Volume 27 | Supplement 2

The Swedish Exhibition & Congress Center, Gothenburg, Sweden

June 15-18, 2019

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".

Posters P01 Reproductive Genetics/Prenatal Genetics

P01.01A Prenatal whole exome sequencing in agenesis of the corpus callosum

S. Heide1, B. Keren1, M. Moutard2, T. Billette de Villeumeur2, M. Spentchian1, C. Garel3, C. Mignot1, J. Buratti1, V. Layet4, V. Tsatsaris5, S. Moutton6, M. Milh7, M. Gorce8, M. Spodenkiewicz9, G. Quenum Miraillet10, S. Chantot-Bastaraud10, d. Vincent11, L. Guibaud12, J. Jouannic13, S. Valence2, D. Heron1

1APHP, Department of Genetics, Armand-Trousseau and Pitié Salpêtrière hospital, Reference Center for Intellectual disability of Rare Causes, Paris, France, 2APHP, Department of pediatric neurology, Armand Trousseau hospital, Paris, France, 34. APHP, Department of pediatric radiology, Armand Trousseau hospital, Paris, France, 4Department of genetics, Le Havre hospital, Le Havre, France, 5APHP, Departement of obstetrics, Cochin hospital, Paris, France, 6Reference Center for Developmental Anomalies, Department of Medical Genetics, Dijon University Hospital, Dijon, France, 7APHM, Department of pediatric neurology, La Timone hospital, Marseille, France, 8Department of clinical genetics, CHU d’Angers, Angers, France, 9Department of clinical genetics, CHU de Reims, Reims, France, 10APHP, Department of cytogenetics, Trousseau hospital, Paris, France, 11HCL, Department of pediatric neurology, HFME, Bron, France, 12HCL, Department of radiology, HFME, Bron, France, 1314. APHP, Fetal Medicine Department, Trousseau Hospital, Sorbonne Medicine University, Paris, France

Agenesis of the corpus callosum (ACC) is usually diagnosed by prenatal ultrasound examination. In case of isolated ACC (iACC), neurodevelopment is within normal range in 80 % of cases whereas 20 % of children present mild to severe intellectual disability (ID). Among genetic etiologies, only chromosomal causes are investigated (karyotyping and microarray) during prenatal period while ACC with ID is due to a single gene mutation in most cases. Thus, parents make the decision to continue or terminate the pregnancy on statistics.

Our study aims to evaluate the feasibility of prenatal testing of all known ACC genes using whole exome sequencing (WES).

Eighteen fetuses with ACC were included (ongoing inclusions): 14 iACC and 4 cases of ACC associated to other anomalies (aACC). Trio WES were performed on fetal DNA extracted from amniotic fluid sampling. Only pathogenic variants in known ACC genes were considered. Variants of unknown significance (VUS) and secondary findings were not reported. WES results were available within an average of 21 days. Thirteen WES (72%) were normal (11 iACC, 2 aACC). A pathogenic variant in an ACC with ID gene was identified in two cases (11%): BRAT1 (aACC) and PPP2R1A (iACC). A pathogenic 6q27 deletion was identified in one case (aACC). We identified a VUS in 2 cases.

Our preliminary results suggest feasibility of prenatal WES in fetuses with prenatal diagnosis of ACC. In case of etiological diagnosis, WES helps the parents to make a decision for the pregnancy. However, WES in the prenatal period arises ethical questions.

S. Heide: None. B. Keren: None. M. Moutard: None. T. Billette de Villeumeur: None. M. Spentchian: None. C. Garel: None. C. Mignot: None. J. Buratti: None. V. Layet: None. V. Tsatsaris: None. S. Moutton: None. M. Milh: None. M. Gorce: None. M. Spodenkiewicz: None. G. Quenum Miraillet: None. S. Chantot-Bastaraud: None. D. Vincent: None. L. Guibaud: None. J. Jouannic: None. S. Valence: None. D. Heron: None.

P01.02B Results of karyotype analysis of 8361 pregnancies in prenataly identified cases with amniocentesis from south of Turkey

A. Pazarbasi1, D. Alptekin1, I. N. Uslu1, N. S. Ilgaz1, L. Ozpak1, G. Comertpay1, G. Ay1, N. Cetinel1, E. Akbal-Isık1, G. Evyapan1, S. Kocaturk-Sel1, U. Luleyap1, M. B. Yilmaz1, S. Buyukkurt2

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

Objectives: 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. Methods: A retrospective review of our amniocentesis database for the period from January 2000 to February 2019 was carried out. The karyotyping of 8361 fetuses was carried out in Department of Medical Biology from the samples of amniotic fluids which were sent from Department of Obstetrics and Gynecology of Balcali Hospital. A standard nomenclature has been developed to describe each of types of abnormality found in human chromosomes.

Results: A total of 8361 amniocentesis specimens were processed during the study period. 601 fetuses (7.18%) had various chromosomal abnormalities. 54.4% of abnormal karyotypes (329 cases) were numerical and 43.09% (259 cases) were structural. Both numerical and structural chromosomal aberrations were observed in 13 cases (2.16%). The ratios were as: trisomy 21 (48.93%), trisomy 18 (17.93%), monosomy X (9.72%), trisomy 13 (6.99%), Triploidy (4.86%), Klinefelter Syndrome (3.34%), Trisomy X (1.21%), XYY Syndrome (0.91%), and the others in all numerical abnormalities. The frequent structural abnormalities were as: 46,XX/XY, inv(9) (p11;q12)/(p11;q13)(29.34%), 46,XX/XY, 1qh(+)(11.58%), 46,XY, Yqh(-)(7.33%), 46,XX/XY, 16qh(+)(7.33%), 46,XX/XY, 9qh(+)(4.63%) and 46,XY, Yqh(+)(4.24%). Balanced and unbalanced translocations, deletions and duplications were also found in less ratio.

Conclusions: 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 7.18%. This ratio emphasizes the importance of prenatal diagnosis.

A. Pazarbasi: None. D. Alptekin: None. I.N. Uslu: None. N.S. Ilgaz: None. L. Ozpak: None. G. Comertpay: None. G. Ay: None. N. Cetinel: None. E. Akbal-Isık: None. G. Evyapan: None. S. Kocaturk-Sel: None. U. Luleyap: None. M.B. Yilmaz: None. S. Buyukkurt: None.

P01.03C Biallelic variants in AMPD2 gene cause prenatal isolated agenesis of corpus callosum

L. Mouthon1, S. Heide1, C. Depienne2, C. Nava3, A. Rastetter4, D. Lacombe5, T. Attie Bitach6, D. Héron1

1UF Génétique Médicale, APHP Hôpital Pitié Salpêtrière, Paris, France, 2Institüt für Humangenetik, Universitätsklinikum, Essen, Germany, 3UF de Génomique du Développement, APHP Hôpital Pitié Salpêtrière, Paris, France, 4Institut du Cerveau et de la Moelle épinière, Paris, France, 5Service de Génétique Clinique Médicale, CHU Bordeaux, Bordeaux, France, 6Département de Génétique, Hôpital Necker -Enfants Malades, Paris, France

Introduction: Recessive mutations in AMPD2 gene cause Pontocerebellar Hypoplasia (PCH) type 9. This severe neurodevelopmental disease is characterized by intellectual disability associated with epilepsy and spasticity. Brain imaging shows the association of pontocerebellar anomalies, agenesis of corpus callosum (ACC) and microcephaly. The 2 fetuses already reported with mutations in this gene had HPC associated with ACC on prenatal ultrasound or MRI. We report two cases of AMPD2 mutations in sibling fetuses with prenatal diagnosis of isolated ACC (iACC).

Cases: Male fetus 1 : prenatal iACC at 32 gestational weeks (GW) ; termination of pregnancy (top) at 37GW, normal CGH array; postnatal iACC (neuropathological examination). Male fetus 2: prenatal iACC ; top at 31 GW; normal CGH array; postnatal iACC (neuropathological examination).

Method: Whole exome sequencing (2 fetuses and 2 parents).

Results: Identification of two compound heterozygous AMPD2 mutations (c.751C>T, p.Arg251Trp; c.1648G>A, p.Glu550Lys) in the 2 fetuses.

Discussion: We report here an unusual prenatal presentation of PCH9 in 2 sibling fetuses, limited to a prenatal and postnatal iACC. ACC is one of the most frequent brain malformations, usually detected by prenatal ultrasound. Prognosis is variable and correlates with the presence or absence of other malformations. When isolated, the neurodevelopment is within a normal range in 80 % of cases whereas 20 % have mild to severe intellectual disability. Establishing an etiological diagnosis of a prenatal apparently iACC is essential to assess neurodevelopmental prognosis. AMPD2 should be considered as a gene responsible for isolated prenatal ACC.

L. Mouthon: None. S. Heide: None. C. Depienne: None. C. Nava: None. A. Rastetter: None. D. Lacombe: None. T. Attie Bitach: None. D. Héron: None.

P01.04D Rare autosomal abnormalities detected using noninvasive prenatal screening

J. Shubina, I. Y. Barkov, O. K. Stupko, L. V. Kim, T. O. Kochetkova, A. Y. Goltsov, I. S. Mukosey, M. V. Kuznetsova, N. A. Karetnikova, N. K. Tetruashvili, V. A. Bakharev, D. Y. Trofimov

National medical research center for obstetrics, gynecology and perinatology named after academician, Moscow, Russian Federation

Introduction: Noninvasive prenatal DNA screening (NIPS) is believed to be highly sensitive to common aneuploidy detection. Since some NIPS technologies use whole genome sequencing, aneuploidies and large CNV of any chromosome could be detected. Although detection of rare aneuploidies and large CNV is possible it is not recommended in Obstetricians and Gynecologists guidelines. Majority of NIPS providers do not report this information.

Materials and Methods: NIPS was carried on Ion S5 sequencer with in-house developed data analysis pipeline. 1300 samples collected at 11-20 weeks of gestation were analyzed. NIPS results were confirmed with karyotyping of invasively obtained samples or blood cells obtained postnatally. Four biopsies of placental tissue were performed to assess placental karyotype.

Results: High aneuploidy risk was detected in 39 cases. 14 - trisomy 21, 3 - trisomy 13, 15 sex chromosome abnormalities (4 due to maternal mosaicism) and 7 rare autosomal abnormalities (trisomies 7, 8, 10, large CNV). In one case large CNV was confirmed in the fetus. In two cases of rare trisomies, placental tissue was available, in both cases, mosaic trisomy was confirmed in the placenta.

Conclusions: High risk of rare autosomal abnormality could be associated with miscarriage, confined placental mosaicism, true fetal mosaicism, and uniparental disomies. Whole genome analysis may identify pregnancies at risk of miscarriage and other complications.The study is supported by the Ministry of Healthcare of Russian Federation

J. Shubina: None. I.Y. Barkov: None. O.K. Stupko: None. L.V. Kim: None. T.O. Kochetkova: None. A.Y. Goltsov: None. I.S. Mukosey: None. M.V. Kuznetsova: None. N.A. Karetnikova: None. N.K. Tetruashvili: None. V.A. Bakharev: None. D.Y. Trofimov: None.

P01.05A Array CGH and next-generations sequencing in diagnostics of fetal skeletal dysplasia

I. Dimova1, V. Peycheva1, P. Chaveeva2, R. Bozhilova1, K. Mihova1, K. Kamenarova1, A. Kanev3, A. Todorova1, V. Dimitrova4, R. Kaneva1

1Medical University Sofia, Sofia, Bulgaria, 2SAGBAL "Dr Shterev", Sofia, Bulgaria, 3Military Medical Academy, Sofia, Sofia, Bulgaria, 4SBALAG "Maichin dom", Sofia, Sofia, Bulgaria

Background: Array CGH is routinely used in prenatal diagnostics of structural fetal defects where the possibility to find genomic aberrations is high. This technique is especially suitable in a history of repeated pregnancy failure together with structural defects.

Materials and Methods: We report here the results from genomic analysis in fetal skeletal dysplasia (SD) - we performed arrayCGH in cases with repeated abortions and fetal skeletal anomalies and next-generation sequencing (NGS) - in cases with lack of diagnosis after aCGH or phenotypes, strongly suggestive for monogenic disorder.

Results: The findings from arrayCGH and NGS analyses are presented in the Table below.

Clinical manifestation

Genomic/gene aberration

Limbs anomalies and 3 previous abortions

7q21.3(94,214,445 - 95,257,297)x1 (1.04Mb) Split hand/foot malformation

3rd pregnancy with phocomely and limbs aplasia

1q21.1 (145425395 -146507577)x1 (1.08 Mbp) TAR syndrome

Skeletal dysplasia and 2 previous abortions

17q21.33(48,264,999-48,277,988)x3 (12.99 Кbp, including COL1A1 gene)

2nd pregnancy with arthrogryposis

Xq28(154,974,667-155,208,354)x0 (234 Kbp) VOUS: c.357T>G, p.Ile119Met (NM_017946.3) in FKBP14 gene

1st pregnancy with dwarfism

Possibly damaging variants: c.6117C>G (p.Ile2038Met) in HSPG2 gene; c.7223C>G (p.Ala2408Gly) in PCNT gene

Conclusion: Genetic diagnosis of SDs is strongly needed because there are so many diseases with complex phenotypes and identification of the responsible gene(s) is important to understand the diseases themselves. We demonstrated here the diagnostic utility of aCGH, which could be the first-line test in prenatal settings. NGS is a very powerful technique in diagnostics of very rare diseases, but needs much longer time for analysis and additional steps for elucidations of the found variants.

I. Dimova: None. V. Peycheva: None. P. Chaveeva: None. R. Bozhilova: None. K. Mihova: None. K. Kamenarova: None. A. Kanev: None. A. Todorova: None. V. Dimitrova: None. R. Kaneva: None.

P01.06B Application of array comparative genomic hybridization (aCGH) for identification of lethal chromosomal aberrations in spontaneous abortion

K. Sobecka, M. Smyk, M. Chojnacka, B. Wiśniowiecka-Kowalnik, E. Michalak, T. Klepacka, B. Nowakowska

Institute of Mother and Child, Warsaw, Poland

Spontaneous abortions occur in 8-20% cases of recognized pregnancies. Most often, the miscarriage take place in the first trimester (7-11 weeks). There are many causes of pregnancy loss, but the most important (~75%) is the presence of chromosomal aberrations. A high percentage of chromosomal aberration is caused by the abnormal genetic material of germ cells. Research showed that 20-30% of ova and 6-8% of sperm showed chromosomal abnormalities. This percentage significantly increases with advancing age in women and in cases of abnormalities of sperm in men.

We present the results of oligonucleotide array application in a cohort of 31 cases of mothers with adverse pregnancy histories. DNA was extracted from trophoblast and umbilical cord. Array CGH was performed using 4x180K microarrays from Oxford Gene Technology (CytoSure ISCA, v3).

The detection rate in our cohort was 61,3% (19/31). The most commonly found (73,7%) were aneuploidies: trisomy of chromosomes 14, 16, 18, 21, 22, Turner syndrome and triploidy. Other chromosomal abnormalities included structural aberrations: deletion 7p22.3p12.3 and duplication 9p24.3p13.2 inherited from normal father, deletion 3q13.31q22.2, deletion 3q22.3q23, duplication 17p12 inherited from father with foot malformations, deletion 17p13.1 inherited from normal mother, deletion 5q14.3 and de novo deletion 1q21.1q21.2.

Our research shows that microarray is the only method permitting the identification of all unbalanced aberrations (number and structure) with a much higher resolution than karyotype.

The work was supported by project 2016/23/N/NZ2/02364 from the National Science Centre, Poland.

K. Sobecka: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Significant; National Science Centre, Poland. M. Smyk: None. M. Chojnacka: None. B. Wiśniowiecka-Kowalnik: None. E. Michalak: None. T. Klepacka: None. B. Nowakowska: None.

P01.08D Screening by Multiple ligation-dependent probe amplification assay of AZF a, b, c regions in primary infertile men with azoospermia

K. Haziyeva1, O. Cilingir1, B. Durak Aras1, I. Ure2, E. Erzurumluoglu1, E. Tosumoglu1, S. Artan1

1Eskisehir Osmangazi University, Faculty of Medicine, Department of Medical Genetics, Eskısehır, Turkey, 2Eskisehir Osmangazi University, Faculty of Medicine, Department of Urology, Eskısehır, Turkey

Y chromosome microdeletions are the most common cause of azoospermia and occur in 5-15% of infertile men with azoospermia. Microdeletions/partial deletions of the azoospermia factor(AZF) regions on the Y chromosome are a well-known genetic cause of male infertility, resulting in impairment of spermatogenesis. We sought to determine the frequency and the character of AZF region microdeletions/partial deletions in infertile men with azoospermia by using Multiplex Ligation-Related Probe Amplification (MLPA). In total, 50 azoospermic infertile men without gene and chromosome mutations, were screened for Y chromosome microdeletions in AZF regions and 50 fertile men assigned to control group. The total frequency of the microdeletions was 16%. Most deletions (10%) were seen in the AZFc followed by the AZFb (4%). The partial BPY2 gene deletions, located at AZFc region were detected in five patients while partial EIF1AY gene deletions, located in AZFb locus were seen in two patients. The combined AZFb and AZFc loci deletion was revealed in a patient. No AZFa region deletion was detected among the azoospermic cases. In the literature, it has been shown that BPY2 gene is effective in male germ cell development and loss of EIF1AY gene function can result in azoospermia sporadically. The present findings suggest that MLPA is more appropriate compared to mPCR because it investigates partial and complete deletions of AZF regions for male sterility compared to previous methods. Our study shows that microdeletion and partial deletions, which are mentioned in the literature as the reason of azoospermia, can be detected by MLPA method at the same time.

K. Haziyeva: None. O. Cilingir: None. B. Durak Aras: None. I. Ure: None. E. Erzurumluoglu: None. E. Tosumoglu: None. S. Artan: None.

P01.09A Characterisation of Bardet Biedl Syndrome in a foetus by post-mortem microfocus computed tomography

A. Beleza-Meireles1,2, S. C. Shelmerdine3,4, M. Singh3, I. Simcock3,4, A. D. Calder3, M. Ashworth5, N. J. Sebire5,4, O. J. Arthurs3,4

1Guy’s Hospital, London, United Kingdom, 2Centre for Craniofacial & Regenerative Biology, King’s College London, London, Austria, 3Department of Radiology, Great Ormond Street Hospital for Children NHS Foundation Trust, London, UK, London, United Kingdom, 4UCL Great Ormond Street Institute of Child Health, London, United Kingdom, 5Department of Histopathology, Great Ormond Street Hospital for Children NHS Foundation Trust, London, United Kingdom

Confirmation of antenatally detected foetal anomalies is vital following termination of pregnancy due to congenital anomalies. Phenotypic information is essential for genetic testing and counselling. However antenatal ultrasonography identifies only up to 68% of autopsy findings, which are not always available.

Post-mortem imaging using microfocus computed tomography (micro-CT) provides an alternative, or complementary tool, to autopsy. It provides high-resolution, 3D digital images, which may enable accurate diagnosis of complex multisystem syndromes, in particular in small early gestation fetuses.

We describe the case of a 33 year old primigravida, who attended for antenatal sonography at 12+5 weeks gestation. An increased nuchal translucency and hypoplastic left heart were observed; multicystic kidneys suspected. Array-CGH was normal. The couple opted to terminate the pregnancy at 15+6 weeks.

Due to small foetal size, whole-body micro-CT was performed. Image analysis revealed an atrioventricular septal defect, mitral valve stenosis, left ventricular hypoplasia, aortic atresia and ascending and aortic arch hypoplasia. Multiple renal cortical cysts and bilateral upper and lower limb post-axial polydactyly were observed. Renal histology confirmed extensive cystic dilatation of the renal tubules.

Genetic testing identified compound heterozygous mutations in BBS7 [c.187G>A; p.(Gly63Arg)mat and c.973G>T; p.(Glu325Ter)pat], consistent with Bardet-Biedl Syndrome. Given a recurrence risk for future pregnancies at 25%, the couple received counselling regarding future preimplantation genetic testing.

In conclusion, micro-CT is a non-invasive highly detailed method for assessing early gestation foetal anatomy, and provides an adjunctive or alternative tool to the standard foetal autopsy technique.

A. Beleza-Meireles: None. S.C. Shelmerdine: None. M. Singh: None. I. Simcock: None. A.D. Calder: None. M. Ashworth: None. N.J. Sebire: None. O.J. Arthurs: None.

P01.11C Preconception expanded carrier screening in a developing country: where does science exactly meet bioethics?

S. Klumsathian1, B. Panthan1, N. Iemwimangsa1, I. Sensorn1, A. Charoenyingwattana1, T. Chareonsirisuthigul2, Y. Worakijthamrongchai3, M. Sukpraserrt3, W. Chantratita1, O. Trachoo4,1

1Center for Medical Genomics, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bangkok, Thailand, 2Department of Pathology, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bangkok, Thailand, 3Department of Obstetrics-Gynecology, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bangkok, Thailand, 4Department of Medicine, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bangkok, Thailand

Introduction: Expanded carrier screening (ECS) was recently introduced to Thailand and other developing countries in Asia by a large number of international laboratories. Several incidences were reported concerning negative psychosocial impact, i.e., anxiety and difficulty in making decision on reproductive option.

Material and Methods: Forty healthy Thai couples visiting preconception clinic were enrolled. ECS was performed using 630-disease panel covering most autosomal recessive and X-linked disorders. Thereafter, attitudes on ECS and clinical application were interviewed. Genetic data were then combined with previous in-house database, pooling up to 355 unrelated individuals. Carrier identification were interpreted using our developed bioinformatic and clinical criteria suitable for legal and ethical issues.

Results: The most common carrier frequency in our cohort belonged to beta thalassemia (1/5) which was consistent with national statistics. Interestingly, primary hemochromatosis H63D, which have never been recognised in Southeast Asian populations, became the second common (1/16). Other carriers detected as common in Thais included alpha thalassemia, Gilbert syndrome, 21-hydroxylase-deficient congenital adrenal hyperplasia and spinal muscular atrophy. More than 95% of the subjects had a positive attitude to ECS. They accepted preimplantation genetic diagnosis as their reproductive option if they were identified as couples at risk, either mild or severe condition.

Conclusions: ECS would be useful if the selection criteria were clear and consistent with national regulation and ethics. Pre-test genetic counseling was necessarily required for the couples to understand further outcome.

Grants: Faculty of Medicine Ramathibodi Hospital and Center of Excellence on Medical Biotechnology, Mahidol University.

S. Klumsathian: None. B. Panthan: None. N. Iemwimangsa: None. I. Sensorn: None. A. Charoenyingwattana: None. T. Chareonsirisuthigul: None. Y. Worakijthamrongchai: None. M. Sukpraserrt: None. W. Chantratita: None. O. Trachoo: None.

