in CLCN7 were detected. Subsequent whole exome sequencing identified the novel homozygous splice-site mutation c.261G>A in the gene LRRK1. cDNA sequencing showed nearly complete skipping of exon 3 leading to a frameshift (p.Ala34Profs*33), very likely leading to a loss of function. LRRK1 encodes a protein kinase and its dysfunction can cause osteosclerotic metaphyseal dysplasia (OSMD; OMIM # 615198). Osteoclasts differentiated in vitro from the patient’s peripheral blood monocytes formed quickly and became unusually large. In resorption assays these cells formed large areas with superficial pseudo-resorption but excavated only few resorption pits (resorption pit surface surface [%] 1.3 + 0.6 vs. 15 + 3.6; p < 0.01). Phosphorylation of L-plastin was strongly reduced in patient-derived osteoclasts, thus corroborating a LRRK1 loss of function (0.26 + 0.1 vs. 1 + 0.08; p < 0.01). Our analysis indicates a strong overlap of LRRK1-related OSMD with other late-onset forms of osteopetrosis, but an exceptionally leaky osteoclast resorption.

A. Howaldt: None. A. Hennig: None. T. Rolvien: None. U. Roessler: None. A. Knaus: None. S. Boettger: None. S. Geißler: None. R. Oheim: None. M. Amling: None. H. Howaldt: None. U. Kornak: None.


The lrp5 knockout zebrafish as a model to study osteoporosis

P. J. Coucke, J. Bek, H. De Saffel, A. De Clercq, A. Boel, A. Willaert

Center for Medical Genetics, Gent, Belgium

Osteoporosis-pseudoglioma syndrome (OPPG) is an autosomal recessive disorder characterized by reduced bone mass and strength, caused by loss-of-function mutations in LRP5 (low-density lipoprotein receptor-related protein 5), a co-receptor in the WNT signaling pathway. Here we describe the first reliable genetically induced form of osteoporosis in the zebrafish in a lrp5 knockout (KO). Molecular analysis confirmed reduced lrp5 transcripts and absence of Lpr5 protein. Reduced survival was observed in lrp5 KO larvae after the onset of mineralization. The fraction of larvae that did survive, displayed delayed ossification as was shown via mineral staining. Subsequently, these fish were grown to adulthood and analyzed via µCT. This revealed decreased bone volume and decreased bone mineralization in the vertebral column, which are hallmark features of osteoporosis. Next, we performed CRISPR/Cas9 mediated mutagenesis targeted at lrp5 followed by F0 phenotype analysis (crispant screening). Miseq sequencing of the mosaic larvae revealed out-of-frame mutations in on average 90% of the reads. Consequently, we showed a similar skeletal phenotype in the F0 ‘crispants’ to that of the stable lrp5-/- larvae, showing that crispant screening in zebrafish is a promising approach to functionally screen a large set of osteoporosis candidate genes. In summary, we present lrp5−/− zebrafish as the first genetic osteoporosis zebrafish model that can be used as a screening platform for potential therapeutics. Additionally, we show that F0 crispant screening can be used for functional validation of candidate genes for osteoporosis as high rates of out-of-frame mutations in the lrp5 gene are sufficient to induce phenotypes.

P.J. Coucke: None. J. Bek: None. H. De Saffel: None. A. De Clercq: None. A. Boel: None. A. Willaert: None.


The p.R321C mutation in p62 protein, associated to Paget’s Disease of Bone, leads to a relocation of the protein in human osteoclasts

N. Gestoso-Uzal1,2, R. Usategui-Martín1,2, C. Gutiérrez-Cerrajero2,1, A. I. Turrión Nieves1,3, J. Del Pino-Montes1,3, R. González-Sarmiento2,1,4

1Institute of Biomedical Research of Salamanca (IBSAL), Salamanca, Spain, 2Molecular Medicine Unit, Department of Medicine, University of Salamanca, Salamanca, Spain, 3Rheumatology Service, University Hospital of Salamanca, Salamanca, Spain, 4Institute of Molecular and Cellular Biology of Cancer (IBMCC), University of Salamanca-CSIC, Salamanca, Spain

Background: The most important known genetic factor predisposing to Paget´s disease of bone (PDB) is mutation in SQSTM1 gene, which encodes p62 protein. We detected the c.961C>T SQSTM1 gene mutation in three PDB patients, that causes the p.R321C mutation. It has been reported that p.R321C mutation causes a blockade of autophagy and an activation of NF-kB pathway. The aim of this study was to characterize the effect of the p.R321C mutation in human osteoclasts.

Methods: Peripheral blood mononuclear cells (PBMCs) were isolated from blood samples for generating human osteoclasts. Cells were differentiated by adding M-CSF and RANKL for 3 weeks. Human osteoclasts were transiently transfected with total plasmid DNA (pCEFL-Flag-SQSTM1-321C and pCEFL-Flag-SQSTM1-321R constructs). Finally, we performed a co-immunofluorescence assay to detect Flag and p62.

Results and discussion: Our results showed that, while 321R-p62 protein remained scattered though the cytoplasm, 321C-p62 was concentrated in the periphery of osteoclasts. This relocation of p62 protein caused by p.R321C mutation could be a consequence of autophagy blockade, which has been reported that could have a crucial role in the etiology of PDB. The p.R321C mutation causes a blockade of autophagy and an activation of NF-kB pathway, therefore the alteration of the cell metabolism could be the cause of the relocation of p62 protein in human osteoclasts.

Conclusion: The p.R321C mutation in p62 protein leads to a relocation of the protein in human osteoclasts. This reinforces the hypothesis that an alteration of autophagy could be involved in PDB etiology.

This work was supported by FIS-FEDER:PI16/01920.

N. Gestoso-Uzal: None. R. Usategui-Martín: None. C. Gutiérrez-Cerrajero: None. A.I. Turrión Nieves: None. J. Del Pino-Montes: None. R. González-Sarmiento: None.


Penttinen syndrome and infantile myofibromatosis in a patient carrying the tyrosine kinase-activating variant Asn666Ser in PDGFRB

D. Horn1, A. T. Abad-Perez1, V. Stephan2

1Institute of Medical Genetics and Human Genetics, Charité-Universitätsmedizin, Berlin, Germany, 2Sana Children´s Hospital Lichtenberg, Berlin, Germany

Introduction: Penttinen syndrome belongs to the group of progeroid disorders and is characterized by progressive subcutaneous lipoatrophy, sclerotic skin lesions and acro-osteolysis. This very rare condition is caused by a few distinct missense mutations of PDGFRB, other distinct mutations of this gene have been shown to be associated with different conditions such as infantile myofibromatosis.

Material and Methods: Here, we describe a 5-year-old male patient with infantile myofibromatosis diagnosed in his first month of life who in addition developed a prematurely aged appearance with lipoatrophy, scar-like skin nodules on the hands and joint contractures.

Results: Sanger sequencing revealed the heterozygous de novo mutation c.1997A>G; p.Asn666Ser in the PDGFRB gene. Recently, this mutation has been described in two patients affected with a severe form of Penttinen syndrome presenting a more pronounced connective tissue destruction.

Conclusions: This is the first report of an individual affected with both conditions, infantile myofibromatosis and Penttinen syndrome. This mutation lead to increased phosphorylation of downstream signalling proteins such as PTPN11. Imatinib is a strong inhibitor of phosphorylation of these targets representing a treatment option.

D. Horn: None. A.T. Abad-Perez: None. V. Stephan: None.


Blood secreted miRNA hsa-miR-374b-5p inhibits osteoblast-differentiating gene UHMK1 and is upregulated in periodontitis patients

R. Wiehe1, D. Bajric2, A. El Sharawy3, A. Keller4, H. Dommisch1, A. Schaefer1

1Charité-Universitymedicine Berlin, Institute for Dental and Craniofacial Sciences, Department of Periodontology and Synoptic Dentistry, Berlin, Germany, 2University Medical Center Schleswig-Holstein, Campus Kiel, Department of Operative Dentistry and Periodontology, Kiel, Germany, 3Institute for Clinical Molecular Biology, Christian-Albrechts-University Kiel, Kiel, Germany, 4Clinical Bioinformatics, Saarland University, Saarbrücken, Saarbrücken, Germany

microRNAs interact with the 3’UTRs of target mRNAs to suppress gene expression. MicroRNAs act tissue specifically but some are secreted into blood. Recent studies reported upregulation of specific miRNAs in the inflamed oral mucosa of periodontitis patients. The regulatory targets of these miRNAs may reveal genes that are conducive to oral health. If secreted into blood, these miRNAs could serve as biomarkers to diagnose periodontitis before clinical manifestation. To identify such miRNAs, we quantified 863 miRNAs in the blood of 18 periodontitis patients and 70 periodontitis-free controls (Geniom Biochip array). miRNA hsa-miR-421 and hsa-miR-374b-5p were most significantly upregulated in blood of periodontitis cases (Padjusted < 5x10−6). Two previous studies that profiled microRNAs in the inflamed and healthy oral mucosa independently reported >2fold upregulation of hsa-miR-374b-5p, but not hsa-miR-421. Using mirVana miRNA mimics (ThermoFisher Scientific), we overexpressed hsa-miR-374b-5p in primary gingival fibroblasts (3 biological and 3 technical replicates) and quantified the mRNA levels in 24h transfected and mock-transfected cells (Clariom D Array, ThermoFisher Scientific). The gene U2AF homology motif kinase 1 (UHMK1) was significantly downregulated in the miRNA mimic transfected cells (Fold Change= -1.8, P=0.00025). Two binding sites of hsa-miR-374b-5p were predicted at the 3’UTR of UHMK1. To validate the functional mechanism, we cloned the respective 3’UTR of UHMK1 into a reporter plasmid (pGL4.24, Promega). After co-transfection of the plasmid and hsa-miR-374b-5p mimic into HeLa cells for 24 hours, the reporter gene expression was significantly reduced (Fold Change= -1.5, p=0.02, 3 independent replicates). UHMK1 has a role in the coordination of osteoblast differentiation.

R. Wiehe: None. D. Bajric: None. A. El Sharawy: None. A. Keller: None. H. Dommisch: None. A. Schaefer: None.


POP1-Skeletal Dysplasias : Description of two new families.


CHU Liège, Liège, Belgium

Background: POP1 mutations should be considered in anauxetic dysplasia (AD) patients without RMRP mutations but also in skeletal dysplasia (SD) of different severities. To date, only 5 patients were reported.

Methods: Cartilage Hair Hypoplasia (CHH) was suspected in two siblings based on clinical and radiological evaluation. In family 1, a 4 years boy was investigated. His older sister is also affected by SD but currently refuses to pursue genetic testing. In family 2, two sisters presented a severe short stature : the older has 120 cm as final adult height. The second aged of 13 also suffered from an immune deficiency.

Results: A skeletal dysplasia gene panel revealed in the POP1 gene:Family N°1 : a composite heterozygous variant c.1846A>C, class3 (paternally inherited) and c.1747G>T, class 4 (maternally inherited). Family N°2 : a homozygous variant c.632G>A, class 4.

Discussion: POP1 patients are characterized by severe short stature of prenatal-onset and very short adult height (classically less than 1 meter, but our adult patients were 120 cm and near 160 cm), hypodontia, midface hypoplasia, mild intellectual disability and radiological specific findings including systematic bullet-shaped middle phalanges. Notably, none of the 5 published cases were reported with immune deficiency.

Conclusions: We describe two new families with POP1 mutations encountered in patients clinically suspected for CHH and achondroplasia, enhancing that the phenotypical spectrum is larger than AD type 2. Larger cohorts of patients are needed to improve the clinical characterization, especially the risk of having associated symptoms such as immune deficiency.

J. Harvengt: None. S. Alkan: None. B. Florkin: None. S. Bulk: None. V. Bours: None.


Coding variants of the CARD14 NF-kB adaptor are associated with palmar plantar pustulosis

A. Niaouris1, S. Haddad1, A. PushpaRajah2, N. Benzian-Olsson1, P. Baum3, S. Visvanathan4, T. he APRICOT and PLUM study team2, J. Barker2, C. Smith2, F. Capon1

1King’s College London, London, United Kingdom, 2St John’s Institute of Dermatology, Guy’s and St Thomas’ NHS Foundation Trust, London, United Kingdom, 3Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach, Germany, 4Boehringer Ingelheim Pharma GmbH & Co. KG, Ridgefield, CT, United States

Caspase recruitment domain containing protein 14 (CARD14) is a soluble NF-kB adaptor protein, which is mostly expressed in keratinocytes. In the presence of inflammatory stimuli, free CARD14 molecules oligomerise, recruit a BCL10/MALT1 signalosome and activate NF-kB signalling. Gain-of-function CARD14 variants have previously been associated with inflammatory skin diseases such as psoriasis vulgaris and generalised pustular psoriasis. However, the involvement of CARD14 in palmar plantar pustulosis (PPP), a localised form of pustular psoriasis, remains unclear. Here, we analysed CARD14 changes in 160 PPP cases (125 females, 35 males; mean age of onset: 41 years) ascertained across the UK. We identified 11 rare variants predicted to have a deleterious effect on protein function. A burden test (comparing PPP cases vs controls sequenced by the ExAC consortium) demonstrated a significant association between these changes and PPP (OR: 2.5; 95% CI: 1.39-4.65; p = 0.0023). We also detected a low-frequency allele with deleterious potential (E422K) that occurred more frequently in affected individuals compared to controls (OR: 1.7; 95% CI: 1.07-2.69; p= 0.025). To assess the effect of disease-associated variants, CARD14 FLAG-tagged plasmids were mutagenized and transfected into HEK293 cells. The depletion of free CARD14 from the cytoplasm (which is an indirect measurement of protein oligomerization) was then analysed by western blotting. This confirmed that most variants promote the formation of insoluble CARD14 oligomers. These findings implicate CARD14 in the pathogenesis of PPP. They also suggest that the proteins encoded by disease alleles are prone to constitutive oligomerization, which will cause abnormal NF-kB activation.

A. Niaouris: None. S. Haddad: None. A. PushpaRajah: None. N. Benzian-Olsson: None. P. Baum: None. S. Visvanathan: None. T. he APRICOT and PLUM study team: None. J. Barker: None. C. Smith: None. F. Capon: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Significant; 3 Boehringer Ingelheim Pharma GmbH & Co.


Further delineation of Roifman syndrome: report of four new Spanish patients

I. Valenzuela1, I. Cusco1, I. Paramonov1, F. López-Grondona1, S. Garcia-Miñaur2, M. Palomares-Bralo2, A. Martin1, J. Riviere1, M. Garcia-Prat1, P. Soler1, R. Colobran1, E. Garcia-Arumí1, E. Tizzano1

1Hospital Vall d’Hebron, Barcelona, Spain, 2Hospital La Paz, Madrid, Spain

Roifman Syndrome (RS, OMIM 300258) is a rare disorder characterized by pre- and postnatal growth retardation, cognitive delay, spondyloepiphyseal dysplasia, antibody deficiency and facial dysmorphism. Biallelic mutations in RNU4ATAC have been reported as the molecular cause. Our series expand the phenotypic spectrum of Roifman patients. We report four patients from three unrelated families with RS. Due to the complexity of variant annotation in RNU4ATAC, molecular studies are not usually straightforward. Indeed, in families 1 and 2 exome sequencing was inconclusive. In patient 1 the diagnosis was finally uncovered by genome sequencing, in sisters 2 and 3 by Sanger sequencing upon clinical suspicion and in patient 4 by a customized gene panel. Main clinical characteristics include skeletal dysplasia (4/4), short stature (4/4), intellectual disability (4/4), antibody deficiency (3/4) and retinal dystrophy (2/4). Main dysmorphic features included markedly long philtrum and thin upper lip. Hepatomegaly was identified in 2/4 and patient 1 presented hepatic cavernoma (unreported so far in Roifamn patients). Molecular results showed variants n.8C>T and n.50 G>A (patient 1); variant n.13C>T and n.17 G>C (sisters 2 and 3) and two novel variants, n.37G>A and n.53C>T (patient 4). Segregation analysis indicated that each variant was inherited from a healthy parent in the three families. The inclusion of new patients with Roifman increases the knowledge of mutational spectrum and it’s essential to delineate the phenotype of this syndrome. The recognition of the syndrome is indispensable for the orientation of molecular diagnosis, prompt follow up and counseling.

I. Valenzuela: None. I. Cusco: None. I. Paramonov: None. F. López-Grondona: None. S. Garcia-Miñaur: None. M. Palomares-Bralo: None. A. Martin: None. J. Riviere: None. M. Garcia-Prat: None. P. Soler: None. R. Colobran: None. E. Garcia-Arumí: None. E. Tizzano: None.


Identification of the deregulated NEK1 protein network in skeletal ciliopathies

C. Vogl1, K. Kessler1, A. Giessl2, P. Kirchner1, C. Büttner1, A. B. Ekici1, A. Reis1, C. T. Thiel1

1Institute of Human Genetics, FAU Erlangen-Nürnberg, Erlangen, Germany, 2Department of Ophthalmology, FAU Erlangen-Nürnberg, Erlangen, Germany

The primary cilium is an organelle, which is present on almost all vertebrate cells. Cilia consist of the axoneme and the basal body and present various ciliary membrane receptors, which receive extracellular signals. Those signals are transmitted into the cell, where they modulate different intracellular signaling pathways. Mutations in ciliary proteins are involved in formation, maintenance and function of the primary cilium and associated with developmental defects, like brain malformations, polydactyly, kidney cysts and skeletal abnormalities. This phenotypic spectrum is present among patients with short-rib polydactyly syndromes (SRPS). This group constitutes the most frequent lethal autosomal recessive osteochondrodysplasias.

Diallelic mutations in NEK1, encoding for a basal body protein, are the underlying cause of SRPS II. To gain further insight into the pathogenic mechanism, we used two approaches: RNA sequencing of CRISPR/Cas9 mediated genomic edited HEK293T cells and scRNASeq in limb buds of E13 embryos of the Nek1 knock down mouse, and respectively compared the knock out and the wildtype in order to find differentially expressed genes. Analysis of significantly enriched pathways revealed an activation of genes in early growth response and cell cycle control and silencing of different collagens and ribosomal and histone proteins. One of the main findings was a severely reduced expression of Acan, coding for Aggrecan the most abundant cartilaginous protein, explaining the severe growth phenotype.

These results underline that the NEK1 associated defect of the primary cilium causes the deficiency in cell cycle and gene expression, mainly aggrecan, in the growth plate.

C. Vogl: None. K. Kessler: None. A. Giessl: None. P. Kirchner: None. C. Büttner: None. A.B. Ekici: None. A. Reis: None. C.T. Thiel: None.


Novel variant in PLAG1 in a familiar case of Silver-Russell syndrome

A. Pereda1, Y. Vado2, I. Llano-Rivas3, N. Gorria-Redondo4, I. Díez5, G. Perez de Nanclares2

1Rare Diseases Research Group, Molecular (Epi)Genetics Laboratory, BioAraba National Health Research Institute, Vitoria-Gasteiz, Spain, 2Rare Diseases Research Group, Molecular (Epi)Genetics Laboratory, BioAraba National Health Research Institute, Vitoria-Gasteiz, Araba, Spain, 3Service of Genetics, BioCruces Health Research Institute, Hospital Universitario Cruces., Barakaldo, Bizkaia, Spain, 4Service of Paediatric Neurology, Araba University Hospital, Vitoria-Gasteiz, Araba, Spain, 5Department of Pediatric Endocrinology. BioAraba Health Research Institute, Araba University Hospital-Txagorritxu, Vitoria-Gasteiz, Araba, Spain

Introduction: Silver-Russell syndrome is a rare imprinting disorder characterized by prenatal (SGA) and postnatal growth retardation (PNGR), relative macrocephaly at birth, a prominent forehead, body asymmetry and feeding difficulties and/or low BMI. The most common underlying mechanisms are hypomethylation at H19/IGF2:IG-DMR and UPD(7)mat. Rarely, rearrangements involving 11p15, or inactivating point variants have been identified (maternal CDKN1C or paternal IGF2 variants). Recently PLAG1 and HMGA2 genes, which participate in the IGF2 pathway, have been associated with this syndrome.

Patients and Methods: We present a 9 years-old girl who was initially referred to pediatric endocrinology because of short stature. A clinical geneticist suggested the clinical diagnosis of SRS (NHC score=4: SGA, PNGR, prominent forehead and problems for feeding manifested as poor food intake). She also showed dental crowding with delayed teeth eruption, triangular face, small and bulbous nose, prominent maxillary, high ojival palate, fine voice, fine hair and learning difficulties. Her mother and maternal grandmother presented the same phenotype. After discarding alterations in 11p15 and chromosome 7 (MS-MLPA), a custom NGS panel for genes associated with growth was run.

Results: The study of the NGS panel revealed an heterozygous novel variant within exon 5 of PLAG1 [NM_001114634.2:c.551delA; p.(Lys184Serfs*45)], bioinformatically predicted as likely pathogenic (ACMG: PVS1; PM2; PP1). Familial studies by Sanger sequencing confirmed cosegregation.

Conclusions: We report the second familial case of SRS caused by a novel PLAG1 frameshift alteration, and we expand the clinical spectrum to the previously described. Funding: Instituto de Salud Carlos III (PI16/00073); Basque Department of Health (GV2017/111040).

A. Pereda: None. Y. Vado: None. I. Llano-Rivas: None. N. Gorria-Redondo: None. I. Díez: None. G. Perez de Nanclares: None.


Increased yield of genetic diagnoses in skeletal ciliopathies using massively parallel sequencing, structural variant and RNA analyses

A. Hammarsjo1, M. Pettersson1, D. Chitayat2,3, A. Handa4, F. Taylan5, D. Batkovskyte5, B. Anderlid1, A. E. Lin6, K. Shimizu7, A. Beleza-Meireles8,9, B. Chung10,11, U. Voss12, A. Nordgren1, G. Nishimura13, A. Lindstrand1, G. Grigelioniene1

1Department of Molecular Medicine and Surgery, Center for Molecular Medicine, Karolinska Institutet, and Clinical Genetics, Karolinska University Laboratory, Karolinska University Hospital, Stockholm, Sweden, 2Division of Clinical and Metabolic Genetics, The Hospital for Sick Children, University of Toronto, Toronto, ON, Canada, 3The Prenatal Diagnosis and Medical Genetics Program, Department of Obstetrics and Gynecology, Mount Sinai Hospital, University of Toronto, Toronto, ON, Canada, 4Department of Radiology, University of Iowa Hospitals and Clinics, Iowa City, IA, United States, 5Department of Molecular Medicine and Surgery, Center for Molecular Medicine, Karolinska Institutet, Stockholm, Sweden, 6Medical Genetics, MassGeneral Hospital for Children, Boston, MA, United States, 7Division of Medical Genetics, Saitama Children’s Medical Center, Saitama, Japan, 8Department of Clinical Genetics and Genomics, Guy’s Hospital, Guy’s and St Thomas’ NHS Foundation Trust, London, United Kingdom, 9Centre for Craniofacial and Regenerative Biology, King’s College London, London, United Kingdom, 10Department of Pediatrics and Adolescent Medicine, The University of Hong Kong and Shenzhen Hospital, Futian District, Shenzhen, China, 11Department of Pediatrics and Adolescent Medicine, Queen Mary Hospital, The University of Hong Kong, Hong Kong, China, 12Department of Pediatric Radiology, Karolinska University Hospital, Stockholm, Sweden, 13Department of Pediatric Imaging, Tokyo Metropolitan Children’s Medical Center, Tokyo, Japan

Introduction: Cilia are important for cell migration and signaling using multiple pathways and proteins essential for development of many body organs and systems, including the skeleton. Skeletal ciliopathies are a heterogenous group of disorders with overlapping features, caused by pathogenic variants in at least 30 genes, coding for different structural cilia proteins.

Materials and Methods: We examined 28 probands with radiographic findings of skeletal ciliopathies. For single nucleotide variant (SNV) analysis, we used whole exome and genome sequencing with in silico filtering for variants in known skeletal ciliopathy genes.

Results: We identified biallelic variants in 23 patients in DYNC2H1, KIAA0753, WDR19, C2CD3, TTC21B, EVC and EVC2. Subsequent examination of four potential splice variants showed that cDNA analyses are important in facilitating molecular diagnoses in a significant proportion (20%) of the patients and that aberrant splicing in DYNC2H1, EVC and KIAA0753 can be confirmed with RNA extracted from blood. Copy number variant (CNV) analyses was performed for the same cohort and we found one individual with an intragenic deletion of DYNC2H1 and another with 1q24q25-microdeletion syndrome.

Conclusions: In conclusion, combined SNV, CNV and RNA analyses result in a high yield of genetic diagnoses (86%) in a cohort of patients with a clinical, autopsy and radiological findings consistent with skeletal ciliopathy.

Grants: AnH - Sällskapet Barnavård and Karolinska Institutet. GG - Stiftelsen Samariten, Promobilia and Stiftelsen Frimurare. GG, AL and AN - Stockholm County Council. AN and AL - The Swedish Research Council. AN - The Swedish Childhood Cancer Foundation.