P01.12D Interest in expanded carrier screening among prospective parents: systematic review of the literature

D. Chokoshvili, E. Van Steijvoort, P. Borry

University of Leuven, Leuven, Belgium

Expanded carrier screening (ECS) is a reproductive genetic test aimed at identifying prospective healthy parents who are at risk of conceiving a child affected with a recessive disorder. ECS tests screen for large numbers of recessive disorders and are currently available through various genetic testing laboratories. However, the extent to which ECS can be successfully implemented in the context of reproductive healthcare depends largely on prospective parents’ interest in ECS. In this systematic review, we synthesized evidence from empirical studies exploring interest in ECS among individuals and couples in the general population. Studies included in this review reported prospective parents’ intentions to undergo a (hypothetical) ECS test, uptake of an actual ECS offer, or both. Four databases (Pubmed, Web of Science, CINAHL, Cochrane Library) were systematically searched for relevant publications and 12 empirical studies were included in the review. Owing to the novelty of ECS, all the included studies were relatively recent, having been published during 2015-2019. In the included studies, 33%-66% of respondents were interested in a (hypothetical) ECS test, while uptake rates for actual ECS offers were reported at 10%-50%. The highest uptake was observed in a study where ECS was offered to pregnant women. By contrast, studies focusing on the preconception population reported lower uptake rates, even though some of these preconceptional ECS tests were provided free of charge. Our findings suggest that there may be discrepancies between prospective parents’ self-reported intentions to undergo ECS and their actual test-taking behavior, particularly during the preconception period.

D. Chokoshvili: None. E. Van Steijvoort: None. P. Borry: None.

P01.14B Prenatal clubfoot increases the risk for clinically significant chromosomal microarray (CMA) results - analysis of 269 singleton pregnancies

A. Singer1, I. Maya2, B. Ehud3, H. Baris4, C. Vinkler5, C. Vinkler5, S. Ben-Shachar6, A. Bar-Shira6, L. Sagi-Dain7

1Community Genetics, Public Health Services, Ministry of Health, Jerusalem, Israel, 2Recanati Genetics Institute, Beilinson Hospital, Rabin Medical Center, Petach Tikva, Israel, 3Genetic Institute Kaplan Med Cntr, Rehovot, Israel, 4 The Genetics Institute, Rambam the Technion, Haifa, Israel, 5Genetic Institute Wolfson Med Cntr, Holon, Israel, 6Genetic Institute Tel Aviv Sourasky Medical Center, Tel Aviv, Israel, 7Genetic Institute Carmel medical Cntr, Haifa, Israel

Clubfoot, also known as talipes equinovarus, is a well-recognized congenital foot deformity. It is diagnosed in about 1:1000 pregnancies. Clubfoot can present as an isolated defect or as non-isolated, also called "complex" or "syndromic", clubfoot, associated with additional anomalies.

Objective: This work was done in order to examine the detection rate of clinically significant chromosomal microarray analysis (CMA) results in singleton pregnancies with clubfoot.

Methods: Data from all CMA tests performed due to sonographic abnormal findings between January 2013 and September 2017 were retrospectively obtained from Ministry of Health computerized database. All singleton pregnancies with sonographic diagnosis of clubfoot (talipes equinovarus) and documentation of CMA result were included.

Results: Of the 5750 CMA tests, a total of 269 (4.7%) were performed due to demonstration of fetal clubfoot. Of the 229 cases with isolated deformity, nine (3.9%) clinically significant CMA results were detected. This detection rate is significantly increased compared CMA results in normal pregnancies as previously reported. In 40 pregnancies with syndromic clubfoot, seven (17.5%) clinically significant CMA results were detected, a significantly higher frequency compared to isolated clubfoot cases.

Conclusion: Sonographic diagnosis of clubfoot, whether isolated or associated with additional sonographic anomalies, seems to increase the risk for abnormal CMA findings. Thus, CMA analysis, in conjunction with thorough sonographic anatomic survey, should be recommended in such pregnancies.

A. Singer: None. I. Maya: None. B. Ehud: None. H. Baris: None. C. Vinkler: None. C. Vinkler: None. S. Ben-Shachar: None. A. Bar-Shira: None. L. Sagi-Dain: None.

P01.15C The influence of prenatal chromosomal microarray analysis on the decision on termination of pregnancy

T. Reischer, J. Brandstetter, R. Drahonsky, C. Fast-Hirsch, B. Streubel

Medical University of Vienna, Vienna, Austria

Introduction: Chromosomal microarray analysis (CMA) was introduced to prenatal genetic diagnosis a couple of years ago. There have been many publications before demonstrating the benefit of CMA in detecting microduplications and microdeletions in the prenatal setting. Aim of this study is to evaluate the association between prenatal CMA and the decision on termination of pregnancy.

Materials and Methods: This is a retrospective study of a single prenatal center in Austria between 2011 and 2017, including all fetuses that had prenatal diagnosis with CMA.

Results: In general highest rates of pathogenic CNVs were found if the indication for prenatal diagnosis was congenital heart disease or brain malformations. There was a statistical significant association between pathogenic CNV and the decision on termination of pregnancy (p<0,05). But comparing pregnancy outcomes with prenatal CMA and without CMA, irrespective of the result of the CMA, we found significant fewer terminations of pregnancies in the group with CMA, even if the severity of malformations of the fetuses were comparable.

Conclusions: In summary the decision on termination of pregnancy is individual and there are many influencing factors to consider. However our data suggests that the result of additional CMA can be a crucial factor in the decision on termination of pregnancy.

T. Reischer: None. J. Brandstetter: None. R. Drahonsky: None. C. Fast-Hirsch: None. B. Streubel: None.

P01.16D Congenital anomalies in children born after assisted reproduction in the Czech Republic: Population based study

A. Sipek Jr1,2, V. Gregor2,3, A. Sipek Sr2,3,4,5, J. Klaschka6,7, M. Maly6,8, J. Jirova9

1Institute of Biology and Medical Genetics, First Faculty of Medicine, Charles University, Prague, Czech Republic, 2Department of Medical Genetics, Thomayer Hospital, Prague, Czech Republic, 3Department of Medical Genetics, Pronatal Sanatorium, Prague, Czech Republic, 4Institute of Medical Genetics, 3rd Faculty of Medicine, Charles University, Prague, Czech Republic, 5GENNET, Prague, Czech Republic, 6Institute of Computer Science of the Czech Academy of Sciences, Prague, Czech Republic, 7Institute of Biophysics and Informatics, First Faculty of Medicine, Charles University, Prague, Czech Republic, 8National Institute of Public Health, Prague, Czech Republic, 9Institute for Health Information and Statistics, Prague, Czech Republic

Introduction: Assisted reproduction (AR) is nowadays commonly-used method for treating various fertility problems. However, several studies have shown a higher incidence of selected types of congenital anomalies among AR-conceived children. Our goal was to study this association on a large population cohorts (over 3 millions of live-births) using data from our population based registries.

Methods: Our retrospective epidemiological study is based on the official data from the National Registry of Congenital Anomalies and National Registry of assisted Reproduction (run by the Institute of the Health Information and Statistics of the Czech Republic). The registration process is population-wide and compulsory by national law. We evaluated the incidence of congenital anomalies (ICD-10 diagnoses Q00-Q99) in AR-conceived children and compared it to the incidence of congenital anomalies in naturally-conceived children. Time period: 2013-2015.

Results: The overall incidence of congenital anomalies was slightly higher in the AR-children group however, the difference was not statistically significant. The congenital anomalies were more common in twins (p<0.005). The incidence of congenital anomalies in AR-twins was significantly higher than the incidence of congenital anomalies in non-AR-twins (p<0.005) while the difference of congenital anomalies incidence in AR and non-AR singletons was not significant.

Discussion: We have found that the incidence of congenital anomalies was higher especially in twins born after assisted reproduction while in singletons the difference was not significant. We will further analyze this finding during the next phases of our population study.

Acknowledgments: 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); Significant; Thomayer Hospital - Research grant TN, 00064190. V. Gregor: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Modest; Thomayer Hospital - Research grant TN, 00064190. A. Sipek Sr: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Modest; Thomayer Hospital - Research grant TN, 00064190. J. Klaschka: None. M. Maly: None. J. Jirova: None.

P01.17A Carriers for deafness and vision loss are identified as common autosomal recessive conditions in 40 healthy Thai couples obtaining expanded carrier screening

S. Klumsathian1, W. Lorlipiwong1, K. Sararat1, N. Poolthong1, N. Iemwimangsa1, I. Sensorn1, B. Panthan1, A. Charoenyingwattana1, T. Chareonsirisuthigul1,2, O. Trachoo1,3, W. Chantratita1

1Center for Medical Genomics, Faculty of Medicine Ramathibodi Hospital Mahidol University, Bangkok, Thailand, 2Department of Pathology, Faculty of Medicine Ramathibodi Hospital Mahidol University, Bangkok, Thailand, 3Department of Medicine, Faculty of Medicine Ramathibodi Hospital Mahidol University, Bangkok, Thailand

Introduction: One of the applications for next-generation sequencing (NGS) is expanded carrier screening (ECS) which offer prospective parents to learn their carrier status of many autosomal recessive (AR) and x-linked recessive (XLR) diseases at once. However, designing an ECS panel for Thais can be difficult due to insufficient variant frequency data. Therefore, we conduct a study to gather the frequency of pathogenic variants in the associated diseases by offering ECS to a Thai couple who plan to conceive.

Methods: DNA samples of 40 non-consanguineous Thai couples were underwent sequencing by NGS in 540 genes associated with AR and XLR diseases. We classified the variants detected by NGS using population databases (1000 Genomes Project, gnomAD, and our in-house Thai exome database) and diseases databases (Clinvar and HGMD professional) to determine their pathogenicity.

Results: 76 variants with strong evidence for pathogenicity are identified. Deafness is the most common AR diseases with six individuals carry deafness-related variants in these genes: SLC26A4, USH2A, OTOF, and MYO3A followed by five carriers of vision-loss variants in GRM6, USH2A, EYS, and ABCA4 gene. One couple shared a different pathogenic mutation in the ABCA4 gene.

Conclusions: Though deafness and vision-loss are the two most common AR carrier found in the Thai population, consideration need to be taken if they were in the ECS panel because these phenotypes are not severe enough for a prenatal diagnosis.

Grant: The study is supported by the Faculty of Medicine Ramathibodi Hospital, Mahidol University and Center of Excellence on Medical Biotechnology (CEMB).

S. Klumsathian: None. W. Lorlipiwong: None. K. Sararat: None. N. Poolthong: None. N. Iemwimangsa: None. I. Sensorn: None. B. Panthan: None. A. Charoenyingwattana: None. T. Chareonsirisuthigul: None. O. Trachoo: None. W. Chantratita: None.

P01.18B High risk - what’s next? Decisional conflict, regret and satisfaction among pregnant women making choices about further prenatal testing after a high probability result from the combined test

C. Ingvoldstad Malmgren1,2,3, T. Schlaikjær Hartwig4, C. Borregaard Miltoft5, A. Tabor5, F. Stener Jørgensen6

1Dept of Clinical Science, Intervention and Technology, Division of Obstetrics and Gynaecology, Stockholm, Sweden, 2Department of Public Health and Caring Science, Uppsala University,, Uppsala, Sweden, 3Department of Women’s and Children’s Health, Uppsala University, Uppsala, Sweden, 4Fetal Medicine Unit, Department of Obstetrics and Gynecology, Copenhagen University Hospital Hvidovre, Denmark, Copenhagen, Denmark, 5Fetal Medicine Center, Department of Obstetrics, Copenhagen University Hospital Rigshospitalet, Copenhagen, Denmark, 6Fetal Medicine Unit, Department of Obstetrics and Gynecology, Copenhagen University Hospital Hvidovre, Copenhagen, Denmark

Introduction: The aim was to investigate decision-making among Danish high-risk pregnant women when choosing between non-invasive prenatal testing (NIPT), invasive testing or no further testing. Women with a high-risk result from the combined first trimester screening were invited to fill in two online questionnaires at GA 12-14(Q1) and GA 24(Q2). The scales used were Decisional Conflict and Regret Scales, Satisfaction with genetic Counselling Scale and Health-Relevant Personality Inventory.

Results: In total, 339 women were included, and the response rates were 76 % on Q1 and 88% on Q2, respectively. Of the participants, 75.4% chose an invasive test and 23.8% chose NIPT. The median DCS score among all participants was within the level associated with implementing decisions, whereas 13.3% had a high level of decisional conflict. Choosing NIPT was associated with a high decisional conflict; receiving genetic counselling the same day was associated with a high decisional conflict; and a high satisfaction with the genetic counselling was associated with low decisional conflict. Furthermore, ‘alexithymia’, the personality sub-trait that describes a disinterest or inability in identifying and understanding feelings, was associated with low decisional conflict. High decisional regret was associated with high decisional conflict and low satisfaction with genetic counselling.

Conclusion: The results from this study show that satisfaction with, and timing of counselling are essential factors to limit decisional conflict. Also, the results indicate that women choosing NIPT have more decisional conflict compared to women choosing invasive testing. There was a significant association between high decisional conflict and later decisional regret.

C. Ingvoldstad Malmgren: None. T. Schlaikjær Hartwig: None. C. Borregaard Miltoft: None. A. Tabor: None. F. Stener Jørgensen: None.

P01.19C A novel diagnostic panel of 15 STR markers used to confirm the detection of Duchene Muscular Dystrophy, aneuploidy screening, and sample authenticity testing

Z. Sharifi1,2, F. Golnabi1,2, F. Rahiminejad1, S. Amini1, H. Farahzadi3, S. Zeinali1,4

1Dr. Zeinali’s Medical Genetics Laboratory, Kawsar Human Genetics Research Center, Tehran, Iran, Islamic Republic of, 2Department of Genetics, Faculty of Advanced Science and Technology, Tehran Medical Sciences, Islamic Azad University, Tehran, Iran, Islamic Republic of, 3Proteomics Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran, Islamic Republic of, 4Department of Molecular Medicine, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran, Islamic Republic of

Introduction: Duchenne (DMD) and Becker muscular dystrophy (BMD) are one of the most common genetic muscular dystrophies. Prenatal diagnosis (PND) or Pre-implantation Genetic Diagnosis (PGD) could be attractive options to prevent the birth of new affected cases. Aneuploidies are the cause of over 50% of all miscarriages. Early aneuploidy screening in conjunction with PND or PGD can decrease the subsequent complication of pregnancy termination.

Methods: This study aimed to develop a novel panel to confirm detection of DMD/BMD and aneuploidy screening simultaneously. The panel functions on the principle of hemizygosity mapping of the 6 novels STR (Short Tandem Repeat) markers linked to dystrophin gene. Additionally, this includes autosomal STR markers for the critical regions of Chromosomes 21, 18, 13, X and Y. These markers were amplified in a time-saving and cost-effective multiplex PCR reaction.

Results: Allele frequency and heterozygosity assessment of STR markers were studied in 250 unrelated healthy individuals. Totally, 85 alleles were detected. Heterozygosity of markers was 75.7%-89.8%. Genotype frequencies of markers were found to be in agreement with the Hardy–Weinberg equilibrium (P ≥ 0.1899). For further confirmation direct mutation analysis was also performed. The results were compatible. The panel was used for more than 150 PND cases and 15 PGD candidates and the results were successful.

Conclusion: This panel increases the accuracy and sensitivity of diagnosis. It can be easily applied for PGD/PND of DMD, aneuploidy screening and sex determination. Also provides extra advantages like ruling out maternal cell contamination, paternity testing and avoidance of sample cross-contamination.

Z. Sharifi: None. F. Golnabi: None. F. Rahiminejad: None. S. Amini: None. H. Farahzadi: None. S. Zeinali: None.

P01.20D Relative telomere length in women with early pregnancy losses

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

1Institute of Hereditary Pathology NAMS of Ukraine, Lviv, Ukraine, 2Rzeszów University of Technology, Department of Biochemistry and Biotechnology, Rzeszów, Poland

Introduction: Early pregnancy loss (EPL), a spontaneous abortion before 14 weeks of gestation, occurs in ~ 15% of clinically-recognized pregnancies and is the most common complication of pregnancy. Recurrent early pregnancy loss (REPL) affects 1-2% of couples and has a complex etiology. Approximately half of miscarriages from EPL cases are caused by chromosomal abnormalities in the embryo. Spontaneously lost pregnancies are characterized by shortened telomeres, especially in embryos with aneuploidies. Thus, short telomere length may be more frequent in women with REPL.

Material and Methods: Relative telomere length (RTL) was measured in DNA isolated from the blood samples using a real-time polymerase chain reaction approach. RTL was examined in three groups of women with EPL (N=171): (i) patients with single EPL (EPL1) (N=52), (ii) patients with two EPL (EPL2) (N=68), (iii) patients with REPL (N=49); and control group (C) - women who had healthy pregnancies with no history of infertility or miscarriage (N=113).

Results: The EPL group had significantly lower RTL than control (EPL: 1.39±0.06 versus C: 2.23±0.01, P=0.0000001). Average RTL were similar in EPL2 and REPL groups (1.36 ±0.04 in EPL2 and 1.31±0.07 in RPL) and were significantly lower compared to control (EPL2: 1.36 ±0.04 vs C: 2.23±0.01, P=0.000001 and REPL: 1.31±0.07 vs C: 2.23±0.01, P=0.000001).

Conclusions: Women experiencing two or more EPL have shorter telomeres. We assume that short telomeres in women are involved in complex factors that provoke early pregnancy loss.

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

P01.21A Endometrial receptivity revisited: endometrial transcriptome adjusted for tissue cellular heterogeneity

M. Suhorutshenko1,2, V. Kukushkina3, A. Velthut-Meikas2, S. Altmäe2,4, M. Peters2,1, R. Mägi3, K. Krjutškov2,5, M. Koel2,6, F. M. Codoñer7, J. Martinez-Blanch7, F. Vilella8, C. Simón8,9,10, A. Salumets2,1,11, T. Laisk2,1,3

1Department of Obstetrics and Gynecology, Institute of Clinical Medicine, University of Tartu, Tartu, Estonia, 2Competence Centre on Health Technologies, Tartu, Estonia, 3Estonian Genome Center, Institute of Genomics, University of Tartu, Tartu, Estonia, 4Department of Chemistry and Biotechnology, Tallinn University of Technology, Tallinn, Estonia, 5Research Program of Molecular Neurology, Research Programs Unit, University of Helsinki, Helsinki, Finland, 6Department of Cell Biology, Institute of Molecular and Cell Biology, University of Tartu, Tartu, Estonia, 7Lifesequencing SL, Valencia, Spain, 8Igenomix Foundation/INCLIVA, Valencia, Spain, 9Research Department, Igenomix SL, Valencia, Spain, 10Department of Pediatrics, Obstetrics and Gynecology, Valencia University, Valencia, Spain, 11Department of Obstetrics and Gynecology, University of Helsinki and Helsinki University Hospital, Helsinki, Finland

Introduction: The proportions of epithelial and stromal cells vary in endometrial tissue during the menstrual cycle. Each cell type has its unique gene expression profile, that compose whole-tissue expression pattern.

Materials and Methods: Using cell-type specific transcriptome data and computational deconvolution approach, we estimated the epithelial and stromal cell proportions in whole-tissue biopsies taken during early secretory (ES) and mid-secretory (MS) phases. The estimated proportions were used as covariates in whole-tissue differential gene expression (DGE) analysis. For RNA sequencing we used paired ES and MS endometrial biopsies, obtained from 35 healthy, fertile volunteers (23 - 36 years). DGE analysis was performed using two approaches - with and without deconvolution step, and results compared with each other.

Results: The estimated average proportions of stromal and epithelial cells in ES phase were 65% and 35%, and during MS phase 46% and 54%, that correlated with histological evaluation (r=0.88, p=1.1×10-6). Endometrial DGE analysis showed 26% differentially expressed transcripts (n=946) in receptive endometrium in both cell-type unadjusted and adjusted analyses. However, the other 74% (n=2,645) become statistically non-significant after cell-type adjustment, underlining the impact of tissue heterogeneity on DGE analysis. The results suggest new mechanisms involved in endometrial maturation involving genes like LINC01320, SLC8A1 and GGTA1P, described for the first time in endometrial receptivity context.

Conclusion: The better understanding of molecular processes during transition from pre-receptive to receptive endometrium serves to improve the effectiveness of assisted reproduction protocols. Biopsy cellular composition should be taken into account in future endometrial ‘omics’ studies.

M. Suhorutshenko: None. V. Kukushkina: None. A. Velthut-Meikas: None. S. Altmäe: None. M. Peters: None. R. Mägi: None. K. Krjutškov: None. M. Koel: None. F.M. Codoñer: None. J. Martinez-Blanch: None. F. Vilella: None. C. Simón: None. A. Salumets: None. T. Laisk: None.

P01.22B A prospective evaluation of exome sequencing in 51 fetuses with multiple congenital anomalies and lessons for future prenatal implementation

A. Yeung1,2,3, F. Chan4,3, A. Vasudevan5, J. Collett4,3, S. Prystupa6, Y. Chan6, G. McGillivray1,5,3

1Victorian Clinical Genetics Services, Murdoch Children’s Research Institute, Parkville, Australia, 2Monash Genetics, Monash Health, Melbourne, Australia, 3Melbourne Genomics Health Alliance, Melbourne, Australia, 4Department of Anatomical Pathology, Royal Women’s Hospital, Parkville, Australia, 5Department of Clinical Genetics, Royal Women’s Hospital, Parkville, Australia, 6Department of Anatomical Pathology, Monash Health, Melbourne, Australia

Introduction: Exome sequencing (ES) is a powerful adjunct to post-mortem examination for investigating fetuses with congenital anomalies. However, with the implementation of rapid “in-utero” sequencing becoming eminently feasible, fetal phenotyping may, in future, rely on antenatal imaging alone.

Aim: To determine whether the diagnostic yield of ES in fetuses with congenital anomalies following autopsy is comparable to the yield of ES implemented at the time of antenatal ultrasound detection.

Method: ES was prospectively performed on 51 fetal probands following autopsy as part of the Perinatal Autopsy Flagship of the Melbourne Genomics Health Alliance Demonstration Project. Candidate variants concordant with phenotypic findings from fetal autopsy were classified by multidisciplinary review using ACMG guidelines. Independently, a phenotype-driven virtual gene panel for each proband was derived from antenatal imaging reports by clinicians blinded to autopsy findings. Where a pathogenic variant fell within this “pre-autopsy” virtual gene panel, this was viewed as a diagnosis that would have been made by combining WES with antenatal imaging alone.

Results: The diagnostic rate in this cohort was 17/51 (33%) with 4 further variants awaiting functional validation. Phenotypes with the highest diagnostic yield included skeletal dysplasias (100%); multiple malformations (33%) and hydrops fetalis (33%). 14/17 of diagnoses would have been made on antenatal findings alone while 3/17 additional diagnoses were made with findings only apparent on autopsy.

Conclusion: ES in pregnancy following the detection of structural anomalies on ultrasound is feasible and returns comparable diagnostic yields to ES combined with autopsy.

A. Yeung: None. F. Chan: None. A. Vasudevan: None. J. Collett: None. S. Prystupa: None. Y. Chan: None. G. McGillivray: None.

P01.23C From diagnostic yield to clinical impact: implementation of prenatal exome sequencing in routine car

M. A. de Koning1, M. C. Haak2, P. N. Adama van Scheltema2, C. M. P. C. Peeters-Scholte3, T. T. Koopman1, E. A. R. Nibbeling1, E. Aten1, N. S. den Hollander1, C. A. L. Ruivenkamp1, M. J. V. Hoffer1, G. W. E. Santen1

1Dept. of Clinical Genetics, Leiden University Medical Center, Leiden, Netherlands, 2Dept. of Obstetrics, Leiden University Medical Center, Leiden, Netherlands, 3Dept. of Neurology, Leiden University Medical Center, Leiden, Netherlands

Introduction: Exome sequencing (ES) is an efficient tool to diagnose genetic disorders postnatally. Recent studies show that it may have a considerable diagnostic yield in fetuses with structural anomalies on ultrasound. We report on the clinical impact of the implementation of prenatal ES (pES) for ongoing pregnancies in routine care.

Material and Methods: We retrospectively analyzed the impact of pES on pregnancy outcome and pre- or perinatal management in the first 22, consecutively referred patients to our department for pES because of one or more structural anomalies on fetal ultrasound.

Results: In two cases, a diagnosis was made by chromosomal microarray analysis after ES counselling. The remaining 20 cases were divided in three groups; (1) pES to aid parental decision making (n = 12), (2) pES in the context of late pregnancy termination requests (n = 5) and (3) pES to guide prenatal or perinatal management (n = 3). pES had a clinical impact in 75% (9/12), 40% (2/5) and 100% (3/3) respectively, showing an overall clinical impact of pES of 70% (14/20).

Conclusion: We show that clinical implementation of pES is feasible and affects parental decision making or prenatal and perinatal management supporting further implementation of ES in the routine prenatal setting.

M.A. de Koning: None. M.C. Haak: None. P.N. Adama van Scheltema: None. C.M.P.C. Peeters-Scholte: None. T.T. Koopman: None. E.A.R. Nibbeling: None. E. Aten: None. N.S. den Hollander: None. C.A.L. Ruivenkamp: None. M.J.V. Hoffer: None. G.W.E. Santen: None.

P01.24D Diagnosis of fetal structural abnormalities using whole exome sequencing: a single centre study

S. Drury1, L. Reed1, V. Ramachandran1, Y. Patel1, A. Haworth1, J. Rizvi1, R. Dubis1, J. Anton1, J. Short2, E. Dempsy2, S. Mansour2, T. Homfray2

1Congenica Ltd, Hinxton, United Kingdom, 2South West Thames Regional Genetics Service, St George’s University Hospitals NHS Foundation Trus, London, United Kingdom

Introduction: Fetal abnormality detected on ultrasound occurs in up to 5% of fetuses. In the absence of aneuploidy or cytogenetic causes, single gene disorders can be responsible; a recent review of 31 prenatal exome sequencing studies by Best et al., (2018) showed a diagnostic rate of 6.2% to 80%. We have undertaken exome sequencing at a single centre for cases of unexplained fetal anomaly in both ongoing and retrospective pregnancies.

Materials and Methods: Patients with a fetal abnormality detected on ultrasound were referred to clinical genetics at St George’s Hospital, London. Fetal DNA was from CVS, amniotic fluid, fetal blood or tissue. Maternal cell exclusion was performed where relevant. DNA was enriched using the Agilent SureSelect CREv2 and sequenced using Illumina NextSeq 500. Variant analysis performed using Sapienita™. Gene panels and variant filter settings including Exomiser prioritisation were preconfigured to expedite analysis.

Results: In the first year (2018), 42 families were referred for exome sequencing due to fetal anomaly. A diagnosis was made in 15 cases (36%); 9/17 singletons (BICD2, FGFR2, LZTR1, NIPBL, PTPN11, RAF1, RMRP, SLC26A2, TSC1), 1/1 duo (UBE2A), 4/23 trios (CHD7, PIEZO1, POMGNT1, SLC6A9, TUBA1A) and 0/1 quad. Updated figures will be presented.

Conclusions: The diagnostic rate for this selected group is consistent with the literature and is higher than recent unselected cohort studies (Petrovski et al 2019, Lord et al 2019). In several cases the molecular diagnosis was not the primary suspected clinical diagnosis emphasising the continued importance of expanding and publishing prenatal genotype-phenotype associations.

S. Drury: A. Employment (full or part-time); Significant; Congenica Ltd. L. Reed: A. Employment (full or part-time); Significant; Congenica Ltd. V. Ramachandran: A. Employment (full or part-time); Significant; Congenica Ltd. Y. Patel: A. Employment (full or part-time); Significant; Congenica Ltd. A. Haworth: A. Employment (full or part-time); Significant; Congenica Ltd. J. Rizvi: A. Employment (full or part-time); Significant; Congenica Ltd. R. Dubis: A. Employment (full or part-time); Significant; Congenica Ltd. J. Anton: A. Employment (full or part-time); Significant; Congenica Ltd. J. Short: A. Employment (full or part-time); Significant; St George’s University Hospitals NHS Foundation Trust. E. Dempsy: A. Employment (full or part-time); Significant; St George’s University Hospitals NHS Foundation Trust. S. Mansour: A. Employment (full or part-time); Significant; St George’s University Hospitals NHS Foundation Trust. T. Homfray: A. Employment (full or part-time); Significant; St George’s University Hospitals NHS Foundation Trust.

P01.25A Chorionic villi sampling and QF-PCR + SNP-array routinely offered in early pregnancy losses

A. Borrell1, M. Pauta2, M. Grande3, V. Borobio1, C. Illanes1, L. Rodriguez-Revenga4, A. Soler4, C. Badenas4

1BCNatal. Hospital Clinic Barcelona, Barcelona, Catalonia, Spain, 2BCNatal. IDIBAPS. Hospital Clinic Barcelona, Barcelona, Catalonia, Spain, 3IDIBAPS. Hospital Clinic Barcelona, Barcelona, Catalonia, Spain, 4Biomedical Diagnostic Center. Hospital Clinic Barcelona, Barcelona, Catalonia, Spain

Background: Genetic investigation is not routinely offered in early pregnancy loss due to its high failure and maternal cell contamination rates.

Objective: To identify the genetic causes of early pregnancy losses, with the use of QF-PCR and SNP-array in chorionic villi samples obtained previously to evacuation.

Methods: Women with a pregnancy loss before 13 weeks were offered transcervical chorionic villi sampling prior to surgical or medical uterine evacuation. During a 3-year study period (September 2015-October 2018) 584 women consented. A first round of QF-PCR including chromosomes 21, 18, 13, X, Y and a second round with chromosomes 15, 16, and 22 were assessed. Concurrent karyotyping was also performed and SNP-array when the chromosomes were normal.

Results: Fifty-five (9%) samples were excluded after microscopic inspection because only maternal decidua could be retrieved. Among the 529 samples suitable for analysis, a chromosomal anomaly was found in 159 (30%) cases at the first QF-PCR round, and 128 (24%) at the second round. The karyotype revealed 66 (12%) anomalies undetected by QF-PCR. Among the 176 remaining early losses with a normal karyotype, SNP-array was performed in 90 cases with sufficient DNA ad 6 (2.4%) submicroscopic anomalies were found.

Conclusions: QF-PCR and SNP-array in chorionic villi samples are able to provide a result in 91% of early pregnancy losses. Two rounds of QF-PCR detected a chromosomal anomaly in 54% of the cases. If karyotyping is replaced by SNP-array in QF-PCR normal cases, further 12% chromosomal anomalies, 2.4% submicroscopic anomalies and molar pregnancies will be revealed.

A. Borrell: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Modest; Illumina. M. Pauta: None. M. Grande: None. V. Borobio: None. C. Illanes: None. L. Rodriguez-Revenga: None. A. Soler: None. C. Badenas: None.

P01.26B NGS based multigene panel analysis in cases of fetal brain malformations

D. Liebrecht, C. Daumer-Haas, C. Bagowski, N. Hirschberger, S. Minderer, T. Schramm, K. P. Gloning, M. Shoukier

Praenatal-Medizin München, Munich, Germany

Introduction: Identifying the genetic cause of fetal brain malformations is a diagnostic challenge due to the genetic heterogeneity. We present here the results of multigene panel analysis (MGPA) of 65 fetuses with congenital brain malformations.

Materials and Methods: From January to December 2018, 65 prenatal cases with fetal brain malformations (10 with minor extracerebral anomalies) were investigated by targeted Next Generation Sequencing of the coding regions of 262 genes. Genes were selected from the developmental brain disorders database (DBDB) and results reported within two weeks. Familial co-segregation analysis for variant pathogenicity assessment was carried out, when necessary. Karyotypes and array-CGH results of fetuses were normal.

Results: Pathogenic variants were identified in 10 (15%) out of the 65 cases. Mutations were found in a total of 8 genes: TUBA1A and ASPM (2 cases respectively) as well as in GPSM2, FKRP, KAT6B, OFD1, TSEN54 and TUBB (1 case each). In addition variants of unclear significance were detected in 4 cases (6%). No mutation was identified in a total of 51 fetuses (79%). Our findings show a strong correlation between the detection rate of causative mutations and complexity of the fetal phenotype. Additionally, Trio-based whole exome sequencing should be considered in cases with negative MGPA results to further improve the detection rate.

Conclusions: MGPA facilitates the identification of genetic causes with a good diagnostic yield and should be used for routine molecular genetic diagnosis of fetal brain malformations.

D. Liebrecht: None. C. Daumer-Haas: None. C. Bagowski: None. N. Hirschberger: None. S. Minderer: None. T. Schramm: None. K.P. Gloning: None. M. Shoukier: None.

P01.27C A case of fetal hydrops and FOXP3 - another argument for using exomes in pregnancy for isolated hydrops

S. Ringsted1, L. Andreassen1, C. Kamper2, N. Becher1,3, I. Vogel1,3,4

1Departement for Clinical Genetics, Aarhus University Hospital, Aarhus, Denmark, 2Department for Obstetrics and Gynecology, Aarhus University Hospital, Aarhus N, Denmark, 3Center for Fetal Diagnostics, Aarhus University Hospital, Aarhus, Denmark, 4Biomedicine, Aarhus University, Denmark, Denmark

Introduction: Whole Exome Sequencing (WES) is currently entering prenatal genetic diagnosis for unsolved fetal malformations. Diagnostic rates vary significantly, but with low diagnostic rates in isolated increased nuchal translucency (NT) or cystic hygroma [Best et al 2018].

Materials and Methods: G5P2 with severe hydrops in 2 male fetuses (missed abortion week 19 and 20) and healthy children. Both hydrope fetuses presented with normal first trimester scans. Cytomegalovirus (CMV) was the presumed cause for the first case of hydrops, as the mother seroconverted just after the pregnancy. However, PCR for CMV was negative in tissue from the aborted fetuses. Array-CGH was carried out on DNA obtained from amniotic fluid from both pregnancies, but leaving both cases unresolved. WES was performed as trio on DNA extracted from amniotic fluid from the second pregnancy, and both parents. Results were validated by Sanger sequencing.

Results: A novel variant in FOXP3 was identified in hemizygous state in the second pregnancy, inherited from the mother, and subsequently confirmed in cell culture from the first pregnancy. This variant is currently under further validation.

Conclusions: Germline FOXP3 mutations cause immune dysregulation, polyendocrinopathy and enteropathy, X-linked (IPEX) syndrome. This syndrome is well characterized in patients postnatally. Prenatally, mutations have been reported to cause the loss of male fetuses as a result of fetal hydrops [Reichert et al. 2015]. This case adds to the body of knowledge suggesting that WES can improve our understanding of prenatal presentation of not only fetal malformations but also isolated hydrops.

S. Ringsted: None. L. Andreassen: None. C. Kamper: None. N. Becher: None. I. Vogel: None.

P01.28D Absence of AGG interruptions is a risk factor for a full mutation expansion among ethnically diverse FMR1 premutation carriers

N. Domniz1, L. Ries-Levavi1, Y. Cohen1, L. Marom Haham1, M. Berkenstadt1, E. Pras1, A. Glicksman2, N. Tortora2, G. J. Latham3, A. G. Hadd3, S. L. Nolin2, S. Elizur1

1Sheba Medical Center, Tel Hashomer, Ramat Gan, Israel, 2New York State Institute for Basic Research in Developmental Disabilities, New York, NY, United States, 3Asuragen, Austin, TX, United States

Introduction: Genetic counseling given to FMR1 premutation carriers is currently based upon the number of CGG repeats. However, recent studies revealed that AGG interruptions may decrease the risk of intergenerational expansion.

Materials and Methods: All FMR1 premutation carriers who underwent chorionic villus sampling (CVS) or amniocentesis (AC) at Sheba Medical Center during the period of 2011-2017 were included in this study. To increase the accuracy of our results, we combined results from Israel with those from the New York State Institute for Basic Research in Developmental Disabilities (IBR). FMR1 PCR and Asuragen Kit were used to determine the number of CGG repeats and AGG interruptions in all women and fetuses.

Results: The combined data included 1471 transmissions of maternal premutation alleles: 369 (25.1%) stable and 1,102 (74.9%) unstable transmissions. Full mutation expansions were identified in 20.6% (303/1471) of transmissions. A total of 97.4% (388/397) of transmissions from alleles with no AGGs were unstable, compared to 79.6% (513/644) in alleles with 1 AGG and 46.7% (201/430) in alleles with 2 or more AGGs. 40% (159/397) of alleles with no AGGs expanded to a full mutation, compared to 20.2% (130/644) for alleles with 1 AGG and only 3.2% (14/430) in alleles with 2 AGGs or more.

Conclusions: Based on this ethnically diverse data we recommend that the risk estimates for a full mutation expansion for FMR1 premutation carriers will include the number of AGG interruptions as well as CGG repeat size. Study funding/competing interest(s) - Azrieli foundation, Canada-Israel

N. Domniz: None. L. Ries-Levavi: None. Y. Cohen: None. L. Marom Haham: None. M. Berkenstadt: None. E. Pras: None. A. Glicksman: None. N. Tortora: None. G.J. Latham: None. A.G. Hadd: None. S.L. Nolin: None. S. Elizur: None.

P01.29A Targeted next-generation sequencing and CNV analysis identifies two novel mutations in the FRAS1 gene

M. Kuhn1, M. Burkert1, S. Tschürtz2, K. Mehnert1, D. Gläser1

1genetikum - Center for Human Genetics, Neu-Ulm, Germany, 2Medical Office for Prenatal Medicine, Munich, Germany

Introduction: We report on a case of a nonconsanguineous couple with one healthy child and two spontaneous abortions (one at 22 and one at 36 weeks of gestation). Test results of the couple’s chromosome analysis were normal, and there was no evidence of genetically relevant diseases in their families. Frozen skin cells of the first abort had an insufficient quality to perform gene panel analysis.

Materials and Methods: Clinical exome sequencing (CES) was performed on the parents’ DNA followed by screening for carrier status in 34 relevant genes for intrauterine fetal death.

Results: In the maternal DNA sample, we identified a heterozygous novel mutation c.2423-1G>T in the FRAS1 gene affecting a highly conserved donor splice site and leading most probably to aberrant splicing. Various splice site mutations in the FRAS1 have been described before. In the paternal DNA sample, copy number variation (CNV) analysis based on next-generation sequencing data showed a novel heterozygous deletion of the last three exons of the FRAS1 gene. Junction fragment PCR confirmed this deletion and sequencing of the PCR products revealed the breakpoints (c.11092+658_*12039+6368del). Although the material of the first abort had poor quality, Sanger sequencing and junction fragment PCR on frozen skin cells were able to identify these two novel variants as compound heterozygous.

Conclusion: Therefore, clinical exome sequencing in combination with CNV analysis on a couple’s DNA is an appropriate method to detect possible causes of multiple stillbirths, especially when no fetal DNA is available.

M. Kuhn: None. M. Burkert: None. S. Tschürtz: None. K. Mehnert: None. D. Gläser: None.

P01.30B Identification of compound heterozygous FTO-mutations in a severe malformation syndrome

U. Siebers-Renelt, Y. Stratis, J. Seggewiß, S. Ledig, J. Horvath, P. Wieacker

Institut für Humangenetik, Muenster, Germany

We report on a non-consanguineous couple who presented in the 19th week of their 6th pregnancy with ultrasound abnormalities of the fetus (broad neck with increased nuchal translucency, multicystic dysplasia of the kidney, short femur, fetal clubfoot). The couple has got just one healthy child. Two spontaneous abortions occurred and one pregnancy with trisomy 21 was terminated. Another child also exhibited a broad neck and additional omphalocele early in pregnancy. Hydrocephaly was first visible in the 32nd week. After delivery in the 35th week, she required artificial respiration. MRI showed a complex brain malformation. That child deceased at the age of one month. Chromosome analysis and microarray were normal in both malformed children. After termination of pregnancy we performed a clinical trio-exome (Agilent SureSelect Inherited Disease Panel). We identified two loss-of-function mutations in compound heterozygosity in the FTO gene not yet described (ACMG class 4). The deceased sibling carried both mutations and the healthy sister none. Homozygous FTO loss-of-function mutations have been associated with multiple malformations including short broad neck, different brain malformations, severe developmental delay, failure to thrive and high risk of death in early childhood (Boissel 2009, Daoud 2016). In summary, the FTO-mutations are very likely to be causative for the disease because of the type of mutation, the phenotype and the segregation data. Nevertheless, prenatal molecular analysis should be accompanied by a detailed ultrasound work-up. Moreover, the couple has to be aware that the absence of the FTO-mutations does not completely exclude the disease.

U. Siebers-Renelt: None. Y. Stratis: None. J. Seggewiß: None. S. Ledig: None. J. Horvath: None. P. Wieacker: None.

P01.31C Early prediction of preeclampsia (PE) severity in pregnant women with preexisting type 1 diabetes mellitus (T1DM): new implements for genetic markers

T. Avramenko1, A. Hrybanov1,2,3

1State Institution "Institute of pediatrics, obstetrics and gynecology by the National Academy of Medical Sciences of Ukraine", Kyiv, Ukraine, 2Maternity Hospital #2, Mykolaiv, Ukraine, 3Public Health Board by Mykolaiv Region State Administration, Mykolaiv, Ukraine

Background: Severe PE strongly correlates with persistent disability, long-term cardiovascular complications in mothers and prematurity, IUGR or BPD in neonates. The study objective was to establish reliable clinical, genetic markers associated with PE severity in T1DM women.

Materials and Methods: Group 1 included patients with mild (n=15), moderate (n=11), severe (n=4) PE; Group 2 - 30 patients, in whom pregnancy was not complicated with PE. Polymorphisms in eNOS (4b/4a, G894T), ACE (I/D), AT2R1 (A1166C), MGP (Thr83Ala, T138C) and PON1 (C108T) genes were detected using PCR-RFLP. Data was analyzed with SPSS17.0 using paired sample t-test, Pearson’s chi-squared test with 95%CI calculation for OR. Binary logistic regression was used to generate statistical models and evaluate their prognostic value. ROC curve analysis was performed in order to establish the influence of polymorphic variants of genes on PE severity.

Results: Main contributors to moderate/severe PE development: pre-existing vascular complications (nephropathy [χ2=10,05, p=0,004, OR=11,23 95%CI 2,13-59,26], lower extremities angiopathy [χ2=7,39, p=0,007, OR=16,00 95%CI 1,86-137,61], retinopathy [χ2=4,27, p=0,039, OR=6,50 95%CI 1,25-33,91]) and their combinations (nephropathy + retinopathy; nephropathy + lower extremities angiopathy) and ACE DD-genotype. In PON1_108CT carriers clinical parameters exerted modifying effect: when combined with nephropathy, or T1DM duration < 8,5 years, or BMI < 23,8 kg/m2, mild PE develops; moderate/severe PE develops when diabetes duration is > 13,5 years or BMI is > 25,17 kg/m2. Combination ACE_II/PON1_CC was protective against PE development, while ACE_ID/PON1_CT is associated with mild PE. Conclusion: Genetic markers are helpful in identification of women at high risk for severe PE.

T. Avramenko: None. A. Hrybanov: None.

P01.32D Case of 8p and 18p genetic imbalance in a subfertile female patient without pronounced physical and mental abnormalities

Y. V. Shilenkova, A. A. Pendina, O. A. Efimova, A. V. Tikhonov, O. G. Chiryaeva, V. S. Dudkina, L. I. Petrova, I. D. Mekina, O. V. Malysheva, E. S. Shabanova, T. E. Ivashchenko, A. M. Gzgzyan, I. Y. Kogan

D.O. Ott Research Institute of Obstetrics, Gynecology and Reproductology, Saint Petersburg, Russian Federation

Introduction: We report an unusual case of unbalanced karyotype female carrier with no physical and mental abnormalities and her reproductive history.

Materials and Methods: Conventional karyotyping (GTG and QFH/AcD-banding techniques) was performed on the chorionic villi from aborted material and on PHA-stimulated lymphocytes of the patient, her husband and patient’s parents. FISH and aCGH (CGXv1.1 8x60K,PerkinElmer) were performed on the patient’s PHA-stimulated lymphocytes and whole blood sample, respectively. Standard IVF was performed with an embryo biopsy on day 3 (6-8-cell stage) for PGT-SR by FISH.

Results: The patient - 28-years old women - sought genetic counseling after loss of two naturally conceived pregnancies. The first miscarriage was not karyotyped. The second had karyotype 45,ХХ,der(8)t(8;18)(p23;p11.3),-18mat. The patient appeared to have the same karyotype: 45,ХХ,der(8)t(8;18)(p23;p11.3),-18dn. aCGH revealed terminal deletions combined with microduplication: del(8)(p23.1p23.3) (6.718Mb), dup(8)(p22p22) (4.937Mb), del(18)(p11.31p11.32) (3.693Mb), dup(18)(q23q23) (0.060Mb). The patient’s husband and parents had normal karyotypes. The patient had only slight dysmorphic face features and no pronounced physical and mental abnormalities. Subsequently, the patient underwent four IVF cycles with a total of 25 oocytes obtained. Only 13 of them reached MII (52% versus expected 75-90%). Only one out of 10 embryos was cytogenetically balanced. The embryo was transferred, but the pregnancy was not registered.

Conclusions: The present case demonstrates a genotype-phenotype disparity in the carrier of unbalanced chromosomal rearrangement. Considering an unpredictable effect of the genetic imbalance on the offspring phenotype, a personalized approach to the patient’s genetic counseling is required in order to achieve pregnancy with cytogenetically balanced embryo.

Y.V. Shilenkova: None. A.A. Pendina: None. O.A. Efimova: None. A.V. Tikhonov: None. O.G. Chiryaeva: None. V.S. Dudkina: None. L.I. Petrova: None. I.D. Mekina: None. O.V. Malysheva: None. E.S. Shabanova: None. T.E. Ivashchenko: None. A.M. Gzgzyan: None. I.Y. Kogan: None.