A. Hammarsjo: None. M. Pettersson: None. D. Chitayat: None. A. Handa: None. F. Taylan: None. D. Batkovskyte: None. B. Anderlid: None. A.E. Lin: None. K. Shimizu: None. A. Beleza-Meireles: None. B. Chung: None. U. Voss: None. A. Nordgren: None. G. Nishimura: None. A. Lindstrand: None. G. Grigelioniene: None.


Lack of bone mineralization in a TANGO1 deficient patient

B. Guillemyn1, S. Nampoothiri2, O. Foresti3, I. Raote3, V. Malhotra3, F. Malfait1, S. Symoens1

1Center for Medical Genetics Ghent, Ghent University Hospital, Department of Biomolecular Medicine, Ghent, Belgium, 2Department of Pediatric Genetics, Amrita Institute of Medical Sciences & Research Center, Cochin, India, 3Center for Genomic Regulation, The Barcelona Institute of Science and Technology, Barcelona, Spain

Introduction: TANGO1 (Transport and Golgi organization-1) encodes a transmembrane protein, located at Endoplasmic Reticulum (ER) exit sites. TANGO1 binds collagen in the lumen, COPII-coats in the cytoplasm and recruits membranes from the ERGIC (ER-Golgi intermediate compartment) to create an export route for bulky cargos like collagen. Recently aberrant expression of truncated TANGO1 was linked to a severe collagenopathy associated with primary obesity, diabetes mellitus and intellectual disability. We show that complete loss of TANGO1 results in human embryonic lethality with near-total bone loss.

Material and Methods: Fetal DNA and parental blood/fibroblast samples were obtained. Whole exome sequencing (WES) was performed. RTqPCR, Western blot, immunocytochemical staining and collagen secretion assays were conducted.

Results: Ultrasound examination of a 13-weeks-old fetus of healthy Indian consanguineous parents revealed tetramicromelia, increased nuchal translucency, absent nasal bone, pingpong appearance of the skull, shortened limb segments, and extreme skeletal hypomineralization. The pregnancy was terminated and the infantogram showed an almost boneless fetus. Fetal WES detected a homozygous 4-basepair deletion in TANGO1, heterozygously present in both parents. Parental fibroblast studies demonstrated decreased TANGO1 expression and protein, and severe defects in collagen secretion.

Conclusions: Our findings underscore the importance of TANGO1 in cellular protein secretion. While absence of TANGO1 results in human embryonic lethality, aberrant expression of truncated TANGO1 results in a severe collagenopathy. This further supports the physiological role of TANGO1 in collagen export, which is necessary for bone mineralization, skin and tissue biogenesis. Moreover, the data clearly reveal the severity of human pathologies in association with levels of functional TANGO1 expression.

B. Guillemyn: None. S. Nampoothiri: None. O. Foresti: None. I. Raote: None. V. Malhotra: None. F. Malfait: None. S. Symoens: None.


TAR syndrome: clinical and molecular characterization of a cohort of 26 patients and description of novel non-coding variants of RBM8A

S. Boussion1,2, F. Escande2,3, A. Jourdain2,3, T. Smol2,4, P. Brunelle2,3, C. Duhamel2, Y. Alembik5, T. Attié-Bitach6, G. Baujat7, A. Bazin8, M. Bonnière6, P. Carassou9, D. Carles10, L. Devisme2,11, C. Goizet12, A. Goldenberg13, S. Grotto14, A. Guichet15, P. Jouk16, L. Loeuillet17, C. Mechler18, C. Michot7, F. Pelluard19, A. Putoux20,21, S. Whalen22, J. Ghoumid1,2, S. Manouvrier1,2, F. Petit1,2

1CHU Lille, Clinique de Génétique, Centre de Référence Anomalies du Développement, Lille, France, 2Université de Lille, EA7364, Lille, France, 3CHU Lille, Service de Biochimie et Oncologie Moléculaire, Lille, France, 4CHU Lille, Institut de Génétique Médicale, Lille, France, 5Service de Génétique Médicale, CHU Strasbourg, Strasbourg, France, 6Unité d’Embryofoetopathologie, Service Histologie Embryologie et Cytogénétique, Hôpital Necker-Enfants Malades, AP-HP, Paris, France, 7Service de Génétique Clinique, Hôpital Necker – Enfants Malades, AP-HP, INSERM UMR 1163, Institut IMAGINE, Paris, France, 8Service de Diagnostic Anténatal, Hôpital René Dubois, Pontoise, France, 9Service d’Hématologie, CHR Metz-Thionville, Metz, France, 10Service d’Anatomo-Pathologie, CHU Bordeaux, Bordeaux, France, 11Institut d’Anatomo-Pathologie, CHU Lille, Lille, France, 12Service de Génétique Médicale, CHU Bordeaux, et laboratoire MRGM, INSERM U1211, Univ. Bordeaux, Bordeaux, France, 13Département de Génétique, et centre de référence anomalies du développement et syndromes malformatifs, CHU de Rouen, Centre Normand de Génomique et de Médecine Personnalisée, Rouen, France, 14Département de Génétique, Hôpital Robert Debré, AP-HP, Paris, France, 15Service de Génétique, CHU Angers, Angers, France, 16Département de Génétique, CHU Grenoble-Alpes, Grenoble, France, 17Service d’Anatomo-Cytopathologie, Hôpital Cochin, AP-HP, Paris, France, 18Unité de Foetopathologie, Hôpital Robert Debré, AP-HP, Paris, France, 19INSERM U1053-UMR BaRITOn et Unité de Foetopathologie, Service de pathologie, Hôpital Pellegrin, CHU Bordeaux, Bordeaux, France, 20Service de Génétique, Hospices Civils de Lyon, Lyon, France, 21GENDEV Team, CRNL, INSERM U1028, CNRS UMR 5292, UCBL1, Lyon, France, 22UF de Génétique Clinique, Centre de Référence Maladies Rares des Anomalies du développement et syndromes malformatifs, Hôpital Armand Trousseau, AP-HP, Paris, France

Thrombocytopenia-Absent Radius (TAR) syndrome is characterized by radial defect and neonatal thrombocytopenia. It is caused by bi-allelic variants of RBM8A gene (1q21.1) with the association of a null allele and a hypomorphic non-coding variant. RBM8A encodes Y14, a core protein of the Exon Junction Complex involved in mRNA maturation. To date, only two hypomorphic variants have been identified. We report on a cohort of 26 patients affected with TAR syndrome. Half patients carried a 1q21.1 deletion and one of the two known hypomorphic variants. Four novel non-coding variants of RBM8A were identified in the remaining patients. We developed experimental models enabling their functional characterization in vitro. Two variants, located respectively in the 5’-UTR and 3’-UTR regions, are responsible for a diminished expression whereas two intronic variants alter splicing. Our results bring new insights into the molecular knowledge of TAR syndrome and enabled us to propose genetic counseling for patients’ families.

S. Boussion: None. F. Escande: None. A. Jourdain: None. T. Smol: None. P. Brunelle: None. C. Duhamel: None. Y. Alembik: None. T. Attié-Bitach: None. G. Baujat: None. A. Bazin: None. M. Bonnière: None. P. Carassou: None. D. Carles: None. L. Devisme: None. C. Goizet: None. A. Goldenberg: None. S. Grotto: None. A. Guichet: None. P. Jouk: None. L. Loeuillet: None. C. Mechler: None. C. Michot: None. F. Pelluard: None. A. Putoux: None. S. Whalen: None. J. Ghoumid: None. S. Manouvrier: None. F. Petit: None.

P05 Cardiovascular Disorders


Improving family screening of abdominal aorta aneurysm patients

R. L. Lechner, A. S. IJpma, D. Heijsman, J. W. Roos-Hesselink, H. J. M. Verhagen, S. ten Raa, R. M. W. Hofstra, D. F. Majoor-Krakauer

Erasmus MC, Rotterdam, Netherlands

Relatives of abdominal aneurysm patients (AAA) have an increased risk of developing aneurysms. To improve early detection and reduce risk of rupture in relatives, guidelines recommend screening of first degree relatives. Current guidelines do not specify the method or extend of aorta screening in relatives. In practice, mostly male relatives are referred for abdominal echographic screening of the abdominal aorta, possibly missing thoracic aneurysms (TAA) in relatives. To determine in which part of the aorta affected relatives of AAA patients have an aneurysm or dilatation, we reviewed the results of family history taking and family screening of families of 471 AAA patients with familial aneurysms of which medical records and/or CT images relatives were available. In total 475 affected relatives (311 male, 164 female) in 237 families had an aorta dilatation or aneurysm: 258 (54,3%) had abdominal and 147 (31%) thoracic aneurysm. Male relatives were more likely to have AAA (40% vs 28%, p < 0.05), females to have TAA (32% vs 17%, p < 0.05). Co-occurrence of AAA and TAA was observed in 7% of relatives. In 17% an iliac and in 2% a popliteal dilation occurred. In conclusion: relatives of AAA patients have an increased risk for dilatation of the abdominal and the thoracic aorta. To prevent underdiagnosis of aortic aneurysm in relatives and improve early detection of aortic aneurysms, a CT-scan of the total aorta and the iliac arteries should be considered as the method of choice for family screening.

Conflicts of interest: none. Funding: Lijf en Leven Foundation

R.L. Lechner: None. A.S. IJpma: None. D. Heijsman: None. J.W. Roos-Hesselink: None. H.J.M. Verhagen: None. S. ten Raa: None. R.M.W. Hofstra: None. D.F. Majoor-Krakauer: None.


Yield of whole exome sequencing of aneurysm genes in 858 patients with an abdominal aorta aneurysm

R. L. Lechner, A. S. IJpma, D. Heijsman, J. W. Roos-Hesselink, H. J. M. Verhagen, S. ten Raa, R. M. W. Hofstra, D. F. Majoor-Krakauer

Erasmus MC, Rotterdam, Netherlands

A growing number of genes are associated with risk for aortic aneurysms. Similar mechanisms of remodeling of the extracellular matrix influencing the integrity of the aortic wall may play a role in abdominal and thoracic aneurysms. This helps to explain co-occurrence of AAA and TAA (25%), relatives with AAA in familial TAA and relatives with TAA in familial AAA.

To establish the role of aneurysm genes in AAA, the yield of diagnostic whole exome sequencing (WES) of a panel of aneurysm genes in AAA patients is presented.

As part of an ongoing genetic AAA study consecutively diagnosed patients at the department of Vascular Surgery were invited for genetic counseling. In this way WES DNA testing was performed in 858 AAA patients (738 unique families, 345 with familial AAA).

A pathogenic (PV) variant occurred in 20 (3%) AAA index cases and 7 family members and 301 AAA patients (41%) had a variant of unknown clinical significance (VUS). PV occurred in FBN1 (n = 4), COL3A1 (n = 6), DSCH1 (n = 1), TGFBR2 (n = 3), NOTCH1 (n = 1), PLOD1 (n = 1), MYLK (n = 1), MFAP5 (n = 1) SMAD6 (n = 1) and SLC2A10 (n = 1, heterozygous). VUS were observed in FBN2, COL5A1, MYH11, PLOD1, TGFB2, COL1A1 and COL1A2. There was no difference between familial and sporadic AAA in the WES yield. The outcome was comparable to that found in thoracic aneurysm patients.

The conclusion is that DNA diagnostics may improve stratification of familial risk for aortic aneurysms and distinguish relatives who benefit most from close surveillance.

Funding Lijf en Leven Foundation

Conflicts of interest: none

R.L. Lechner: None. A.S. IJpma: None. D. Heijsman: None. J.W. Roos-Hesselink: None. H.J.M. Verhagen: None. S. ten Raa: None. R.M.W. Hofstra: None. D.F. Majoor-Krakauer: None.


ADAMTS19 associated heart valve defects: novel genetic variants consolidating a recognizable cardiac phenotype

S. Massadeh1,2, A. AlHashem3, I. van de Laar4, F. Alhabshan5, N. Ordonez6, S. Alawbathani6, S. Khan6, M. Kabbani5, F. Chaikhouni5, A. Sheereen1, I. Almohammed1,2, B. Alghamdi1, S. Ahmad7, I. Frohn-Mulder8, C. Beetz6, A. Rolfs6,9, P. Bauer6, M. Wessels4, M. Alaamery1,2, A. Bertoli-Avella6

1Developmental Medicine Department, King Abdullah International Medical Research Center, King Saud Bin Abdulaziz University for Health Sciences, King Abdulaziz Medical City, Ministry of National Guard Health Affairs, Riyadh, Saudi Arabia, 2The Joint Center of Excellence for Biomedicine Between King Abdulaziz City for Science and Technology (KACST) and Brigham & Women’s Hospital (BWH), Joint Centers of Excellence Program, King Abdulaziz City for Science and Technology (KACST), Riyadh, Saudi Arabia, 3Division of Pediatric Genetics, Department of Pediatrics, Prince Sultan Military Medical City, Riyadh, Saudi Arabia, 4Department of Clinical Genetics, Erasmus University Medical Center, Rotterdam, Netherlands, 5Cardiac Sciences Department, Ministry of the National Guard - Health Affairs, King Abdullah International Medical Research Center, King Saud bin Abdulaziz University for Health Sciences, Riyadh, Saudi Arabia, 6CENTOGENE AG, Rostock, Germany, 7Cardiac Center, Prince Sultan Military Medical City, Riyadh, Saudi Arabia, 8Department of Pediatric Cardiology, Erasmus University Medical Center, Rotterdam, Netherlands, 9University of Rostock, Rostock, Germany

Recently, ADAMTS19 was identified as a novel causative gene for heart valve disease (HVD), affecting mainly the aortic and pulmonary valves as described in four patients from two families and supported by a mouse knockout model.

Exome sequencing and data repository (CentoMD®) analyses were performed to identify patients with ADAMTS19 variants (two families). A third family was recognized based on cardiac phenotypic similarities and SNP array homozygosity. Three novel loss of function (LoF) variants were identified in six patients from three families. Clinically, all patients presented anomalies of the aortic/pulmonary valves, which included thickening of valve leaflets, stenosis and insufficiency. Three patients had (recurrent) subaortic membrane, suggesting that ADAMTS19 is the first gene identified related to discrete subaortic stenosis. In contrast to previous publication, none of these patients were reported as having a bicuspid aortic valve. However, one case presented a bi-commissural pulmonary valve. All patients displayed some degree of atrioventricular valve insufficiency. Other cardiac anomalies included atrial/ventricular septal defects, persistent ductus arteriosus, and mild dilated ascending aorta.

Our findings confirm that biallelic LoF variants in ADAMTS19 are causative of a specific and recognizable cardiac phenotype. We recommend considering ADAMTS19 genetic testing in all patients with multiple semilunar valve abnormalities, particularly in the presence of subaortic membrane. ADAMTS19 screening in patients with semilunar valve abnormalities is needed to estimate the frequency of the HVD related phenotype, which might be not so rare.

S. Massadeh: None. A. AlHashem: None. I. van de Laar: None. F. Alhabshan: None. N. Ordonez: A. Employment (full or part-time); Significant; CENTOGENE AG. S. Alawbathani: A. Employment (full or part-time); Significant; CENTOGENE AG. S. Khan: A. Employment (full or part-time); Significant; CENTOGENE AG. M. Kabbani: None. F. Chaikhouni: None. A. Sheereen: None. I. Almohammed: None. B. Alghamdi: None. S. Ahmad: None. I. Frohn-Mulder: None. C. Beetz: None. A. Rolfs: A. Employment (full or part-time); Significant; CENTOGENE AG. P. Bauer: A. Employment (full or part-time); Significant; CENTOGENE AG. M. Wessels: None. M. Alaamery: None. A. Bertoli-Avella: A. Employment (full or part-time); Significant; Centogene AG.


Aortic aneurysm and dissection, an underestimated finding in JAG1 mutation carriers

I. Van Gucht1, J. R. Bento1, A. Van Berendoncks2, E. Van Craenenbroeck2, J. Saenen3, S. Laga3, J. Meester1, A. Verstraeten1, N. Boeckx1, L. Van Laer1, B. Loeys1

1Center of Medical Genetics Antwerp, University of Antwerp, University Hospital Antwerp, Edegem, Belgium, 2Department of Cardiac Surgery, University Hospital Antwerp, University of Antwerp, Edegem, Belgium, 3Department of Cardiology, University Hospital Antwerp, University of Antwerp, Edegem, Belgium

Loss-of-function Jagged-1 mutations classically lead to Alagille syndrome (AGS), a dominantly inherited disorder affecting liver, heart, eye, face and skeleton. Pulmonary artery stenosis, present in two thirds of AGS patients, is considered as a hallmark of the condition. Other vascular anomalies are identified in up to 10% of AGS patients. We describe a unique family with a JAG1 missense variant that segregates with a wide range of cardiovascular anomalies. Whole exome sequencing in a fetus with left hypoplastic heart and left renal agenesis revealed a missense variant (c.2242T>C; p.Cys748Arg) in JAG1, which was absent from gnomAD, predicted likely pathogenic by prediction programs and maternally inherited. The mother underwent surgery for aortic coarctation at age 10. The JAG1 variant was present in the mother’s monozygotic twin-sister and brother but absent in another sister. Subsequent familial clinical evaluation revealed normal eye exam (no embryotoxon), normal ultrasound of kidneys and absence of hemivertebrae in all mutation carriers. Echocardiographic evaluation revealed a normal aorta in the mother, borderline aortic dilatation (40 mm) in her monozygotic twin-sister and a huge aortic sinus aneurysm (58 mm) in the brother. The brother also has scoliosis. Remarkably, in literature another JAG1 variant affecting a Cys-residu has been linked to severe congenital heart disease (Tetralogy of Fallot). Aortic aneurysm was previously reported in four patients, three of which were only diagnosed on autopsy after fatal dissection. Our family data warrant regular echocardiographic follow-up in JAG1 mutation carriers as severe aortic aneurysms remain asymptomatic and predispose to fatal aortic dissection.

I. Van Gucht: None. J.R. Bento: None. A. Van Berendoncks: None. E. Van Craenenbroeck: None. J. Saenen: None. S. Laga: None. J. Meester: None. A. Verstraeten: None. N. Boeckx: None. L. Van Laer: None. B. Loeys: None.


Expanding the genetic and phenotypic spectrum of ACTA2 -related vasculopathies in a Dutch cohort

L. van den Bersselaar1, E. Rompen1, S. Kurul1, M. Kempers2, A. Houweling3, E. Overwater3, Y. Hilhorst4, D. Barge-Schaapveld4, I. Krapels5, E. Dulfer6, M. Wessels1, B. Loeys2, J. Bekkers1, H. Bruggenwirth1, A. Maugeri3, J. Roos-Hesselink1, J. Verhagen1, I. van de Laar1

1Erasmus Medical Center, Rotterdam, Netherlands, 2Radboud University Medical Center, Nijmegen, Netherlands, 3Amsterdam University Medical Center, Amsterdam, Netherlands, 4Leiden University Medical Center, Leiden, Netherlands, 5Maastricht University Medical Center, Maastricht, Netherlands, 6University Medical Center Groningen, Groningen, Netherlands

Introduction: Heterozygous variants in the ACTA2 gene, encoding the smooth muscle cell specific isoform of alpha-actin, confer a high risk for thoracic aortic aneurysms and aortic dissections. The purpose of this retrospective multicenter study was to review the clinical and genetic data of 47 new individuals with a pathogenic or likely pathogenic ACTA2 variant and elucidate the clinical outcome of ACTA2-related vasculopathies.

Material and Methods: Index patients and relatives with a pathogenic or likely pathogenic variant in the ACTA2 gene were included in this study. Data were collected through retrospective review of available medical records using a standardized questionnaire.

Results: Forty-seven individuals from 26 families participated in our study. Mean age at last evaluation was 50.8 ± 17.0 years. A total of 21 different ACTA2 variants were detected in our cohort, including 19 missense variants, 1 splice site variant and 1 frameshift variant. Four variants are novel. Aortic events occurred in 30/47 (63.8%) of patients. Mean age at first aortic event was 55.6 ± 13.7 years. Male gender and the presence of hypertension emerged as significant predictors of aortic events. Men were also at higher risk of aortic aneurysms compared to women. Other clinical findings included congenital heart disease in 6/47 and iris flocculi in 6/47.

Conclusions: We provide further insight into the genotype and phenotype of ACTA2-related vasculopathies. Male gender and hypertension are predisposing factors for aortic events in individuals with a pathogenic ACTA2 variant.

Supported by Dutch Heart Foundation (2014T007) and Erasmus University Rotterdam Fellowship to IvdL.

L. van den Bersselaar: None. E. Rompen: None. S. Kurul: None. M. Kempers: None. A. Houweling: None. E. Overwater: None. Y. Hilhorst: None. D. Barge-Schaapveld: None. I. Krapels: None. E. Dulfer: None. M. Wessels: None. B. Loeys: None. J. Bekkers: None. H. Bruggenwirth: None. A. Maugeri: None. J. Roos-Hesselink: None. J. Verhagen: None. I. van de Laar: None.


Prevalence of TTR mutations associated with transthyretine amyloidosis (ATTR) estimated from national exome sequencing data.

D. Monies1,2, D. Mohty3,4,5, B. Fadel3,4, B. Meyer1,2, I. Alayary6, M. Abouelhoda1,2

1Department of Genetics, King Faisal Specialist Hospital and Research Centre, Riyadh, Saudi Arabia, 2Saudi Human Genome Program, King Abdulaziz City for Science and Technology, Riyadh, Saudi Arabia, 3Heart center, King Faisal Specialist Hospital & Research Center, Riyadh, Saudi Arabia, 4Al-Faisal University, College of Medicine, Affiliate Harvard Medical School International, Riyadh, Saudi Arabia, 5CHU, Limoges, France, 6Pfizer Inc, Jeddah, Saudi Arabia

Introduction: Amyloidosis is a rare, frequently under-diagnosed systemic disease due to protein deposition that develops when a native protein misfolds into insoluble amyloid fibrils. These fibril deposits alter function of most affected organs, including the myocardium. Transthyretin amyloidosis (ATTR) is one of the two main causes of cardiac amyloidosis and occurs as a result of mutations in TTR. Under or misdiagnosis of affected individuals and incomplete penetrance make estimates of prevalence challenging. While worldwide prevalence is estimated to be around 50,000 individuals, with TTR V142I being the most common cardiac ATTR associated mutation, no data on TTR mutations or their frequency in Middle Eastern countries are available to date.

Materials and Methods: Blinded from clinical information, we undertook data mining of 13,906 exomes of unrelated Saudi individuals, sequenced between 2015 and 2019 as part of the Saudi Human Genome Project (SHGP). Pathogenic or highly pathogenic TTR variants were identified following filtration to remove common, synonymous, non-coding and other benign variants.

Results and conclusion: We identified seven missense variants (known or predicted pathogenic) in TTR, three of which have been previously reported in amyloidosis patients worldwide. The most frequent variants were c.424G>A:p.V142I (1:435) and c.370C>T:p.R124C (1:2,318). Prevalence of TTR variants in the general Saudi population was much higher than the actual estimated rate of ATTR cases nationally (1:10,000), which may be explained by incomplete penetrance or sub-clinical manifestation of the disease.

D. Monies: None. D. Mohty: None. B. Fadel: None. B. Meyer: None. I. Alayary: None. M. Abouelhoda: None.


Investigation of NOTCH1mutation frequency in bicuspid aortic valve disease

S. E. Hamby1,2, R. Debiec1,2,3, M. Nath1,2, P. D. Jones1,2, S. Coolman1,2, M. Asiani1,2, S. Kharodia1,2, G. J. Skinner3, N. J. Samani1,2, A. Bolger1,2,3, T. R. Webb1,2

1University of Leicester Department of Cardiovascular Sciences, Leicester, United Kingdom, 2NIHR Leicester Biomedical Research Centre, Leicester, United Kingdom, 3East Midlands Congenital Heart Centre, Glenfield Hospital, Leicester, United Kingdom

Background: Bicuspid Aortic valve (BAV) is a common congenital heart defect present in around 1% of the population. Familial BAV has an autosomal dominant pattern of inheritance with reduced penetrance, however, the majority of BAV cases are sporadic. Pathogenic and likely pathogenic NOTCH1 variants have been reported in familial, as well as up to 4% of sporadic cases.

Aim: To investigate the frequency of causative NOTCH1 mutations in a large cohort of patients of sporadic and familial BAV disease recruited to the Bicuspid aoRtic vAlVe gEnetic research (BRAVE) study.

Methods: Whole exome sequencing (WES) was performed in 12 families (30 affected and 25 unaffected family members). Targeted NOTCH1 sequencing was carried out in 383 sporadic BAV cases. NOTCH1 variants were filtered by gnomAD allele frequency (< 0.001) and potential deleterious effect (Combined Annotation Dependent Depletion (CADD) score>20). We also compared cumulative frequency of rare NOTCH1 variants between sporadic BAV patients and 1102 apparently healthy controls.

Results: One BAV family had a truncating NOTCH1 variant (c.873C>G/p.Tyr291*). There were no candidate NOTCH1 variants in any of the other 11 families. We identified seven rare and potentially deleterious NOTCH1 variants in the sporadic patient group. Among which, there were no protein truncating mutations. There was no enrichment of NOTCH1 variants between cases and controls (1.83% vs 3.09%, p-value 0.18).

Conclusions: Our data suggest that whilst occasionally responsible for familial cases of BAV, NOTCH1 mutations are an infrequent cause of sporadic forms of the disease.