P01.33A Trans-ethnic meta-analysis meta-analysis of gestational diabetes reveals shared genetic background with type 2 diabetes

N. Pervjakova1,2, J. P. Cook3, A. P. Morris3,4, T. Ferreira5,6, R. Mägi1

1Estonian Genome Center, Institute of Genomics, University of Tartu, Tartu, Estonia, Tartu, Estonia, 2Genomics of Common Disease, Division of Diabetes, Endocrinology and Metabolism, Department of Medicine, Imperial College London, UK, London, United Kingdom, 3Department of Biostatistics, University of Liverpool, Liverpool, UK, Liverpool, United Kingdom, 4Estonian Genome Center, Institute of Genomics, University of Tartu, Tartu, Estonia, 5Big Data Institute, Li Ka Shing Center for Health for Health Information and Discovery, Oxford University, Oxford, UK, Oxford, United Kingdom, 66. Wellcome Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, UK, Oxford, United Kingdom

Gestational diabetes mellitus (GDM), defined as glucose intolerance first recognized in pregnancy, has important implications for both mother and child. Offspring of mothers with GDM have an increased risk of birth complications associated with higher risk for developing metabolic syndrome, type 2 diabetes (T2D) and cardiovascular disease in later life. To date, the only genome-wide association study (GWAS) for GDM, in 1,399 affected Korean women, revealed associations at two loci: MNTR1B and CDKAL1. We conducted trans-ethnic meta-analysis of 21 GWAS in 5,374 cases and 346,506 controls of diverse ancestry (62.0% European, 16.5% East Asian, 3.2% Mexican-American and 18.3% Afro-Caribbean), each imputed up to reference panels from the 1000 Genomes Project or Haplotype Reference Consortium. The trans-ethnic meta-analysis, performed with MR-MEGA to allow for heterogeneity in allelic effects between ancestries, included 13,980,490 variants, after excluding those with minor allele count <5 and imputation quality <0.4 in each GWAS. We replicated both known GDM associations at genome-wide significance (P<5x10-8): MTNR1B (rs10830963, P=2.3×10-49) and CDKAL1 (rs9348441, P=9.7×10-15). We also identified three additional novel loci: TCF7L2 (rs7903146, P=1×10-14), CDKN2A/B (rs10811660, P=1.9×10-9) and LOC105369513 (rs143421658, P=4.1×10-8). Allelic effects of GDM association signals were mostly homogeneous across ethnic groups. The exception was at the CDKAL1 locus (Phet=3.68×10-9), where the signal was driven by East Asian ancestry GWAS (OREast-asian=1.67(1.46-1.89); OREuropeans=1.06(1.01-1.12)). Four of the loci (MTNR1B, CDKAL1, TCF7L2, and CDKN2A/B) have also been robustly associated with T2D, and lead variants are identical to those we have identified for GDM, supporting a shared underlying genetic contribution to both diseases.

N. Pervjakova: A. Employment (full or part-time); Modest; AnteGenes OÜ. J.P. Cook: None. A.P. Morris: None. T. Ferreira: None. R. Mägi: None.

P01.34B Genome-wide analysis of losses and gains a better understanding of idiopathic male infertility

A. Hodžić1, N. Trošt1, B. Zorn2, D. Plaseska-Karanfilska3, P. Noveski3, T. Kunej4, K. Urh4, L. Lovrečić1, B. Peterlin1

1Clinical Institute of Medical Genetics, University Medical Centre Ljubljana, Ljubljana, Slovenia, 2Andrology Unit, Reproductive Unit, Department of Obstetrics and Gynecology, University Medical Centre Ljubljana, Ljubljana, Slovenia, 3Research Centre for Genetic Engineering and Biotechnology "Georgi D. Efremov" Macedonian Academy of Sciences and Arts, Skopje, Macedonia, The Former Yugoslav Republic of, 4Department of Animal Science, Biotechnical Faculty, University of Ljubljana, Ljubljana, Slovenia

Introduction: Diagnosting workup of infertile man, which include screening for chromosomal aberrations and Y chromosomal azoospermia factor deletions, have not been changed for years and still the etiology of most of the infertile cases remains unknown. Aiming to achieve progress in elucidating genetic causes of male infertility and to examine suitability of novel genetic testing methods in clinical routine of infertile man, we investigated man with unexplained infertility with Array Comparative Genomic Hybridisation method.

Materials and Methods: We enrolled 90 patients with severe male factor infertility, defined as idiopathic azoospermia or oligoasthenoteratozoospermia, and analyzed DNA using SurePrint G3 Unrestricted CGH 4x180K microarrays. We have selected candidate Copy Number Variants (CNVs) according 2 criterias: 1st criteria was that identified CNVs are not present in currently available databases of genomic variants, and 2nd criteria was that identified CNVs are not present in our control data set of men with proven fertility.

Results: We identified 5 CNVs which included genes previously related to male infertility (CLCA4, USF1, FCER1G, FNTA, HOTAIR). We have also identified 7 CNVs not presented in currently available databases of genomic variants or in our control data set. The identified CNVs spanning genes with noticed expression in testis, however, their possible functional impact on infertility are currently not known.

Conclusion: We provide a several candidate CNVs, possibly implicated in male infertility. Taken together with previous research, these findings are one more step forward to implementation of new testing methods into routine clinical practice of infertile man.

A. Hodžić: None. N. Trošt: None. B. Zorn: None. D. Plaseska-Karanfilska: None. P. Noveski: None. T. Kunej: None. K. Urh: None. L. Lovrečić: None. B. Peterlin: None.

P01.35C Detection of de novo copy-number variations from exome sequencing of 108 infertile patient-parents trios

F. K. Mastrorosa1, M. J. Xavier1, A. Mikulasova2, M. S. Oud3, R. M. Smits4, G. Astuti3, B. Alobaidi1, S. J. Cockell5, J. Coxhead6, C. Gilissen3, L. Ramos4, J. A. Veltman1,3

1Institute of Genetic Medicine, Newcastle University, Newcastle upon Tyne, United Kingdom, 2Institute of Cellular Medicine, Newcastle University, Newcastle upon Tyne, United Kingdom, 3Department of Human Genetics, Donders Institute for Brain, Cognition and Behaviour, Radboudumc, Nijmegen, Netherlands, 4Department of Obstetrics and Gynaecology, Radboudumc, Nijmegen, Netherlands, 5Bioinformatics Support Unit, Newcastle University, Newcastle upon Tyne, United Kingdom, 6Genomics Core Facility, Newcastle University, Newcastle upon Tyne, United Kingdom

Introduction: Approximately 7% of all men are infertile and genetics is known to play an important role in the most severe forms of infertility such as azoospermia and extreme oligozoospermia. However, most patients remain undiagnosed. In this study, we explored the role of de novo copy-number variations (CNVs) in whole-exome sequencing (WES) data from a unique cohort of 108 infertile men and their parents.

Material and Methods: Three different software, CoNIFER, XHMM and an optimized GATK4 pipeline were used to detect rare de novo CNVs. XHMM and GATK4 reduced the inherent noise in the exome coverage data best, resulting in the most accurate CNVs detection. Additionally, the GATK4 pipeline allowed to integrate allele frequency analysis, confirming the loss of heterozygosity (LOH) in deletions.

Results: Application of these tools revealed two rare de novo deletions in two different patients. One deletion occurred on chromosome 11 and partially overlapped a deletion previously reported in an infertile man. The second, affected NXT2 on chromosome X, a gene evolutionary conserved and highly expressed in testis. Both CNVs were validated by microarrays and/or Q-PCR.

Conclusions: These first data indicates that de novo CNVs may play an important role in severe male infertility. However, replication and functional studies are required to further validate the impact of our findings. By further improving CNV detection from WES data we may be also able to identify additional de novo CNVs that are currently missed, as well as maternally inherited CNVs that may play a role in male infertility.

F.K. Mastrorosa: None. M.J. Xavier: None. A. Mikulasova: None. M.S. Oud: None. R.M. Smits: None. G. Astuti: None. B. Alobaidi: None. S.J. Cockell: None. J. Coxhead: None. C. Gilissen: None. L. Ramos: None. J.A. Veltman: None.

P01.36D Identification of a Novel Genetic Cause of Familial Nonobstructive Azospermia

S. G. TEMEL1, B. Turkgenç2, K. Teralı3, M. Ergören4, M. Cetinkaya5, M. Başar6, S. Kahraman5

1UNIVERSITY OF ULUDAG, FACULTY OF MEDICINE, DEPARTMENT OF MEDICAL GENETICS, BURSA, Turkey, 2Acibadem Genetic Diagnostic Genetic Center, labgen, ISTANBUL, Turkey, 3Near East University, Faculty of Medicine, Department of Biochemistry, Nicosia, Cyprus, 4University of Near East, Faculty of Medicine, Department of Medical Biology, NICOSIA, Cyprus, 5Assisted Reproductive Technologies and Reproductive Genetics Centre, Istanbul Memorial Hospital, Istanbul, Turkey, 6Department of Urology & Andrology, Memorial Sisli Hospital, Istanbul, Turkey

Infertility is a global reproductive health problem, and in males it is usually because of the reduced number or the complete absence of sperm cells in semen (oligozoospermia and azoospermia, respectively). Nonobstructive azoospermia (NOA) is the most severe form of male infertility affecting ~0.6% of men from the general population and ~10% of infertile men. Its etiology remains largely unknown. To identify the genetic cause of NOA in four affected members from a consanguineous family, we performed whole-exome sequencing (WES). WES revealed a homozygous c.1166C>T (p.Pro389Leu) variation in the M1AP gene. The segregation of the M1AP variant with NOA in this family was confirmed by Sanger sequencing. The 3D structure of the mutant protein was predicted computationally. Sequence- and structure-based in silico studies and subsequent preliminary gene expression studies imply that the M1AP variant has severe implications for protein structure and function. Further functional studies to corroborate our findings are ongoing. Overall, M1AP is a novel candidate gene for male infertility and, to the best of our knowledge, this is the first report identifying M1AP as a cause for human familial NOA. And our pedigree analysis suggests an autosomal recessive mode of inheritance for NOA due to M1AP in the present family.

S.G. Temel: None. B. Turkgenç: None. K. Teralı: None. M. Ergören: None. M. Cetinkaya: None. M. Başar: None. S. Kahraman: None.

P01.39C Genetic analysis of 24 candidate genes validates TEX14, TEX11, NR5A1 and DMRT1 as clinically relevant for non-obstructive azoospermia

A. Röpke1, N. Köckerling1, M. J. Wyrwoll1, J. Emich1, M. Wöste2, M. Dugas2, A. Pilatz3, H. Schuppe3, T. Diemer3, D. Fietz4, C. Krallmann5, S. Kliesch5, C. Friedrich1, F. Tüttelmann1

1Institut für Humangenetik, Münster, Germany, 2Institut für Medizinische Informatik, Münster, Germany, 3Klinik und Poliklinik für Urologie, Kinderurologie und Andrologie, Gießen, Germany, 4Institute of Veterinary Anatomy, Histology and Embryology, Gießen, Germany, 5Centre of Reproductive Medicine and Andrology, Department of Clinical and Surgical Andrology, Münster, Germany

Male infertility is a clinically and genetically highly heterogeneous disease, mostly characterised by spermatogenetic failure, clinically noted as oligo- or azoospermia. The most common form of the latter phenotype is non-obstructive azoospermia (NOA), which can be caused by various genetic defects such as chromosomal aberrations, Y-chromosomal AZF microdeletions or monogenic defects. In this study, we systematically analysed 24 genes reported to be associated with NOA in the OMIM database. Variants in these genes were detected in whole exome sequencing data from 484 well-phenotyped infertile patients (azoospermia, N=94; cryptozoospermia, N=52; mixed atrophy, N=93; Sertoli-Cell-Only syndrome, N=179; meiotic arrest, N=43; other arrest, N=23). Other clinical causes for their infertility such as previous chemo-/radiotherapy, as well as karyotype aberrations and AZF deletions had been excluded. Exclusively novel or rare coding variants were assessed concerning their potential pathogenicity. Out of the 24 genes listed in OMIM, TEX14, TEX11, NR5A1 and DMRT1 were validated as clinically relevant genes causing NOA with strong evidence. Specifically, we identified eight patients carrying TEX11 variants. Ten patients demonstrated compound heterozygous or homozygous TEX14 variants. Five patients carried NR5A1 and five others DMRT1 variants. This is the first report of concise exome sequencing in a large group of infertile males. Our results clearly demonstrate that the TEX14, TEX11, NR5A1 and DMRT1 genes have reached a sufficient level of evidence to be prioritised for clinical analyses. This work was carried out within the frame of the DFG Clinical Research Unit “Male Germ Cells: from Genes to Function” (CRU 326).

A. Röpke: None. N. Köckerling: None. M.J. Wyrwoll: None. J. Emich: None. M. Wöste: None. M. Dugas: None. A. Pilatz: None. H. Schuppe: None. T. Diemer: None. D. Fietz: None. C. Krallmann: None. S. Kliesch: None. C. Friedrich: None. F. Tüttelmann: None.

P01.40D Maternal copy number variations in the DMD gene as incidental findings in non-invasive prenatal testing

N. Brison1, J. Storms1, K. Claeys2, L. Dehaspe1, E. Dimitriadou1, C. Melotte1, T. de Ravel1, L. De Waele3, N. Goemans4, E. Legius1, H. Peeters1, H. Van Esch1, V. Race1, J. Vermeesch1, K. Devriendt1, K. Van Den Bogaert1

1Department of Human Genetics, Leuven, Belgium, 2Department of Neurology, Leuven, Belgium, 3Department of Pediatric Neurology, Leuven, Belgium, 4Department of Neurosciences, Leuven, Belgium

Noninvasive prenatal testing (NIPT) using shallow whole-genome sequencing also reveals maternal copy number variations (CNV’s). Some of those variants are clinically actionable or could be harmful for the fetus. CNV’s in the DMD gene, potentially causing dystrophinopathies, are amongst the most commonly observed maternal CNV’s. We analyzed the data of maternal CNV’s detected in the DMD gene by NIPT. Out of 26.123 NIPT analyses, 16 maternal CNV’s in the DMD gene were detected (1/1.632 pregnant women). Variant classification regarding pathogenicity and phenotypic severity was based on public databases, segregation analysis in the family and a prediction of the effect on the reading frame. Ten CNV’s were classified as pathogenic, 4 as benign whereas 2 remained unclassified. We present our experience with the detection of maternal CNV's in the DMD gene and propose a scheme for the interpretation and the returning of these CNV’s detected by NIPT. We show that genome-wide NIPT leverages CNV screening in the general population of pregnant women and that interrogating the maternal CNV landscape can improve overall pregnancy management.

N. Brison: None. J. Storms: None. K. Claeys: None. L. Dehaspe: None. E. Dimitriadou: None. C. Melotte: None. T. de Ravel: None. L. De Waele: None. N. Goemans: None. E. Legius: None. H. Peeters: None. H. Van Esch: None. V. Race: None. J. Vermeesch: None. K. Devriendt: None. K. Van Den Bogaert: None.

P01.41A Validation of SNP-based noninvasive prenatal screening test to detect maternal X chromosome abnormalities

V. Kantor1, R. Dhamankar1, E. Valenti1, D. Lyons1, M. T. Trefogli1, I. Balosbalos1, C. Kao2, H. Hakonarson2, K. A. Martin1

1Natera, Inc., San Carlos, CA, United States, 2Center for Applied Genomics, Children’s Hospital of Philadelphia, Philadelphia, PA, United States

Introduction: Maternal chromosome abnormalities (CA) are known incidental findings with non-invasive prenatal screening (NIPS), contributing to false positives with quantitative methodologies. SNP-based NIPS can distinguish the maternal and fetal SNP allele distributions. This study validates the performance of SNP-based NIPS to detect maternal CA on chromosome X.

Methods: Plasma samples from singleton pregnancies were obtained (April-December 2018) and analyzed by SNP-based NIPS. Samples were included in the analysis if the algorithm could not return a result (below threshold for reporting) for suspected CA. These samples were stratified into suspected maternal (group A, n=107) and fetal origin (group B, n=110). Associated maternal buffy coats were blinded and sent to CHOP for cytogenetic analysis using the Global Screening Array (GSA, Illumina).

Results: Of 107 group A samples, 101 were confirmed by GSA to have maternal X CA (PPV: 94.4%; 97.5% confidence interval (CI), 88.2-100%); in the remaining 6 samples, 3 were discordant (GSA unable to detect suspected maternal CA), and in the other 3 samples, maternal CA was suspected, but too ambiguous to call as present. In 67.3% (68/101) cases, NIPS vs. GSA interpretations matched; and 32.7% (33/101) showed similar but varied interpretations due to ambiguities arising from mosaicism and/or specific X abnormality. No maternal X CA were found in group B by GSA (NPV: 100%; 97.5% CI, 96.7-100%).

Conclusions: The study supports the reporting of maternal X CA suspected by SNP-based NIPS, which was confirmed in 94.4% of suspected cases. No maternal abnormalities were identified when a fetal abnormality was suspected.

V. Kantor: A. Employment (full or part-time); Significant; Full-time Employee, Natera, Inc.. E. Ownership Interest (stock, stock options, patent or other intellectual property); Significant; Stock, Natera, Inc. R. Dhamankar: A. Employment (full or part-time); Significant; Full-time Employee, Natera, Inc.. E. Ownership Interest (stock, stock options, patent or other intellectual property); Significant; Stock, Natera, Inc. E. Valenti: A. Employment (full or part-time); Significant; Full-time Employee, Natera, Inc.. E. Ownership Interest (stock, stock options, patent or other intellectual property); Significant; Stock, Natera, Inc.. D. Lyons: None. M.T. Trefogli: A. Employment (full or part-time); Significant; Full-time Employee, Natera, Inc.. E. Ownership Interest (stock, stock options, patent or other intellectual property); Significant; Stock, Natera, Inc. I. Balosbalos: A. Employment (full or part-time); Significant; Full-time, Natera, Inc.. E. Ownership Interest (stock, stock options, patent or other intellectual property); Significant; Stock, Natera, Inc.. C. Kao: None. H. Hakonarson: None. K.A. Martin: None.

P01.42B Genetic signals for use of hormone replacement therapy in post-menopausal women suggest potential drug targets and reflect changes in health practice

K. S. Ruth, R. N. Beaumont, J. Tyrrell, A. R. Wood, S. E. Jones, M. N. Weedon, T. M. Frayling, A. Murray

University of Exeter Medical School, Exeter, United Kingdom

Introduction: Hormone replacement therapy (HRT) is an effective treatment for vasomotor symptoms around menopause, but in the UK HRT usage dropped following the publication of health risks in 2002.

Materials and Methods: To understand the biology of HRT use, we carried out genome-wide analyses of self-reported HRT phenotypes (ever taken, age started, age stopped and time taken) in up to 153,152 white European women aged 40-70 years from the UK Biobank.

Results: We identified 15 independent signals. A signal (AF=95%) in TACR3, the receptor for neurokinin B, was associated with raised odds of using HRT (1.18 per allele; P=1×10-26) and with vasomotor symptoms in a previous genome-wide analysis (Crandall et al 2017). Additionally, a putative signal (AF=0.1%) near ALDH8A1 (gene involved in tryptophan catabolism) was associated with taking HRT for longer (0.5 SD per allele; P=4×10-8). Tryptophan is a precursor of serotonin, which has been implicated in hot flushes, as has neurokinin B.

Of the signals, 13/15 were associated with starting HRT younger at P<5×10-8 and 11 with earlier menopause. A genetically-predicted one year earlier menopause raised the odds of HRT use in women starting treatment before 2002 (OR=1.12, 95% CI=1.10,1.13) but not after 2002 (OR=1.02, 95% CI=0.95,1.00). In contrast, the signals near TACR3 and ALDH8A1 were not associated with menopause timing.

Conclusions: Genetic associations for HRT are largely driven by menopause timing and are affected by changes in health practice. We identified two genetic signals, which are not affected by health practice, near plausible candidate genes for vasomotor symptoms.

K.S. Ruth: None. R.N. Beaumont: None. J. Tyrrell: None. A.R. Wood: None. S.E. Jones: None. M.N. Weedon: None. T.M. Frayling: None. A. Murray: None.

P01.43C Prenatal diagnostics: the utility of molecular karyotyping

F. Sachinidi, E. Panou, D. Mpouzarelou, C. Billi, L. Florentin

Alfalab, Genetics and Genomics Center, Athens, Greece

Introduction: the aim of this study is to highlight the use of molecular karyotyping in the context of prenatal diagnostic testing.

Materials and Methods: Molecular karyotype was applied on 4012 prenatal samples (CVS, amniotic fluids) as the first line diagnostic tool. Specimens have been analyzed using Agilent SurePrint G3 8x60K ISCA design and Cytogenomics software.

Results: women who underwent prenatal molecular karyotype were referred for ultrasound findings, increased NT, advanced maternal age, previous pregnancy with aneuploidy, positive biochemical or NIPT, anxiety etc.

Molecular karyotype shows an increased diagnostic yield compared to conventional karyotype varying from 1,4% to 3,4% depending on the referral reason with the highest added value in fetuses with abnormal U/S findings. Among all abnormal CGH samples with normal karyotype, 72,5% of clinically significant imbalances would have escaped detection from NIPT since they concern rare syndromes (25%), susceptibility to-loci (60%) and backbone abnormalities (15%) related to the referral reason.

Conclusion: Since more than 70% of the abnormal CGH cases would not be detected neither by conventional karyotype nor by NIPT, we suggest that every pregnant woman of any age should be offered the informed choice to undergo molecular karyotype prenatal testing.

F. Sachinidi: None. E. Panou: None. D. Mpouzarelou: None. C. Billi: None. L. Florentin: None.

P01.44D Prenatally diagnosed megalencephaly-polymicrogyria-polydactyly-hydrocephalus syndrome

K. Tael1, K. Ridnõi2,3, E. Kurvinen1, P. Ilves2,4, L. Makarenkova3, S. Pajusalu1,2,5, M. H. Wojcik6,7, K. Õunap1,2,7, T. Reimand1,2

1Department of Clinical Genetics, United Laboratories, Tartu University Hospital, Tartu, Estonia, 2Institute of Clinical Medicine, University of Tartu, Tartu, Estonia, 3East-Tallinn Central Hospital, Tallinn, Estonia, 4Clinic of Radiology, Tartu University Hospital, Tartu, Estonia, 5Yale University School of Medicine, Department of Genetics, New Haven, CT, United States, 6Division of Genetics and Genomics, Department of Medicine, Boston Children’s Hospital, Harvard Medical School, Boston, MA, United States, 7Broad Institute of MIT and Harvard, Cambridge, MA, United States

Introduction:Megalencephaly-polymicrogyria-polydactyly-hydrocephalus (MPPH) syndrome is characterized by megalencephaly, polymicrogyria and a variant in one of three genes: CCND2, AKT3 or PIK3R2. To the best of our knowledge, there is only one prenatally reported MPPH case. We present a new case of MPPH with a variant in CCND2 that was diagnosed prenatally.

Case report: Chorionic biopsy was performed during the 1st trimester of the pregnancy due to increased NT value (3.39 cm) to a 29-years-old patient. The karyotype of the fetus was 46,XY. Ultrasonography and magnetic resonance imaging of the fetus showed extensive bilateral cortical dysplasia of the supratentorial cerebral parenchyma with a suspected focal acute lesion in the right parietotemporal region. Dysgenesis of the corpus callosum as well as asymmetry of the hemispheres of cerebellum were also visualized. The radiological changes were nonspecific but indicated either intrauterine infection or genetic brain malformation. The pregnancy was ultimately terminated, and an autopsy revealed multiple congenital cerebral anomalies: abnormal cranial occlusion, abnormal additional clefts on right in the occipital and parasagital regions, dysgenesis of the corpus callosum, and a collapsed and hypoplastic septum pellucidum. There were no polydactyly and signs of inflammation in the placental and fetal tissues. To investigate the underlying etiology of this phenotype, trio exome sequencing (ES) analysis was done.

Result: ES revealed a pathogenic de novo missense variant: NM_001759.3(CCND2):c.839C>A p.(Thr280Asn) rs587777620, confirmed by Sanger sequencing.

Conclusions: MPPH syndrome should be considered when prenatally cerebral dysgenesis is visualized on US and/or MRI.

Funding: Estonian Research Council grants PRG471 and PUTJD827.

K. Tael: None. K. Ridnõi: None. E. Kurvinen: None. P. Ilves: None. L. Makarenkova: None. S. Pajusalu: None. M.H. Wojcik: None. K. Õunap: None. T. Reimand: None.

P01.46B Robust strategy for preimplantation genetic testing of myotonic dystrophy type 1 by bidirectional triplet-primed PCR combined with multi-microsatellite haplotyping following whole genome amplification

M. Lian1,2, C. G. Lee3,4,5, S. S. Chong3,1,2

1National University Health System, Singapore, Singapore, 2National University Hospital, Singapore, Singapore, 3National University of Singapore, Singapore, Singapore, 4Duke-NUS Graduate Medical School, Singapore, Singapore, 5National Cancer Center, Singapore, Singapore

Myotonic dystrophy type 1 (DM1) is caused by moderate to very large expansions of the DMPK CTG trinucleotide repeat. Disease transmission to offspring can be avoided through prenatal diagnosis or preimplantation genetic testing for monogenic disorders (PGT-M). We describe a robust PGT-M strategy that can be applied to virtually any couple at risk for DM1, including carriers of large expanded alleles with non-CTG interruptions at either the 5’ or 3’ end of the repeat. This strategy utilizes whole-genome amplification by multiple displacement amplification, followed by bidirectional triplet-primed PCR (TP-PCR) sizing of the DMPK CTG repeat, in parallel with single-tube genotyping and haplotype phasing of 12 closely linked and highly polymorphic microsatellite markers. Bidirectional TP-PCR and dodecaplex marker PCR assays were optimized and validated on whole-genome amplified single lymphoblasts isolated from DM1 reference cell lines, and tested on a simulated PGT-M case comprising a parent-offspring trio and three simulated embryos. DMPK TP-PCR reliably detects repeat expansions regardless of allele size, and employing TP-PCR in both directions ensures successful expansion detection even when non-CTG interruptions occur at either the 5’ or 3’ end of the expanded allele. Misdiagnoses and diagnostic ambiguity due to allele dropout or exogenous DNA contamination can be easily detected through the use of tightly linked microsatellite markers, minimizing the exclusion of potentially unaffected embryos for uterine transfer. The highly polymorphic multi-marker panel also maximizes the likelihood of marker informativeness in at-risk couples, thus minimizing the need for couple-specific assay customization.

M. Lian: None. C.G. Lee: None. S.S. Chong: None.

P01.47C Nicotine and resveratrol alter sox2 and sox4 genes expression levels in human amniotic cell culture

G. Cömertpay, Ü. H. Lüleyap, B. M. Yılmaz, P. Pazarcı

Cukurova University, Medical Faculty, ADANA, Turkey

Introduction: The aim of this research is to investigate the effects of nicotine on expression levels of SOX2 and SOX4 master genes in human amniotic cell cultures to indicate the risks of smoking in pregnancy. The effects of resveratrol on expression levels of these genes in human amniotic cell cultures, which are treated by nicotine were also examined in this study.

Materials and Methods: Twenty patients were included in our study and for each patient; control, nicotine treated and nicotine + resveratrol treated cell culture groups are formed. The expression levels of SOX2 and SOX4 genes are examined in each group by using real time RT-PCR.

Results: According to the results of our study, change in expression levels of SOX2 and SOX4 genes in nicotine treated group were found to be statistically significant. Also, when groups treated with nicotine and nicotine + resveratrol were compared the difference was found to be statistically significant.

Conclusion: In conclusion, nicotine increased the expression levels of SOX2 and SOX4 genes by 60% in human amniotic cell cultures and resveratrol was found to be an important antioxidant that reduces the increased expression levels of SOX2 and SOX4 genes caused by nicotine treatment.

This study was supported by the Scientific Research Project Unit of the Çukurova University

G. Cömertpay: None. Ü.H. Lüleyap: None. B.M. Yılmaz: None. P. Pazarcı: None.

P01.48D A placental trisomy 2 detected by NIPT evolved in a fetal small Supernumerary Marker Chromosome (sSMC)

J. Domaradzka1, M. Deperas1, E. Obersztyn1, A. Kucińska-Chahwan2, N. Brison3, K. Van Den Bogaert3, T. Roszkowski2, M. Kędzior1, M. Bartnik-Głaska1, A. Łuszczek1, K. Jakubów-Durska1, J. Vermeesch3, B. Nowakowska1

1Medical Genetics Department, The Institute of Mother and Child, Warsaw, Poland, Warsaw, Poland, 2Department of Obstetrics and Gynecology, Witold Orlowski Public Teaching Hospital, Warsaw, Poland, Warsaw, Poland, 3Centre for Human Genetics, KU Leuven, Leuven, Belgium, Leuven, Belgium

Objective: Here we report a prenatally detected mosaicism of a small supernumerary marker chromosome (sSMC) derived from chromosome 2. The 38-year-old woman underwent amniocentesis because of a high risk of trisomy 2 revealed by the Non-Invasive Prenatal Test (NIPT).

Methods and results: A genome-wide NIPT detected a trisomy of chromosome 2. The amniocentesis was performed to verify the NIPT result. Array comparative genomic hybridization (aCGH) from uncultured amniocytes revealed a duplication of 14,83 Mb on chromosome 2q11.1q13. Interphase fluorescence in situ hybridization (FISH) revealed three signals of centromere 2 in 30% of the cells. GTG-banded metaphases confirmed the abnormal karyotype (47,XX,+mar[21]/46,XX[19]), indicating 52% mosaicism of the cell line with the sSMC. The ultrasound examination did not reveal abnormalities. The pregnancy has been terminated. Cytogenetic analyses (FISH, aCGH and conventional karyotype) on fetal skin biopsies were performed and confirmed the genomic gain of the centromeric region of chromosome 2. In the placenta, three cell lines were detected: a normal cell line, a cell line with trisomy 2 and a third one with only the sSCM derived from chromosome 2. Conclusion: Whole-genome NIPT allows not only the identification of common fetal trisomies (13, 18, 21) but also diagnosis of rare chromosomal abnormalities. Especially in such cases, it is extremely important to perform not only NIPT verification on a sample of material other than trophoblast, but also to apply appropriate research methods. Such conduct allows detailed analysis of the detected aberration, thus appropriate clinical validity.

J. Domaradzka: None. M. Deperas: None. E. Obersztyn: None. A. Kucińska-Chahwan: None. N. Brison: None. K. Van Den Bogaert: None. T. Roszkowski: None. M. Kędzior: None. M. Bartnik-Głaska: None. A. Łuszczek: None. K. Jakubów-Durska: None. J. Vermeesch: None. B. Nowakowska: None.

P01.49A Non-Invasive Prenatal Testing pitfalls: exceptional report of multiple discrepancies between noninvasive screening for fetal trisomy 21, karyotype, array CGH and fetal ultrasound

F. Kundul1, K. Cassinari1, G. Joly-Hélas1, N. Le Meur1, J. Coursimault1, M. Castelain1, A. Diguet2, E. Verspyck2, S. Torre3, B. Macé1, T. Frebourg1, P. Chambon1

1Normandie Univ, UNIROUEN, Inserm U1245 and Rouen University Hospital, Department of Genetics, F76000, Normandy Centre for Genomic and Personalized Medicine, Rouen, France, 2Department of Gynecology and Obstetrics, Rouen University Hospital-Charles Nicolle, Rouen, France, 3Department of Neonatal Pediatrics, Intensive Care and Neuropediatrics, Rouen University Hospital, Rouen, France

Introduction: Pitfalls of noninvasive prenatal test (NIPT) for fetal trisomy 21 on circulating cell-free DNA are currently well known and are linked to confined placental mosaicism, maternal or fetal mosaicism, maternal neoplasia, evanescent twin, or low fetal DNA fraction. Unbalanced chromosomal rearrangements may also interfere with NIPT. We report here a rare anomaly identified through NIPT.

Case: A NIPT was performed on a 39-year-old woman with abnormal first trimester serum markers. This NIPT (Clarigo, Multiplicom Agilent) did not show any trisomy 21 but was evocative of trisomy 18. An amniocentesis was performed at the 22th week of gestation (WG) and FISH analyses (Aneucyte, Cytocell) on native amniotic fluid were compatible with a trisomy 18 in a female fetus. Unexpectedly, concomitant ultrasound control didn’t detect any morphological abnormality on a male fetus. Then, fetal karyotype and array-CGH confirmed the female chromosomal sex and revealed a very partial trisomy 18, linked to a small supernumerary marker chromosome, in mosaic, which was derivative from a chromosome 18. In addition, SRY locus was found on the short arm of one X chromosome. Genetic counseling being less alarming in this context than with a complete trisomy 18, this patient pursued her pregnancy and delivered a boy at 36 WG with normal neonatal examination. A regular monitoring of his psychomotor development is under way.

Discussion: This observation underlines the necessary precautions while interpreting NIPT and the necessity of a strong cytogenetic confirmation. This case also illustrates that incidental findings are to be expected with NIPT.

F. Kundul: None. K. Cassinari: None. G. Joly-Hélas: None. N. Le Meur: None. J. Coursimault: None. M. Castelain: None. A. Diguet: None. E. Verspyck: None. S. Torre: None. B. Macé: None. T. Frebourg: None. P. Chambon: None.

P01.50B Pilot study of locally performed noninvasive prenatal testing NIPT in Bulgaria

R. RAYNOVA, S. Bichev, S. Andonova, N. Yaneva, C. Kercheva, I. Bradinova, A. Savov

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

Introduction: With the introduction of noninvasive prenatal testing (NIPT), the prenatal screening for chromosomal aneuploidies has been expanded beyond ultrasound examination and combined first-trimester screening (cFTS). NIPT using cell-free DNA (cfDNA) circulating in maternal blood presents as an early, accurate, and safe prenatal testing. Since August 2018 NIPT for chromosomes 21, 18, 13 and sex determination was introduced in-situ in the University Hospital of Obstetrics and Gynecology ”Maichin dom”, Sofia. The analysis was discussed with pregnant women after performing cFTS.

Materials and Methods: A total of 134 pregnant women were screened for chromosomal aneuploidies by NIPT using ion semiconductor sequencing platform (Ion Proton™, Thermo Fisher Scientific) and IONA® test (Premaitha Health plc, Manchester, UK). The patients were at a mean age of 34.5 years. Plasma samples were collected between 10+5 and 24+5 g.w.

Results: Due to an intermediate risk (1/100 to 1/1000) NIPT was performed on 68 (47.76%) pregnant women. Others were tested because of maternal anxiety or advanced age. An average of 10.6% (from 3 to 24%) fetal fraction (FF) was achieved. One report was unsuccessful due to a low fetal fraction in patient with BMI=43.03. We obtained 132 “low risk” reports and one “high risk” - for trisomy 21, confirmed by amniocentesis followed by QF-PCR.

Conclusions: This is the first introduction of locally performed NIPT in Bulgaria. With the accumulation of more samples and data a possible correlation between cFTS results and NIPT; FF or/and gestational week and NIPT results could be established.

R. Raynova: None. S. Bichev: None. S. Andonova: None. N. Yaneva: None. C. Kercheva: None. I. Bradinova: None. A. Savov: None.

P01.51C Unexpected finding of uniparental disomy mosaicism in term placentas: Is it a common feature in trisomic placentas?

D. Van Opstal1, K. E. M. Diderich1, M. Joosten1, L. C. P. Govaerts1, J. Polak1, M. Boter1, J. J. Saris1, W. Cheung1, S. van Veen1, R. M. van der Helm1, A. T. J. I. Go1, M. F. C. M. Knapen1, D. N. M. Papatsonis2, A. Dijkman3, F. A. T. de Vries1, R. H. Galjaard1, L. H. Hoefsloot1, M. I. Srebniak1

1Erasmus MC, Rotterdam, Netherlands, 2Amphia Hospital, Breda, Netherlands, 3Reinier de Graaf Hospital, Delft, Netherlands

Objective: Non‐invasive prenatal testing (NIPT) detects placental chromosome aberrations. When amniocentesis reveals a normal karyotype, confined placental mosaicism (CPM) may be assumed. In order to confirm this, placental cytogenetic studies were performed.

Method: NIPT was conducted in the course of the DutchTRIDENT study. Placentas of 10 cases with NIPT results indicating an autosomal trisomy and showing a normal (N = 9) or low mosaic karyotype (N = 1) in amniotic fluid (AF) were investigated. The cytotrophoblast as well as the mesenchymal core of two to four placental chorionicvilli biopsies were studied with single nucleotide polymorphism (SNP) array. Clinical outcome data were collected.

Results: In 10/10 cases, CPM was proven. In 3/10 cases trisomy/uniparental disomy (UPD)/biparental disomy (BPD) mosaicism was discovered. In 2/3 cases, all three cell lines were present in the placenta, whereas BPD was found in AF. In 1/3 cases trisomy 22/UPD22 was present in AF while trisomy 22/BPD22 mosaicism was found in the placenta. Five of 10 pregnancies were affected with pre‐eclampsia, low birth weight, preterm delivery, and/or congenital malformations.

Conclusion: The presence of trisomy/UPD/BPD mosaicism in 3/10 cases that we investigated proves that trisomic zygote rescue may involve multiple rescue events during early embryogenesis. UPD mosaicism, when present in crucial fetal tissues, may explain the abnormal phenotype in undiagnosed cases.

D. Van Opstal: None. K.E.M. Diderich: None. M. Joosten: None. L.C.P. Govaerts: None. J. Polak: None. M. Boter: None. J.J. Saris: None. W. Cheung: None. S. van Veen: None. R.M. van der Helm: None. A.T.J.I. Go: None. M.F.C.M. Knapen: None. D.N.M. Papatsonis: None. A. Dijkman: None. F.A.T. de Vries: None. R.H. Galjaard: None. L.H. Hoefsloot: None. M.I. Srebniak: None.

P01.53A Non-Invasive Prenatal Testing: Laboratory Clinical Experience: 20000 Clinical Samples

G. Savarese, L. De Falco, P. Savarese, R. Ruggiero, L. D’Amore, T. Suero, L. Circelli, R. D’Angelo, C. Ramiro, I. Pisano, A. Di Carlo, E. Evangelista, G. Furino, M. Bruno, C. Vicedomini, A. Fico

AMES Genetic Lab, Polidiagnostic Instrumental Centre, Casalnuovo di Napoli, Italy

Introduction: Whole-genome sequencing (WGS) of maternal plasma cell-free DNA (cfDNA) can potentially evaluate all 24 chromosomes to identify abnormalities of the placenta, fetus, or pregnant woman. The objective of this study is to give a complete and robust clinical picture of the current performance of NIPT for trisomy 13, 18, and 21 and sex chromosomes aneuploidies as well as for the other chromosomes.

Materials and Methods: All data were generated in our AMES accredited laboratory from January 2017 to January 2019 in 20000 samples. The pipeline included automated library preparation (VeriSeq NIPT Microlab STAR, Illumina) and WGS sequencing on a Next550 (Illumina). VeriSeq NIPT Assay Software (www.illumina.com/NIPTsoftware) was used for data analysis of aneuploidy status of 13, 18, 21, X and Y chromosomes and fetal fraction of cffDNA. An in house algorithm was optimized to analyze other aneuploidies and subchromosomal aberrations.

Results: The main results were showed in Table 1. We also reported 19 rare autosomal trisomies (RATs) and 6 structural abnormalities identified in 10500 samples, mainly trisomy 15 (n = 5), followed by trisomy 8 (n = 4) and trisomies 16 and 22 (n=2).

Conclusions: The performance characteristics were established in samples in which we analysed all 24 chromosomes with a minimum fetal fraction of 4%, and has been confirmed by our extensive clinical experience in the same clinical population.

Table 1: Clinical performance based on clinical Experience

Overall performance n=20000




Sensitivity (%(95% Cl))

Specificity (%(95% Cl))









(99.97, 100.0)








(91.97, 100.0)

(99.93, 100.0)









(99.95, 100.0)

Sex chromosome aneuploidies








(99.88, 99.96)










Multiple gestation n =441




Sensitivity (%(95% Cl))

Specificity (%(95% Cl))









(99.13, 100.0)








(99.13, 100.0)

ART pregnancies n=984




Sensitivity (%(95% Cl))

Specificity (%(95% Cl))









(99.61, 100.0)









(99.61, 100.0)








(99.13, 100.0)

Sex chromosome aneuploidies








(99.10, 99.69)

G. Savarese: None. L. De Falco: None. P. Savarese: None. R. Ruggiero: None. L. DAmore: None. T. Suero: None. L. Circelli: None. R. DAngelo: None. C. Ramiro: None. I. Pisano: None. A. Di Carlo: None. E. Evangelista: None. G. Furino: None. M. Bruno: None. C. Vicedomini: None. A. Fico: None.

P01.54B High total DNA contributes to low fetal fraction in NIPT and tends to resolve on redraw

A. Ryan, S. Krinshpun

Natera, San Carlos, CA, United States

Introduction: Low fetal fraction (FF) is a common cause of test failures in NIPT and is correlated with maternal weight, gestational age, and aneuploidy[1,2]. We show that low FF can also be associated with high DNA concentration (DC) which frequently reverts toward average after redraw.

Materials and Methods: Sample DC was estimated by comparing to a reference sequence added before amplification. 99,936 eligible samples were collected between 2012 and 2014. Additionally, 1,374 received a redraw after test failure and changes in DC and FF between the two draws were observed.

Results: Samples with low DC (below 10th percentile) have average FF 12.4% and samples with high DC (above 90th percentile) have average FF 8.6%. The average FF is 10.1%. Samples with test failure were twice as likely to have high DC. After redraw, cases with high initial DC had 38 times larger median reduction in DC compared to overall. The 10% of cases with greatest reduction in DC had median FF increase 1.2%, compared to FF increase 0.4% for cases without significant change in DC. Change in DC was a significant parameter (p<10-5) in a logistic regression model for redraw success.

Conclusions: Samples with high DC are overrepresented in test failures and have lower average FF. High DC tends to reduce after redraw, producing larger FF increase. This supports the option for repeat NIPT after a failure due to low FF.

[1] Pergament et al. Obstet Gynecol. 2014 Aug;124(2 Pt 1):210-8.

[2] McKanna et al. Ultrasound Obstet Gynecol. 2019 Jan;53(1):73-79.

A. Ryan: A. Employment (full or part-time); Significant; Natera. S. Krinshpun: A. Employment (full or part-time); Significant; Natera.

P01.55C Microarray findings in pregnancies with oligohydramnios - a retrospective cohort study and literature review

S. Sagi1, L. Sagi-Dain2, I. Maya3, S. Ben-Shachar4, A. Singer5

1Bnai Zion Medical Center, Haifa, Israel, 2Carmel Medical Center, Haifa, Israel, 3Rabin Medical Center, Petah Tikva, Israel, 4Souraski Medical Center, Tel Aviv, Israel, 5Ministry of Health, Jerusalem, Israel

Introduction: This study was performed following a statement in national position paper defining the advised management of pregnancies with abnormal amniotic fluid volume. The proposed statement recommended to refer all pregnancies with oligohydramnios to genetic counseling, except for proven rupture of membranes, evidence of placental insufficiency or oligohydramnios diagnosed after 37 weeks of gestational age. Thus, the objective of this study was to explore the risk for abnormal chromosomal microarray analysis (CMA) findings in pregnancies with oligohydramnios.

Methods: Data from all CMA analyses performed due to oligohydramnios between January 2013 and September 2017 were retrospectively obtained from the Ministry of Health database. In addition, a search was conducted through the Pubmed database from inception to February 2018 for English articles exploring the issue.

Results: Fifty CMA analyses were performed due to oligohydramnios. Of these, in 21 tests it constituted an isolated anomaly, 13 cases were associated with anatomic defects, and the remaining 16 pregnancies were diagnosed with intra-uterine growth restriction (IUGR) as well. All CMA tests were normal, except for one pathogenic finding in the IUGR group - a 16p11.2 duplication sized 722Kb. Literature search yielded 394 titles, of which two relevant articles were found. One of these, published at 1998, yielded 31 (20.1%) abnormal karyotypes in 154 pregnancies with oligohydramnios (11 of these as an isolated findings), while another (1995) did not find any aberrations in 28 pregnancies with isolated oligohydramnios.

Discussion: Current evidence does not support invasive prenatal testing in pregnancies with isolated oligohydramnios.

S. Sagi: None. L. Sagi-Dain: None. I. Maya: None. S. Ben-Shachar: None. A. Singer: None.

P01.57A Early experiences with screening for aneuploidy in preimplantation genetic testing for inherited disorders

T. Diemer1, C. L. F. Toft2, C. Hnida3, B. Degn2, H. Okkels2, A. Ernst2, H. J. Ingerslev3, I. S. Pedersen2

1Department of Clinical Genetics, Aalborg University Hospital, Aalborg, Denmark, 2Department of Molecular Diagnostics, Aalborg University Hospital, Aalborg, Denmark, 3Fertility Unit, Aalborg University Hospital, Aalborg, Denmark

Preimplantation genetic testing (PGT) is divided into three categories based on the indication: PGT-M (Monogenic), PGT-SR (Structural rearrangements) and PGT-A (Aneuploidy). A large study of more than 15000 trophectoderm biopsies found the lowest rate of aneuploidy to be 25% in young women increasing with maternal age to more than 90%. Thus at least 1 in 4 embryos are expected to be aneuploid, possibly leading to implantation failure, miscarriage or an abnormal fetus. Hence, selection of euploid embryos should in theory increase pregnancy rates and decrease miscarriage rates, but this is still a subject of intense international discussion. In our center we don’t offer PGT-A, but as our PGT-SR setup is based on Shallow Whole Genome Sequencing, the ploidy status is revealed, and thus used for prioritizing embryos. Our PGT-M setup includes fragment analysis of short tandem repeats, and SNaPshot analysis of the specific mutation when relevant. Since PGT-M setup does not detect aneuploidy, we would expect a higher rate of implantation when performing PGT-SR. Mean maternal age is similar in the two groups (30.96 in the PGT-M and 32.56 in the PGT-SR group). We examined the rate of positive hCG and ongoing pregnancies (OPR) per transfer (SET) on embryos (159 PGT-M and 27 PGT-SR) transferred between 01-01-2017 and 01-12-2018. Surprisingly, we found no significant difference in outcome in the two groups in terms of positive hCG (49.7 vs. 48.1, P = 0.881) or OPR (34.6 vs. 37.0, P = 0.805), but the number of cases is still rather low.

T. Diemer: None. C.L.F. Toft: None. C. Hnida: None. B. Degn: None. H. Okkels: None. A. Ernst: None. H.J. Ingerslev: None. I.S. Pedersen: None.

P01.58B Counseling conundrum: sex discordance identification following preimplantation genetic testing or noninvasive prenatal testing using SNP-based methodologies

K. L. Howard, M. K. Maisenbacher, K. Merrion, S. Leaonard, W. DiNonno

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

Introduction: Preimplantation genetic testing (PGT) and noninvasive prenatal testing (NIPT) can determine fetal sex with high accuracy prior to/during pregnancy. However, additional prenatal screening or clinical presentation may indicate sex discrepancy requiring medical follow-up for an infant’s health.

Methods: Cases of possible sex discrepancy after PGT or NIPT at a single lab were retrospectively reviewed. For PGT, genotyping was performed using Illumina Cyto12 SNP-based microarray with informatics. For NIPT, cell-free DNA was isolated and amplified by massively-multiplexed PCR targeting 13,392 SNPs covering chromosomes 13, 18, 21, X and Y. Only cases with testing to identify a cause for discrepancy were included.

Results: Four of 23,297 (0.02%) PGT and 49 of 1,081,541 (0.005%) NIPT cases had discrepant sex by prenatal screening or postnatal exam. For PGT, child concordance with parental samples and remaining embryos revealed 2 (50%) resulted from incorrect embryo transfers and 2 (50%) resulted from natural conception around the time of embryo transfer. For NIPT, phlebotomy labeling errors comprised 6 (12.2%); confined placental mosaicism, 10 (20.4%); ultrasound errors, 13 (26.6%); and disorders of sexual development (DSD), 20 (40.8%). No discrepancies were due to lab error for PGT/NIPT cases.