S.E. Hamby: None. R. Debiec: None. M. Nath: None. P.D. Jones: None. S. Coolman: None. M. Asiani: None. S. Kharodia: None. G.J. Skinner: None. N.J. Samani: None. A. Bolger: None. T.R. Webb: None.


Bicuspid aortic valve: a pilot candidate gene-based approach in a representative Czech cohort

P. Votýpka1, P. Peldová1, A. Krebsová2, V. Zoubková1, M. Balaščaková1, 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

Aim: Analysis of the genetic basis in a representative cohort of Czech patients with familiar bicuspid aortic valve (BAV) with / without aortic aneurysm (MIM# 109730).

Materials and Methods: Altogether 100 unrelated cases were clinically examined, underwent genetic counselling and positive cases were subjected to “cascade screening” of first-degree relatives. Massively parallel sequencing (MiSeq platform; was utilised for a custom-made panel comprising either 136, 229 or 100 cardiac/aortic conditions-related candidate genes (Sophia Presence of pathogenic variants was validated by Sanger sequencing and via their segregation in respective families.

Results: Pathogenic/likely pathogenic variant (ACMG Class ≥ 4) were found in 3/100 patients (3 %) within TGFB2, SMAD6 and FBN1. Variants of unknown significance (Class 3) were detected in 9/100 patients (9 %) comprising genes FLNA, FBN1, MYH11, TAB2, TGFB2 and GATA5. A family with concurrent familial dilated cardiomyopathy, BAV and ectopia lentis was analysed as well. Likely causative DNA variants in MYBPC3 and FBN1 were identified, but FBN1 variants did not co-segregate with BAV.

Conclusions: The yield of genetic testing in familial forms of BAV is rather low in our cohort. Moreover, the identification of pathogenic variant in BAV-related gene is not a guarantee of their causality, hence detected variants should be carefully interpreted. The genetic basis for the familial forms of BAV will be further studied, e.g. by whole exome analysis together with improved clinical stratification of our patient cohort.

Supported by Ministry of Health of the Czech Republic, grant Nr. NV18-02-00237

P. Votýpka: None. P. Peldová: None. A. Krebsová: None. V. Zoubková: None. M. Balaščaková: None. M. Macek, Jr.: None.


Electrophysiological characterization of a Brugada syndrome SCN5A Belgian founder mutation in induced pluripotent stem cell cardiomyocytes

E. Simons1, A. Nijak1, B. Vandendriessche1, D. Van de Sande2, E. Sieliwończyk1, A. J. Labro2, D. Snyders2, D. Schepers1, B. Loeys1,3, M. Alaerts1

1Centre of Medical Genetics, faculty of Medicine and Health Sciences, University of Antwerp & Antwerp University Hospital, Antwerp, Belgium, 2Laboratory of Molecular Biophysics, Physiology & Pharmacology, Department of Biomedical Sciences, University of Antwerp, Antwerp, Belgium, 3Department of Human Genetics, Radboud University Medical Centre, Nijmegen, Netherlands

Introduction: SCN5A encodes the α-subunit of voltage-gated cardiac sodium channel Nav1.5. Mutations in SCN5A are identified in about 20% of patients with Brugada syndrome (BrS), an inherited cardiac arrhythmia. We have identified an SCN5A founder mutation (c.4813+3_4813+6dupGGGT), leading to a loss-of-function of Nav1.5 in 25 different families. Mutation carriers show variable expression of the phenotype: from asymptomatic to syncopes and SCD. We used induced pluripotent stem cell derived cardiomyocytes (iPSC-CM) to investigate the underlying pathophysiology.

Material & Methods: Dermal fibroblasts of three patients with different severity, and one unrelated healthy control were reprogrammed using the CytoTune™-iPS 2.0 Sendai Reprogramming Kit. iPSC-CMs were differentiated following a published protocol. T3 hormone addition was tested to improve maturity of the cells. We performed several differentiation rounds and investigated expression of cardiac markers using qPCR and immunocytochemistry and electrophysiological properties using patch-clamping and calcium imaging.

Results: All iPSC-CMs expressed tested markers. We observed reduction in sodium current density in patient iPSC-CMs, together with shortening of the action potential and calcium transient duration compared to the control. However, our data display variability between the differentiation batches, as well as between clones generated from one donor. Addition of T3 improved organization of the sarcomeres and decreased result variability.

Conclusions: We established iPSC-CM models for a unique Belgian SCN5A founder mutation. Despite observed variability, we could detect expected differences in electrophysiological properties of patient cells compared to controls. We prove that T3 addition improves the maturity of the cells, with a positive impact on the variability.

E. Simons: None. A. Nijak: None. B. Vandendriessche: None. D. Van de Sande: None. E. Sieliwończyk: None. A.J. Labro: None. D. Snyders: None. D. Schepers: None. B. Loeys: None. M. Alaerts: None.


Network-based identification of novel Shox2-dependent genes in cardiac rhythm control

S. Hoffmann1,2, S. Schmitteckert1, K. Raedecke1,2, D. Rheinert1, S. Diebold3, R. Roeth1, B. Weiss1, M. Granzow4, B. Niesler1,2, A. Griesbeck1, V. Eckstein5, W. H. Zimmermann6,7, S. Just3, G. A. Rappold1,2

1Department of Human Molecular Genetics, Institute of Human Genetics, University of Heidelberg, Heidelberg, Germany, 2DZHK, German Center for Cardiovascular Research, Partner Site Heidelberg/Mannheim, Heidelberg, Germany, 3Department of Internal Medicine II, University of Ulm, Ulm, Germany, 4Department of Human Genetics, Institute of Human Genetics, University of Heidelberg, Heidelberg, Germany, 5FACS Core Facility, Department of Medicine V, University Hospital Heidelberg, Heidelberg, Germany, 6Institute of Pharmacology, University Medical Center Goettingen, Goettingen, Germany, 7DZHK, German Centre for Cardiovascular Research, Partner Site Goettingen, Goettingen, Germany

The transcription factor SHOX2 is involved in the development and function of the sinoatrial node (SAN), which is crucial for initiating and controlling heart rhythm. SHOX2 has been associated with cardiac arrhythmias such as atrial fibrillation (AF) and sinus node dysfunction. Yet, our knowledge on co-regulatory networks associated with cardiac rhythm control in health and disease still remains limited. To dissect the role of Shox2-dependent networks, we utilized a murine embryonic stem cell (ESC) model that allowed us to investigate molecular pathways involving Shox2 in SAN-like cardiomyocytes. Differential RNA-seq-based expression profiling of Shox2+/+ and Shox2-/- ESCs revealed 94 regulated transcripts in Shox2-/- SAN-like cells. Fifteen putative target genes were selected for further validation based on comparative expression analysis of our dataset with publicly available RNA-seq data of SAN and right atria enriched genes at different mouse embryonic stages. Network-based analyses integrating data from the Mouse Organogenesis Cell Atlas and the Ingenuity pathway analysis tool as well as validation in mouse and zebrafish confirmed a species conserved regulation for Shox2 target genes in cardiac rhythm control. Our results indicate that co-regulatory networks involving SHOX2 participate in the development of conduction traits through regulatory mechanisms, thereby affecting the expression of multiple genes. This work was supported by grants from the DFG (RA 380/14-4) and DZHK (81X2500110 and 81X2500171).

S. Hoffmann: None. S. Schmitteckert: None. K. Raedecke: None. D. Rheinert: None. S. Diebold: None. R. Roeth: None. B. Weiss: None. M. Granzow: None. B. Niesler: None. A. Griesbeck: None. V. Eckstein: None. W.H. Zimmermann: None. S. Just: None. G.A. Rappold: None.


Diagnostic yield of targeted versus broad next generation sequencing data analysis in inherited cardiomyopathy and arrhythmia syndromes

K. Kolokotronis1, N. Pluta1, E. Klopocki1, S. Störk2, S. Rost1, B. Gerull2

1Institute of Human Genetics, University of Wuerzburg, Wuerzburg, Germany, 2Comprehensive Heart Failure Center and Department of Medicine I, University Hospital Würzburg, Wuerzburg, Germany

Introduction: Although next generation sequencing has drastically changed cardiogenetics, the fact that many cases with positive family history remain unsolved after routine panel diagnostics suggests that some genetic causes remain to be discovered. In this study we examined the diagnostic yield of targeted gene panels compared to whole exome data analysis.

Methods: The cohort consisted of 61 consecutive patients with a diagnosis of cardiomyopathy or primary arrhythmia syndrome, with DCM being the most common diagnosis. The genetic analysis involved whole exome sequencing with subsequent stepwise bioinformatic analysis starting from virtual gene panels till whole exome data. Genetic variants were classified according to ACMG-criteria.

Results: Overall, in 39 out of 61 (64%) patients a variant of interest was detected. The detection rate was highest in patients with a diagnosis of HCM (7/9; 77%) or DCM (25/36; 69%). In DCM the diagnostic yield was better compared to current literature. The majority of variants of interest were found in the disease-specific panel (46%; 28/61), while the further analysis of an extended panel and whole exome data led to an additional diagnostic yield of 13% and 5%, respectively. Whole exome data analysis also detected variants in thus far not well characterized candidate genes whose expression and function profile suggested a probable pathogenetic role.

Conclusions: Targeted gene panel analysis led to the identification of the genetic cause in the majority of solved cases. The additional diagnostic yield of whole exome data analysis was low, enabled though the detection of potentially causal variants in candidate genes.

K. Kolokotronis: None. N. Pluta: None. E. Klopocki: None. S. Störk: None. S. Rost: None. B. Gerull: None.


Diagnostic yield of core panels for cardiomyopathy- and arrhythmia genes versus a larger panel of heart-disease related genes

D. M. E. I. Hellebrekers1, B. J. C. van den Bosch1, G. R. F. Claes1, I. Roozen1, Y. E. G. Barrois1, N. Vergoossen1, A. T. M. Hendrickx1, W. van Dijk1, C. L. M. Marcelis2, M. Kempers2, Y. M. Hoedemaekers2, A. T. J. M. Helderman-van den Enden1, E. K. Vanhoutte1, I. P. C. Krapels1, A. van den Wijngaard1

1Maastricht University Medical Center, Maastricht, Netherlands, 2Radboud University Medical Center, Nijmegen, Netherlands

Introduction: We evaluated the prevalence of (likely) pathogenic variants in our cardiomyopathy- and arrhythmia core panels in patients with cardiac disease. Furthermore, the additional diagnostic value of a larger panel of heart disease-associated genes was assessed.

Methods: This study included a large population of ~2000 patients suspected of a heritable cardiac condition who underwent cardiogenetic testing at the laboratory of the Clinical Genetics Department in Maastricht. Sequencing a diagnostic panel of 46 cardiomyopathy genes and/or 27 arrhythmia genes was performed using single-molecule molecular inversion probes (smMIPs). For 347 of these patients, whole exome sequencing was performed for the analysis of a panel of 350 heart disease-associated genes.

Results: A (likely) pathogenic variant was found in 20% (336/1719) and 8% (34/408) of patients tested for the cardiomyopathy or arrhythmia gene panel, respectively. The majority of these variants were found in the MYBPC3, MYH7, LMNA, TNNT2, MYL2 and PKP2 genes for cardiomyopathy and SCN5A, KCNQ1, RYR2 and KCNH2 genes for arrhythmia. Sequencing the larger panel of 350 genes in 347 patients identified an additional pathogenic variant explaining the clinical phenotype in only 3 patients.

Conclusions: Our data support parallel sequencing of core genes as the preferred strategy for genetic testing of patients suspected of having a genetic cardiomyopathy or arrhythmia. The incremental diagnostic yield of a larger panel of heart disease-associated genes is minimal. Therefore, focus should not be on analysing more genes, but on developing functional assays to predict pathogenicity of variants of unknown significance (VUS’s) in the core panel genes.

D.M.E.I. Hellebrekers: None. B.J.C. van den Bosch: None. G.R.F. Claes: None. I. Roozen: None. Y.E.G. Barrois: None. N. Vergoossen: None. A.T.M. Hendrickx: None. W. van Dijk: None. C.L.M. Marcelis: None. M. Kempers: None. Y.M. Hoedemaekers: None. A.T.J.M. Helderman-van den Enden: None. E.K. Vanhoutte: None. I.P.C. Krapels: None. A. van den Wijngaard: None.


Dominant mild Carvajal syndrome: arrhythmogenic cardiomyopathy caused by the heterozygous p.R1113* nonsense variant in the DSP gene.

A. T. J. M. Helderman-vd Enden1, J. G. Post2, K. J. A. F. van Kaam1, R. H. Lekanne Deprez3, D. Dooijes4, A. A. M. Wilde5, I. P. C. Krapels1

1Department of Clinical Genetics, Maastricht University Medical Center, Maastricht, Netherlands, 2Department of Clinical Genetics, University Medical Center Utrecht, Utrecht, Netherlands, 3Department of Clinical Genetics, Amsterdam University Medical Center, Amsterdam, Netherlands, 4Department of Clinical Genetics, University Medical Center Utrecht, Utrecht, Netherlands, 5Department of Cardiology, Amsterdam University Medical Center, Amsterdam, Netherlands

Introduction: Arrhythmogenic cardiomyopathy (ACM) is a dominant inherited cardiac disease characterized by progressive fibrofatty replacement of the myocardium that predisposes to ventricular tachycardia and sudden death. Carvajal syndrome is a rare recessive form of ACM associated with woolly hair and palmo-plantar keratoderma and pathogenic variants in the desmoplakin (DSP) gene. Some families have been described with a dominantly inherited DSP variant with features of Carvajal.

Materials and Methods: In a four generation family seggregation analysis was done for a dominantly inherited DSP p.R1113* variant. The phenotypes are described and the results are compared to literature.

Results: In the large sibship (4M + 8F) at least three siblings are obligate carrying the DSP pathogenic variant and have offspring with cardiac abnormalities. Five family members died suddenly, 2/5 obligate carriers, 3/5 the status is unknown. The curly hair is co-seggregating with the variant. Not all carriers have keratoderma. A number of individuals with the variant have no cardiac complaints so far, cardiological investigations are planned. An overview will be presented at the conference and compared to the six families described by Maruthappu et al in 2019, one of these families also has the p.R1113* variant.

Conclusion: The p.R1113* variant in the DSP gene leads to a well recognizable phenotype of mild Carvajal syndrome. Drawing the attention to this phenotype by presenting this large four generation family may help in identifying other families with this disease and in preventing sudden death.

A.T.J.M. Helderman-vd Enden: None. J.G. Post: None. K.J.A.F. van Kaam: None. R.H. Lekanne Deprez: None. D. Dooijes: None. A.A.M. Wilde: None. I.P.C. Krapels: None.


Chromosome 22q11 copy number variations in paediatric and adult patients with congenital heart defects

G. K. ZODANU, D. Nagy, M. Széll, M. Oszlánczi, K. Havasi, A. Kalapos, M. Katona


Introduction: Congenital heart defect (CHD) is the most common birth defect, affects ~1% of newborns. Chromosome 22q11 microdeletion syndrome is often associated with CHDs and is the most common human microdeletion, occurs in ~1/4000 live births in the general population and in 2.8-14.9% of CHD patients. 22q11 copy number variants (CNVs) are caused by non-allelic homologous recombination events in the flanking low copy repeat regions (LCRs), labeled A-H.

Materials and Methods: Our aim was to evaluate the prevalence of 22q11 CNVs in Hungarian CHD patients. 175 blood samples were obtained from children and adults with different isolated and combined CHDs. Samples were analyzed using multiplex ligation-dependent probe amplification (MLPA) to detect CNVs in the given chromosomal region. Positive MLPA results were confirmed with locus-specific FISH. Genotype-phenotype comparison and family screening was also performed.

Results: CNVs were detected in overall 10 cases (5.7%). Six cases with typical microdeletion (between LCRs A and D) also known as DiGeorge syndrome, 2 cases with microduplication and 2 cases with combined microdeletion and microduplication were detected. The phenotypical appearance of patients was highly variable. CNVs were familiar in 3 cases.

Conclusions: Until present this was the largest systemic genetic screening of Hungarian CHD patients. Since the clinical diagnosis of 22q11 microdeletion/microduplication syndrome may be challenging due to its variable manifestation, systematic genetic testing may be of utmost benefit in the early diagnosis and proper management of these patients.

Funding grant: GINOP-2.3.2-15-2017-00039

G.K. Zodanu: None. D. Nagy: None. M. Széll: None. M. Oszlánczi: None. K. Havasi: None. A. Kalapos: None. M. Katona: None.


Genetic variation in participants of the 100,000 Genomes Project with congenital heart disease

S. L. Baross1, S. G. Williams1, K. E. Hentges2, A. D. Sharrocks3, B. D. Keavney1

1Division of Cardiovascular Sciences, University of Manchester, Manchester, United Kingdom, 2Division of Evolution and Genomic Sciences, University of Manchester, Manchester, United Kingdom, 3Division of Molecular and Cellular Function, University of Manchester, Manchester, United Kingdom

Congenital heart disease is the most common birth defect, affecting approximately 1% of live births globally. Congenital heart disease shows a high degree of heritability but the genetic causes are still poorly understood.

The 100,000 Genomes Project provides whole genome sequences for patients with rare disease or cancer and their families. Over 600 participants were recruited to the project under the congenital heart disease arm of the project, almost all of whom have no known causative variant. In addition, analysis of phenotyping and clinical data identified an additional ~2,500 participants with congenital heart disease who were recruited to the project for other conditions.

We will present rare, likely pathogenic single nucleotide, short insertions/deletions and structural variants, both transmitted and de novo, from this cohort of 3,100 participants with congenital heart disease. Short variants are filtered for variants with moderate or high impact on protein function; CADD (combined annotation dependent depletion) score ≥ 20; and absent in gnomAD v3.

This research was made possible through access to the data and findings generated by the 100,000 Genomes Project (

Grant reference: BHF FS/16/58/32734.

S.L. Baross: None. S.G. Williams: None. K.E. Hentges: None. A.D. Sharrocks: None. B.D. Keavney: None.


Mutation spectrum of congenital heart disease in 73consanguineous Turkish families

W. Dong1, H. Kaymakcalan2, N. Diab1, S. Chih Jin1, C. Tanıdır3, A. Yalcin2, S. Mane1, K. Bilguvar1, M. Brueckner1, R. Lifton1

1Yale University, New Haven, CT, United States, 2Demiroglu Bilim University, Istanbul, Turkey, 3Mehmet Akif Ersoy Hospital, istanbul, Turkey

Mutation spectrum of congenital heart disease in 73 consanguineous Turkish families

Introduction: Congenital heart disease (CHD) is the most frequent defect among live births and the leading cause of mortality from a birth defect. Nearly 90% of CHD cases have a suspected genetic etiology, but in 56% of cases the genetic etiology is unknown. Few gene discovery efforts have been made with consanguineous CHD cohorts. We recruited 74 CHD patients from 73 consanguineous families in Turkey and used whole exome sequencing to identify affected genes in these patients. In 13.7% of the cohort, we could identify a genetic etiology and we discovered four new genes that are putatively causative for CHD. This represents an important advance in our understanding of the genetic consequences of consanguinity in CHD.

Materials and methods: 74 samples from 73 families were recruited from two pediatric cardiology clinics in Istanbul, Turkey. Cardiac phenotypes were classified into 5 major categories and whole exome sequencing was performed at the Yale Center for Genomic Analysis using Illumina.

Result: 7 out of 73 (9.6%) patients carried damaging homozygous variants in known CHD genes and 3 out of 73 (4.1%) of them harbored other types of CHD-related genomic alterations, which explained 13.7% of the cohort.

Conclusions: Our findings is consistent with the hypothesis that high consanguinity is more likely to be associated with recessive cardiac abnormalities. Additionally, it fits the perception that the heritability for severe defects is higher than milder malformations.

Grant: Yale-National Institutes of Health Center for Mendelian Genomics

W. Dong: None. H. Kaymakcalan: None. N. Diab: None. S. Chih Jin: None. C. Tanıdır: None. A. Yalcin: None. S. Mane: None. K. Bilguvar: None. M. Brueckner: None. R. Lifton: None.


Altered microRNA and target gene expression related to Tetralogy of Fallot

M. Grunert, S. Appelt, S. Rickert-Sperling

Charité - Universitätsmedizin Berlin, Berlin, Germany

MicroRNAs (miRNAs) play an important role in guiding development and maintaining function of the human heart. Dysregulation of miRNAs has been linked to various congenital heart diseases including Tetralogy of Fallot (TOF), which represents the most common cyanotic heart malformation in humans. Several studies have identified dysregulated miRNAs in right ventricular (RV) tissues of TOF patients. In the study presented here, we profiled genome-wide the whole transcriptome and analyzed the relationship of miRNAs and mRNAs of RV tissues of a homogeneous group of 22 non-syndromic TOF patients. Observed profiles were compared to profiles obtained from right and left ventricular tissue of normal hearts. To reduce the large list of predicted target genes of dysregulated miRNAs, we applied a stringent target prediction pipeline integrating probabilities for miRNA-mRNA interaction. The final list of disease-related miRNA-mRNA pairs comprises novel as well as known miRNAs including miR-1 and miR-133, which are essential to cardiac development and function by regulating KCNJ2, FBN2, SLC38A3 and TNNI1. Overall, our study provides additional insights into post-transcriptional gene regulation of malformed TOF hearts.

M. Grunert: None. S. Appelt: None. S. Rickert-Sperling: None.


Circulating lncRNA ANRIL a potential biomarker in Tanzanian patients with coronary artery disease

G. AKAN1, E. Nyawawa2, B. Nyangasa2, M. K. Turkcan3, E. Mbugi1, O. Ozgen4, M. Janabi2, F. Atalar1

1MUHAS Genetics Laboratory, Biochemistry Department, School of Medicine, Muhimbili University of Health and Allied Science, Dar es Salaam, Tanzania, United Republic of, 2Muhimbili National Hospital Jakaya Kikwete Heart Institute, Dar es Salaam, Tanzania, United Republic of, 3Department of Electrical Engineering, Columbia University,, New York, NY, United States, 4Molecular Medicine Department, Aziz Sancar Experimental Medicine Research Institute, Istanbul University, Istanbul, Turkey

Background: 9p21.3 locus was identified as a hot spot locus associated with coronary artery disease(CAD) and it contains CDKN2A and CDKN2B genes which encode the cell cycle regulators and a non-coding RNA ANRIL gene. Role of 9p21.3 locus variations in molecular mechanisms of CAD pathogenesis and the expression of neighbour-genes remain unknown. The study aims to assess of expressions of CDKN2A, CDKN2B, ANRIL and ANRIL splices variants association with CAD susceptibility, and to investigate the impact of 9p21.3 locus variations on the expression of studied genes in peripheral blood, Epicardial Adipose Tissue(EAT), Mediastinal Adipose tissue(MAT) and Subcutaneous Adipose Tissue(SAT) in Tanzanian CAD patients.

Methods&Results: Expression of CDKN2A, CDKN2B, ANRIL, and its splice variants (circANRIL, NR003529, EU741058 and DQ485454)were measured by QRT-PCR in peripheral blood, EAT, MAT and SAT of 25 patients undergoing coronary artery bypass grafting due to CAD and 25 non-CAD patients undergoing heart valve surgery. ANRIL gene expression levels were significantly up-regulated while expression levels of circANRIL were significantly down-regulated in peripheral blood of CAD patients. Moreover, increased expression levels of circANRIL were associated with less severity of CAD. ROC analysis data has also confirmed that circANRIL could act as a potential biomarker for detecting CAD.

Conclusions: Our results are the first data presenting the expressions of ANRIL and its splice variants in cardiac adipose tissues of CAD patients. We demonstrate circANRIL has a synergistic effect with ANRIL and plays a protective role in CAD. circANRIL could possibly be used as a biomarker for CAD in Tanzania.

G. Akan: None. E. Nyawawa: None. B. Nyangasa: None. M.K. Turkcan: None. E. Mbugi: None. O. Ozgen: None. M. Janabi: None. F. Atalar: None.


Members of subgingival plaque bacterial complexes are present in blood of patients with coronary artery disease

A. Suarez-Molina1, C. Fong2, L. Cifuentes-C2, S. Guauque-Olarte2

1Instituto Departamental de Salud de Nariño, Pasto, Colombia, 2Universidad Cooperativa de Colombia, Pasto, Colombia

Introduction: It has been hypothesized that oral bacteria can migrate, through the blood, from the mouth to the artery plaques exacerbating atherosclerosis. This study compared the bacteria present in peripheral blood of individuals with and without coronary artery disease.

Materials and Methods: RNA sequences from blood downloaded from GEO (GSE58150) correspond to 8 cases with coronary artery calcium (CAC) >514 and 8 controls (CAC=0). After quality controls, the sequences were aligned to the hg38 reference genome using Hisat2. The unmapped sequences were fed into Kraken to determinate bacterial taxa. Ecological indices were calculated using Vegan. The counts of bacteria between groups was compared using a Mann-Whitney U test.