Conclusions: Causes of discordant sex after PGT or NIPT can include sample swap, ultrasound errors, PGT/NIPT result errors, natural conception around the time of embryo transfer, vanished twin on non-SNP-based NIPT, embryo mosaicism, confined placental mosaicism and various DSDs. A thorough investigation can provide reassurance and guide appropriate medical management and counseling about cause and recurrence risk.

K.L. Howard: A. Employment (full or part-time); Significant; Full Time Employee, Natera, Inc.. E. Ownership Interest (stock, stock options, patent or other intellectual property); Significant; Stock, Natera, Inc. M.K. Maisenbacher: A. Employment (full or part-time); Significant; I am full time employee, Natera, Inc.. E. Ownership Interest (stock, stock options, patent or other intellectual property); Significant; Stock, Natera, Inc. K. Merrion: A. Employment (full or part-time); Significant; Full Time Employee, Natera, Inc.. E. Ownership Interest (stock, stock options, patent or other intellectual property); Significant; Stock, Natera, Inc. S. Leaonard: A. Employment (full or part-time); Significant; Full Time Employee, Natera, Inc. W. DiNonno: A. Employment (full or part-time); Significant; Full Time Employee, Natera, Inc.. F. Consultant/Advisory Board; Significant; Stock Options, Natera, Inc..

P01.59C Preimplantation genetic testing of monogenic disease: experience in Russia

S. O. Zhikrivetskaya, Y. L. Volkova, E. V. Musatova, Y. V. Sofronova, N. A. Shirokova, E. A. Pomerantseva

Center of Genetics and Reproductive Medicine GENETICO LLC, Moscow, Russian Federation

Introduction: Preimplantation genetic testing of monogenic disease (PGT-M) is an alternative to prenatal testing for couples with high risk of having offspring with single-gene disorder. PGT-M is technically challenging, because of extremely small amount of biomaterial. We report here our experience of PGT-M in “Genetico” center in Russia.

Material and Methods: A retrospective analysis of all requests and cycles of PGT-M referred to our center was performed. Personalized PGT-M assays combine direct diagnosis of the pathogenic variants and linkage analysis of highly heterozygous STRs. PGT-A by NGS or aCGH was performed for unaffected embryos upon patient’s request.

Results: Of 109 couples referred to our center for PGT-M, 92 completed preliminary test for PGT-M for 42 genetic condition: 24 autosomal-dominant, 54 autosomal-recessive, 13 X-linked requests. The most frequent indication was spinal muscular atrophy (17). We performed 85 PGT-M cycles with 413 embryos. The whole genome amplification failed in 16 cases (3,9%). Median number of markers in test-systems was 12 and for embryo analysis it was 10. These highly informative test systems contributed to low number of inconclusive results - only for 7 samples (1,8%). For 156 (58,6%) unaffected embryos PGT-A was performed and 91 (34,1%) were suitable for transfer. At the moment we have information about 43 transfers, 19 pregnancies and 7 healthy births and no affected pregnancy or birth.

Conclusions: Highly informative test system and accurate analysis of results can lead to both - high accuracy of obtained results and decreased number of embryos, that were rejected because of inconclusive results.

S.O. Zhikrivetskaya: None. Y.L. Volkova: None. E.V. Musatova: None. Y.V. Sofronova: None. N.A. Shirokova: None. E.A. Pomerantseva: None.

P01.60D Identifying eQTL influence on gene expression through microRNAs

R. Inno, S. Sõber, M. Laan

Institue of Biomedicine and Translational Meidicine, Tartu, Estonia

Introduction: microRNAs drive coordinated expressional changes of their target genes and trigger functional shift in cells. Placental microRNAs are specifically involved in trophoblast differentiation and function. Single nucleotide variants (SNVs) associated with the expression level of genes are defined as expression quantitative trait loci (eQTLs). The aim of my PhD project is to identify placental eQTLs modulating the expression of microRNAs and to understand their downstream effect on the placental transcriptome.

Materials and Methods: Placental miRSeq (unpubl. data) and genotyping (Kasak et al 2015) datasets were subjected to genetic association testing for eQTL discovery (n = 40), implicated in PLINK v1.07 (Purcell et al 2007). microRNAs and their eQTLs were cross-referenced with newbors growth parameters. Additionally association testing between identified miRNA eQTLs and placental expression levels of predicted target genes were analyzed. Correlations between the expression profile of placental miRNAs (miRSeq dataset) and transcripts (RNA-Seq dataset; Sõber et al 2015) were analyzed using DESeq2 platform (Love et al 2014). eQTL and newborn growth parameter association was validated in combined REPROMETA and Happy Pregnancy cohort (n = 2100)

Results: In total, 11 placental microRNAs were detected that were expressionally modulated by eQTLs. Four of these microRNAs and their eQTLs show assosiation with newborns growth parametes. Several novel target genes and biological pathways were identified for these microRNAs.

Conclusions: miRNA eQTLs may represent additional modulators of the placental transcriptome, placental function and pregnancy course.

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

R. Inno: None. S. Sõber: None. M. Laan: None.

P01.62B Whole exome-based preconception carrier test (PCT) for consanguineous couples: first results from clinical practice

S. C. E. H. Sallevelt1, B. de Koning1, C. E. M. de Die-Smulders1, C. Gilissen2, A. P. A. Stegmann1, H. G. Brunner1,2, A. D. C. Paulussen1

1Maastricht University Medical Centre+, Maastricht, Netherlands, 2Radboud University Nijmegen Medical Centre, Nijmegen, Netherlands

Introduction: consanguineous partners are at increased risk of both being carrier of the same autosomal recessive (AR) disorder, with 25% risk of affected offspring as a consequence. Until recently, no comprehensive preconception carrier test was available to identify the very rare disease-causing mutations these couples may carry. We developed such a test for consanguineous couples and implemented this in our clinical practice.

Materials and Methods: both individuals of a couple undergo whole exome sequencing (WES). First the couple’s sequence data are merged: only variants present in the same gene in both of them and with a population frequency <5%, remain in the dataset. Subsequently, this dataset is filtered against a gene panel, consisting of >1900 genes associated with known AR diseases (OMIM-based). Only combinations of likely pathogenic or pathogenic mutations (class IV or V variants) in both partners are reported. Carriership in only one individual is not detected.

Results: thus far 22 consanguineous couples were included. For 7 (32%), ‘unexpected’ pathogenic variants were reported conferring risk of severely affected offspring, allowing these couples to opt for prenatal or preconception diagnostic choices. Disease examples are: sulfite oxidase deficiency, AR epidermolysis bullosa dystrophica, restrictive dermatopathy, infantile epileptic encephalopathy. None of the disease associations were linked to any known disease in the family, demonstrating the innovative value of this approach.

Conclusions: our WES-based preconception carrier test (PCT) provides a powerful diagnostic tool for identification of serious disease carrier status in consanguineous couples. Outcomes provide significant reproductive choices that no other test currently offers.

S.C.E.H. Sallevelt: None. B. de Koning: None. C.E.M. de Die-Smulders: None. C. Gilissen: None. A.P.A. Stegmann: None. H.G. Brunner: None. A.D.C. Paulussen: None.

P01.63C CarrierTest - the expanded preconception carrier screening

F. Lhota, F. Zembol, L. Dohnalova, Z. Vilimova, M. Bittoova, I. Soldatova, B. Honysova, M. Famfulikova, M. Koudova, D. Stejskal

Gennet, Clinic of genetics and reproductive medicine, Prague, Czech Republic

We have developed a NGS panel which screens (i) 889 mutations causing 68 severe genetic disorders that can affect progeny of healthy individuals (genetic compatibility test), (ii) mutations associated with blood hypercoagulability influencing infertility treatment and pregnancy, (iii) inherited ovarian response to gonadotrophin stimulation. The test is designated for patients undergoing an IVF program and for gamete donors.

CarrierTest is a custom NGS panel with locally assembled bioinformatic pipeline and variant database. To replace MLPA and fragmentation analysis we implemented specific design adjustments and sequencing data analysis allowing detection of large deletions of SMN1, CFTR genes and also microdeletions on chromosome Y (AZF region) within one laboratory test. The report contains comparative analysis of detected variants for evaluation of residual risk and preconception compatibility of couple for consideration of preimplantation (PGT-M) or prenatal diagnostics.

So far 8258 samples were analyzed. These include: 3196 couples before conception, 1388 gamete donors and 478 patients with reproduction impairment without compatibility testing. Frequent occurrence of carriers was observed in the commonly screened genes (SMN1 2,4%, CFTR 3,7%, GJB2 6,2%) but also in other genes previously not tested (e.g. ABCA4 4,2%, DHCR7 2,7%, SERPINA1 2,7%, PAH 2,5%, ACADM 1,5%, ATP7B 1,2%, AR 1,1%). We identified 79 pairs (2,4%) with a reproduction risk, which is twofold increase detection rate in comparison with only CFTR/SMN1/GJB2 testing.

CarrierTest facilitates informed decision about reproduction. Screening of couples and gamete donors allows to elucidate higher number of carriers of severe disorders in order to offer appropriate preconception and prenatal care.

F. Lhota: A. Employment (full or part-time); Significant; Gennet. F. Zembol: A. Employment (full or part-time); Significant; Gennet. L. Dohnalova: A. Employment (full or part-time); Significant; Gennet. Z. Vilimova: A. Employment (full or part-time); Significant; Gennet. M. Bittoova: A. Employment (full or part-time); Significant; Gennet. I. Soldatova: A. Employment (full or part-time); Significant; Gennet. B. Honysova: A. Employment (full or part-time); Significant; Gennet. M. Famfulikova: A. Employment (full or part-time); Significant; Gennet. M. Koudova: A. Employment (full or part-time); Significant; Gennet. D. Stejskal: A. Employment (full or part-time); Significant; Gennet.

P01.64D The prevalence of genetic and anatomic fetal defects: data from Happy Pregnancy Study

K. Rull1,2,3, E. Hanson1,2, M. Laan3

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

Introduction: The study aimed to assess the prevalence of genetic and anatomic abnormalities among the participants of the prospective Happy Pregnancy study (“Development of novel non-invasive biomarkers for fertility and healthy pregnancy").

Methods: The pregnancy course and outcome including the first trimester serum test and ultrasound scans at 11-14 and 19-21 weeks were documented in the cohort of 2320 women visiting the Women’s Clinic of Tartu University Hospital, Estonia in 2013-2015. Targeted FISH, karyotyping and SNP-based array was applied in cases of abnormal findings after 12 gestational weeks.

Results: In 63 cases (2.7%), the pregnancy resulted with early (<12 weeks) /late (>12 weeks) loss (n=42 /8) or termination of pregnancy (TOP) before 22 weeks (n=13). The genetic disorders were detected in 16 cases (0.7%), the anatomic defects occurred in 53 cases (2.3%). Serious anatomic defects, resulted with TOP, fetal death or postnatal surgical intervention during the first year after birth occurred in 14 cases (0.6%); anomalies two were not antenatally diagnosed: univentricular heart and cleft lip.


Antenatal diagnosis

Diagnosis after birth


9 (7)*

7 (1 trisomy 18)




Cleft lip/palate

1 (1)*


Cardiac mild/severe

1/4 (1)*






4 (1)*



4 (1)*






37 (11)*


*termination of pregnancy

Summary: The overall prevalence of the serious genetic and/or anatomic among unselected pregnant women in Happy Pregnancy cohort was 0.97%.

Funding: The European Regional Development Fund (3.2.0701.12-0047), Estonian Research Council (grants IUT34-12).

K. Rull: None. E. Hanson: None. M. Laan: None.

P01.65A Selecting fetuses in ambiguous setting: Preimplantation Genetic Diagnosis (PGD) for variants of unknown significant (VUS)

S. Zuckerman1, K. Rotshenker Olshinka2, O. Weiss1, N. Srebnik2, S. Shaviv1, O. Freireich1, R. Segel1, T. Eldar Geva2, G. Altarescu1

1Medical Genetics Institute, Shaare Zedek Medical Center,, Jerusalem, Israel, 2IVF Unit, Division of Obstetrics and Gynecology, Shaare Zedek Medical Center, Jerusalem, Israel

The introduction of new technologies of chromosomal microarrays (CMA) and whole exome sequencing is leading to wider awareness of people to their genetic background and influence on offspring. The decision to perform PGD to select an embryo with no inherited VUS, considering the inherent ambiguity of VUS which is not unequivocally linked to a disorder, may give false hope and lead to frustration if an affected child is born. In order to evaluate Israeli VUS carriers’ intentions regarding PGD for VUS findings, we have analyzed database from a large PGD center. Twenty three couples required PGD for VUS detected, in at least one spouse, by CMA analysis. Five couples had two VUS detected. Eight couples (35%) had previous pregnancy termination (TOP) solely due to the detection of VUS in an embryo. Thirteen couples (57%) had no other indication for PGD. The interpretation of VUS in the time of counseling was ‘likely-pathogenic’ in 10 cases (36%), ‘VUS’ in 9 cases (32%) and ‘likely-benign’ in 9 cases (32%). PGD was performed in 14 couples (61%): 9/10 likely-pathogenic, 5/9 VUS and 1/9 likely-benign VUS. Although most couples performed PGD for a likely-pathogenic VUS, some couples regard likely-benign VUS as an indication for TOP and PGD. Future demand for such controversial PGD applications will be growing and challenge the medical milieu also considering continuous re-interpretation of VUS. Setting guidelines for VUS’ interpretations, proper counselling regarding VUS’ meanings and defining the justified applications of PGD uses are crucial steps in practicing PGD for VUS findings.

S. Zuckerman: None. K. Rotshenker Olshinka: None. O. Weiss: None. N. Srebnik: None. S. Shaviv: None. O. Freireich: None. R. Segel: None. T. Eldar Geva: None. G. Altarescu: None.

P01.67C Preimplantation genetic test in couples with history of infertility and pregnancy loss

S. Y. Yaneva Staykova1, G. Stamenov2, R. Staneva1,2, M. Pancheva2, M. Serafimova2, K. Nikolova2, O. Antonova1, D. Toncheva1, S. Hadjidekova1,2

1Medical University of Sofia, Medical Faculty, Department of Medical Genetics, Sofia, Bulgaria, 2Nadezhda Hospital, Sofia, Bulgaria

Introduction: Preimplantation genetic test (PGT) is a cutting-edge technology of early genetic disease detection in embryos prior to their implantation in the uterus. The purpose of our study is to show the value of PGT in couples with infertility; its ability to reduce the risk of birth of chromosomally unbalanced offspring and pregnancy loss; to increase the chance of successful pregnancy and birth of an unaffected child.

Materials and Methods: We describe 185 couples with a history of infertility and pregnancy loss, who after extensive genetic counseling opted for in vitro fertilization procedure with PGT. Trophectoderm biopsy was carried out on 497 blastocyst stage embryos originating from 231 oocyte retrieval cycles. DNA was extracted and subjected to whole genome amplification. Array-based comparative genomic hybridization with microarray platforms 24sure v3/24sure+ and next-generation sequencing with VeriSeqPGS LibraryPrep protocol were performed. Results were analyzed by BlueFuse Multi version 4.3 software.

Results: 196 embryos had a balanced profile (39.44%), 292 embryos showed an unbalanced profile (58.75%) and 9 embryos could not be interpreted (1.81%). Embryo transfer was conducted in 109 cases (58.92%) and chemical pregnancy was detected in 33.03% of the women. Pregnancy loss occurred in 3.67% of cases. Live birth rate was 29.36%.

Conclusions: PGT reduces the number of failed transfers and eliminates the trauma of terminating desired pregnancy and possible medical complications. In couples with reproductive failures, PGT can be recommended in order to considerably increase the chance of conceiving with a chromosomally balanced embryo and live birth of a healthy offspring.

S.Y. Yaneva Staykova: None. G. Stamenov: None. R. Staneva: None. M. Pancheva: None. M. Serafimova: None. K. Nikolova: None. O. Antonova: None. D. Toncheva: None. S. Hadjidekova: None.

P01.68D Rapid whole exome sequencing to identify the underlying genetic cause in fetuses with sonographic anomalies

I. Feenstra1, A. C. Deden2, M. I. Nelen1, K. Neveling1, S. Castelein3, C. F. Gilissen3, R. P. Pfundt4, M. W. Elting5, T. K. Rinne4, K. E. Diderich6, S. C. Sallevelt7, N. Corsten-Janssen8, K. D. Lichtenbelt9, T. Gardeitchik4, L. Vissers4, H. G. Yntema4, W. A. van Zelst-Stams1

1Department of Human Genetics, Radboud University Medical Center, Radboud Institute for Health Sciences, Nijmegen, Netherlands, 2Department of Human Genetics, Radboud University Medical Center, Nijmegen, Netherlands, 3Department of Human Genetics, Radboud University Medical Center, Radboud Institute for Molecular Life Sciences, Nijmegen, Netherlands, 4Department of Human Genetics, Donders Institute for Brain, Cognition, and Behaviour, Radboud University Medical Center, Nijmegen, Netherlands, 5Department of Genetics, Amsterdam Medical University, Amsterdam, Netherlands, 6Department of Genetics, Erasmus University Medical Centre, Rotterdam, Netherlands, 7Department of Clinical Genetics, Maastricht University Medical Center, Maastricht, Netherlands, 8Department of Genetics, University Medical Centre Groningen, Groningen, Netherlands, 9Department of Genetics, University Medical Centre Utrecht, Utrecht, Netherlands

Rapid whole exome sequencing (rWES) in critically ill newborns with a presumed genetic disorder has shown to increase diagnostic yield earlier in life, which leads to improved patient care. Due to this impact, rWES has become a routine genetic test in this group of patients. We hypothesize that rWES could have the same effect even before birth. Therefore we studied the use of rWES in fetuses with a wide range of sonographic anomalies representing the clinical practice.

We performed a retrospective analysis of the first 54 cases referred to our laboratory for prenatal rWES following the detection of fetal sonographic anomalies. Parental and fetal DNAs were sequenced simultaneously in 53 of the cases and as singleton analysis in one case due to a deceased parent. The most common identified sonographic anomalies were (multiple) congenital anomalies, skeletal dysplasia and intracerebral structural anomalies.

We were able to identify a causative pathogenic molecular variant in 12 of the 54 cases (22%) and a likely causative pathogenic molecular variant in another five cases (9%), bringing the molecular diagnostic yield in this cohort to 31%. Pathogenic and likely pathogenic variants were detected in fetuses with skeletal dysplasias (n=11, 65%), multiple congenital anomalies (n=4, 23%) or intracerebral structural anomalies (n=2, 12%).

These results suggest that rWES is likely to improve prenatal diagnosis of fetuses with ultrasonic abnormalities.

I. Feenstra: None. A.C. Deden: None. M.I. Nelen: None. K. Neveling: None. S. Castelein: None. C.F. Gilissen: None. R.P. Pfundt: None. M.W. Elting: None. T.K. Rinne: None. K.E. Diderich: None. S.C. Sallevelt: None. N. Corsten-Janssen: None. K.D. Lichtenbelt: None. T. Gardeitchik: None. L. Vissers: None. H.G. Yntema: None. W.A. van Zelst-Stams: None.

P01.69A Half decade experience: karyotyping, aCGH or NIPT - changes in prenatal testing strategy

E. Kuznetsova, V. Gnetetskaya, M. Ermakova, M. Kurtser, Y. Tarasova

Mother and Child, Moscow, Russian Federation

Over 5 years the strategy of genetic counseling significantly has changed after introduction of new prenatal diagnostic methods of detection chromosomal abnormalities. In 2013 our laboratory had possibility to use cell-based FISH and karyotyping for pregnancies at risk (maternal serum screening, advanced age, ultrasound examination (US), family history, patient’s anxiety). Since 2014 patients in first trimester with NT>=2,5 mm or/and US abnormalities were recommended chorionic villus sampling followed by karyotyping and DNA extraction which allowed to perform aCGH (array-based comparative genomic hybridization) in case of normal fetus karyotype. In second trimester after the amniocentesis mostly aCGH/FISH analysis were recommended due to time limit with compare to cell cultivation of amniotic fluid for standard karyotyping. Chromosome imbalances by aCGH let us reveal about 20 families where one of the parents had a balanced rearrangement invisible by standard karyotyping. For these aims subtelomeric FISH analysis have been performed. When a noninvasive prenatal testing (NIPT) appeared in 2016 the whole amount of invasive procedures decreased up to 50%. NIPT should be recommended to women without US abnormalities and family history (Table). Otherwise only invasive prenatal testing is acceptable due to limits of NIPT. Since 2013 more than 12000 prenatal analyses were done. aCGH increased the detection of pathogenic chromosomal aberrations on 12%. Prenatal counseling is the most important tool for clarifying the appropriate strategy in each case.

Table. Comparison of NIPT results.

NIPT test



False positive

False negative

Refused invasive procedure

Low risks

High risk

Panorama (Natera, since 2016)




58 (38%)

1 (0,6%)


Harmony (Ariosa Diagnostics, since 2016)




14 (20%)



VERASITY (NIPD genetics, since 2017)



31 (100%)











E. Kuznetsova: None. V. Gnetetskaya: None. M. Ermakova: None. M. Kurtser: None. Y. Tarasova: None.

P01.70B Correlation between reason to apply a prenatal diagnosis for aneuploidy and the results of test - A retrospective study on 2,881 foetus investigated in last 15 years by FISH method in “Cuza Voda” Maternity, Iasi, Romania

V. Martiniuc1, M. Gramescu2, S. Popa2, R. Popescu2, A. Paduret1, I. Resmerita2, L. Caba2, L. Butnariu2, M. Panzaru2, C. Rusu2, E. GORDUZA1,2

1"Cuza Voda" Obstetrics and Gynecology Hospital, Iasi, Romania, 2"Grigore T. Popa" University of Medicine and Pharmacy, Iasi, Romania

We analysed retrospectively 2,881 cases investigated by prenatal diagnosis, using FISH method in the last 15 years in Iasi, Romania. We used probes for chromosomes 13, 18, 21, X and Y and we found 215 foetus with aneuploidy (7.46%). In 642 cases with abnormal double test we identified 17 chromosomal anomalies [2.64%] (12 cases with trisomy 21 and 1 case with trisomy 18, trisomy 13, trisomy XXY and trisomy XYY). In 577 cases with abnormal triple test we identified 8 chromosomal anomalies [1.38%] (4 cases with trisomy 21 and 4 cases with trisomy 18). In 42 cases with abnormal double and triple test we did not identified chromosomal anomalies. In 759 cases with congenital anomalies identified by ultrasonography we found 162 chromosomal anomalies [21.34%] (78 cases with trisomy 21, 52 cases with trisomy 18, 13 cases with trisomy 13, 10 cases with monosomy X and 9 cases with triploidy). In 732 cases with advanced maternal age (> 35 years) we identified 16 chromosomal anomalies [2.18%] (14 cases with trisomy 21 and 1 case with trisomy 13, and triploidy). We applied FISH test in 11 cases with positive NIPD and we confirmed 9 cases with trisomy 21 and one case with monosomy X. In 118 cases we made prenatal diagnosis for other reason (usually for chromosomal pathology in antecedents) and we found only two cases with trisomy 21 [1.69%]. In conclusion, prenatal diagnosis of chromosomal disorders is very useful in cases with congenital anomalies and positive NIPD.