Results: The mean number of species was different between cases (433.4) and controls (226.1) (p-value = 0.0001). A total of 129 species were found only in cases and 74 were exclusive of controls. According to the Jaccard similarity index 16% of the species identified is not shared between cases and controls (p=0.001). The Alcaligenaceae family was more frequent in cases than in controls (FDR: 0.048). The red complex bacteria Treponema denticola was detected only in 2 cases. The mean count of bacteria belonging to subgingival plaque bacterial complexes was higher in cases (573.5) than in controls (249) (p-value = 0.0206).

Conclusions: This study identified periodontopathogens in blood of patients with coronary artery disease. The list of microorganisms presented only in cases will be key to identify new periodontopathogens that can relate periodontitis and this cardiovascular disease. SGO received funding by CONADI-Universidad Cooperativa de Colombia, grant 1839.

A. Suarez-Molina: None. C. Fong: None. L. Cifuentes-C: None. S. Guauque-Olarte: None.


Molecular genetic screening in patients with spontaneous coronary artery dissection

M. Antonutti1, F. Baldan2, L. Spedicato1, D. Zanuttini1, T. Bisceglia1, E. Favaretto1, C. Tioni1, D. Sut1, M. Ghidina1, C. Lanera3, D. Gregori3, D. Fabbro1, G. Damante1, A. Proclemer1

1ASUFC, Udine, Italy, 2University of Udine, Udine, Italy, 3DCTVPH, Padova, Italy

Spontaneous coronary artery dissection (SCAD) is increasingly recognized as an important cause of myocardial infarction, particularly in women often with few cardiovascular risk factors. SCAD is defined as a spontaneous tear in the coronary arterial wall that is not traumatic or iatrogenic. Studies are still underway to investigate the role of genetics in promoting SCAD; genetic diseases frequently associated with SCAD are connective tissue diseases. Considering the role of SCAD for sudden cardiac death and the increased risk for future cardiac events, an early diagnosis is crucial. We enrolled 60 patients with nonatherosclerotic angiographic diagnosis of SCAD. All patients’ genomic DNA was analyzed by NGS with gene panel involved in collagenopathies and arteriopathies (ACTA2, CBS, COL3A1, COL5A1, COL5A2, ELN, FBN1, FBN2, LTBP2, MYLK, NOTCH1, MYH11, SMAD3, SMAD4, SLC2A10, TGFB2, TGFB3, TGFBR1, TGFBR2, PLOD1). Seven patients result to carry potential SCAD-related mutations. In particular, we observed a pathological COL3A1 mutation; 3 different VUS mutations in: LTBP2 (two patients), MYLK and NOTCH1. Moreover, we found a new nonsense mutation in SMAD3 and a missense alteration in COL5A2; both not enlisted in public database, but that could be categorized as pathogenetic and VUS, respectively, according to the ACMG classification. These data support the use of sequencing of genes inclusive of collagenopathies and arteriopathies as an approach to evaluate genetically mediated SCAD. However,, considering that only a minority of patients have clear SCAD-causative mutations, these results remark the need of further studies to define genetic mechanisms of SCAD.

M. Antonutti: None. F. Baldan: None. L. Spedicato: None. D. Zanuttini: None. T. Bisceglia: None. E. Favaretto: None. C. Tioni: None. D. Sut: None. M. Ghidina: None. C. Lanera: None. D. Gregori: None. D. Fabbro: None. G. Damante: None. A. Proclemer: None.


Comparison of variant detection rate in 100 candidate genes between two representative cohorts of Czech living patients versus victims of sudden cardiac death with clinical. or post mortem diagnosis of dilated-, arrhythmogenic- and hypertrophic cardiomyopathy.

A. Krebsova1, P. Votýpka2, P. Peldová2, K. Ruecklova3, V. Zoubkova2, S. Pohlova-Kucerova4, A. Pilin5, M. Kulvajtova6, M. Kubanek1, M. Bilek5, T. Tavacova7, J. Petrkova8, M. Dobias9, P. Tomasek10, M. Macek Sr.2, M. Macek Jr.2, J. Janousek7, J. Kautzner1

1Institute for Clinical and Experimental Medicine, Prague, Czech Republic, 2Department of Biology and Medical Genetics, 2nd Faculty of Medicine, Charles University and Motol University Hospital, Prague, Czech Republic, 3Paediatric Department, 3rd Faculty of Medicine, Charles University, Prague, Czech Republic, 4Department of Forensic Medicine, Faculty of Medicine in Hradec Králové, Charles University, Hradec Kralove, Czech Republic, 5Institute for forensic medicine and toxicology, 1st Faculty of Medicine, Charles University, Prague, Czech Republic, 6Institute for Forensic Medicine, 3rd Faculty of Medicine, Charles University, Prague, Czech Republic, 7Children´s Heart Centre, 2nd Faculty of Medicine, Charles University and Motol University Hospital, Prague, Czech Republic, 8Department of Cardiology, Faculty Hospital Olomouc and Palacký University, Olomouc, Czech Republic, 9Institute for Forensic Medicine, Faculty Hospital Olomouc, Olomouc, Czech Republic, 10Institute for Forensic Medicine, Hospital Bulovka, 2nd Faculty of Medicine, Charles University, Prague, Czech Republic

Introduction: Hereditary cardiomyopathy is associated with risk of sudden cardiac death (SCD). Genetic aetiology of SCD in a representative Czech cohort with post mortem diagnosis of cardiomyopathy was studied and results compared to those in living cases.

Patients and Methods: Altogether 47 victims of SCD with post mortem diagnosis of hypertrophic- (HCM; 18/47), arrhythmogenic- (ACM; 19/47) and dilated cardiomyopathy (DCM; 10/47) were ascertained. Concurrently, genetic testing was performed in 114 living patients (HCM 54/114, ACM 22/114, DCM 38/114). NGS (MiSeq platform; was utilised for a custom-made panel comprising 100 candidate genes (Sophia Genetics, Switzerland).

Results: The causative detection rate (according to classes 4 or 5) in SCD victims with DCM was 60 % (6/10) and in living patients with DCM 47.4 % (18/38). Variants in TTN, RBM20, DES and FLNC prevailed. Detection rate in ACM was 5 % (1/19 in SCN5A gene) in SCD victims and 31.8 % (7/22) [JJ1] in living patients. The detection rate in SCD victims with HCM was 16 % (3/18) and in living patients 35 % (19/54). The most prevalent gene was MYBPC3, while PRKAG2 was detected in SCD victim and in cardiac arrest survivor.

Conclusion: Post-mortem genetic analysis in DCM yields a high detection rate and allows effective primary prevention of SCD in at risk relatives. In contrast, molecular autopsy of HCM and ACM renders a much lower yield which could reflect more complex genetic aetiology in SCD. Supported by Ministry of Health of the Czech Republic, grant Nr. NV18-02-00237 and 00064203/6003

A. Krebsova: None. P. Votýpka: None. P. Peldová: None. K. Ruecklova: None. V. Zoubkova: None. S. Pohlova-Kucerova: None. A. Pilin: None. M. Kulvajtova: None. M. Kubanek: None. M. Bilek: None. T. Tavacova: None. J. Petrkova: None. M. Dobias: None. P. Tomasek: None. M. Macek Sr.: None. M. Macek Jr.: None. J. Janousek: None. J. Kautzner: None.


Genome wide association analysis in dilated cardiomyopathy revealed two new susceptibility loci for systolic heart failure

S. Garnier1,2, M. Harakalova3,4, S. Weiss5,6, M. Mokry3,7,8, J. van Setten3, C. Proust9, L. Duboscq-Bidot1,2,10, A. Boland11,12, J. Deleuze11,12,13, M. Dörr6,14, F. Asselbergs3,15,16, F. Cambien9, E. Villard1,2,10, D. Trégouët9, P. Charron1,2,10,17

1Sorbonne Université, INSERM, UMR-S1166, research unit on cardiovascular disorders, metabolism and nutrition, Team Genomics & Pathophysiology of Cardiovascular Diseases, Paris, France, 2ICAN Institute for Cardiometabolism and Nutrition, Paris, France, 3Department of Cardiology, Division Heart & Lungs, University Medical Center Utrecht, Utrecht University, Utrecht, Netherlands, 4Regenerative Medicine Utrecht (RMU), University Medician Center Utrecht, Utrecht, Netherlands, 5Interfaculty Institute for Genetics and Functional Genomics, Department of Functional Genomics, University Medicine Greifswald, Greifswald, Germany, 6DZHK (German Centre for Cardiovascular Research), partnersite Greifswald, Greifswald, Germany, 7Laboratory of Clinical Chemistry and Haematology, University Medical Center, Heidelberglaan 100, Utrecht, Netherlands, 8Laboratory for Experimental Cardiology, University Medical Center Utrecht, Heidelberglaan 100, Utrecht, Netherlands, 9INSERM UMR_S 1219, Bordeaux Population Health Research Center, University of Bordeaux, Bordeaux, France, 10APHP, Pitié-Salpêtrière Hospital, Cardiology Department, Paris, France, 11Centre National de Recherche en Génomique Humaine (CNRGH), Institut de Biologie François Jacob, CEA, Université Paris-Saclay, Evry, France, 12Laboratory of Excellence GENMED (Medical Genomics), Paris, France, 13Centre d’Etude du Polymorphisme Humain, Fondation Jean Dausset, Paris, France, 14Department of Internal Medicine B, University Medicine Greifswald, Greifswald, Germany, 15Institute of Cardiovascular Science, Faculty of Population Health Sciences, University College London, London, United Kingdom, 16Health Data Research UK and Institute of Health Informatics, University College London, London, United Kingdom, 17AP-HP, Département de génétique, Centre de Référence Maladies Cardiaques Héréditaires, Hôpital Pitié-Salpêtrière, Paris, France

We conducted the largest Genome Wide Association Study performed so far in Dilated Cardiomyopathy (DCM), a leading cause of systolic heart failure and cardiovascular death. Using a discovery phase of 2,719 cases and 4,440 controls and a replication phase of 584 independent cases and 966 controls, we identified and replicated two new DCM-associated loci one on chromosome 3p (meta-analysis p = 5.3 10−13) and the second on chromosome 22q (meta-analysis p = 5.0 10−10) while confirming the two previously identified DCM loci on chromosome 10 and 1, BAG3 and HSPB7 for an estimated heritability of 31% ± 8%. The genetic risk score constructed from the number of lead risk-alleles at these 4 loci revealed a 27% risk increased in individuals with 8 risk-alleles compared to the 5 risk alleles reference group. The two association signals were then fine-mapped by combining in silico and functional genomics investigations. While a few genes remain candidates at the second locus and deserve further investigations, our work clearly identified one gene as responsible for the association at the first locus whose role in the pathophysiology of DCM is supported by recent observations in human and mice. As the biological pathway in which this gene is involved is a potential target for pharmacological agents, our finding opens novel therapeutic perspectives for treating or preventing heart failure. These results provide new findings that add both on the understanding of the genetic architecture of heart failure and on potential new players involved in the pathophysiology of this devastating disease.

S. Garnier: None. M. Harakalova: None. S. Weiss: None. M. Mokry: None. J. van Setten: None. C. Proust: None. L. Duboscq-Bidot: None. A. Boland: None. J. Deleuze: None. M. Dörr: None. F. Asselbergs: None. F. Cambien: None. E. Villard: None. D. Trégouët: None. P. Charron: None.


Utility of genetics for risk stratification in paediatric dilated cardiomyopathy

J. C. Herkert1, M. H. van der Meulen2, S. L. den Boer2, G. J. du Marchie Sarvaas3, N. Blom4,5, A. D. J. ten Harkel4, H. M. P. J. Breur6, L. A. J. Rammeloo7, R. Tanke8, C. Marcelis9, I. M. B. H. van de Laar10, J. M. A. Verhagen10, A. Baas11, A. Sammani11, I. Christiaans1, D. Q. C. M. Barge-Schaapveld12, R. H. Lekanne dit Deprez13, J. P. van Tintelen11, M. Dalinghaus2

1University of Groningen, University Medical Center Groningen, Department of Genetics, Groningen, Netherlands, 2Erasmus MC, University Medical Center Rotterdam, Department of Paediatric cardiology, Rotterdam, Netherlands, 3University of Groningen, University Medical Center Groningen, Department of Paediatric cardiology, Groningen, Netherlands, 4University of Leiden, Leiden University Medical Center, Department of Paediatric cardiology, Leiden, Netherlands, 5Amsterdam University Medical Center, location AMC, Department of Paediatric cardiology, Amsterdam, Netherlands, 6University of Utrecht, Wilhelmina Children’s Hospital, University Medical Center Utrecht, Department of Paediatric cardiology, Utrecht, Netherlands, 7Amsterdam University Medical Center, location Free University Medical Center, Department of Paediatric cardiology, Amsterdam, Netherlands, 8Radboud University Medical Center, Department of Paediatric cardiology, Nijmegen, Netherlands, 9Radboud University Medical Center, Department of Genetics, Nijmegen, Netherlands, 10Department of Clinical Genetics, Erasmus MC, University Medical Center Rotterdam, Rotterdam, Netherlands, 11University of Utrecht, University Medical Center Utrecht, Department of Genetics, Utrecht, Netherlands, 12University of Leiden, Leiden University Medical Center, Department of Clinical Genetics, Leiden, Netherlands, 13Amsterdam University Medical Center, location AMC, Department of Clinical Genetics, Amsterdam, Netherlands

Introduction: Dilated cardiomyopathy (DCM) in children may have an underlying genetic cause in a substantial number of cases. We describe the current practice and results of genetic evaluation in children with DCM and evaluate genotype-phenotype correlations that may guide prognosis.

Methods: We performed a multicentre prospective observational study in children diagnosed with DCM from 2010-2017.

Results: One hundred forty-four patients were included. Initial diagnostic categories consisted of idiopathic DCM in 67 children (47%), familial in 18 (13%), neuromuscular in 7 (5%), inborn error of metabolism in 4 (3%), malformation syndrome in 2 (1%), myocarditis in 23 (16%) and ‘other’ in 23 (16%). Median follow-up time was 2.1 years [IQR 1.0-4.3]. Hundred-seven patients (74%) underwent genetic testing. A likely pathogenic (LP) or pathogenic (P) variant was found in 39 children (36%); the majority in MYH7 (n = 9). Three patients had more than one LP/P variant. In at least 5/39 patients (13%) the variant occurred de novo. During the study, 39 patients (27%) reached a study endpoint (SE: all-cause death or heart transplantation). Transplant-free survival was significantly lower in patients with a LP/P variant (P = 0.005). Children who carried a LP/P variant were 2.8 times more likely to reach a SE compared to children without, while clinical characteristics at diagnosis did not differ (hazard ratio 2.8; 95%CI 1.3-5.8, P = 0.007).

Conclusions: Genetic testing is useful to predict clinical outcome in children with DCM and should be incorporated into the initial work-up. Patients with a LP/P variant have a poorer prognosis than patients without LP/P variant.

J.C. Herkert: None. M.H. van der Meulen: None. S.L. den Boer: None. G.J. du Marchie Sarvaas: None. N. Blom: None. A.D.J. ten Harkel: None. H.M.P.J. Breur: None. L.A.J. Rammeloo: None. R. Tanke: None. C. Marcelis: None. I.M.B.H. van de Laar: None. J.M.A. Verhagen: None. A. Baas: None. A. Sammani: None. I. Christiaans: None. D.Q.C.M. Barge-Schaapveld: None. R.H. Lekanne dit Deprez: None. J.P. van Tintelen: None. M. Dalinghaus: None.


Non-segregation of truncating TTN variants in families with dilated cardiomyopathy (DCM)

A. G. Fleming, L. Brett, S. Wilkinson, S. John, L. Briggs, M. Edwards, D. J. Morris-Rosendahl

Clinical Genomics Laboratory, London, United Kingdom

DCM is a highly heterogeneous condition with over 30 genes implicated in inherited forms, including TTN. TTN truncating variants are found in ~2% of the general population (Roberts et al. (2015) Sci Transl Med. 7(270); 270) but there is a higher association with DCM for truncating variants in cardiac isoforms of titin; therefore, only truncating variants in exons which are spliced into >90% of TTN cardiac transcripts are reported as potentially pathogenic in DCM by our laboratory. We have identified TTN truncating variants that have been classified as either likely pathogenic or pathogenic in 82 of 613 DCM patients in our cohort. Due to the prevalence of TTN truncations in healthy individuals, predictive testing for truncating TTN variants is offered with caution, and instead segregation testing in affected relatives is first undertaken. Within our cohort we have found several variants which do not segregate with DCM. Out of a total of 36 individuals, across 25 families, we have 5 cases of non-segregation of truncating TTN variants; 3 frameshift, 1 splice site and one exonic deletion. All of these non-segregating variants are absent from populations databases and occur in exons which are spliced into 100% of cardiac TTN isoforms; and were therefore initially classified as likely pathogenic. These findings highlight the importance of a cautious approach when interpreting TTN truncating variants, as well as the risks of predictive testing; especially if this results in clinical discharge for individuals who do not carry the familial truncating TTN variant.

A.G. Fleming: None. L. Brett: None. S. Wilkinson: None. S. John: None. L. Briggs: None. M. Edwards: None. D.J. Morris-Rosendahl: None.


Optimization and refinement of the diagnosis of genetic dyslipidemias by targeted NGS

L. Zuurbier, O. R. F. Mook, M. A. Haagmans, J. C. Defesche

Amsterdam University Medical Centers, Amsterdam, Netherlands

Introduction: Genetic and acquired dyslipidemias are very frequent in the general population and strongly associated with premature atherosclerosis, myocardial infarctions, arterial complications and sudden death at young age. Genetic lipoprotein abnormalities can be explained by a limited number of genes. For optimal clinical management, risk assessment and prevention of cardiovascular disease an accurate diagnosis is an absolute requirement, but the interconnection between the metabolic pathways of different lipoprotein fractions jeopardizes this when only lipid levels are assessed.

Materials and Methods: In order to pinpoint the culprit lipoprotein fraction of clinical dyslipidemia in 4277 patients, we evaluated NGS and CNV data, including 29 dyslipidemia-associated genes that enabled to examine all separate lipoprotein components simultaneously. With this approach 10 types of dyslipidemia can be discriminated. Moreover, this panel includes a genetic risk factor for statin intolerance.

Results: A genetic cause for dyslipidemia was found in 32% of the patients. Of these, 4% carried a CNV. The specific initial clinical diagnoses of hypercholesterolemia or hypertriglyceridemia were confirmed in only 17 and 35% of patients, respectively. However, unsolicited findings causal for a second or an unexpected type of dyslipidemia, were encountered in 17% of cases, and suggest a broader role than initially anticipated for variants causing dysbetalipoproteinemia or hypertriglyceridemia.

Conclusions: The use of the NGS-Dyslipidemia gene panel accurately separated genetic from acquired dyslipidemias and within the detected genetic dyslipidemias the diagnosis was refined based on unsolicited findings in a substantial number of patients. This allows targeted and optimal treatment to reduce cardiovascular risk.

L. Zuurbier: None. O.R.F. Mook: None. M.A. Haagmans: None. J.C. Defesche: None.


Hypermethylation of NRG1 gene correlates with the presence of heart defects in Down’s syndrome

A. Dobosz, A. Grabowska, M. Bik-Multanowski

Department of Medical Genetics, Jagiellonian University Medical College, Krakow, Poland

Congenital heart defects can decrease the quality of life and life expectancy in affected individuals, and constitute a major burden for the health care systems. Endocardial cushion defects are among the most prevalent heart malformations in the general population, and are extremely frequent (approximately a 100-fold higher prevalence) in children with Down syndrome. Several genes have been proposed to be involved in the pathogenesis of these malformations, but no common pathogenic DNA variants have been identified so far. Here, we focussed on constitutive, epigenetic alterations of function of selected genes, potentially important for endocardial cushion development. We used two types of microarrays, dedicated for assessment of gene promoter methylation and whole genome expression.

First, we compared the gene promoter methylation profiles between two groups of Down syndrome patients, with and without heart defects of endocardial cushion-type. Then, to determine the functional role of the detected methylation alterations, we assessed the expression of the genes of interest. We detected significant hypermethylation of the NRG1 gene promoter region in children with heart defects. NRG1 is a key factor in maturation of endocardial cushions. Supplementary gene expression assessment revealed significantly decreased activity of the ERBB3, SHC3 and SHC4 genes in children with heart defects. The above three genes are closely related to the NRG1 gene and are crucial elements of the NRG/ErbB pathway. The results of this pilot study show that hypermethylation of the NRG1 gene promoter can reflect the functional genome alteration contributing to development of congenital heart defects of endocardial cushion-type.

A. Dobosz: None. A. Grabowska: None. M. Bik-Multanowski: None.


How Effective is Genetic Testing for Familial Hypercholesterolaemia Patients in Tayside?

C. Leung, E. Gellatly, J. Berg

Ninewells Hospital and Medical School, University of Dundee, Dundee, United Kingdom

Background: DNA analysis for Familial Hypercholesterolaemia (FH), including the LDLR, PCSK9 and ApoE genes, is available to Scottish individuals with high cholesterol who fulfil Simon Broome criteria. The aim of mutation identification is to allow more effective management of the proband, as well as cascade testing and identification of at risk individuals. The ultimate aim is early statin treatment of affected individuals with prevention of cardiovascular morbidity.

Method: 60 index patients with causative LDLR mutations were identified between 2010 and 2018 in NHS Tayside (Scotland). We used record linkage to assess the effect of genetic testing on cholesterol levels on probands and the number of subsequent cascade tests performed on family members. The main endpoint we used was achieving a 50% reduction of LDL cholesterol levels, as recommended by the UK National Institute of Clinical Excellence (NICE).

Results: 49/189 (25.9%) eligible first degree relatives received cascade testing. Where data was available, 6/22 (27.3%) patients met the 50% reduction target in LDL-C levels. There was no demonstrable relationship between cholesterol reduction and attendance at a cardiovascular risk clinic.

Conclusions: The number of cascade tests following diagnosis in a proband is low. Even if a causative LDLR mutation was identified, there was limited impact on the clinical improvement of cholesterol levels, with only a minority of patients achieving recommended cholesterol lowering following diagnosis. A better mechanism for cascade testing and management of FH is required to gain the maximum clinical benefit from DNA analysis in Familial Hypercholesterolaemia.

C. Leung: None. E. Gellatly: None. J. Berg: None.


Mutation spectrum and polygenic score in German patients with familial hypercholesterolemia

L. Rieck1, F. Bardey1, T. Grenkowitz1, L. Bertram2, J. Helmuth3, C. Mischung3, J. Spranger1, T. Bobbert1, U. Kassner1, I. Demuth1

1Charité Universitätsmedizin Berlin, Berlin, Germany, 2Lübeck Interdisciplinary Platform for Genome Analytics, Institutes of Neurogenetics and Cardiogenetics, Lübeck, Germany, 3Department Molecular Diagnostics, Labor Berlin, Berlin, Germany

Introduction:Autosomal-dominant familial hypercholesterolemia (FH) is a genetic disorder characterized by increased plasma concentrations of LDL-Cholesterol and a substantially elevated risk for cardiovascular disease. Mutations in three major genes are known to cause FH: the LDL receptor gene (LDLR), the apolipoprotein B gene (APOB) and the proprotein convertase subtilisin/kexin 9 gene (PCSK9). Our aim is to analyze the mutation spectrum and to determine a polygenic risk score in a cohort of hypercholesterolemic patients.

Methods: We screened 336 patients who were clinically assessed by the Dutch Lipid Clinic Network (DLCN) score and suspected to have FH for disease causing variants and genotyped six single nucleotide polymorphisms (SNPs) to calculate the polygenic risk score for the FH patients and a control group of 1,946 healthy participants.

Results: We identified an FH causing variant in 117 of the 336 patients (12 novel variants) from the FH cohort. Most sequence variants were found in the LDLR gene (84.9%) out of which 44.4% were of the missense type. The discriminatory power of the DLCN score distinguished FH mutation carriers from non-carriers with an AUC = 0.76 (p < .001) in a receiving operator characteristic (ROC) curve, similar to the value for native LDL-C (AUC = 0.77 (p < .001)). Similar to previous studies, the mean polygenic risk score was significantly higher in FH mutation negative subjects than in FH mutation positive patients (p < .05) and healthy controls (p < .001).

Conclusions: This study substantiates the known clinical and genetic variability of FH for German hypercholesterolemic patients and examines a possible polygenic aetiology.

L. Rieck: None. F. Bardey: None. T. Grenkowitz: None. L. Bertram: None. J. Helmuth: None. C. Mischung: None. J. Spranger: None. T. Bobbert: None. U. Kassner: None. I. Demuth: None.


FLNC gene in Hypertrophic Cardiomyopathy: an effort to accurate variant classification

E. Cuesta-Llavona, R. Lorca, B. Díaz-Molina, J. Lambert, B. Alonso, S. Iglesias-Álvarez, J. Reguero, E. Coto, J. Gómez

Hospital Universitario Central de Asturias, Oviedo, Spain

Hypertrophic Cardiomyopathy (HCM) is the most common inherited heart disease. Recent studies have identified the Filamin C (FLNC) gene as a HCM-associated gene. The aim of this study is to assign the percentage of HCM cases which could be relationated with FLNC variants.

We sequenced 924 index cases of HCM for a comprehensive cardiovascular gene panel of 194 genes, in an Ion GeneStudio S5 Sequencer. We identified 365 patients (39%) with variants in the sarcomeric and FLNC genes. Among them, 330 (90%) were carriers of sarcomeric variants, and 35 (10%) harbored rare FLNC variants (either not described or ≤ 10 cases in gnomAD). Eleven patients were carriers of both sarcomeric and FLNC variant, thus their were excluded to the further analysis and variant classification since their pathogenicity is complex to measure. In this way, we finally selected 24 carriers of 20 different FLNC variants. Family studies were performed in 16/24 cases. In total, we identified 55 relatives who harbor a FLNC variant, thirty nine of them have a HCM diagnosis (71% penetrance). According to the ACMG criteria, and the performed family studies, seven variants (35%) have been classified as pathogenic or likely pathogenic, twelve as variants of unknown significance (60%), and a variant classified as benign or likely benign (5%). In conclusion, at least 1.2% of HCM index cases harbor a FLNC likely pathogenic variant. However, further co-segregation and functional studies have to be performed in order to accomplish an accurate variant classification, specially in VUS variants.

E. Cuesta-Llavona: None. R. Lorca: None. B. Díaz-Molina: None. J. Lambert: None. B. Alonso: None. S. Iglesias-Álvarez: None. J. Reguero: None. E. Coto: None. J. Gómez: None.


A founder truncating mutation in GDF1 causes autosomal-recessive right isomerism in multiplex Arab kindreds.

D. Yagel1,2, Y. Bolkier3,2, O. Barel4, A. Vardi3,2, D. Mishali3,2, A. Veber1, N. Shalva1, U. Katz3,2, S. Abudi1,2, N. Kol4, A. Raas-Rothschild5,2, G. Rechavi4,2,6, B. Pode-Shakked1,7,2, Y. Anikster1,2,6

1Metabolic Disease Unit, Edmond and Lily Safra Children’s Hospital, Sheba Medical Center, Ramat Gan, Israel, 2Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv, Israel, 3Department of Pediatric Cardiac Intensive Care, Edmond Safra International Congenital Heart Center, Edmond and Lily Safra Children’s Hospital, Sheba Medical Center, Ramat Gan, Israel, 4Sheba Cancer Research Center, Sheba Medical Center, Ramat Gan, Israel, 5The Institute for Rare Diseases, The Danek Gertner Institute of Human Genetics, Sheba Medical Center, Ramat Gan, Israel, 6The Wohl Institute for Translational Medicine, Sheba Medical Center, Ramat Gan, Israel, 7Talpiot Medical Leadership Program, Sheba Medical Center, Ramat Gan, Israel

The genetic basis of congenital heart malformations associated with disruption of left-right (L-R) asymmetry is broad and heterogenous, with variants in over twenty five genes implicated thus far. Of these, deleterious mutations in the Growth/Differentiation Factor 1 (GDF1) gene have recently been shown to cause heterotaxy with varied complex heart malformations of left-right patterning, in 23 individuals reported to date, either in monoallelic or biallelic state. We report three unrelated individuals exhibiting heterotaxy with congenital heart defects, each originating from consanguineous kindreds of Arab-Muslim descent. Using Whole exome sequencing, a shared novel homozygous truncating c.608G>A (p.W203*) mutation in the GDF1 gene, was revealed as the molecular basis of their disease. Subsequently, targeted sequencing of this variant showed full segregation with the disease in these families, with over 15 reportedly affected individuals, enabling genetic counselling, prenatal diagnosis and planning of future pregnancies. Our findings further confirm the association of GDF1 variants, heterotaxy and congenital heart defects of left-right patterning, and expand the previously described phenotypic spectrum and mutational profile. Finally, given the high rate of consanguinity and high frequency of the p.W203* mutation in the Arab-Muslim population residing near the city of Hebron, we suggest targeted screening for this variant in relevant clinical circumstances.

D. Yagel: None. Y. Bolkier: None. O. Barel: None. A. Vardi: None. D. Mishali: None. A. Veber: None. N. Shalva: None. U. Katz: None. S. Abudi: None. N. Kol: None. A. Raas-Rothschild: None. G. Rechavi: None. B. Pode-Shakked: None. Y. Anikster: None.


Pathogenic variationin the copper binding domain of lysyl oxidase causes aortic dilation modified by sex and mechanical forces

K. Tsang1, R. H. Knutsen1, C. J. Billington Jr1, E. Lindberg1, H. Steenbock2, A. Wardlaw-Pickett1, C. K. E. Bleck1, J. Brinckmann2, B. A. Kozel1

1National Institutes of Health, Bethesda, MD, United States, 2University of Luebeck, Luebeck, Germany

Mutations in lysyl oxidase (LOX) contribute to familial thoracic aortic aneurysm. GnomAD reveals variation throughout LOX, with more variants reported in the region encoding the N-terminal pro-domain. While ClinVar showed disease-associated stopgain and frameshift variants throughout LOX, the majority of pathogenic missense mutations were identified near the copper binding domain, within the relatively variant-poor C-terminal catalytic domain.

We then studied theLoxb2b370.2Clo (c.G854T; p.C285F) mouse whose mutationfalls within the copper binding domain. The LoxC285F protein was stably secreted but exhibited decreased enzymatic activity and altered elastin crosslinks. Structural analysisby two photon and scanning electron microscopyrevealed holes in the elastic lamellar sheets that increased with age and blood pressure. As in humans, the rate of aortic dilation was higher in males, but was also amplified by pregnancy. Lox mutants showed altered transcription of matrisome genes and their aortas showed increased susceptibility to elastase.

Taken together, these findings suggest that missense variation surrounding the LOX copper binding domain increases risk for aortic aneurysm while missense variation in the region encoding the N-terminal pro-domain appears largely tolerated. Novel gene-first studies are underway to test this hypothesis in a large hospital-based cohort. Our mouse studies confirm that copper binding domain LOX variants cause disease through the production of a dysfunctional extracellular matrix that is unusually susceptible to proteolytic damage. In both humans and mice, the progressive weakening of the connective tissue leads to worsening aortic disease, modified by sex and blood pressure.

Funding: NIH HL006244, USA

K. Tsang: None. R.H. Knutsen: None. C.J. Billington Jr: None. E. Lindberg: None. H. Steenbock: None. A. Wardlaw-Pickett: None. C.K.E. Bleck: None. J. Brinckmann: None. B.A. Kozel: None.


Rare variants in HCN4 identified in the general population are associated with complete atrioventricular (AV) block, 1. degree AV block and bundle branch block, results from 50.000 exomes

J. S. Jensen1,2, O. B. Vad1,2, C. Paludan-Müller2,1, P. R. Lundegaard1, J. H. Svendsen2,3, S. Haunsø2,3, H. Bundgaard2,3, M. S. Olesen1,2

1Department of Biomedical Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark, 2Laboratory for Molecular Cardiology, Department of Cardiology, Centre for Cardiac, Vascular, Pulmonary and Infectious Diseases, Rigshospitalet, Copenhagen University Hospital, Copenhagen, Denmark, 3Department of Clinical Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark

Introduction: Cardiac conduction disorders cover disorders such as atrioventricular (AV) block and bundle branch block (BBB). Genome-wide association studies have identified more than 100 genetic loci for atrial fibrillation, including HCN4 loci associated with duration of the PR interval, a proxy for AV dysfunction. Recent candidate studies suggest an association between HCN4 variants and AV-block. We seek to determine the contribution of rare genetic variants in HCN4 to complete AV-block, 1. degree AV-block and BBB in the general population.

Materials/methods: The UK Biobank is a population-based study of 500 000 individuals including a subset with genome-wide genotyping and exome sequencing (n = 45,596). In this case-control study, we included persons of genetically determined white-European ancestry and analyzed their exome data. Analyses were performed using a logistic mixed-effects model. A gene-based burden analysis and single variant test were performed to examine the relationship between HCN4 variants and complete AV-block, 1. degree AV-block and BBB in the general population.

Results: The study included 43,371 persons. In an analysis of the HCN4 genes a significant association between rare variants (MAF < 0.01) in HCN4 gene was found (complete AV-block, P=2.5 x 10−5, 1. degree AV-block, P=1.3 x 10−3 and BBB, P=0.01). The association to complete AV block was mostly driven by the variants Ser835Phe (P=2.7e−3), Glu153Gly (P=3.5e−3) and Arg378Cys (P=6.3e−3).

Conclusions: Rare HCN4 variants contribute to the risk of complete AV-block, 1. degree AV-block and BBB in the general population. These HCN4 variants seem to confer a substantial penetrance. Clinical screening for some of these variants seems appropriate.

J.S. Jensen: None. O.B. Vad: None. C. Paludan-Müller: None. P.R. Lundegaard: None. J.H. Svendsen: None. S. Haunsø: None. H. Bundgaard: None. M.S. Olesen: None.


MicroRNAs as biomarkers of heart failure

A. M. Peterlin1, K. Pocivavsek2, D. Petrovic1, B. Peterlin3

1Institute of Histology and Embriology, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia, 2Department of Cardiovascular Surgery, University Medical Centre Ljubljana, Ljubljana, Slovenia, 3Clinical Institute of Genomic Medicine, University Medical Centre Ljubljana, Ljubljana, Slovenia

Introduction: MicroRNAs are highly investigated for their role in the pathogenesis of cardiovascular diseases. Nevertheless, evidence for clinical implementation is still lacking. In our systematic review, we evaluated the potential of microRNA as pathophysiological and diagnostic biomarkers of heart failure.

Materials and methods: The literature search was conducted in the PubMed database up to August 2019. Newcastle-Ottawa quality assessment scale was used to assess the quality of included research papers. Using miRTarBase, we identified all known gene targets for selected miRNAs that were differentially expressed in research papers included in the systematic review. We then comprised the list of all target genes and performed gene set enrichment analysis using Enrichr. Results from KEGG, BioPlanet, and Panther databases were analyzed.

Results: We identified 72 differentially expressed microRNA molecules among groups of heart failure patients and control groups. There was no substantial overlap of differentially expressed miRNAs among different studies, only five miRNAs (miR-1228, miR-122, miR-423-5p, miR-142-3p, and exosomal miR-92b-5p) were differentially expressed in more than one included study. Gene set enrichment analysis, based on the gene targets of microRNAs presented in included studies, showed that gene targets of differentially expressed miRNAs were enriched in MAPK signaling pathway, TGFβ signaling pathway, PI3K-Akt signaling pathway, IL-2 signaling pathway, apoptosis pathway, p53 activity regulation, angiogenesis pathway, and PDGF signaling pathway.

Conclusions: Results of our systematic review show that currently, there is insufficient support for the use of any of the presented miRNAs as a pathophysiological or prognostic biomarker in the clinical setting.

A.M. Peterlin: None. K. Pocivavsek: None. D. Petrovic: None. B. Peterlin: None.


Germline variants in transcription factor HEY2 functional domains lead to congenital heart defects and thoracic aortic aneurysms

E. S. van Walree1,2, G. Dombrowsky3, I. E. Jansen2, M. Umićević Mirkov2, R. Zwart4, A. Ilgun4, D. Guo5, S. B. Clur6, A. S. Amin7, J. E. Savage2, A. C. van der Wal8, Q. Waisfisz9, A. Maugeri9, A. Wilsdon10, F. A. Bu’lock11, M. E. Hurles12, S. Dittrich13, F. Berger14, E. Audain Martinez3, M. Hitz3, D. M. Milewicz5, D. Posthuma2, H. Meijers-Heijboer1, A. V. Postma1,4, I. B. Mathijssen1

1Department of Clinical Genetics, Amsterdam UMC, University of Amsterdam, Meibergdreef 9, Amsterdam, Netherlands, 2Department of Complex Trait Genetics, Center for Neurogenomics and Cognitive Research, Amsterdam Neuroscience, Vrije Universiteit Amsterdam, de Boelelaan 1085, Amsterdam, Netherlands, 3Department of Congenital Heart Disease and Pediatric Cardiology, Universitätsklinikum Schleswig-Holstein Kiel, Kiel, Germany, 4Department of Medical Biology, Amsterdam UMC, University of Amsterdam, Meibergdreef 9, Amsterdam, Netherlands, 5Department of Internal Medicine, McGovern Medical School, University of Texas Health Science Center at Houston, 6431 Fannin, Houston, TX, United States, 6Department of Pediatric Cardiology, Emma Children’s Hospital, Amsterdam UMC, University of Amsterdam, Meibergdreef 9, Amsterdam, Netherlands, 7Department of Clinical and Experimental Cardiology, Amsterdam UMC, University of Amsterdam, Meibergdreef 9, Amsterdam, Netherlands, 8Department of Pathology, Amsterdam UMC, University of Amsterdam, Meibergdreef 9, Amsterdam, Netherlands, 9Department of Clinical Genetics, Amsterdam UMC, Vrije Universiteit Medisch Centrum, de Boelelaan 1118, Amsterdam, Netherlands, 10School of Life Sciences, University of Nottingham, Queen’s Medical Centre, Nottingham, United Kingdom, 11East Midlands Congenital Heart Centre and University of Leicester, Glenfield Hospital, Leicester, United Kingdom, 12Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, United Kingdom, 13Department of Pediatric Cardiology, University of Erlangen-Nürnberg, Loschgestrasse 15, 91054, Erlangen, Germany, 14German Heart Center Berlin, Department of Congenital Heart Disease, Pediatric Cardiology, Berlin, Germany

Background: Affecting approximately 1% of all newborns, cardiovascular defects (CVDs) are the most common congenital anomaly worldwide. Here, we present a large family from a genetically isolated population presenting with CVDs, including congenital heart defects (CHD) and familial thoracic aortic aneurysms (FTAA), caused by a loss-of-function mutation in the Notch target gene HEY2, which hasn’t been associated with hereditary CVDs in humans previously.

Methods: The index patient, who has severe CVD, underwent trio WES. The identified variant was subsequently screened in 30 members of this patient’s family, which has a high prevalence of CVDs. Additionally, 3011 unrelated cases and 4940 controls from two datasets (one with CHD and one with FTAA) were screened for rare risk variants in HEY2. To investigate how the various identified variants influence gene function, the regulatory activity of mutated and wildtype HEY2 on a target gene was measured with luciferase assays and qPCR.

Results: A homozygous frameshift variant (p.G108*) in HEY2 was identified in three individuals with life-threatening CVDs. Twenty family members were found to be heterozygous for this variant, of whom 80% had CVD. Two additional CVD datasets show enrichment for rare, likely deleterious variants in HEY2 after meta-analysis. Lastly, we demonstrate that these variants induce failure of repression of HEY2 target genes.

Conclusions: We find that rare HEY2 variants predispose to a broad spectrum of CVDs and advise clinicians to consider HEY2 as a causative gene in unsolved CVD patients. Identifying a potentially deleterious variant in HEY2 warrants clinical follow-up.

E.S. van Walree: None. G. Dombrowsky: None. I.E. Jansen: None. M. Umićević Mirkov: None. R. Zwart: None. A. Ilgun: None. D. Guo: None. S.B. Clur: None. A.S. Amin: None. J.E. Savage: None. A.C. van der Wal: None. Q. Waisfisz: None. A. Maugeri: None. A. Wilsdon: None. F.A. Bu’lock: None. M.E. Hurles: None. S. Dittrich: None. F. Berger: None. E. Audain Martinez: None. M. Hitz: None. D.M. Milewicz: None. D. Posthuma: None. H. Meijers-Heijboer: None. A.V. Postma: None. I.B. Mathijssen: None.


The Unknown Genetic Architecture of Severe Hypercholesterolemia in Finland

N. Junna1, S. Ruotsalainen1, S. Ripatti1,2, E. Widén1

1FIMM, Helsinki, Finland, 2Department of Public Health, Clinicum, Faculty of Medicine, University of Helsinki, Helsinki, Finland

While severe hypercholesterolemia is a significant risk factor for ischemic heart disease (IHD), Finnish patients remain inadequately identified and only a part of their mutation load is known. To elucidate the genetic architecture of severe hypercholesterolemia in Finland, we have screened participants of the GeneRISK-study (n = 7 342) originating from South-eastern Finland for novel mutations and utilized the FinnGen-cohort (n = 110 654) to assess the mutation prevalence, impact, and nationwide distribution.

Five percent (n = 366) of GeneRISK-participants had severe hypercholesterolemia (LDL ≥ 5 mmol/l) and exome-sequencing revealed 3 carriers of previously known mutations (LDLR R594Q, LDLR P309Kfs and APOB R3527Q, which has not previously been reported in Finland). We also identified a novel, likely pathogenic mutation, LDLR R574L. We assessed the geographical distribution of the mutation and the cardiometabolic status of mutation carriers in the nationwide FinnGen-cohort and compared them with 5 previously described hypercholesterolemia-mutations (LDLR R594Q, LDLR G844D, LDLR L401H, LDLR D579N, LDLR C352W). The novel LDLR R574L-mutation was unevenly distributed in the country with a high prevalence among individuals born in the Southeast (the ceded Karjala region) and prevalence of cardiovascular disease among mutation carriers was elevated (22% had been diagnosed with IHD and 14% with stroke). The data further showed that less than half of mutation carriers (both the novel and previously known mutations) were on statin therapy.

Our data show that the genetic spectrum of severe hypercholesterolemia in Finland is more diverse than previously anticipated, and further highlight the urgent need for better recognition and treatment of this disease.

N. Junna: None. S. Ruotsalainen: None. S. Ripatti: None. E. Widén: None.


Mendelian randomisation identifies pleiotropy between uric acid and cardiovascular diseases

E. M. Wigmore1, O. Chazara1, . FinnGen Consortium2, K. Carss1, A. Matakidou1, C. Haefliger1

1AstraZeneca, Cambridge, United Kingdom, 2University of Helsinki, Helsinki, Finland

Elevated levels of uric acid (UA), hyperuricaemia, leads to the formation of urate crystals that cause gout. Hyperuricaemia has also been linked to cardiovascular and metabolic diseases but, unlike gout, causality has not been ascertained. Here we utilised biomarker data from the UK Biobank (UKB, n = ~500,000) and health outcome data from a Finnish biobank, FinnGen (n = ~135,000) to explore the causal relationship between biomarkers and disease. Genome-wide association studies (GWAS) of 48 available biomarkers were conducted in UKB and FINEMAP was applied to ascertain putative causal variants. We assessed the genetic correlation of these biomarkers with 1,485 disease endpoints in FinnGen using LD score regression. Those with a significant correlation following FDR correction were assessed for causal associations using Mendelian randomisation (MR). GWAS of UA corrected for allopurinol treatment (a UA-lowering medication) was completed on 389,114 individuals and FINEMAP identified 320 putative causal signals. Significant genetic correlations were found with multiple diseases (n = 183) including hypertensive diseases (rg=0.25, PFDR=6.2x10−38), type 2 diabetes (rg=0.29, PFDR=6.2x10−38), cardiovascular disease (rg=0.23, PFDR=9.6x10−29) and gout (rg=0.70, PFDR=8.3x10−21). Results from MR analyses confirmed the known causal association of UA with gout (IVW estimate=1.6, PFDR=7.8x10−62) as well as with polyarthropathies (IVW estimate=0.37, PFDR=1.9x10−12) but not with other diseases. A significant causal association was found with hypertension (IVW estimate=0.20, PFDR=0.0015) but horizontal pleiotropy was also detected (MR-Egger intercept P= 0.0069). Our finding suggests that although MR does not support the direct association between UA and cardiovascular disease, we observe pleiotropic effects which could support therapies targeting shared pathways.

E.M. Wigmore: A. Employment (full or part-time); Significant; AstraZeneca. O. Chazara: A. Employment (full or part-time); Significant; AstraZeneca. .. FinnGen Consortium: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Significant; AstraZeneca, Abbvie, Biogen, Celgene, Genentech, GSK, Merck, Pfizer, Sanofi. K. Carss: A. Employment (full or part-time); Significant; AstraZeneca. A. Matakidou: A. Employment (full or part-time); Significant; AstraZeneca. C. Haefliger: A. Employment (full or part-time); Significant; AstraZeneca.


Pathogenic variant in the MYBPC3 gene with founding effect in Galician population

A. Blanco-Verea1,2, R. Cruz3,2, M. Álvarez-Barredo4,1,5, B. López-Abel6,1, R. Gil1,2, E. Ramos-Luis1,2, L. Martínez-Campelo1,2, I. Quintela7,2, J. González-Juanatey4,5, Á. Carracedo8,2,3,7, M. Brion1,2,5

1Xenética Cardiovascular, Instituto de Investigación Sanitaria de Santiago, Santiago de Compostela, Spain, 2Grupo de Medicina Xenómica, Universidade de Santiago de Compostela, Santiago de Compostela, Spain, 3CIBER Enfermedades Raras, Santiago de Compostela, Spain, 4Complejo Hospitalario Universitario de Santiago, Servicio de Cardiología, Santiago de Compostela, Spain, 5CIBER Cardiovascular, Santiago de Compostela, Spain, 6Complejo Hospitalario Universitario de Santiago, Servicio de Pediatría, Santiago de Compostela, Spain, 7Centro Nacional de Genotipado (CEGEN-PRB3-ISCIII), Santiago de Compostela, Spain, 8Fundación Pública Galega de Medicina Xenómica, Santiago de Compostela, Spain

Introduction: The Myosin Binding Protein C3 gene (MYBPC3) is one of the main genes in which mutations involved in familial hypertrophic cardiomyopathy (HCM) appear. One of these pathogenic variants is the deletion rs397515926 that appears in the gnome AD database in only one individual of Latino origin (Allele Frequency=0.000004913). However, we have found 18 unrelated patients with HCM from Galicia with this variant, which has led us to think about a possible founding effect of this variant in the Galician population.

Materials and Methods: Trying to unmask a possible founding effect or a distant family relationship between patients, we made a genotyping with the Applied BiosystemsTM AxiomTM Spain Biobank Array in 6 of these patients and in a cohort of healthy Galician and Spanish population. Principal components analysis and discriminant analysis of principal components were performed in Plink and adegenet package of R software.

Results: discriminant analysis of principal components taking into account rare variants of the array and comparing our patients with the general Spanish and Galician population clearly differentiate our patients with respect to the rest.

Conclusions: Considering the results of the analysis of the genotypes between our cases and the general population, the practically null bibliographic mention and very scarce presence in databases of this variant, we could assume a possible common origin for the presence of this variant in the Galician population, highlighting a possible founding effect. Founding: Instituto de Salud Carlos III and Fondo Europeo de Desarrollo Regional (PI16/00903, CB16/11/00226, CB03/07/0088).

A. Blanco-Verea: None. R. Cruz: None. M. Álvarez-Barredo: None. B. López-Abel: None. R. Gil: None. E. Ramos-Luis: None. L. Martínez-Campelo: None. I. Quintela: None. J. González-Juanatey: None. Á. Carracedo: None. M. Brion: None.


The potential diagnostic value of circulating serum-derived miRNAs in ischemic heart disease (IHD) as a complication of type 2 diabetes mellitus (T2DM).

A. Bielska1, W. Bauer1,2, I. Sidorkiewicz1, A. Szalkowska1, J. Raczkowska1, D. Ostrowski1, P. Prokopczuk3, S. Dobrzycki3, M. Niemira1, A. Kretowski1,4

1Clinical Research Centre, Medical University of Bialystok, Bialystok, Poland, 2Clinical Research Centre, Medical University of Bialystok, Białystok, Poland, 3Department of Invasive Cardiology, Medical University of Bialystok, Bialystok, Poland, 4Department of Endocrinology, Diabetology and Internal Medicine, Medical University of Bialystok, Białystok, Poland

Introduction: Currently, IHD is the most common cause of death in patients with diabetes and it develops much earlier before clinical diagnosis. Therefore, there is an urgent need for the detection of sensitive and specific early biomarkers to predict the risk of the IHD development. We postulate that non-coding miRNAs may have higher diagnostic value for early detection of IHD than inflammatory mediators like chemokine CXCL12 and macrophage migration-inhibitory factor (MIF).

Materials and Methods: We obtained serum samples from 43 T2DM patients (24 with IHD and 19 without IHD). The levels of 798 miRNAs were analysed using NanoString nCounter Technology. Additionally, we predicted the key miRNA regulatory network and miRNAs targets and pathways that may be critical in IHD development using the Ingenuity Pathway Analysis software. The concentration of MIF and CXCL12 was estimated by enzyme-linked immunosorbent assay. Receiver operating characteristic analysis was used to assess the diagnostic value of each miRNA.

Results: Our data showed 9 miRNAs (miRNA-1224-5p, miRNA-1303, miRNA-3147, miRNA-4455, miRNA-498, miRNA-548b-3p, miRNA-548d-3p, miRNA-615-3p, miRNA-651-5p) significantly upregulated in T2DM IHD group compared to T2DM noIHD (FC>1.4 and FDR < 0.05). Furthermore, all miRNAs showed high diagnostic value (AUC>0.85). Targeted genes were identified in canonical pathways involved in the pathology of the cardiovascular system. No statistical differences were observed in the concentration of MIF and CXCL12 between tested groups.

Conclusions: Our findings suggest that circulating miRNAs might have a pivotal role in the development of IHD in T2DM patients and may be used as non-invasively biomarkers for early diagnosis.