V. Martiniuc: None. M. Gramescu: None. S. Popa: None. R. Popescu: None. A. Paduret: None. I. Resmerita: None. L. Caba: None. L. Butnariu: None. M. Panzaru: None. C. Rusu: None. E. Gorduza: None.

P01.71C Applicational of the array comparative genomic hybridization (aCGH) in the prenatal diagnostics of fetuses with increased risk of aneuploidy

M. Chojnacka1, K. Sobecka1, M. Bartnik-Głaska1, M. Smyk1, I. Plaskota1, B. Wiśniowiecka-Kowalnik1, M. Kędzior1, J. Bernaciak1, K. Jakubów-Durska1, E. Obersztyn1, T. Roszkowski2, A. Kucińska-Chahwan2, P. Kretowicz3, B. Nowakowska1

1Institute of Mother and Child, Warsaw, Poland, 2Clinic of Obstetrics and Gynecology, Professor Witold Orłowski Independent Public Clinical Hospital - Postgraduate Medical Education Centre, Warsaw, Poland, 3II Department of Obstetrics and Gynecology, Medical Center of Postgraduate Education (CMKP), Warsaw, Poland

The occurrence of aneuploidy is estimated at 5 - 11 % of all fetuses. The most common are trisomies of chromosomes 13, 18, 21 and monosomy of the chromosome X. Noninvasive prenatal screening tests allow the risk calculation of the aneuploidies based on the patients age (above 35), biochemical markers (free β-hCG) and ultrasonographic markers (nuchal translucency parameter). The results suggesting a high risk of aneuploidy give strong indications for further invasive diagnostics.

The aim of this project was to evaluate the frequency of chromosome aberrations other than aneuploidies in the group of 204 patients with high risk of trisomy based on the screening tests, where the ultrasound test did not show any abnormalities. To perform the array diagnostic evaluation DNA from uncultured amniotic fluid, trophoblast or cultured amniocytes was used.

Aneuploidy was identified in 34/204 fetuses (17%). Trisomy 21 was the most frequent abnormality (66%). The trisomy 13 and 18 were detected in 3% and 11%, respectively, followed by X monosomy (11%), X mosaic monosomy (3%) and sex chromosomes disomy (11%). In 10/204 cases (5%) structural aberrations of 115 kb to 41 Mb were identified. Two of them were classified as potentially pathogenic and 8 as pathogenic, where 4 were localized in the regions of known microdeletion and microduplication syndromes.

Therefore, we postulate that CGH is the most reliable and the fastest method for identification of genomic imbalances, even in the cohort of patients with normal results of ultrasound examination.

M. Chojnacka: None. K. Sobecka: None. M. Bartnik-Głaska: None. M. Smyk: None. I. Plaskota: None. B. Wiśniowiecka-Kowalnik: None. M. Kędzior: None. J. Bernaciak: None. K. Jakubów-Durska: None. E. Obersztyn: None. T. Roszkowski: None. A. Kucińska-Chahwan: None. P. Kretowicz: None. B. Nowakowska: None.

P01.72D Prenatal whole exome sequencing detects a novel Fukutin (FKTN) mutation in a fetus with a suspected recurrence of Dandy-Walker malformation

A. Traversa1, E. Marchionni2, A. Giovannetti2, M. Genovesi2, S. Bernardo2, D. Guadagnolo2, N. Panzironi2, G. Napoli2, B. Torres3, A. Paiardini4, L. Bernardini3, T. Mazza5, M. Carella1, V. Caputo2, A. Pizzuti2,1

1Laboratory of Medical Genetics, Fondazione IRCCS Casa Sollievo della Sofferenza, San Giovanni Rotondo (FG), Italy, 2Department of Experimental Medicine, Sapienza University of Rome, Rome, Italy, 3Laboratory of Cytogenetics, Fondazione IRCCS Casa Sollievo della Sofferenza, San Giovanni Rotondo (FG), Italy, 4Department of Biochemical Sciences "A. Rossi Fanelli", Sapienza University of Rome, Rome, Italy, 5Laboratory of Bioinformatics, Fondazione IRCCS Casa Sollievo della Sofferenza, San Giovanni Rotondo (FG), Italy

Introduction: Posterior fossa malformations are among the most frequent Central Nervous System anomalies prenatally detected. We report on a 17 weeks male fetus with an ultrasonographic suspicion of Dandy-Walker Malformation (DWM) recurrence. The previous pregnancy of the couple had been interrupted at 22 gestational weeks following a diagnosis of DWM. We identified the molecular cause using Next Generation Sequencing in prenatal diagnosis.

Materials and Methods: Whole exome sequencing (WES) has been performed on fetal genomic DNA. After reads pre-processing, mapping, variant calling and annotation, a filtering strategy based on allelic frequency, recessive inheritance and phenotypic ontologies has been applied. A fetal MRI at 18 gestational weeks has been performed. In silico analysis of a potential causative variant in the Fukutin protein has been carried out through a structural modeling approach.

Results: We identified a novel homozygous missense mutation (NM_006731.2, c.898G>A, p.Gly300Arg) in Fukutin gene (FKTN), which is associated to Muscular Dystrophy-Dystroglycanopathies. Fetal MRI supported molecular findings. Structural modeling analyses indicated a potential pathogenetic mechanism of the variant, through a reduced activation of the sugar moieties, which in turn impairs transfer to α-dystroglycan and thus its glycosylation. These findings pointed to a redefinition of the ultrasonographic suspicion of DWM recurrence to a Muscular Dystrophy-Dystroglycanopathy type A.

Conclusions: The present case confirmed WES as a reliable tool for prenatal identification of molecular bases of Central Nervous System phenotypes. Moreover, it highlights the importance of considering Muscular Dystrophy-Dystroglycanopathies when a posterior fossa anomaly is early prenatally described.

Grant: Italian Ministry of Health, RC1801GC11

A. Traversa: None. E. Marchionni: None. A. Giovannetti: None. M. Genovesi: None. S. Bernardo: None. D. Guadagnolo: None. N. Panzironi: None. G. Napoli: None. B. Torres: None. A. Paiardini: None. L. Bernardini: None. T. Mazza: None. M. Carella: None. V. Caputo: None. A. Pizzuti: None.

P01.73A The advantage of comparative genomic hybridization (aCGH) over quantitative fluorescence PCR (QF-PCR) in genetic testing of chorions after miscarriage

K. Matuszewska1,2, M. Piechota1, B. Wieckowska3, K. Milanowska1, N. Kochalska1, A. Stachowiak1, K. Lacna1, P. Puacz4, T. Olejniczak5, A. Latos-Bielenska1,2

1Centers for Medical Genetics GENESIS, Poznan, Poland, 2Department of Medical Genetics, Poznan University of Medical Sciences, Poland, Poznan, Poland, 3Department of Medical Statistics and Information Technologies, Poznan University of Medical Sciences, Poland, Poznan, Poland, 4Department of Mother and Child Health, Poznan University of Medical Sciences, Poland, Poznan, Poland, 5Division of Perinatology and Women’s Diseases, Poznan University of Medical Sciences, Poland, Poznan, Poland

At least 60% of spontaneous abortions occur due to genetic disease (most often chromosomal aberration) in the embryo and fetus. Genetic testing of miscarriage material is very important in order to determine the etiology of miscarriage and to identify couples at risk.

In our laboratory two methods QF-PCR (for chromosomes: 13, 15, 16, 18, 21, 22, X, Y) - 827 chorions and aCGH (with resolution 12x135k or 8x60k) - 417 chorions were used.

Chromosomal aberrations (numerical or structural) were found in 56,5% of chorions analyzed by aCGH and in 52,5% tested by QF-PCR. Among chromosome aberrations most frequent were trisomies (65,7%; most frequent trisomy 16; trisomy 14 with the same frequency as trisomy 18), 45,X (14%) and triploidy (13,1%). The higher number of miscarriages identified in an individual case, the smaller percentage of abnormal results were observed.

Structural chromosomal aberrations including CNVs were identified in 13,6% of noneuploid miscarriages examined by aCGH. Some CNVs were repeated and contained genes that could be candidate genes critical for maintaining healthy pregnancy. The most common micro-aberration (6 noneuploid chorions analyzed by aCGH) concerned the locus 3p21.31, and involved the DAG1 gene. The DAG1 is expressed in the placenta during pregnancy. Literature data correlate deficiency of alpha dystroglycan encoded by DAG1 gene with embryonic lethality.

Introduction of aCGH to the diagnostics of miscarriages, enabled the dynamic development of research on genetic causes of recurrent pregnancy losses including other than chromosomal aberrations.

K. Matuszewska: None. M. Piechota: None. B. Wieckowska: None. K. Milanowska: None. N. Kochalska: None. A. Stachowiak: None. K. Lacna: None. P. Puacz: None. T. Olejniczak: None. A. Latos-Bielenska: None.

P01.74B Two years’ experience with molecular diagnostics of RASopathies in prenatal cases

E. Svobodová1, M. Matyášová1, J. Kadlecová1, D. N. Grochová1, P. Vlašín2

1Cytogenetická laboratoř Brno, s.r.o., Brno, Czech Republic, 2Centrum prenatální diagnostiky, s.r.o., Brno, Czech Republic

Introduction: RASopathies are a group of diseases with similar symptoms caused by disruption of RAS/MAPK pathway function. The most abundant diseases are Noonan syndrome and neurofibromatosis type 1.

Materials and Methods: Resequencing of 20 selected genes was performed by massive parallel sequencing approach on samples with normal karyotype originated from amniotic fluid or chorionic villi tissue.

Results: Samples were indicated on the basis of ultrasound findings. From a total of 104 prenatal samples causative variant was found in 10 of them (detection rate 9,6 %). All those positive samples had severe ultrasound finding – increased nuchal translucency, hygroma coli, cardiac abnormalities, excess amniotic fluid, etc. Most frequently causative variants were found in PTPN11 gene (4/10). Surprisingly, in two cases (2/10) we have detected variant in RIT1, one of the recently reported gene for Noonan syndrome. Especially in prenatal diagnostic variants of uncertain significance (VOUS) pose a significant problem. There is not enough information to sort them out into classificatory categories, so they complicate consecutive genetic consultation. In our group VOUS was identified in 14 samples (14/104; 13,5 %). However, segregation analysis of the variants in family members helped to clarify their significance in eight of them.

Conclusions: The aim of this study was to evaluate clinical benefits of massive parallel sequencing in prenatal diagnostics of cases suspected for RASopathy (i.e. detection rate) and compare them to the fraction of detected VOUS. In general, our results establish this approach as effective but with non-negligible number of uncertain findings.

E. Svobodová: None. M. Matyášová: None. J. Kadlecová: None. D.N. Grochová: None. P. Vlašín: None.

P01.75C 1 out of 4 healthy individuals are carriers of a severe recessive or X-linked disease after using a panel of 15 genes

M. Sánchez-Soler1,2, A. Urbano1,2,3,4, E. Montoya1,2,3,4, I. Ochando1,2,3,4, R. Vázquez1,2, J. Rueda1,2,3,4

1Unidad de Genética, Hospital HLA Vistahermosa, Alicante, Spain, 2Cátedra de Biomedicina Reproductiva Vistahermosa, UMH, Alicante, Spain, 3Departamento Histología, Facultad de Medicina, UMH, Alicante, Spain, 4E-GENETICARE, UMH, Parque Científico, Elche, Spain

Preconceptional tests use Next Generation Sequencing (NGS) to identify carriers of recessive and X-linked diseases, with the aim of reducing the risk of offspring born with these alterations. The goal of the present study is to show the results of the implementation of a reduced carrier screening test analyzed by NGS in a population of 1097 healthy individuals who attend a reproduction center. This test includes 15 genes associated with the 16 most prevalent autosomal recessive and X‐linked diseases in Caucasian population, following the current recommendations of scientific societies (high prevalence, severe, early onset and with clear genotype-phenotype relation diseases). Among the total of individuals, 809 (73.7%) are women and 288 (26.3%) are men. 25.4% are carriers of at least 1 pathogenic or probably pathogenic alteration (22.5% have a single mutation and 2.9% have two). Diseases with highest probability of being a carrier are: cystic fibrosis and congenital bilateral aplasia of vas deferens (1/13), GJB2-related DFNB 1 non-syndromic hearing loss and deafness (1/16), familial Mediterranean fever (1/42), spinal muscular atrophy (1/50) and phenylketonuria (1/52). Out of the 809 women, 2.1% are carriers of a premutation (45-200 CGG repeats) in the FMR1 gene, causative of X-fragile syndrome. In conclusion, 1 out 4 patients without other diseases who attend a reproduction center is a carrier of a pathogenic alteration in one of the screened genes. This test identifies the most frequent alterations that are at risk of being transmitted to the offspring to promote the autonomy of patients to make reproductive decisions.

M. Sánchez-Soler: None. A. Urbano: None. E. Montoya: None. I. Ochando: None. R. Vázquez: None. J. Rueda: None.

P01.77A Genomic analyses in cases with reduced fertility and recurrent fetal anomalies

K. Belemezova1,2,3, M. Rizov1, R. Kaneva4, V. Peicheva4, A. Kanev5, M. Hristova-Savova1, T. Milachich1, A. Shterev1, I. Dimova1,4

1Medical complex „Dr Shterev“, Sofia, Bulgaria, 2University Hospital “St. Ivan Rilski”, Department of Clinical Laboratory and Clinical Immunology, Medical University of Sofia, Sofia, Bulgaria, 3Tissue Bank BulGen, Sofia, Bulgaria, 4Center of Molecular medicine, Medical University of Sofia, Sofia, Bulgaria, 5Military Medical Academy, Sofia, Bulgaria

Objective: Genomic aberrations are usually associated with reduced fertility, recurrent abortions or fetal anomalies. Establishment of the exact genetic diagnosis in such cases has a great impact on the determination of the reproductive risk and making a decision for reproductive options.

Design: In our study we have collected patient samples in the following clinical cases of disturbed reproduction: infertility or repeated abortions, structural fetal anomalies and intrauterine death.

Materials and Methods: We performed cytogenetic analysis on lymphocytes from couples in all the listed above cases (1425 samples) and array CGH analysis of abortion samples in cases of disturbed fetal development (40 samples).

Results: Chromosomal alterations were established by cytogenetic analysis in 93 patients (6.5%), distributed as follows - 19 chromosomal translocations (1.3%), 7 numerical X/Y aberrations (0.5%), 10 cases of X/Y mosaicism (0.7%), 2 cases of mosaic marker (0.1%), 25 cases of chr9 inversion (1.75%), 20 cases of acrocentric satellite polymorphisms (1.4%), 5 cases of pericentric heteromorphism 1qh+ and 9qh+ (0.4%), 5 structural Y-chromosome aberrations (0.4%), 2 deletions of Xqter and 1 ring21 chromosome. In cases of fetal anomalies array CGH revealed 17р13.3 microdeletion (Miller-Dieker syndrome), 1p36 microdeletion, 22q11.21 microdeletion (Di George syndrome), 22q11.1 microduplication (Cat-eye syndrome), 10q26.3 microdeletion (2 cases), 22q11.21 microduplication (2 cases), 15q11.2 microdeletion (3 cases), 17p11.2 microdeletion (Smith-Magenis syndrome).

Conclusions: In 6.5% of couples with reproductive failure chromosomal alterations were detected. There is a high probability (about 30%) to reveal microstructural genomic aberrations by array CGH in cases of recurrent fetal anomalies.

K. Belemezova: None. M. Rizov: None. R. Kaneva: None. V. Peicheva: None. A. Kanev: None. M. Hristova-Savova: None. T. Milachich: None. A. Shterev: None. I. Dimova: None.

P01.79C Using Preimplantation Genetic Diagnosis (PGD) for retinoblastoma survivors: cost-effectiveness and quality of life improvements

M. Zeppel1, D. Schofield1, S. Staffieri2, R. Shrestha1, D. Jelovic3, R. Jamieson4

1GenIMPACT, North Ryde, Australia, 2Dept of Ophthalmology, Royal Children’s Hospital, Parkville, Australia, 3Eye Genetics Research Unit, Children’s Hospital at Westmead, Children’s Medical Research Institute, Save Sight Institute, University of Sydney, Sydney, NSW, Australia, 4Eye Genetics Research Unit, Children’s Hospital at Westmead, Children’s Medical Research Institute, Save Sight Institute, University of Sydney, Sydney, Australia

Background: Retinoblastoma (Rb) is a paediatric cancer, leading to loss of vision, eye(s) or life. Approximately 40% of patients have a heritable form of the disease, caused by a mutation in RB1. Offspring of these individuals are at a 50% risk of inheriting the disease. Aggressive and invasive treatments in the first five years of life under general anaesthetic, followed by regular monitoring until age 18, impact the patient, family, hospital resources. Preimplantation genetic diagnosis (PGD) offers alternative reproductive choices for individuals with a heritable mutation. Costs of genomic sequencing are rapidly declining, with increasing availability of reproductive technologies.

Methods and Materials: We undertook a cost-effectiveness study of access to PGD for survivors with heritable retinoblastoma. We modelled the cost of reproductive technology, number of affected/unaffected births, and quality of life gains, for parental uptake rates of PGD from 0-100%. We included the costs of hospital visits from 0-18 years, and costs of IVF and PGD using three cycles (1 fresh, 2 frozen).

Results: In a cohort of retinoblastoma survivors, using in-vitro fertilization (IVF) and PGD always led to cost-savings, and quality of life improvements, even at low uptake rates. Cost-savings were $156,538 and 20.58 Quality Adjusted Life years were gained for ten couples with 50% using PGD compared to the natural pregnancy pathway.

Conclusions: IVF and PGD were always less expensive, with higher quality-of-life compared with taking the natural pregnancy approach. Affordable and accessible PGD will lead to savings for families and health systems for families with heritable retinoblastoma.

M. Zeppel: None. D. Schofield: None. S. Staffieri: None. R. Shrestha: None. D. Jelovic: None. R. Jamieson: None.

P01.80D Celiac disease predisposition and Recurrent pregnancy loss: HLA- genotyping

K. Sosnina1, O. Terpyliak1, D. Zastavna1,2

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

In recent years, has been discussed a relationship between RPL and celiac disease (CD), one of the most common intestinal disorders in the small intestine affecting up to 1% of individuals in Western populations. It is known that susceptibility to CD is linked to certain human leukocyte antigen (HLA) class II alleles, especially in the HLA-DQ region. In this context, the aim of this study was to investigate the prevalence of the CD-predisposing alleles in women with RPL.

Results: The presence of appropriate HLA alleles in 32 women with RPL and 33 women of control group was determined. High CD predisposition was considered in individuals with HLA alleles DQA1*0501, DQB1*0201-0203 (encoding HLA-DQ2) and DQB1*0302 (encoding HLA-DQ8), the presence of only the alleles DQA1*0501, DQB1*0201-0203 was considered as medium-high predisposition CD and a low risk CD are related allele DQB1*0302. A significantly higher frequency HLA-DQ2/DQ8 haplotype in women with RPL compared to control was established (21.88% vs. 3.03%, p<0.025). The genotype HLA-DQ2 is established with a significantly higher frequency in women with RPL compared to control (34.8% vs. 6.06%, p <0.005). The frequency of the HLA-DQ8 genotype was almost the same in the study groups (18.75% and 12.12%, p>0.05), and the absence of such CD-predisposing alleles was significantly lower in women with RPL compared to control (25% and 78.79%, p <0.0001).

Conclusions: The study of a possible correlation between HLA-DQ2/DQ8 haplotype and RPL might suggest new diagnostic and therapeutical approaches for RPL women.

K. Sosnina: None. O. Terpyliak: None. D. Zastavna: None.

P01.81A Expanding our non-invasive prenatal diagnosis (NIPD) service using droplet digital PCR

J. Shaw, S. Sheppard, N. Chandler, H. Ahlfors, L. Chitty

North East Thames Regional Genetics Service, London, United Kingdom

Introduction: NIPD where the mother carries a mutation is hindered by the maternal mutant background in cell-free DNA (cfDNA). Relative haplotype dosage analysis is used in clinical laboratories for NIPD of recessive and X-linked disorders, however this technique requires DNA from both parents and a proband. Droplet digital PCR (ddPCR) offers high sensitivity for quantification of cfDNA and has potential for use in carrier mothers via relative mutation dosage using only a maternal sample. We have applied this technology to sickle cell disease and 8 X-linked disorders to determine clinical utility.

Methods: ddPCR assays were designed and validated using gDNA for the pathogenic variant. Testing was performed on cfDNA from maternal plasma samples for 22 sickle cell risk pregnancies and 10 X-linked mutations. Fetal fraction was determined using three different methods. Fetal genotype predictions were generated using sequential probability ratio test, with results confirmed by invasive sampling.

Results: For sickle cell disease, correct predictions of fetal HBB genotype were generated for 11 samples, with 2 incorrect predictions and 9 inconclusive results. Incorrect and inconclusive predictions resulted from low fetal fraction and threshold positioning during analysis. For X-linked conditions, assay design has been successful and development of the analysis pipeline initiated.

Conclusions: ddPCR has the potential to be used for NIPD for maternal mutation carriers using only a maternal sample. We will report testing of over 50 additional ongoing cases to refine and validate the analysis pipeline to further extend the scope of our NIPD clinical service.

J. Shaw: None. S. Sheppard: None. N. Chandler: None. H. Ahlfors: None. L. Chitty: None.

P01.82B Comprehensive chromosome screening of human first polar bodies and oocytes using four different whole genome amplification methods and single-cell next-generation sequencing

A. Sarosiak1,2, I. Minota1, K. Koziol3, 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, 3Novum Fertility Clinic, Warsaw, Poland

Background: Aneuploidy is the most frequent type of chromosome aberration and the most significant clinically. It occurs as a result of meiotic chromosomal malsegregation during formation of a haploid oocyte by subsequent extrusion of two polar bodies: polar body first (PB1) and second (PB2). Based on meiosis mechanism, genome of a single oocyte can be deduced by analyzing its sibling polar bodies.

Material and Methods: Next-generation sequencing technology for comprehensive chromosome screening was used to assess ploidy status of the oocytes and their sibling PB1. A total of 120 single cells (60 PB1 and 60 oocytes) were subjected for whole genome amplification (WGA) using four different commercially available WGA kits and analyzed using VeriSeq PGS kit on the MiSeq sequencer (Illumina). Random samples were validated using genomic arrays (aCGH). Aneuploidy detection with the tested kits was evaluated.

Results: Aneuploidy status was determined for 112 of 120 (93,3%) samples. In the group of well-amplified PB1-oocyte pairs (33/60) a 97% concordance between the chromosomal status of PB1 and the corresponding oocyte was observed. Differences in WGA kits performance including amplification uniformity and aneuploidy calling potential were evaluated and the most optimal kit for PB1 genome WGA was selected. Results from aCGH validation of the randomly selected samples showed full consistency with the NGS results.

Conclusions: NGS-based method used for PB1 genome analysis showed a high predictive potential of PB1 in deducing ploidy status of the corresponding oocyte and is a promising method for genetic preconception testing of oocytes.

Supported by: POIR.02.03.02-14- 0092/17

A. Sarosiak: None. I. Minota: None. K. Koziol: None. M. Oldak: None.

P01.83C What is the actual risk of couples carriers for the mutation DHCR7:c.964-1G>C?