A. Bielska: None. W. Bauer: None. I. Sidorkiewicz: None. A. Szalkowska: None. J. Raczkowska: None. D. Ostrowski: None. P. Prokopczuk: None. S. Dobrzycki: None. M. Niemira: None. A. Kretowski: None.


Derivation and validation of a polygenic risk score associated with ischemic heart failure in multiethnic cohorts

I. Paranjpe1, J. De Freitas1, N. Tsao2, R. Judy2, K. Chaudhary3, I. Forrest4, CBIPM Genomics Team, Regeneron Genomics Team, S. Coca5, J. Narula6, B. Glicksberg1, R. Do3,4, S. Damrauer2, G. Nadkarni1,3,7

1The Hasso Plattner Institute for Digital Health at Mount Sinai, New York, NY, United States, 2Department of Surgery, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States, 3The Charles Bronfman Institute for Personalized Medicine, Icahn School of Medicine at Mount Sinai, New York, NY, United States, 4Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, United States, 5Division of Nephrology, Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, NY, United States, 6Division of Cardiology, Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, NY, United States, 7Renal Program, James J Peters VA Medical Center at Bronx, New York, NY, United States

Introduction: Mortality from heart failure (HF) secondary to coronary artery disease (CAD) is 50% and risk-stratification approaches are limited. We aimed to stratify risk of progression to HF in CAD patients by developing a polygenic risk score (PRS).

Methods: Using summary statistics from a recent GWAS for HF secondary to CAD (ischemic HF), we computed risk scores by varying the rho parameter in LDPred and varying the p-value threshold and r2. We selected the PRS that maximized the association with ischemic HF (PRSiHF) from the multiethnic Mount Sinai BioMe Biobank(N=6276). We validated the association in Penn Medicine BioBank (PMBB; N=6674). We performed association analysis using logistic regression adjusted for age, sex, smoking history, hypertension, BMI, type 2 diabetes, and ten genetic principal components.

Results: In BioMe, one standard deviation (SD) increase in PRSiHF was significantly associated with HF (OR=1.1; p=0.001; 2530 cases, 3746 controls HF risk increased monotonically with PRSiHF in a subset of individuals with CAD but no evidence of obstructive CAD on coronary catheterization (N=4775). In PMBB, PRSiHF was associated with ischemic HF in European Americans (adjusted OR=1.07; p=0.02; 2692 cases, 2472 controls) but not African Africans (adjusted OR=0.94; p=0.3; 921 cases, 589 controls).

Conclusions: We derived and validated a PRS for ischemic HF in two multiethnic cohorts and demonstrated association with and without obstructive CAD. PRSiHF replicated in patients of European ancestry, but not in African ancestry. Although studies on clinical utility and in more diverse populations are needed, our results suggest application in early risk stratification.

I. Paranjpe: None. J. De Freitas: None. N. Tsao: None. R. Judy: None. K. Chaudhary: None. I. Forrest: None. S. Coca: None. J. Narula: None. B. Glicksberg: None. R. Do: None. S. Damrauer: None. G. Nadkarni: None.


Identifying Long QT syndrome (LQTS) patients in primary care, an approach for rare diseases.

W. R. H. Evans1, S. F. Weng1, R. K. Akyea1, A. D. Simms2, J. Kai1, N. Qureshi1

1University of Nottingham, Nottingham, United Kingdom, 2Department of Cardiology, Leeds Teaching Hospital NHS Trust, Leeds, United Kingdom

Introduction: LQTS is a rare (~1 in 2000 births) inherited cardiac arrhythmia that predisposes patients to syncope and sudden cardiac death. Identification and treatment can substantially reduce this risk. We aimed to identify the clinical features that precede the diagnosis of LQTS in primary care and use these to develop a predictive model.

Methods:1495 patients diagnosed with LQTS and 7475 propensity-score matched controls were identified in a database of primary care electronic records (CPRD). A range of clinical features that occurred at greater frequency in cases than control (p <0.05) before diagnosis were identified and incorporated into a multivariable logistic regression model.

Results:18 clinical features were incorporated into the final model, including: epilepsy (1.70 (1.12, 2.56)), palpitations (2.22 (1.80, 2.74)), collapse (1.636 (1.32, 2.03), mitral valve disease (2.64 (1.41, 4.91)), atrial fibrillation (1.91 (1.41, 2.60)) and IBS (1.78 (1.41, 2.26)) (OR (95% CI)). The discriminative accuracy of the model assessed by area under the curve (AUC) was 0.74, (95% CI 0.73 to 0.75), with comparable performance in two more tightly phenotyped subgroups: LQTS diagnosis <45 years and those commenced on a betablocker &/or fitted with an Implantable Cardioverter Defibrillator (ICD) post-diagnosis.

Conclusion: This study demonstrates that it is possible to develop a predictive model for LQTS that performs with similar accuracy to other widely adopted cardiovascular risk models. Furthermore, it shows the potential of primary care records to phenotypically identify patients that are at an increased risk of having a rare disease.

W.R.H. Evans: D. Speakers Bureau/Honoraria (speakers bureau, symposia, and expert witness); Modest; Intrabio inc. F. Consultant/Advisory Board; Modest; S.F. Weng: D. Speakers Bureau/Honoraria (speakers bureau, symposia, and expert witness); Modest; Amgen. R.K. Akyea: None. A.D. Simms: None. J. Kai: None. N. Qureshi: D. Speakers Bureau/Honoraria (speakers bureau, symposia, and expert witness); Modest; Amgen.


Functional investigation of possible pathogenic EPHB4 variants associated with lymphatic related fetal hydrops and hereditary haemorrhagic telangiectasia

E. Sackey, S. Martin-Almedina, K. Ogmen, D. Grigoriadis, S. Mansour, P. Ostergaard

St. George’s University, London, United Kingdom

The use of next generation sequencing (NGS) technologies has increased the capacity of rare and novel variant detection. However, this brings challenges to confirm pathogenicity of the identified variants and evaluate them functionally, both needed for precise clinical diagnostics. This study investigates nine novel possible pathogenic nonsynonymous variants in the EPHB4 (Ephrin Receptor B4) gene. EPHB4 mutations have been associated with lymphatic related fetal hydrops (LRFH) and hereditary haemorrhagic telangiectasia (HHT) both with autosomal dominant inheritance. Non-immune fetal hydrops is characterised by fluid accumulation in at least two extravascular fetal compartments and about 15% of the cases result from a lymphatic abnormality. HHT presents commonly with arteriovenous malformations (AVMs) in the liver and lung, in connection with cutaneous and mucosal telangiectasias. Functional analysis including immunoprecipitation, western blotting, and immunofluorescence assays were performed on nine novel variants on EPBH4 to investigate their pathogenicity. This study demonstrates the pathogenic effect of eight variants out of nine. Most of the LRFH-associated EPHB4 variants presented similar protein expression levels compared to the wildtype protein but reduced tyrosine kinase activity while HHT-associated EPHB4 variants showed a reduction or total absence of protein expression. Based on these results, we suggest there could be two distinct EPHB4-associated disease phenotypes distinguished by two distinctive molecular mechanisms, but further research is necessary to elucidate this.

E. Sackey: None. S. Martin-Almedina: None. K. Ogmen: None. D. Grigoriadis: None. S. Mansour: None. P. Ostergaard: None.


Identifying metabolomic fingerprints of microRNAs in cardiovascular disorders

R. Mustafa1, M. Mens2, R. Pinto1, I. Karaman1, G. Roshchupkin3,4, J. Huang1, P. Elliott1, M. Evangelou5, A. Dehghan1, M. Ghanbari2

1Department of Epidemiology and Biostatistics, Imperial College London, London, United Kingdom, 2Department of Epidemiology, Erasmus Medical Center, Rotterdam, Netherlands, 3Department of Radiology and Nuclear Medicine, Erasmus Medical Center, Rotterdam, Netherlands, 4Department of Medical Informatics, Erasmus Medical Center, Rotterdam, Netherlands, 5Department of Mathematics, Imperial College London, London, United Kingdom

Introduction: microRNAs (miRNAs) are small non-coding RNAs that regulate gene expression. Despite an increasing evidence that supports miRNAs as potential biomarkers in cardiovascular disorders, the underlying mechanisms remain to be elucidated. We aimed to identify metabolites that mediate the effect of miRNAs on cardiovascular disorders.

Methods: We performed two steps of two-sample Mendelian Randomization (MR) to study the causal association between miRNAs and metabolites, and between the metabolites and cardiovascular disorders. We conducted genome-wide association studies (GWAS) on miRNA expression data (N=1,687) in the Rotterdam Study to identify genetic instruments for miRNAs. MR was performed in a hypothesis-free manner between 591 miRNAs and 886 metabolites measured by the Metabolon platform. The association of the genetic instruments with metabolites were examined in the Airwave Study (N=1,942). We further performed MR to investigate the causal role of identified metabolites on cardiovascular disorders using publicly available GWAS summary results.

Results: Our analysis indicated causal associations between 16 miRNAs and 21 metabolites after multiple testing correction. Among the identified metabolites, nominally significant associations were found between androsterone sulfate, cysteine sulfinic acid, and N-palmitoylglycine with stroke, and 3-hydroxy-5-cholestenoic acid with coronary artery disease. Taken together, we were able to identify metabolites that might link miR-1273h-5p, miR-3937, and miR-4753-5p with stroke, and miR-181a-2-3p with coronary artery disease.

Conclusions: Our study has added insight into understanding the role of metabolites in the link between miRNAs and cardiovascular disorders. This approach could offer a way for finding candidate biomarkers and therapeutic targets in cardiovascular disorders.

R. Mustafa: None. M. Mens: None. R. Pinto: None. I. Karaman: None. G. Roshchupkin: None. J. Huang: None. P. Elliott: None. M. Evangelou: None. A. Dehghan: None. M. Ghanbari: None.


The detection of mosaic mutationsin hereditary aortic disease through next-generation sequencing

Z. G. Yan, H. Yang, Y. H. Zhang, Z. Zhou

State Key Laboratory of Cardiovascular Disease, Beijing Key Laboratory for Molecular Diagnostics of Cardiovascular Diseases, Diagnostic Laboratory Service, Fuwai Hospital, National Center for Cardiovascular Diseases, Chinese Academy of Medical Sciences, Beijing, China

Background: The mosaic carriers have been clinically normal or had only minor manifestations when the proportion of mutated cells is low, which makes it difficult to be detected. However, if the mutations were transmitted from asymptomatic carriers to their children, it will cause unexpected occurrence of diseases. So genetic tests are often required.

Methods: We choosed 563 patients with suspected marfan syndrome (MFS) or thoracic aortic aneurysm/dissection, who had been detected by Panel. Among them, we selected 56 patients who carried likely pathogenic (pathogenic) mutations and were not de novo, their parents who harbored the same mutation were clinically normal. For these Trio families, we used the Life PGM TM DX platform to perform deep sequencing of single amplicon (approximately 5000×) on DNA samples extracted from whole blood.

Results: We detected two mosaic mutations in these Trio families. The first patient AD700-1 who had typical MFS features carried the mutation FBN1, C1305F. After deep sequencing,23% mutated alleles were detected in healthy father who harbored the same mutation. The second mutation FBN1, c.2167+1G>A was identified in patient AD1833, who was suspected to be MFS. The mutation was also carried by healthy father, who was found to carry 17% of the mutant allele through deep sequencing.

Conclusions: Mosaic mutations could be a more common phenomenon in aortic diseases than has been realized so far. Therefore, whenever children are identified with harmful mutations, the parents who lack obvious phenotypic characteristics should be detected by genetic testing, which will provide more accurate guidance for genetic counseling.

Z.G. Yan: None. H. Yang: None. Y.H. Zhang: None. Z. Zhou: None.


Familial aortic aneurysm syndrome caused by a +3 variant at a donor splice site of MYH11.

B. Chesneau1,2, A. Plancke1, G. Rolland1, B. Marcheix3, Y. Dulac2, T. Edouard2, M. Aubert-Mucca2,4, S. Julia4, M. Langeois2,4, V. Gaston4, P. Khau Van Kien1

1Unité de génétique médicale et cytogénétique, Hôpital Carémeau, CHU de Nîmes, Nîmes, France, 2Centre de Référence du syndrome de Marfan et des syndromes apparentés, Hôpital des Enfants, CHU de Toulouse, Toulouse, France, 3Département de chirurgie cardiaque, Hôpital Universitaire de Rangueil, Toulouse, France, 4Service de génétique médicale, Hôpital Universitaire de Purpan, Toulouse, France

Pathogenic variants in the myosin smooth muscle heavy chain 11 (MYH11) are associated with familial aortic aneurysm (FAA) and patent ductus arteriosus (PDA) syndrome. We report a family with two siblings who presented with type A aortic dissections. The sister made an aortic dissection at 45 years-old. The brother made an aortic dissection at 22 years-old treated with an aortic graft procedure, at 46 years-old he had revision surgery because of pseudoaneurysms complications. His daughter was diagnosed with a patent ductus arteriosus. Their father suddenly died at 28 years-old. Molecular analysis of a panel of 15 aortic aneurysm genes in the brother revealed only the variant c.4599+3A>C at the 5’ donor splice site (5’ss) of the exon 33 of MYH11 (NM_00104114.1) with a potential splicing impact. This variant was also present in the affected sister. It was absent from GnomAD and internal variant database (>1000 patients) and never reported in the literature. Interestingly, a single-nucleotide polymorphism is described at this position: c.4599+3A>G (rs143288748). Interpretation of variations at the position +3 of the 5’ss is difficult because data of the literature are conflicting with variations reported to induce aberrant splicing in some cases but not in others. We performed transcript analysis with RNA extracted from an aortic biopsy during the brother’s second intervention. RT PCR revealed a skipping of the exon 33 of MYH11 predicting a truncated myosin tail and a dominant negative effect. This report confirms genotype-phenotype correlations for MYH11 and FAA/PDA.

B. Chesneau: None. A. Plancke: None. G. Rolland: None. B. Marcheix: None. Y. Dulac: None. T. Edouard: None. M. Aubert-Mucca: None. S. Julia: None. M. Langeois: None. V. Gaston: None. P. Khau Van Kien: None.


A founder pathogenic variant in MYH7 causing hypertrophic cardiomyopathy in Catalonia

P. Fernández-Álvarez1, M. Codina1, J. Limeres2, C. Serra-Juhé3, M. Masas Castro1, A. Cordero Lopez1, E. García-Arumí1,4, E. Tizzano1,4

1Department of Clinical and Molecular Genetics and Medicine Genetics Group, VHIR, University Hospital Vall d’Hebron, Barcelona, Spain, 2Department of Cardiology, Hospital Universitari Vall d’Hebron, Barcelona, Spain, 3Department of Genetics, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain, 4CIBERER, Centro de Investigación Biomédica en Red de Enfermedades Raras, ISCIII, Madrid, Spain

Pathogenic variants in MYH7 are the second most common cause of inherited hypertrophic cardiomiopathy (HCM). 262 unrelated HCM probands were screened using the Illumina TruSightCardio kit. Twenty-nine patients were heterozygous for MYH7 pathogenic variants, 6 (21%) of them had the same variant c.2674C>A p.(Gln892Lys). This variant has been previously described as a VUS in a HCM patient which harbored another pathogenic variant in MYH7 and in ClinVar, and is absent in GnomAD. In all our six patients p.(Gln892Lys) was the only probably pathogenic variant found. Patients (4 males/2 females) were of Catalan origin and had been diagnosed of HCM around 30 years-old. Familial sudden death before the fifth decade of life were reported. We extended the genetic and cardiologic study to 20 relatives of the probands. Twelve were carriers, 9 of them symptomatic and p.(Gln892Lys) co-segregated with the HCM in all the families. We studied the possibility of a founder effect by haplotype analysis using the SNP data from the TruSightCardio kit. We used 14 SNPs, flanking an area of 49,35 kb before MYH6 and after MYH7. A common haplotype with a calculated haplotype frequency in Iberia population of 0.0187 was shared in the 6 patients and was not found in our remaining 256 patients with HCM. Our results indicate a possible founder effect of this MYH7 pathogenic variant in HCM patients of Catalan origin.

Results of habplotype analysis in the 6 patients
SNPPosition (hg19) chr14 case1Proband 1Proband 2Proband 3Proband 4Proband 5Proband 6Distance from Previous SNP (kb)

P. Fernández-Álvarez: None. M. Codina: None. J. Limeres: None. C. Serra-Juhé: None. M. Masas Castro: None. A. Cordero Lopez: None. E. García-Arumí: None. E. Tizzano: None.


Biallelic variants in MYL3 cause autosomal recessive cardiomyopathy

D. P. S. Osborn1, L. Emrahi2, M. T. Tabrizi3, M. Yazdchi3, N. Mazaheri4, A. Mitchell5, C. Hesse6, H. Goullée7, D. Burkardt5, N. Laing7, A. Y. B. Wan1, H. Galehdari4, R. Maroofian1, G. Shariati4, H. Tajsharghi8, Y. Jamshidi1

1Genetics Centre, London, United Kingdom, 2Tarbiat Modares University, Tehran, Iran, Islamic Republic of, 3Tabriz University of Medical Sciences, Tabriz, Iran, Islamic Republic of, 4Shahid Chamran University of Ahvaz, Ahvaz, Iran, Islamic Republic of, 5Case Western Reserve University, Cleveland, OH, United States, 6University of Gothenburg, Gothenburg, Sweden, 7The University of Western Australia and the Harry Perkins Institute of Medical Research, Nedlands, Australia, 8University of Skovde, Skovde, Sweden

MYL3 encodes the slow-twitch specific myosin essential light chain (ELC), and pathogenic variants in this gene are a rare cause of hypertrophic cardiomyopathy. The majority of cases reported in the literature involve MYL3 missense variants transmitted in an autosomal dominant mode of inheritance. Here, through exome sequencing of four unrelated consanguineous families we identify biallelic MYL3 sequence variants. Affected individuals present with hypertrophic or dilated cardiomyopathy of variable severity from early- to late-onset of cardiomyopathy, and sudden cardiac death in 10 individuals. We report homozygous and compound heterozygous missense variants p.Ala57Asp, p.Met173Lys and p.Glu177Gly, a novel homozygous translational stop codon variant p.Glu36Ter, and a homozygous splice variant c.482-1G>A in MYL3. G0 zebrafish larvae with CRISPR/Cas9 gene disruption or transient knockdown of the major ELC orthologue in zebrafish, cmlc1, display significantly larger ventricular chamber volume and compromised cardiac function, which cannot be rescued by re-introduction of human mutant RNA. Our genetic and functional data expand the mutational and phenotypic spectrum of MYL3 variants, identifying for the first time a homozygous loss of function variant, and highlighting the role of MYL3 in recessive hypertrophic and dilated cardiomyopathy with occurrence of sudden cardiac death.

D.P.S. Osborn: None. L. Emrahi: None. M.T. Tabrizi: None. M. Yazdchi: None. N. Mazaheri: None. A. Mitchell: None. C. Hesse: None. H. Goullée: None. D. Burkardt: None. N. Laing: None. A.Y.B. Wan: None. H. Galehdari: None. R. Maroofian: None. G. Shariati: None. H. Tajsharghi: None. Y. Jamshidi: F. Consultant/Advisory Board; Modest; DiNAQOR AG.


Impact of abdominal obesity on postprandial lipid concentrations

M. R. Christiansen1, T. I. A. Sorensen1,2, T. Hansen1, T. O. Kilpeläinen1

1Novo Nordisk Foundation Center for Basic Metabolic Research, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark, 2Department of Public Health, Section of Epidemiology, University of Copenhagen, Copenhagen, Denmark

Introduction: Humans spend most of the day in a post-absorptive state, and thus prolonged and high postprandial lipemia can have major implications for the risk of cardiovascular disease. Abdominal adiposity may influence postprandial lipemia by influencing lipid metabolism after a meal. To date, >400 genetic loci have been discovered for association with abdominal adiposity, assessed by waist-hip ratio adjusted for body mass index (WHRadjBMI). Whether these variants are associated with postprandial lipid concentrations, remains unknown. We thus examined whether genetic risk score for increased waist-hip ratio adjusted for body mass index (WHRadjBMI) is associated with lipid concentrations at fasting state and after a high-fat meal.

Materials and Methods: 771 individuals of European ancestry and with obesity from the NUGENOB study consumed a liquid high-fat meal (95% fat of the energy content). The postprandial concentrations of HDL cholesterol, LDL cholesterol, free fatty acids, glycerol and triglycerides were measured at fasting state and postprandially for 3 hours. The associations between the WHRadjBMI score and lipid concentrations were examined using linear models.

Results: At fasting state, the genetic risk score for WHRadjBMI was associated with higher fasting triglycerides (P=0.0013) and lower HDL cholesterol (P=0.0059). Postprandially, the genetic risk score was not associated with changes in triglycerides or HDL cholesterol (P>0.05), but was significantly associated with elevated glycerol concentrations (P=0.012).

Conclusions: Genetic predisposition to abdominal adiposity is associated with elevated blood glycerol concentration after a high-fat meal.

M.R. Christiansen: None. T.I.A. Sorensen: None. T. Hansen: None. T.O. Kilpeläinen: None.


Familial pulmonary arterial hypertension by KDR heterozygous loss of function

M. EYRIES1, D. Montani2, B. Girerd2, N. Favrolt3, M. Riou4, L. Faivre5, G. Manaud6, F. Perros6, S. Gräf7, N. Morrell8, M. Humbert2

1Département de génétique, Hôpitaux Universitaires Pitié Salpêtrière - Charles Foix,, Paris, France, 2Service de Pneumologie, Centre de Référence de l’Hypertension Pulmonaire, Hôpital Bicêtre, Le Kremlin Bicêtre, France, 3Service de Pneumologie et Soins Intensifs Respiratoires, Centre de référence constitutif des maladies pulmonaires rares de l’adulte, Centre de compétence de l’hypertension pulmonaire, CHU Dijon-Bourgogne, Dijon, France, 4Service de Pneumologie, Nouvel hôpital civil, Strasbourg, France, 5Centre de génétique, FHU TRANSLAD, Institut GIMI et UMR INSERM 1231, CHU de Dijon et Université de Bourgogne, Dijon, France, 6UMR_S 999, Université Paris-Saclay, INSERM, Hôpital Marie Lannelongue, Le Plessis Robinson, France, 7NIHR Bioresource – Rare Diseases, Department of Medicine, Department of Haematology, Cambridge Biomedical Campus, Cambridge, United Kingdom, 8Department of Medicine, University of Cambridge School of Clinical Medicine, Addenbrooke’s and Royal Papworth Hospitals, Cambridge, United Kingdom

Introduction: Pulmonary arterial hypertension (PAH) is a rare and devastating disease, resulting from progressive obliteration of small caliber pulmonary arteries leading to cardiac failure. PAH can occur in the context of family history or genetic mutations causing heritable PAH, or is considered as idiopathic in the absence of an identified predisposing factor. Beyond the major gene BMPR2, a gene encoding a type 2 receptor of the BMP signaling pathway several new genes predisposing to PAH have been identified during the last decade. Recently, preliminary evidence of the involvement of the KDR gene was found in a large genetic association study.

Methods: We prospectively analysed the KDR gene by targeted panel NGS sequencing in a series of 311 PAH patients referred to a clinical molecular laboratory for genetic diagnosis of PAH.

Results: Two index cases with severe PAH were found to carry a loss-of-function mutation in the KDR gene. These two patients were clinically characterized by low diffusing capacity for carbon monoxide adjusted for hemoglobin (DLCOc) and radiological evidence of parenchymal lung disease. Segregation analysis in one family revealed that all KDR mutations carriers have a low DLCOc that is more pronounced in affected subjects compared to non-affected

Conclusions: Our study provided genetic evidence for considering KDR as a newly identified PAH causing gene since we describe the segregation of KDR mutations with PAH in two families. Furthermore, KDR mutations are associated with a particular form of PAH characterized by low DLCOc and parenchymal lung disease.

M. Eyries: None. D. Montani: None. B. Girerd: None. N. Favrolt: None. M. Riou: None. L. Faivre: None. G. Manaud: None. F. Perros: None. S. Gräf: None. N. Morrell: None. M. Humbert: None.


PHACTR1 mRNA expression is affected by rs9349379 in PBMC of patients six months after the first MI

J. Kuveljic1, T. Djuric1, M. Dekleva2, N. Markovic Nikolic2, A. Stankovic1, D. Alavantic1, M. Zivkovic1

1Vinca Institute of Nuclear Sciences, Laboratory for Radiobiology and Molecular Genetics, University of Belgrade, Belgrade, Serbia, Belgrade, Serbia, 2Department of Cardiology, University Clinical Center “Zvezdara”, Belgrade, Serbia; Faculty of Medicine, University of Belgrade, Belgrade, Serbia, Belgrade, Serbia

Aim: Phosphatase and actin regulator 1 (PHACTR1) was at first associated with early onset myocardial infarction (MI). Later on, it was associated with other vascular disorders, but still the mechanism of its effect is unknown. Rs9349379 is an eQTL for PHACTR1 in coronary arteries, where A allele leads to higher expression. Also PHACTR1 mRNA expression is elevated in the heart after MI, indicating its up-regulation in pathological conditions. The aim of our study was to investigate the association of rs9349379 with the first MI in Serbian population and its effect on PHACTR1 mRNA expression in PBMC of MI patients and controls.

Methods: Blood samples from 171 controls and 156 patients with the first MI were obtained for DNA extraction. For RNA extraction, we collected 32 control samples and 65 samples of patients six months after the first MI. Genotyping and expression analysis were performed with Taqman technology. Statistical analyses were done by Statistica 8 software.

Results: The association of rs9349379 with the first MI was not significant (p=0.13). PHACTR1 mRNA expression was significantly higher in MI patients compared to controls (p=0.02). We have found that rs9349379 has no effect on PHACTR1 mRNA expression in control group (p=0.90), while AA genotype led to higher expression in patient group (p=0.04).

Conclusions: Rs9349379 affects PHACTR1 mRNA expression in PBMC of MI patients, six months after the MI. In order to accurately estimate its association with MI, further analysis and larger sample size are needed. Funded by Serbian Ministry, grant III41028.

J. Kuveljic: None. T. Djuric: None. M. Dekleva: None. N. Markovic Nikolic: None. A. Stankovic: None. D. Alavantic: None. M. Zivkovic: None.


Polygenic risk scores for hypertension risk stratification

O. Borisov1, E. Salvi2, L. Citterio3, C. Lanzani3, C. Barlassina4, N. Glorioso5, P. Krawitz1, D. Cusi4, P. Manunta3, C. Maj1

1Institute for Genomic Statistics and Bioinformatics, Bonn, Germany, 2Neuroalgology Unit, Fondazione IRCCS Istituto Neurologico “Carlo Besta”, Milan, Italy, 3Genomics of Renal Diseases and Hypertension Unit, Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS) San Raffaele Scientific Institute; Vita-Salute San Raffaele University, Milan, Italy, 4Dept. Of Health Sciences, University of Milan, Milan, Italy, 5Hypertension and related diseases Centre Department of Clinical and Experimental Medicine University of Sassari Italy, Sassari, Italy

Introduction: Hypertension (HTN) is a major cardiovascular risk factor and responsible for ~50% of cardiovascular morbidity and mortality. Blood pressure (BP) is also a complex genetic trait with heritability estimates of 30-50%. Genome-wide association studies (GWAS) have identified a high number of genetic loci associated with HTN and BP traits, indicating underlying polygenic architectures. This study proposes to compute polygenic risk scores (PRS) for HTN and BP for potential genetic risk stratification.

Materials and Methods: We investigated the additive effect of common genetic variants on HTN and BP by means of regression models based on PRS. We used genotyping data of 561 cases and 731 controls from Hypergenes cohort. We computed PRS based on clumping and thresholding method using GWAS summary statistics of systolic/diastolic BP measurements from GERA cohort (99,785 health records) and the HTN case-control cohort of the UK Biobank (144,793 cases and 313,761 controls).

Results: PRS for HTN and diastolic/systolic BP are strongly associated with hypertensive status and BP measurements. The highest significance is achieved for the case-control status by considering UK Biobank effect sizes (p=6.4e-28, R2=0.09). The area under the curve is 0.68. The model remains significant after adjusting for sex and body mass index (p=4.1e-27).

Conclusions: PRS modeling shows a strong polygenic component both for HTN status and for BP levels. Despite the limited predictive accuracy expected for BP, which is largely influenced by environmental factors, the strength of associations suggests that PRS could be potentially used for genetic risk stratification.

O. Borisov: None. E. Salvi: None. L. Citterio: None. C. Lanzani: None. C. Barlassina: None. N. Glorioso: None. P. Krawitz: None. D. Cusi: None. P. Manunta: None. C. Maj: None.


Biallelic variants in PPP1R13L cause paediatric dilated cardiomyopathy

H. K. Robinson1, E. Zaklyazminskaya2, I. Povolotskaya3, Y. Surikova4, L. Mallin1, C. Armstrong5, M. R. Chrisant6, P. J. Benke7, M. McDonald8, C. C. Marboe9, K. McWalter10, G. Douglas10, K. Agre11, D. Deyle11, K. M. Balashova3, V. Kaimonov3, N. Shirokova3, E. Pomerantseva3, C. Turner12, S. Ellard1,13

1Exeter Genomics Laboratory, Exeter, United Kingdom, 2Center of Syncope and Cardiac Arrhythmias in Children and Adolescents, Moscow, Russian Federation, 3Centre of Genetics and Reproductive Medicine “Genetico”, Moscow, Russian Federation, 4B.V. Petrovsky Russian National Centre of Surgery, Moscow, Russian Federation, 5Paediatric Cardiac Service, Bristol Royal Hospital for Children, Bristol, United Kingdom, 6Joe DiMaggio Children’s Hospital, Hollywood, FL, United States, 7Charles E. Schmidt College of Medicine, Florida Atlantic University, Hollywood, FL, United States, 8Department of Pediatrics, Division of Medical Genetics, Duke University Medical Center, Durham, NC, United States, 9Department of Pathology and Cell Biology, Columbia University Medical Centre, New York, NY, United States, 10GeneDx Inc., Gaithersburg, MD, United States, 11Mayo Clinic, Department of Clinical Genomics, Rochester, MN, United States, 12Peninsula Clinical Genetics Service, Department of Clinical Genetics, Royal Devon and Exeter NHS Foundation Trust, Exeter, United Kingdom, 13Institute of Biomedical and Clinical Science, College of Medicine and Health, Exeter, United Kingdom

Introduction: Childhood DCM is a leading cause of heart failure requiring cardiac transplantation, with ~5% of cases resulting in sudden death. Knowledge of the underlying genetic cause can aid prognostication and clinical management and enables accurate recurrence risk counselling. Here our aim was to identify the causative genetic variant(s) in families with children affected by severe DCM.

Materials and Methods: Exome/genome sequencing was performed on leukocyte DNA from seven children affected with severe DCM and their unaffected parents from five independent families, followed by inheritance-based gene-agnostic variant filtering. Families were brought together by an international collaborative effort facilitated by GeneMatcher.

Results: DCM presented early (range: 3 months – 9 years) and was progressive, resulting in death (n = 3) or transplant (n = 3), with one child currently awaiting transplant. Biallelic variants in PPP1R13L were identified in the affected children in each family. PPP1R13L encodes inhibitor of apoptosis-stimulating protein of p53 protein (iASPP). iASPP has roles in apoptosis, acts as a regulator of desmosomes and has been implicated in inflammatory pathways. Spontaneous biallelic loss-of-function variants in PPP1R13L orthologues have been shown to underlie bovine and murine models of DCM and biallelic variants were previously reported in a single consanguineous family with paediatric DCM.

Conclusions: Gene-agnostic exome/genome sequencing is valuable for the identification of novel and emerging candidate genes. The identification of these five families now provides sufficient evidence to support a robust gene-disease association between PPP1R13L and severe paediatric DCM. PPP1R13L is therefore recommended for inclusion in panel-based genetic testing for paediatric DCM.

H.K. Robinson: None. E. Zaklyazminskaya: None. I. Povolotskaya: None. Y. Surikova: None. L. Mallin: None. C. Armstrong: None. M.R. Chrisant: None. P.J. Benke: None. M. McDonald: None. C.C. Marboe: None. K. McWalter: A. Employment (full or part-time); Significant; GeneDx Inc. G. Douglas: A. Employment (full or part-time); Significant; GeneDx Inc.. K. Agre: None. D. Deyle: None. K.M. Balashova: None. V. Kaimonov: None. N. Shirokova: None. E. Pomerantseva: None. C. Turner: None. S. Ellard: None.


Identification of ANGPT2 mutations as a novel cause of primary lymphedema

V. Leppänen1, P. Brouillard2, E. Korhonen1, N. Revencu3, G. Koh4, P. Saharinen1, K. Alitalo1, M. Vikkula2,5

1Wihuri Research Institute, Biomedicum Helsinki, Helsinki, Finland, 2Human Molecular Genetics, de Duve Institute, UClouvain, Brussels, Belgium, 3Center for Human Genetics, Cliniques universitaires Saint-Luc, UCLouvain, Brussels, Belgium, 4Center for Vascular Research, Korea Advanced Institute of Science and Technology (KAIST), Daejoen, Korea, Republic of, 5Walloon Excellence in Lifesciences and Biotechnology (WELBIO), UCLouvain, Brussels, Belgium

Purpose: Primary lymphedema (PLE) is caused by developmental and functional defects of the lymphatic system resulting in accumulation of protein-rich fluid in tissues. The 28 currently known genes causing lymphedema explain <30% of cases. We search for additional genes to better understand pathophysiology.

Methods: We used WES to screen 543 patients within our cohort of >900 index patients. We filtered variants using our in-house developed Highlander software. In vitro and in vivo studies were performed to validate functional effects.

Results: We identified a complete angiopoietin-2 (ANGPT2) gene deletion in one family, and four variants of interest in four other families. All are extremely rare or absent in controls and databases, and predicted to be pathogenic by several algorithms. Functional analyses of the substitution-mutants unraveled that 3/4 showed decreased ANGPT2 secretion and even inhibited secretion of the wild-type ANGPT2, suggesting that they have a dominant-negative effect. The fourth mutant, located in the dimerization interphase, showed reduced integrin-α5 binding and its expression in mouse skin promoted hyperplasia and dilation of cutaneous lymphatic vessels.

Conclusions: We identified five mutations in the TIE2-ligand ANGPT2 as a novel cause of primary lymphedema. The mutations alter ANGPT2 structure and/or function. The data underscore loss of ANGPT2 function as the mechanism leading to primary lymphedema, and provide new insights into TIE2 signaling. The ANGPT2 gene is now included in the diagnostic panel used in our hospital to perform diagnostic testing for primary lymphedema. These studies were supported by Walloon Excellence in Lifesciences and Biotechnology (WELBIO).

V. Leppänen: None. P. Brouillard: None. E. Korhonen: None. N. Revencu: None. G. Koh: None. P. Saharinen: None. K. Alitalo: None. M. Vikkula: None.


Novel variants in ABIN2 and TRAF2 implicated in pathogenesis of pulmonary arterial hypertension

N. Gallego1, D. Condon2, S. Pienkos2, V. de Jesús Pérez2, I. Hernández3, C. Pérez-Olivares4, N. Ochoa4, P. Spanish Consortium5, P. Lapunzina6, P. Escribano7, J. Tenorio6

1Instituto de Genética Médica y Molecular, Madrid, Spain, 2Stanford University, Standford, CA, United States, 3Hospital Universitario Fundación Jiménez Díaz, Madrid, Spain, 4Hospital Universitario Doce de Octubre, Madrid, Spain, 5Instituto de Genética Médica y Molecular, Centro de Investigación Biomédica en Red de Enfermedades Raras, Madrid, Spain, 6Instituto de Genética Médica y Molecular, Centro de Investigación Biomédica en Red de Enfermedades Raras, Madrid, Spain, 7Hospital Universitario Doce de Octubre, Centro de Investigación Biomedica en Red en Enfermedades Cardiovasculares, Madrid, Spain

Introduction: Pulmonary Arterial Hypertension (PAH) is an infrequent cardiopathy with poor prognosis despite therapeutic advances. Mutation in several known risk genes predispose to disease however, some cases remain genetically undefined. Increasing knowledge of the underlaying molecular mechanisms may lead to a better understanding of the disease and the opening of new drug targets that can improve the treatment of patients. The advance in massive paralleled sequencing technologies has allowed improving the characterization of the molecular profiling of the patients and a better clinical characterization.

Material and methods: The aim of this project is to study 20 patients and available relatives by whole exome sequencing (WES) technology in order to identify new genes and regions that can be involved in PAH. Variant prioritization was performed through bioinformatic tools and a custom algorithm. In addition, to stablish mechanistic significance, a series of in vitro studies were performed.

Results: Several candidate genes were identified after the variant prioritization. We have identified two genes (TRAF2 and TNIP2) in two unrelated families, which are related to the NFƙβ pathway, associated with the progression of PAH. In vitro studies demonstrate a possible impairment of NFƙβ transcription factors localization of a modulation of NFƙβ-inducible genes after inhibition of ABIN2 and TRAF2.

Conclusions: Although these results have to be validated through more different functional assays, they suggested an association between disease and new genes which may also be considered for inclusion in genetic screening for PAH patients.

Grants: FIS-PI18/01233, Janssen unrestricted grant, FCHP.

N. Gallego: None. D. Condon: None. S. Pienkos: None. V. de Jesús Pérez: None. I. Hernández: None. C. Pérez-Olivares: None. N. Ochoa: None. P. Spanish Consortium: None. P. Lapunzina: None. P. Escribano: None. J. Tenorio: None.


Association of two novel SCN5A intragenic duplications with epilepsy, ventricular tachycardia, fibrillation and Brugada-like Syndrome

N. Marziliano1, A. Medoro2, D. Fiscella3, A. Fiscella4, L. Tursi1, L. Greco1, C. Reverberi5, S. Folzani6, M. Intrieri2

1ASL TARANTO, Taranto, Italy, 2Università degli Studi del Molise, Campobasso, Italy, 3Ospedale Garibaldi, Catania, Italy, 4Fondazione Floresta Longo, Catania, Italy, 5Centro Clinico Gemini, Parma, Italy, 6Poliambulatorio Città di Collecchio, Collecchio, Italy

In the last years, the increasing evidences about the co-existence of genetically based cardiac arrhythmias and epilepsy brings out the emerging concept of cardio-cerebral channelopathy. In this view, we describe two unrelated cases presenting cardiac and epileptic phenotypes in which two novel intragenic duplications in the SCN5A gene (c.1627_1653dup, p.Phe543_Ala551dup and c.1846_1863dup, p.Ser616_Pro621dup, respectively) were identified. The SCN5A gene encodes for the α-subunit of the Nav1.5 channel and mutations in this gene are mainly involved in electrical channelopathies that lead to life-threatening arrhythmias (Brugada syndrome, Long-QT syndrome, Sudden Infant Death syndrome, etc.) or structural changes in the myocardium, such as in the dilated cardiomyopathy (DCM). With the aim of characterizing the biological effects of such genetic defects, we studied the biophysical properties of the Nav1.5 channels revealing that duplication might prompt for a loss-of-function of the Nav1.5-mediated current in a zebrafish model without alterations in the heart structure and function (at least for the first mutation). Taking together, clinical and biochemical data support the idea that the intragenic in-frame duplications within the SCN5A gene could be responsible for both cardiac and epileptic phenotypes.

N. Marziliano: None. A. Medoro: None. D. Fiscella: None. A. Fiscella: None. L. Tursi: None. L. Greco: None. C. Reverberi: None. S. Folzani: None. M. Intrieri: None.


Clonal occurrence of the Hutchinson-Gilford progeria syndrome lamin A mutation in chronic kidney disease

G. Revechon1, H. Helgadottir1, N. Viceconte1, A. Witasp2, A. Sola Carvajal1, D. Whisenant1, D. McGuinness3, G. Artiach4, E. Wallen Arzt1, A. Thorell5, A. Babler6, S. Ziegler6, R. Kramann6, M. Bäck4, P. G. Shiels3, A. Wernerson2, P. Stenvinkel2, M. Eriksson1

1Karolinska Institute, Dept of Biosciences and Nutrition, Huddinge, Sweden, 2Karolinska Institute, Dept of CLINTEC, Huddinge, Sweden, 3University of Glasgow, Institute for Cancer Sciences, Glasgow, United Kingdom, 4Karolinska Institute, Dept of Medicine, Stockholm, Sweden, 5Karolinska Institute, Dept of Clinical Sciences, Dept of Surgery, Ersta hospital, Stockholm, Sweden, 6University hospital RWTH Aachen, Aachen, Germany

Somatic mutagenesis occurs continuously across the life course, resulting in tissues composed of clones with different genetic makeups. While the majority of mutations have no functional significance, some can contribute to disease and aging. The nature of the arterial wall, with its media being composed of clonal smooth muscle cells, makes this tissue particularly sensitive to somatic mutagenesis. Here we analyzed progerin, the protein responsible for the premature aging disorder Hutchinson-Gilford progeria syndrome (HGPS), for its possible involvement in the accelerated vascular aging process observed in chronic kidney disease (CKD), due to similarities in vascular phenotypes. We provide evidence for expression of progerin, and the occurrence of the HGPS-causing LMNA c.1824C>T mutation as a somatic mutation in CKD patient arteries. Clonal populations of vascular cells with the 1824C>T mutation was supported by clusters of progerin positive cells in the media and a high frequency of the mutant allele in several CKD arteries. Further analysis of proliferation and DNA damage in the CKD arteries indicated regeneration and progerin-induced genomic damage. Our results implicate somatic mutations in the functional decline of the vascular wall and support that somatic mutations may become a risk factor during extensive tissue damage.

G. Revechon: None. H. Helgadottir: None. N. Viceconte: None. A. Witasp: None. A. Sola Carvajal: None. D. Whisenant: None. D. McGuinness: None. G. Artiach: None. E. Wallen Arzt: None. A. Thorell: None. A. Babler: None. S. Ziegler: None. R. Kramann: None. M. Bäck: None. P.G. Shiels: None. A. Wernerson: None. P. Stenvinkel: None. M. Eriksson: None.


Enrichment of rare variants in Loeys-Dietz syndrome genes in spontaneous coronary artery dissection but not in severe fibromuscular dysplasia

J. Meester1, A. Verstraeten1, M. Perik1, L. Van Den Heuvel1, M. Kempers2, I. Krapels3, L. Van Laer1, N. Samani4, F. Van Dijk5, A. Persu6, D. Adlam4, B. Loeys1

1Centre of Medical Genetics, University of Antwerp, Antwerp University Hospital, Antwerp, Belgium, 2Department of Human Genetics, Radboud University Nijmegen Medical Center, Nijmegen, Netherlands, 3Department of Clinical Genetics, Maastricht University Medical Centre, Maastricht, Netherlands, 4Department of Cardiovascular Sciences and National Institute for Health Research Leicester Biomedical Research Centre, Glenfield Hospital, Leicester, United Kingdom, 5Ehlers-Danlos Syndrome, National Diagnostic Service, Northwick Park and St. Mark’s Hospitals, Harrow, United Kingdom, 6Pole of Cardiovascular Research, Institut de Recherche Expérimentale et Clinique, Université Catholique de Louvain, and Division of Cardiology, Cliniques Universitaires Saint-Luc, Université Catholique de Louvain, Brussels, Belgium

Spontaneous coronary artery dissection (SCAD) is characterized by separation of the inner intimal lining of the coronary artery from the outer vessel wall. It is the prime cause of acute myocardial infarction in women below the age of 50. Fibromuscular dysplasia (FMD) presents with condensed areas of arterial stenosis, aneurysm, tortuosity and dissection. It has become clear that FMD is prevalent in SCAD cohorts and vice versa. However, the genetic etiology of both conditions remains largely elusive. We aimed to investigate the contribution of rare genetic variants in vascular Ehlers-Danlos and the Marfan, Loeys-Dietz (LDS), Shprintzen-Goldberg syndrome genes to the etiology of SCAD and FMD in a large multi-center patient cohort. Haloplex-based gene panel sequencing of COL3A1, FBN1, TGFB2/3, SMAD2/3, TGFBR1/2 and SKI was performed in 191 SCAD patients with or without FMD and 111 isolated FMD patients. Subsequent variant filtering was performed. The gnomAD database was used as an independent control dataset and filtered identically. Case-control burden analyses were performed. Rare variants in SMAD2 are significantly enriched in SCAD patients compared to controls (p=0.001). A combined LDS-gene burden analysis was performed, revealing a highly significant association (4.5% vs 1.5%; p=0.000003). When performed for FMD, none of the analyses yielded a significant p-value or showed a trend. We here provide evidence for an important contribution of rare genetic variants in LDS genes to the etiology of SCAD. Our findings suggest a strong role for the TGFbeta signaling in the pathogenesis of SCAD but not FMD.

J. Meester: None. A. Verstraeten: None. M. Perik: None. L. Van Den Heuvel: None. M. Kempers: None. I. Krapels: None. L. Van Laer: None. N. Samani: None. F. Van Dijk: None. A. Persu: None. D. Adlam: None. B. Loeys: None.


Genetic association study of eight steroid hormones and implications for sexual dimorphism of coronary artery disease

J. Pott1,2,3, Y. J. Bae4,2, K. Horn1,2, A. Teren2,5, A. Kühnapfel1,2,3, H. Kirsten1,2, U. Ceglarek4,2, M. Loeffler1,2, J. Thiery4,2, J. Kratzsch4,2, M. Scholz1,2,3

1Institute of Medical Informatics, Statistics und Epidemiology, University of Leipzig, Leipzig, Germany, 2LIFE Research Center for Civilization Diseases, University of Leipzig, Leipzig, Germany, 3IFB AdiposityDiseases, University Hospital, Leipzig, Germany, 4Institute of Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics, University Hospital, Leipzig, Germany, 5Heart Center Leipzig, Leipzig, Germany

Introduction: Steroid hormones act as important regulators of physiological processes in the body. Our aim is to identify genetic factors that influence steroid hormone metabolism, to detect genetic sexual dimorphisms and to investigate possible causal implications for atherosclerotic vascular disease phenotypes.

Materials and Methods: In two independent cohorts, we performed genome-wide sex-specific meta-association studies of eight steroid hormones: progesterone, 17-hydroxyprogesterone (17-OHP), androstenedione, testosterone, estradiol, dehydroepiandrosterone sulfate (DHEA-S), cortisol, and aldosterone (LIFE-Adult, LIFE-Heart, max. n = 7667). Genome-wide significant loci (p < 5x10−8) were tested for sex differences in effect size. The causal relationships between hormone levels and atherosclerotic vascular diseases were investigated using sex-specific eQTL analyses and Mendelian Randomization (MR) approaches.

Results: We detected 22 genome-wide significant loci for seven steroid hormones of our panel, of which nine displayed significant sexual dimorphisms. Eight loci relate to genes coding for enzymes involved in steroid biosynthesis, almost completing the list of genes of this pathway with genetic associations. Other loci correspond, e.g., to the WNT4/β-catenin pathway. MR revealed that cortisol, androstenedione, 17-OHP and DHEA-S had sex-specific causal effects on coronary artery disease.

Conclusions: Our study greatly improves insight into genetic regulation of steroid hormones and their dependence on sex. The significant MR estimates support the hypothesis that steroid hormones influence the development of atherosclerosis, which partly explains the higher risk for men. These results could serve as a basis for analyzing sex-dimorphisms in other complex diseases.

This project was supported by the HI-MAG Project Funding for Young Scientists of the Medical Faculty of the University Leipzig.

J. Pott: None. Y.J. Bae: None. K. Horn: None. A. Teren: None. A. Kühnapfel: None. H. Kirsten: None. U. Ceglarek: None. M. Loeffler: None. J. Thiery: None. J. Kratzsch: None. M. Scholz: None.


Genetic post-mortem in drug users with sudden cardiac death

H. Massey, J. Dean, L. Deboys, D. O’Sullivan

Aberdeen royal infirmary, Aberdeen, United Kingdom

Aim: To quantify our ability to make a genetic diagnosis in drug users with sudden cardiac death (SCD).

Background: While there are European recommendations for genetic testing in SCD1, these guidelines do not extend to drug users with positive toxicology at post-mortem. We wanted to gain a better understanding of the genetic pick up rate in this population.

Method: We undertook a 10-year retrospective study looking at genetic diagnoses in drug related deaths discussed at the joint pathology-genetic MDT. This was compared to age and provisional aetiology matched controls.

Results: 60 drug related deaths were identified, and 40 patients underwent genetic testing. 7 variants were found of which 3 were classified as benign and 4 VUS. The most common reason for genetic testing was enlarged heart at post mortem; however the genetic pick up in this group was 0. Patients with structurally normal hearts had the most variants detected. This would coincide with the majority of variants being found in arrhythmogenic genes. Family history did not influence genetic pick up. In the control group variants in arrhythmogenic genes again predominated correlating with the highest genetic pick up in patients with normal post-mortems. However in the control group 17 variants were detected of which 3 were pathogenic.

Conclusions: Our ability to make a genetic diagnosis in drug users with SCD is low. We need to undertake a larger study to characterise those drug users with a positive genetic diagnosis so testing can be rationalised.

H. Massey: None. J. Dean: None. L. Deboys: None. D. O’Sullivan: None.


Insights into the genetic basis of ventricular tachycardia in Kazakhstani patients. Targeted next generation sequencing

A. R. Akilzhanova1, C. Guelly2, Z. Abilova1, S. Rakhimova1, A. Akhmetova1, U. Kairov1, S. Trajanoski2, G. Akilzhanova3, M. Bekbosynova4, Z. Zhumadilov1

1Center for life sciences, National Laboratory Astana, Nazarbayev University, Nur-Sultan, Kazakhstan, 2Center for Medical Research, Medical University of Graz, Graz, Austria, 3Medical University of Semey, Pavlodar branch, Pavlodar, Kazakhstan, 4National Research Cardiac Surgery Center, Nur-Sultan, Kazakhstan

Introduction: Recent advances in genetic screening and the potential of therapeutic intervention in patents with cardiac arrhythmias have garnered this group of disorders much attention in the scientific community. The purpose of the study was to identify the genetic basis of ventricular tachycardia (VT) in Kazakhstani patients.

Material and Methods: using predeveloped a targeted panel of 96 known cardiac disease genes, associated with cardiomyopathy and arrhythmia we screened 92 individuals, diagnosed with VT and dilated cardiomyopathy (VT DCM) or idiopathic VT (iVT). Results. Targeted sequencing and stepwise filtering of the annotated variants identified 307 unique variants in 74 genes totaling up in 456 variants for the overall study group. Variants included one in/del, four splice-site and 451 single-nucleotide variants (SNV) within the coding exonic regions. Seven (0.15%) of the SNVs were unique stop-gain variants, three of those residing in the TTN gene. 168 HGMD mutations (61 unique) were observed in 37 genes. According to ACMG all variants were classified as 9 pathogenic (KCNJ2 R218Q and TTN R5338X in iVT patients, KCNQ1 c.477+1G>A, LMNA Q353X, MYH7 F244L, TTN L17465X and W21011X, DSG2 c.2334+1G>A, GAA W746C in VT DCM patients), 11 likely pathogenic, 97 variants with uncertain significance and rest as benign.

Conclusions: Individuals with VT either secondary to DCM or of idiopathic etiology carry multiple rare mutations and potentially pathogenic sequence variants in cardiac risk genes in a similar pattern and at a comparable frequency. Study support: grant from the Ministry Education and Science, Republic of Kazakhstan (AP05134683).

A.R. Akilzhanova: None. C. Guelly: None. Z. Abilova: None. S. Rakhimova: None. A. Akhmetova: None. U. Kairov: None. S. Trajanoski: None. G. Akilzhanova: None. M. Bekbosynova: None. Z. Zhumadilov: None.


Identification of the major genetic contributors to Tetralogy of Fallot

R. M. Monaghan1, D. J. Paige2, S. G. Williams1, B. D. Keavney1

1University of Manchester, Manchester, United Kingdom, 2Manchester Metropolitan University, Manchester, United Kingdom

Tetralogy of Fallot (TOF) is the most common cyanotic congenital heart defect. Rare genetic variants have been identified as important contributors to the risk of congenital heart disease, but relatively small numbers of TOF cases have been studied. We used whole exome sequencing to assess the prevalence of unique, deleterious variants in the largest cohort of nonsyndromic TOF patients reported to date. The presence of such variants was defined by their absence in the Genome Aggregation Database and bioinformatic prediction of their deleterious effect on function. The enrichment of variants in two genes, NOTCH1 and FLT4, surpassed thresholds for exome-wide significance (P < 5×10-8) after correction for multiple comparisons. NOTCH1 was most frequently found to harbour unique, deleterious variants (4.5%; 95% CI, 3.2%-6.1%). Three NOTCH1 variants were subjected to functional evaluation, and two showed a reduction in Jagged1-induced NOTCH signalling. FLT4 variants were found in 2.4% (95% CI, 1.6%-3.8%) of our cohort. In addition, variants in the well-established TOF gene, TBX1, and the genes were also significantly enriched. Our study underlines the importance of sequencing large cohorts of CHD cases in order to further discover their genetic basis.

Grant reference: BHF (RG/15/12/31616)

R.M. Monaghan: None. D.J. Paige: None. S.G. Williams: None. B.D. Keavney: None.


In search of genetic modifiers that explain the phenotypic variability in SMAD3 -related aortopathy

J. D. Velchev1, M. Perik1, I. M. B. H. van de Laar2, J. Richer3, A. Verstraeten1, M. Alaerts1, B. L. Loeys1,4

1Center of Medical Genetics, University of Antwerp and Antwerp University Hospital, Antwerp, Belgium, 2Department of Clinical Genetics, Erasmus MC, University Medical Center Rotterdam, Rotterdam, Netherlands, 3Department of Medical Genetics, Children’s Hospital of Eastern Ontario, Children’s Hospital of Eastern Ontario Research Institute, Ottawa, ON, Canada, 4Department of Human Genetics, Radboud University Nijmegen Medical Center, Nijmegen, Netherlands

Introduction: Thoracic aortic aneurysm and dissection (TAAD) is a frequent cause of mortality in the Western world. Loeys-Dietz syndrome (LDS), an early-onset TAAD syndrome is caused by mutations in genes coding for components of the TGFbeta signaling, including SMAD3. Remarkably, a fraction of SMAD3 mutation carriers remains cardiovascularly unaffected throughout life, suggesting the existence of genetic modifiers for aortopathy. This study implements a combination of linkage analysis and whole-genome sequencing (WGS) in order to identify genetic modifiers that explain the variability in SMAD3-related aortopathy.

Material and Methods: In two large LDS families (n = 41 mutation carriers) segregating SMAD3 missense mutations and presenting variable TAAD expressivity genome-wide SNP-based linkage analysis was performed. LOD scores were obtained using MERLIN and SUPERLINK and linkage was complemented with WGS of six affected and six unaffected mutation carriers.

Results: In the first family linkage analysis identified a chromosomal region (chr2:149,604,077-155,607,398) with a LOD score of 2.68, suggesting the presence of an aggravating modifier. Linkage peak was observed in the second family with a LOD score of 2.86 (chr18:43,013,157-57,021,550). Additionally, the WGS data analysis of the second family resulted in the discovery of a rare variant in TGFB2, a known TAA causative gene, exclusively present in affected mutation carriers.

Conclusions: The obtained data indicate that variants in known TAAD genes may play a role in modulating a primary causative mutation; chromosomal regions segregate with disease severity suggesting a presence of genetic modifiers and that distinct families may bear different genetic modifiers as indicated by the linkage analysis.

J.D. Velchev: None. M. Perik: None. I.M.B.H. van de Laar: None. J. Richer: None. A. Verstraeten: None. M. Alaerts: None. B.L. Loeys: None.


Identification of Clinically Relevant Variants by Whole Exome Sequencing in Chinese Patients with Sporadic Non-syndromic Type A TAAD

J. Guo, C. Hao, X. Hu, F. Leng, X. Liu

Genetics and Birth Defects Control Center, Beijing Children’s Hospital, Beijing, China

Introduction: Thoracic aortic aneurysm and dissection (TAAD) is a life-threatening disease, of which genetic abnormalities are considered as important risk factors. The present research aims at identifying causal variants in Chinese patients with sporadic non-syndromic type A TAAD (ATAAD).

Materials and Methods: Whole exome sequencing (WES) was performed on 73 sporadic Chinese patients with ATAAD, 30 TAAD associated genes were curated for bioinformatic analyses. Clinical differences were compared between patients with and without causal variants.

Results: 15 pathogenic/likely pathogenic variants were identified (8 novel and 7 previously described) in 4 known TAAD-causal genes (FBN1, TGFBR2, SMAD3 and ACTA2) in 15 individuals, including 11 variants in FBN1 (7 missense, 3 truncating, and 1 splicing variants), 2 missense variants in TGFBR2, 1 ACTA2 frameshift variant and 1 SMAD3 frameshift variant. Significant clinical differences were found between patients with and without causal variants. Patients with TAAD-causal variants proved to have an earlier onset age, a more dilated aorta, and relatively intractable subtypes. Even without risk factor like hypertension, they might still suffer from TAAD with TAAD-causal variants.

Conclusions: The variants identified in our research might not only result in the occurrence of ATAAD, but also add complexities and difficulties to the clinical practice. Our data demonstrated that WES was an effective tool for determining genetic etiologies of non-syndromic ATAAD and could be helpful in genetic counseling for ATAAD patients and their at-risk family members.

J. Guo: None. C. Hao: None. X. Hu: None. F. Leng: None. X. Liu: None.


Investigating the role of pathogenic TTN variants in heart failure by whole-exome sequencing

O. Chazara1, G. Povysil2, S. Deevi1, J. Armisen1, D. B. Goldstein2, C. Haefliger1

1Astrazeneca, Melbourn, United Kingdom, 2Institute for Genomic Medicine, Columbia University Medical Center, New York, NY, United States

Heart failure (HF) affects ~40 million people globally. Estimates of the genetic predisposition of developing HF is 1.69 if one parent is affected and 1.92 if both parents have HF (Framingham cohort study, Lopez et al. 2013).

Recent studies suggest higher cardiovascular genetic risk in patients with and without diagnosed heart failure if they carry a pathogenic loss of function variant in the gene encoding titin (TTN).

Here, we assess the role of pathogenic TTN variants by whole-exome sequencing in 5916 patients with heart failure from two clinical studies CHARM and CORONA, and 268,451 unrelated Europeans from the UK Biobank prospective cohort study, including 4634 individuals with ICD-10 codes for heart failure (HF, based on hospital in-patient diagnoses).

In both types of cohorts, we detect a significant enrichment of pathogenic TTN variants in subjects with HF diagnosis. Furthermore, after restricting the analysis to the variants located in exons that are predominantly spliced in the heart, the genetic risk was increased: from 2.54 (95% CI, 1.96-3.31) to 4.52 (95% CI, 3.10-6.68) in the clinical studies, and from 2.95 (95% CI, 2.42-3.57) to 4.97 (95% CI, 3.94-6.19) in the UKBB. The enrichment observed is similar in clinical trial data and routine care health records.

As expected, individuals with a diagnosis of cardiomyopathy had the highest rate of TTN mutations, but we also observed TTN variants in up to 5% of HF patients due to other aetiologies, such as ischaemic and hypertensive disease. This result highlights the importance of TTN variants beyond cardiomyopathies.

O. Chazara: A. Employment (full or part-time); Significant; Astrazeneca. G. Povysil: None. S. Deevi: A. Employment (full or part-time); Significant; Astrazeneca. J. Armisen: A. Employment (full or part-time); Significant; Astrazeneca. D.B. Goldstein: F. Consultant/Advisory Board; Modest; Astrazeneca. C. Haefliger: A. Employment (full or part-time); Significant; Astrazeneca.


Non-amyloidotic transthyretin cardiomyopathy

J. Gimeno Blanes1, M. Sabater Molina1, I. San Roman1, L. Polo2, I. Garrido3, C. Salas4, F. Lopez Andreu5

1Inherited Cardiac Disease Unit. Universitary Hospital Virgen de la Arrixaca., Murcia, Spain, 2Hospital Universitario Virgen Arrixaca, Department of Pathological Anatomy., Murcia, Spain, 3Department of Cardiology Universitary Hospital Virgen de la Arrixaca., Murcia, Spain, 4Hospital Puerta de Hierro, Department of Pathological Anatomy, Madrid, Spain, 5Hospital General Reina Sofia, Service of Internal Medicine, Murcia, Spain

Introduction: We aim to present an unsolved case of a young patient with unclassified cardiomyopathy associated with a genetic TTR variant predicted to cause a dramatic change in protein structure. Clinical course went from asymmetric hypertrophy with restrictive phenotype to cardiac dilatation and systolic dysfunction. Pulmonary pressures were strikingly elevated.

Case: A 33 years old woman was admitted in 1997 with limiting dyspnoea. Echocardigram showed non-obstructive asymmetrical septal hypertrophy of 17mm maximum at the septum and restrictive physiology. Sarcoid cardiomyopathy was ruled out with negative scintigraphy and unspecific biopsy in the absence of other extracardiac features. Atrio-ventricular ablation and permanent pacing were required in 2002. Clinical deterioration led to frequent heart failure admissions and one episode of syncope. Implantable cardioverter defibrillator was implanted. Emergency transplant was performed with a postoperative complicated course in 2006. Patient remained stable to date. Study of the explanted heart demonstrated macro and microscopic hypertrophy, extensive fibrosis, unspecific vacuolization and disarray. There were no signs of amyloid with usual stains and immunohistochemical typing. Sequencing of the TTR gene led to identification of a de novo frameshift mutation p.S132fs*25(g.6903_6906delinsGTAGGG) causing original stop codon lost, results in prolonged protein. Cardiac evaluation of her brother, sister and parents ruled out any sign of cardiomyopathy. All relatives were free for the TTR variant.

Conclusions: Case of cardiomyopathy associated with a TTR variant. Severe right and left heart failure clinical course with very particularly severe pulmonary hypertension ended up in heart transplant. Mechanisms of disease in this case remain unknown.

J. Gimeno Blanes: None. M. Sabater Molina: None. I. San Roman: None. L. Polo: None. I. Garrido: None. C. Salas: None. F. Lopez Andreu: None.


Searching for genes associated with primary varicose veins using targeted next-generation sequencing

E. Strauss1,2, M. Zmyslowski3, B. Budny4, L. Dzieciuchowicz3, E. Wrotkowska4, M. Ruchala4, K. Ziemnicka4, G. Oszkinis5, Z. Krasinski3

1Institute of Human Genetics of the Polish Academy of Sciences in Poznan, Poznan, Poland, 2Department of Vascular, Endovascular Surgery, Angiology and Phlebology Poznan University of Medical Sciences, Poznań, Poland, 3Department of Vascular, Endovascular Surgery, Angiology and Phlebology Poznan University of Medical Sciences, Poznan, Poland, 4Department of Endocrinology, Metabolism and Internal Medicine, Poznan University of Medical Sciences, Poznan, Poland, 5Department of General and Vascular Surgery, Poznan University of medical Sciences, Poznan, Poland

Introduction: The aim of this study was to search for genes involved in the pathogenesis of the primary varicose veins (PVV) using targeted next-generation gene sequencing. Sporadic cases with the onset of the first symptoms of disease below or equal 40 years of age were considered.

Methods: A cohort of 66 PVV patients was collected and exome sequencing of the 35 candidates´ genes was performed, which focused on the genes encoding structural elements of the vessel wall, metalloproteinases, and their protein inhibitors, proteins involved in the homeostasis of the coagulation system and regulation of sex hormones system, as well as genes previously associated with VV development.

Results: Most of the patients (91.9%) had a positive family history of VV, considering first-, second- or third-degree relatives. 26 potentially pathogenic changes: 12 mutations in 9 genes (7 known and 5 novel), 5 rare SNPs and 9 common SNPs were found. 40% of the patients had a potentially causative mutation in the candidate genes: 33% of them had 1 mutation, 3% had 2 mutations and 1.5% had 3 mutations. The association between one SNP in the MMP-9 gene and the age of the onset of PVV symptoms was also observed.

Conclusions: This report provides evidence for the heterogeneity and complexity of the genetic background of PVV in sporadic cases and shows the influence of genetic factors on the age of the onset of this disease. It also reveals attractive targets for further research in a larger group of patients and functional analyses.

E. Strauss: None. M. Zmyslowski: None. B. Budny: None. L. Dzieciuchowicz: None. E. Wrotkowska: None. M. Ruchala: None. K. Ziemnicka: None. G. Oszkinis: None. Z. Krasinski: None.

P06 Metabolic and Mitochondrial Disorders


Identification of AADC patient through combined determination of 3-OMD biomarker and DDC gene sequencing and copy number variation analysis

C. Pereira1, C. Cozma1, L. Demuth1, G. Morales Torres1, M. Iurascu1, P. Bauer1, A. Rolfs1, C. Lourenco2

1Centogene AG, Rostock, Germany, 2Universidade de São Paulo, Sao Paulo, Brazil

L-amino acid decarboxylase (AADC) deficiency is caused by bi-allelic variants in the DDC gene. This very rare neurometabolic disorder is clinically characterized by vegetative symptoms, oculogyric crises, dystonia, and severe neurologic dysfunction, usually beginning in infancy or childhood. Treatment has become available recently. We quantified 3-OMD (3-O-Methyldopa), a suggested AADC-specific biomarker, in dried blood spots by tandem mass spectrometry. Upon suggestive findings, we subsequently analyzed the DDC gene by amplicon-based next generation sequencing (NGS) and copy number variant (CNV) screening. This two-step approach identified several patients. The ones with at least one hitherto undescribed variant are described here in detail. All three patients had pathological levels of 3-OMD. In patient 1, we identified a novel whole DDC gene duplication in homozygosity. In patient 2, we identified the two missense variants p.(Ser147Ile) and p.(Val60Ala) in compound heterozygosity; interestingly, variant p.(Ser147Ile) was not only novel, but shown to have occurred de novo. In patient 3, p.(Trp121Arg) and p.(Arg347Gln) were identified in compound heterozygosity. The former is novel, while the latter has frequently been described in AADC patients previously. Using a combined biochemical, NGS and CNV approach, a positive identification and diagnosis of AADC deficiency patients is reliably reached. This allows for an early treatment of the patients, leading to an improvement of their quality of life. Moreover, the identification of novel DDC variants and the establishment of correlations with 3-OMD levels can be expected to increase our understanding of the disease.

C. Pereira: None. C. Cozma: A. Employment (full or part-time); Significant; Centogene AG. L. Demuth: A. Employment (full or part-time); Significant; Centogene AG. G. Morales Torres: A. Employment (full or part-time); Significant; Centogene AG. M. Iurascu: A. Employment (full or part-time); Significant; Centogene AG. P. Bauer: A. Employment (full or part-time); Significant; Centogene AG. A. Rolfs: A. Employment (full or part-time); Significant; Centogene AG. C. Lourenco: None.


Patients affected by autosomal recessive or dominant cutis laxa type 3 show diverse degrees of collagen alterations

N. El Choubassi1,2, L. Lugli3, C. Spada4, D. de Silva5, O. Klein1, D. Meierhofer2, D. Horn1, U. Kornak1,2,6, B. Fischer-Zirnsak1,2

1Charité Universitaetsmedizin Berlin, Berlin, Germany, 2Max Planck Institute for Molecular Genetics, Berlin, Germany, 3Division of Neonatal Intensive Care Unit, Department of Pediatrics, University Hospital, Modena, Italy, 4Department of Medical and Surgical Sciences for Mothers, Children and Adults, Post Graduate School of Paediatrics, University of Modena & Reggio Emilia, Modena, Italy, 5Department of Physiology, Faculty of Medicine, University of Kelaniya, Ragama, Sri Lanka, 6Institute of Human Genetics, University Medical Center Göttingen, Göttingen, Germany

Introduction: Mutations in ALDH18A1, encoding the mitochondrial enzyme pyrroline-5 carboxylate synthase (P5CS), lead to a broad spectrum of rare genetic conditions. Besides hereditary spastic paraplegia, ALDH18A1 mutations are causative for autosomal recessive (ARCL3) and dominant (ADCL3) cutis laxa with progeroid features.

Materials and Methods: We performed whole exome (WES) and gene panel sequencing to identify pathogenic variants in our probands. These analyses were complemented by mRNA expression studies, immunofluorescence, immunoblot and mass-spectrometry approaches on patient derived fibroblasts.

Results: We report on novel pathogenic variants in three affected individuals with ARCL3/ADCL3. These variants have variable impacts on the stability of the protein and its localization within the mitochondrial network. However, targeted metabolomics revealed substantial alterations of mitochondrial proline synthesis rates. Additionally, gene expression profiling, comparative proteomics and subsequent immunoblot analyses showed alterations in expression of different collagens.

Conclusions: Our data expand the mutational spectrum of ALDH18A1-related diseases and demonstrate the importance of correct ALDH18A1 localization within the mitochondria for proper functioning. Additionally, they suggest a potential connection of the mitochondrial proline synthesis pathways with the regulation of collagen synthesis.

Funding: The study was supported by the Deutsche Forschungsgemeinschaft (FI 2240/1-1) to BF-Z

N. El Choubassi: None. L. Lugli: None. C. Spada: None. D. de Silva: None. O. Klein: None. D. Meierhofer: None. D. Horn: None. U. Kornak: None. B. Fischer-Zirnsak: None.


Clinical study of the relationship between histamie intolerance and variants in the AOC1/ABP1 Gene

J. Vinas-López1, P. Ojeda2, I. Ojeda2, A. Duelo3, B. García-Velasco1, C. Lenz2, T. Perucho1, M. Berbel3, A. Vásquez3, E. Ruiz-Casares1,4

1Genyca, Majadahonda, Spain, 2Clínica Ojeda de Asma y Alergia Dres. Ojeda, Madrid, Spain, 3Centro de Nutrición AD Dietistas, Barcelona, Spain, 4Universidad San Pablo CEU, Madrid, Spain

Introduction: Histamine is a biogenic amine with many essential physiological activities. However, its deregulation and accumulation can cause a multitude of pathologies such as histamine intolerance. There are two enzymes involved in the metabolism of histamine: histamine N-methyltransferase (HNMT) is responsible for degrading histamine inside cells. Diamine-oxidase (DAO) is the other enzyme involved in the degradation of histamine, mainly exogenous histamine, ingested with the diet. The deficiency of DAO activity could be one of the main causes of histamine intolerance, an alteration in homeostasis that is caused by a reduced intestinal degradation of histamine, with the consequent increase in its plasma levels. Variants in AOC1/ABP1 gene, coding DAO enzyme, have been associated with a reduction in DAO activity and accumulation of histamine.

Objective: The present study aims to demonstrate the negative effect that the variants rs10156191, rs1049742 and rs1049793 have on DAO plasma activity in Caucasian male patients with clinical manifestations associated with histamine intolerance.

Methods: We analyzed 78 white male patients with a clinical manifestation associated with histamine intolerance. Variants analyzed were genotyped by multiplex SNPE (Single Nucleotide Primer Extension).

Results: The variants rs10156191 and rs1049742 reduce significantly DAO plasma activity. Factors such as worsening symptoms after eating a diet rich in histamine or NSAID-type drugs (non-steroidal anti-inflammatory) are associated with a greater number of alternative alleles in the SNVs analyzed, and reduced DAO plasma activity.

Conclusions: These results show the predictive value of this genetic study in patients who have a clinical manifestation associated with histamine intolerance.

J. Vinas-López: C. Other Research Support (supplies, equipment, receipt of drugs or other in-kind support); Modest; Genyca. P. Ojeda: None. I. Ojeda: None. A. Duelo: None. B. García-Velasco: None. C. Lenz: None. T. Perucho: None. M. Berbel: None. A. Vásquez: None. E. Ruiz-Casares: None.


Low prevalence of classic galactosemia in the Sverdlovsk Region (Russian Federation)

O. Lagutina1, S. Deryabina1,2, T. Belyaeva1, N. Nikitina1, E. Nikolaeva1

1Medical Center “Health Care of Mother and Child”, Yekaterinburg, Russian Federation, 2Federal State Autonomous Educational Institution of Higher Education «Ural Federal University named after the first President of Russia B.N. Yeltsin, Yekaterinburg, Russian Federation

Introduction: Classic galactosemia is one of the five hereditary metabolic diseases, which has been included in the State Program of Neonatal Screening of the Russian Federation in 2006.

Aim of the study: To estimate the incidence of galactosemia in the Sverdlovsk Region based on neonatal screening data.

Patients and methods: 698 550 children born in the Sverdlovsk Region from 2006 to 2019 were examined. Initially, the level of total galactose and galactose-1-phosphate was determined in a dry blood spot, then an activity of the enzyme galactose phosphate-uridyl transferase (GALT) has been selected as a biochemical marker of galactosemia. The diagnosis of galactosemia should then be confirmed by genetic analysis.

Results: During the 13-year study period 657 newborns were included in the risk group and assigned for molecular genetic testing. Eight children were identified as having classical galactosemia on the basis of the enzymatic and molecular genetic analysis. Moreover,142 babies had a galactosemia Duarte, another 134 had decreasing activity of the GALT enzyme due to non-genetic reasons. The spectrum of revealed mutations in the GALT gene: K285N (31.3%), Q188R (25.0%), M142K (12.5%), E352Q (12.5%), W316X (6.2%), R262W (6.2%) and one new variant P140L (6.2%), not described earlier. Conclusion. Based on the results of newborn screening programs, the prevalence of classic galactosemia in the Ural population is 1:87 318 live birth and unlikely that it will undergo significant changes with further increase in the screening period.

O. Lagutina: None. S. Deryabina: None. T. Belyaeva: None. N. Nikitina: None. E. Nikolaeva: None.


What is beyond the congenital lactic acidosis? From variants to personalized medicine

I. Bravo-Alonso, R. Navarrete, P. Ruiz-Sala, F. Leal, M. Ugarte, C. Pérez-Cerdá, B. Merinero, B. Pérez, P. Rodríguez-Pombo

Centro de Diagnóstico de Enfermedades Moleculares, Centro de Biología Molecular Severo Ochoa, UAM-CSIC, CIBERER, IDIPAZ, Madrid, Spain

Implementation of Next Generation Sequencing has dramatically increased the diagnosis success-rate of highly heterogeneous inborn-errors of metabolism such as those underlying congenital lactic acidosis. However, the lack of integrated phenotypic data, the scarce number of datasets of global metabolomics and the necessary expertise to analyze the functional effects of variants of unknown significance, are limiting the transfer of genetic data into clinical practice. Our aim in this study was to evaluate the powerful of an adequate clinical-biochemical and genetic analysis to drive a clinical response in a timely manner preventing the use of major invasive techniques. With a pipeline that combined specific datasets from metabolomics sub-sections and phenotypic information, with DNA and/or RNA analysis, we identified likely pathogenic variants in strong-candidate genes in 64% of a cohort of 39 patients with clinical suspicion of congenital lactic acidosis. These variants, 14 of which were new, were found in 19 different nuclear and two mitochondrial genes. Specific biochemical test and/or bioenergetics analysis in fibroblasts including measures of oxygen consumption rate, steady-state levels of OxPhos proteins using Blue-native geles, and changes in mitochondri