H. Daum1, V. Meiner1, R. Michaelson-Cohen2, R. Sukenik-Halevy3,4, M. Levy-Zalzberg5, A. Bar-Ziv6, A. Weiden7, S. Scher8, M. Shohat4,9,10, J. Zlotogora1

1Department of Genetics and Metabolic Diseases, Hadassah-Hebrew University Medical Center, Jerusalem, Israel, 2Medical Genetics Institute, Dept. of Obstetrics & Gynecology, Shaare Zedek Medical Center, Hebrew University of Jerusalem, Jerusalem, Israel, 3Recanati Genetic Institute, Rabin Medical Center, Petah Tikva, Israel, 4Sackler School of Medicine, Tel Aviv University, Tel aviv, Israel, 5Genetic Institute, Soroka, Beer sheva, Israel, 6The Danek Gertner Institute of Human Genetics, Sheba Medical Center, Tel hashomer, Israel, 7Dor Yeshorim, Committee for Prevention of Jewish Genetic Diseases, Jerusalem, Israel, 8Dor Yeshorim, Committee for Prevention of Jewish Genetic Diseases, Brooklyn, NY, United States, 9Bio-informatics department, Cancer center, Sheba Medical center, Tel hashomer, Israel, 10Institute of Medical Genetics, Maccabi HMO, Rehovot, Israel

Introduction: Smith-Lemli-Opitz is an autosomal recessive disorder characterized by variable expression including multiple congenital malformations, dysmorphic features, metabolic derangement and intellectual disability. The founder mutation DHCR7:c.964-1G>C was introduced into the Israeli preconception screening tests for Ashkenazi Jews in 2017 since the carrier frequency of the mutation is high in this population (1.2%). Despite the high carrier rate, the disease itself is not known to be frequent in this population. Discrepancy between the carrier and disease frequency raises the question of the actual risks for affected offspring for couples detected by the screening program.

Methods: We performed a literature review of all publications available with detailed information regarding homozygous DHCR7:c.964-1G>C fetuses/patients were included. We also collected clinical data about the couples identified in the national screening program, such as reproductive history.

Results: Out of 31 homozygous fetuses, six died in-utero (IUFD), ten pregnancies were terminated during the second trimester and 15 children were born. All 15 died between day one till three months of age. Reproductive history of Smith-Lemli-Opitz at-risk couples showed that out of 61 pregnancies, 32 spontaneous miscarriages were reported (52%).

Discussion: Our observations support the previous knowledge that homozygosity for c.964-1G>C in DHCR7 leads to a severe phenotype or early spontaneous abortion. An unexpected observation was the excess of early spontaneous abortions. The reason for this is not clear and awaits further studies.

H. Daum: None. V. Meiner: None. R. Michaelson-Cohen: None. R. Sukenik-Halevy: None. M. Levy-Zalzberg: None. A. Bar-Ziv: None. A. Weiden: None. S. Scher: None. M. Shohat: None. J. Zlotogora: None.

P01.85A Developing quantitative real-time PCR for preconception spinal muscular atrophy carrier screening in Thai population

P. Chit ayanan1, B. Panthan2, S. Klumsathian2, A. Charoenyingwattana2, W. Chantratita2, O. Trachoo3,2

1Panthupark Genetics Clinic, Bangkok, Thailand, 2Center for Medical Genomics, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bangkok, Thailand, 3Department of Medicine, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bangkok, Thailand

Introduction: Spinal muscular atrophy (SMA) is a severe neuromuscular disease and one of the most common autosomal recessive diseases in children, caused by homozygous absence of the survival motor neuron gene (SMN1). Heterozygous exon 7 deletion of SMN1 is defined as a carrier with the reported frequency of 1/40 to 1/50 worldwide. Clinical practice in many countries recommends SMA carrier screening for most couples at the preconception stage.

Materials and Methods: We developed a quantitative real-time PCR protocol to detect SMN1 exon 7 deletion (SMADX7™) as a cost-effective method, aiming to perform carrier screening in 430 Thai individuals visiting preconception clinic. Validation of the method was done in 100 positive and negative controls compared to standard DHPLC method.

Results: The result of control testing was 100% consistent between real-time PCR and DHPLC. Further population screening revealed the SMA carrier frequency of 1/72.

Conclusion: Developing of SMADX7™ is an outstanding strategy for SMA carrier screening in Thai population due to good cost-benefit and rapid turnaround time within 24 hours. Carrier frequency in our cohort seemed to be lower than global frequency; however, additional subjects are required to be enrolled, aiming to obtain the national data that can represent overall carrier frequency.

Grants: Leader Medical Genetics and Genomics, Co., Ltd.

P. Chitayanan: A. Employment (full or part-time); Modest; Leader Medical Genetics and Genomics, Co., Ltd. B. Panthan: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Modest; Leader Medical Genetics and Genomics, Co., Ltd.. S. Klumsathian: None. A. Charoenyingwattana: None. W. Chantratita: None. O. Trachoo: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Modest; Leader Medical Genetics and Genomics, Co., Ltd..

P01.86B Detection and validation of subchromosomal aberrations detected as part of routine noninvasive prenatal testing

M. Sekelská1, A. Izsáková1, K. Kubošová1, P. Tilandyová1, E. Csekes1, Ž. Kúchová1, R. Lukáčková2, D. Landlová2, M. Hýblová2, M. Haršányová3, J. Budiš3, T. Szemes3, P. Križan2, G. Minárik2

1Trisomy test Ltd., Bratislava, Slovakia, 2Medirex Inc., Bratislava, Slovakia, 3Geneton Ltd., Bratislava, Slovakia

Introduction: Noninvasive prenatal testing is based on analysis of circulating DNA from blood of pregnant women. Low coverage whole genome sequencing is able to detect not only most commonly screened chromosomal aneuploidies but also different subchromosomal aberrations.

Aim: Aim of the work was prospective study of detection and validation of subchromosomal aberrations of fetal origin identified as part of routine noninvasive prenatal testing.

Materials and Methods: From April 2016 till December 2018, more than 8000 samples of pregnant women using Illumina NextSeq 500 platform were analysed. Low coverage whole genome sequencing was used in combination with optimized CNV detection algorithm.

Results: In tested cohort of patients different suchromosomal aberrations were detected in 32 samples. Half of patients (16) refused confirmatory testing or we were not able to get sample or feedback from the further management of the patient. In second half of samples (16) DNA from amniocenteses were available for confirmatory testing. Of these cases 5 findings were confirmed and 11 were negative after verification analyses. In samples with detected aberrations fetal fraction varied between 10,3 and 18,26 %. The smallest reported aberration was 1 Mb and the largest one approx. 80 Mb long.

Conclusions: In this prospective study the possibility of utilization of low coverage genomic sequencing for detection of subchromosomal aberrations over the whole genome was confirmed. For proper estimations of sensitivity, specificity, NPV and PPV larger studies are necessary as samples significantly differ in crucial factors that are fetal fraction, position and size of detected aberration.

M. Sekelská: None. A. Izsáková: None. K. Kubošová: None. P. Tilandyová: None. E. Csekes: None. Ž. Kúchová: None. R. Lukáčková: None. D. Landlová: None. M. Hýblová: None. M. Haršányová: None. J. Budiš: None. T. Szemes: None. P. Križan: None. G. Minárik: None.

P01.87C The risk of spontaneous abortion in subfertile couples

G. C. COZARU1,2, M. Aschie1,2, A. Mitroi1,2, C. Brinzan1,2

1CEDMOG - “Ovidius” University of Constanta, Constanta, Romania, 2“Sf Apostol Andrei” Emergency Clinical County Hospital of Constanta, Constanta, Romania

Our research has gone from the observation that women with a history of subfertility have been shown to have increased rates of subclinical early pregnancy loss, as detected via human chorionic gonadotropin, relative to women without a history of impaired fertility.

Objectives: The purpose of this study was to assess the association between subfertility and spontaneous abortion.

Methods: A total of 122 women in Constanta reported 384 pregnancies between 2014 and 2018 and were able to provide an estimate of the waiting time to conception for 276 (71.87% of the 384 eligible pregnancies). We noted retrospective data, including sociodemographic characteristics, obstetric and medical history, genetic risk factors, cigarette smoking, caffeine and alcohol consumption, and occupational information. Rates of spontaneous abortion were determined among women with and without subfertility, and adjusted odds ratios (ORs) and 95% confidence intervals (CIs) were estimated via multiple logistic regression.

Results: Multivariate logistic regression analyses with generalized estimating equations indicated that spontaneous abortion rates were 28.0% in pregnancies preceded by subfertility and 17.0% in pregnancies without impaired fertility (adjusted OR=1.68, 95% CI=1.36, 2.84).

The attributable risk of spontaneous abortion associated with subfertility was 6.9%.

Conclusions: Subfertile women evidence an increased number of spontaneous abortions.

Research supported by CEDMOG, Ovidius University of Constanta, POS CCE 2.21. Project, (ID 1844, SMIS 48750).

G.C. Cozaru: None. M. Aschie: None. A. Mitroi: None. C. Brinzan: None.

P01.88D A molecular strategy increases the PGD accuracy for Taiwan b-thalassemia population

Y. C. Li1,2,3, Y. C. Chen1, H. H. Shih1, E. H. Cheng1, Y. P. Lin1, Y. P. Pai1, M. S. Lee1,3,4

1Lee Women’s Hospital, Taichung, Taiwan, 2China Medical University, Taichung, Taiwan, 3Chung-Shan Medical University, Taichung, Taiwan, 4Chung-Shan Medical University Hospital, Taichung, Taiwan

β-thalassemia carries over 1.1% prevalent rate in Taiwan and has polymorphism variants coordinated with geography. Both β-thalassemia carrier couple would transmit 1/4 risk of thalassemia major to their offspring in every pregnancy. Thalassemia major patients require lifelong transfusions, iron-chelation therapy and, an expensive support system. These bring a great socioeconomic burden to the suffered family. PGD (preimplantation genetic diagnosis) for β-thalassemia is conducted in IVF (in vitro fertilization) to transfer an embryo without thalassemia major to the uterus. The problem of allele dropout (ADO) and preferential amplification highly exist in PGD involving WGA (whole genome amplification) of blastocyst biopsies. It had been reported that a combination of Sanger sequencing and QF-PCR of STR markers increased the detection rate of the mutated β-globin gene in PGD. However, the uninformative STRs and ADO of the mutated gene usually limited the validation of PGD. Moreover, the heterozygosity rate of STRs is highly population different. In this study, we verified several STRs for providing enough informative STRs for Taiwan β-thalassemia population. Our results showed that six linked STRs (D11S4146, D11S1760, D11S1243, D11S4891, D11S2352, and D11S1871) with highly heterozygous polymorphism and low recombination rate in Taiwan population and would achieve up to 99% diagnostic validation in PGD for β-thalassemia. Furthermore, the multifluorescent PCR of STRs conducted in a single tube for the linkage analysis of the mutated β-globin gene provided a simple method for performing the PGD of β-thalassemia major. This study was supported from MOST of Taiwan (107-2314-B-040-019), CSMU (104-OM-A-102), and CSMU-CCH (CSMUCCH-102-06).

Y.C. Li: A. Employment (full or part-time); Significant; Lee Women’s Hospital. B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Significant; Chung-Shan Medical University. Y.C. Chen: A. Employment (full or part-time); Significant; Lee Women’s Hospital. H.H. Shih: A. Employment (full or part-time); Significant; Lee Women’s Hospital. E.H. Cheng: A. Employment (full or part-time); Significant; Lee Women’s Hospital. Y.P. Lin: A. Employment (full or part-time); Modest; Lee Women’s Hospital. Y.P. Pai: A. Employment (full or part-time); Significant; Lee Women’s Hospital. M.S. Lee: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Significant; Chung-Shan Medical University.

P01.89A Maternal and paternal genotypes analysis of thrombophilic gene variants in the risk of reproductive disorders in married couples

Z. I. Rossokha1,2, O. F. Popova1, N. L. Medvedieva1, L. P. Sheiko2, N. G. Gorovenko2

1State Institution “Reference-centre for molecular diagnostic of Public Health Ministry of Ukraine”, Kyiv, Ukraine, 2State Institution «Genetic and Regenerative Medicine of the National Academy of Medical Sciences of Ukraine», Kyiv, Ukraine

Introduction: Inherited thrombophilia is well defined. Factor V Leiden mutation and prothrombin G20210A genetic variants are classic thrombophilic determinants for clinical manifestation of reproductive disorders. But new genetic variants are discussed now. The aim of the study to evaluate the influence of classic and new genetic variants on reproductive disorders in married couples.

Materials and Methods: The genetic variants of Factor V (G1691A, rs 6025), prothrombin (G20210A, rs 1799963), PAI-I (675 5G/4G, rs 1799889), FGB (C148T, G-455A, rs 1800787 and rs 1800790) were analyzed in 206 couples with reproductive disorders (69 with unspecified infertility/137 with recurrent pregnancy loss) and 35 couples of comparison group (who gave birth to one or two healthy children). Frequency genotypes analysis was carried out without taking into account the origin (maternal or paternal) of genotypes. SNP Stats program were used for statistical analysis.

Results: There were found no significant differences for prothrombin, Factor V and FGB genes variants among investigated groups. 5G/5G genotypes were significantly decreased among couples with reproductive disorders (Table). We identified significant dominant inheritance models for PAI-I (5G/4G) gene (χ²=5,16; p=0,023;OR=0,51, 95%CI: 0,29-0,88) for reproductive failure in couples.

Conclusions: We suppose that PAI gene has a beneficial effect on reproductive function due to improved anticoagulant properties of blood when genotype 5G/5G in married couples present or for embryos this genotype are predicted.

Table. Distribution of genes variants


(polymorphic variants)


Basic group, n/%


Comparison group, n/%


Protrombin (G20210A)










Factor V























(-455 G/A)











(-675 5G/4G)







4G /4G



Z.I. Rossokha: None. O.F. Popova: None. N.L. Medvedieva: None. L.P. Sheiko: None. N.G. Gorovenko: None.

P01.90B Droplet digital PCR multiplexing for fetal aneuploidy detection

I. Zednikova1, M. Korabecna1, E. Pazourkova1, S. Santorova1, P. Calda2, M. Brestak2,3, A. Horinek1

1Institute of Biology and Medical Genetics, First Faculty of Medicine, Charles University and General University Hospital in Prague, Prague 2, Czech Republic, 2Department of Obstetrics and Gynecology, First Faculty of Medicine, Charles University and General University Hospital in Prague, Prague 2, Czech Republic, 3Screening Center ProfiG2, Prague, Czech Republic

Background: Nowadays, cell-free fetal DNA (cffDNA) in maternal plasma is routinely applied for non-invasive prenatal testing (NIPT). Detection of the most common fetal aneuploidies is ordinarily performed using NGS technology. Low concentration of cffDNA in maternal circulation prevents the utilization of cheaper and simpler technology - droplet digital PCR (ddPCR). The aim of our study was to overcome this problem by target multiplexing.

Methods: Three genes on chromosome 21 and chromosome 18 were absolutely quantified in one ddPCR reaction (hexaplex). Targets on different chromosomes were distinguished using different labelling (FAM/HEX). Expected FAM/HEX ratio for DNA with normal karyotype is 1. In case of fetal trisomy of 21 or 18, the ratio will be deflected accordingly. Artificial mixtures of euploid and trisomic DNA were used for pilot study.

Results: DNA with normal karyotype was systematically mixed with DNA with trisomy of 21 (0%; 5%; 10%; 20%; 40%); chromosome 18 was used as a reference chromosome in this case. Eight replicates for each mixture were performed. Detected FAM/HEX ratios always corresponded to the expected ratios for respective mixtures. Even the lowest representation of aneuploid DNA (5%) was correctly detected.

Conclusion: After promising results of the initial phase with the artificial mixtures, the optimized workflow will now be applied for the analysis of plasma of pregnant women. This method could be applicable for detection of trisomy 21 or 18 in the same reaction, as both trisomies together have never been observed. Supported by the Ministry of Health of the Czech Republic RVO VFN64165

I. Zednikova: None. M. Korabecna: None. E. Pazourkova: None. S. Santorova: None. P. Calda: None. M. Brestak: None. A. Horinek: None.

P01.91C Clinical implementation of diagnostic whole exome sequencing for fetal multiple congenital anomalies on ultrasound

N. Corsten-Janssen, J. C. D. Diphoorn, K. Bouman, J. El Mecky, J. B. G. M. Verheij, W. S. Kerstjens, A. Scheper, R. Kinds, I. M. van Langen, R. J. Sinke, R. H. Sijmons, B. Sikkema-Raddatz, H. Westers, C. C. van Diemen

Department of Genetics, University Medical Center Groningen, Groningen, The Netherlands, Groningen, Netherlands

Introduction: Identifying the cause of fetal anomalies seen on ultrasound provides important information for perinatal management, but we can diagnose only ~40% of fetuses using conventional genetic tests (QF-PCR and microarray). Whole Exome Sequencing (WES) may improve diagnostic yield, but is challenging due to uncertainties around fetal phenotyping, variant interpretation, ethical/counseling issues of incidental findings and variants of unknown clinical significance, and the requirement of short turnaround times. In this study, we implemented WES in prenatal care to increase our diagnostic yield.

Materials and Methods: We conducted a prospective study of rapid trio WES analysis next to conventional genetic tests for twenty-five fetuses with ultrasound abnormalities. Inclusion criteria were at least two congenital malformations on ultrasound or one congenital malformation in a fetus with a sib with similar phenotype, without known genetic cause. We performed trio WES analysis using a custom virtual gene-panel (~3,800 OMIM genes, excluding late-onset disease genes).

Results: We established an additional genetic WES-based diagnosis in nine fetuses, being: syndromes of MIRAGE (SAMD9), Zellweger (PEX1), Walker Warburg (POMGNT1), Noonan (PTNP11), Kabuki (KMT2D), CHARGE (CHD7); two cases of Osteogenesis Imperfecta type 2 (COL1A1), and one potential diagnosis in a fetus with hydrops (MYH7). In six out of 25 cases the WES diagnosis aided in perinatal management. The mean turnaround time was 10 (range 6-15) working days.

Conclusion: Our prospective study shows that implementing WES as a routine test in the prenatal setting is challenging, but technically feasible and has a promising diagnostic yield and significant clinical relevance.

N. Corsten-Janssen: None. J.C.D. Diphoorn: None. K. Bouman: None. J. El Mecky: None. J.B.G.M. Verheij: None. W.S. Kerstjens: None. A. Scheper: None. R. Kinds: None. I.M. van Langen: None. R.J. Sinke: None. R.H. Sijmons: None. B. Sikkema-Raddatz: None. H. Westers: None. C.C. van Diemen: None.

P01.92D CNVs with unknown significance on chromosome 15 in prenatal samples

K. Markova, R. Mansfeldova, V. Becvarova, H. Pekova, T. Marie, M. Spacirova, B. Laposova, J. Starkova, V. Sucha, P. Sidova, J. Horacek, M. Koudova, D. Stejskal

Gennet, Prague, Czech Republic, Prague, Czech Republic

Introduction: Chromosomal microarray analysis (CMA) is increasingly used in prenatal diagnostics. It provides better resolution than conventional karyotyping, nevertheless, CNVs with variable expressivity and low penetrance require a proper interpretation attitude.

Materials and Methods: More than 2200 prenatal samples (AMC, CVS) were examined at our clinic within the last two years. In 9.8% samples an aneuploidy was detected by QFPCR. The remaining samples were analyzed by CMA as a first-tier test: samples were assessed by array CGH (or SNP-array) and analyzed with AgilentCytoGenomics software. Parental samples were analyzed using CMA to specify the origin of the identified aberrations.

Results: 72 CMA analyzed samples were considered to be pathogenic (out of them 56 below karyotype resolution) while additional 112 cases as variants of unknown significance: class 2, 3 and 4 (likely benign, uncertain clinical relevance, likely pathogenic) or without subclassification. Most frequent VOUS were identified on chromosomes 15 (19), 16 (8) and X (13). Prominent CNVs were del/dup 15q11.2 (BP1BP2) and del/dup 15q13.2q13.3 encompassing significant genes NIPA1 and CHRNA7, respectively. These CNVs are associated with neurodevelopmental disorders, autism and behavioral problems. Penetrance is incomplete and expressivity varies greatly. Databases, literature search and familial genealogy did not prove definite pathogenicity. The most of these CNVs was inherited and all traceable pregnancies with normal ultrasound continued.

Conclusion: All fetuses with VOUS on chromosome 15 except one had normal ultrasound. CNVs identified on chromosome 15 except deletion of CHRNA7, were described as likely benign with respect to prevailing parental origin and normal ultrasound.

K. Markova: None. R. Mansfeldova: None. V. Becvarova: None. H. Pekova: None. T. Marie: None. M. Spacirova: None. B. Laposova: None. J. Starkova: None. V. Sucha: None. P. Sidova: None. J. Horacek: None. M. Koudova: None. D. Stejskal: None.

P01.93A Preconception carrier screening: effect of reporting variants of unknown significance in partners of carriers with clinically significant variants

H. Fridman1,2,3, D. Behar4, S. Carmi2, E. Levy-Lahad1,3

1Medical Genetics Institute, Shaare Zedek Medical Center, Jerusalem, Israel, 2Braun School of Public Health and Community Medicine, The Hebrew University of Jerusalem, Jerusalem, Israel, 3Faculty of Medicine, The Hebrew University of Jerusalem, Jerusalem, Israel, 4Igentify, Tirat Hacarmel, Israel

Background: Expanded preconception carrier screening (ECS) panels can identify at-risk couples for multiple diseases. ECS reports are currently limited to pathogenic/likely-pathogenic variants (PV/LPV). Variants of unknown significance (VOUS) are not reported, even though reporting VOUS is routine in genomic and chromosomal array testing in both postnatal and prenatal settings. This policy misses pairings of PV/LPV carriers with VOUS carriers (PV/LPV*VOUS), who may be at-risk. To evaluate the importance of this omission, we examined the rates and characteristics of PV/LPV*VOUS virtual matings in an Ashkenazi-Jewish (AJ) cohort, a population with well-established preconception screening. Methods: We analyzed 672 AJ genomes (225,456 virtual couples) for variants in different gene-panels (exons ±10bp): AJ (55 genes), pan-ethnic (168 genes) and combined (180 genes), using an ACMG classifier.

Results: Across 180 genes, we identified 4671 variants, among them 144 (3.1%) were PV/LPV and 1963 (42%) were VOUS. The proportion of matings who were PV/LPV*PV/LPV was 2.7-3.8% (depending on the panel), similar to previous reports. The proportion of PV/LPV*VOUS matings was 17-20%. Considering only exonic, non-UTR VOUS resulted in a decreased proportion of PV/LPV*VOUS matings to ≈7%. Most (65-70%) PV/LPV*VOUS matings were at risk for severe/profoundly-severe diseases, while only 6-7% were at risk for mild diseases.

Conclusions: Non-reporting of VOUS in ECS may miss at-risk couples for severe diseases. Reporting PV/LPV*VOUS couples would triple the proportion of at-risk couples, even if considering only exonic, non-UTR VOUS. Even if only 10% of VOUS are ultimately reclassified as PV/LPV, reporting PV/LPV*VOUS would increase the yield of ECS by ≈25%.

H. Fridman: None. D. Behar: A. Employment (full or part-time); Significant; Igentify. E. Ownership Interest (stock, stock options, patent or other intellectual property); Significant; Igentify. S. Carmi: None. E. Levy-Lahad: None.

P01.94B Incidence of Y chromosome microdeletion in different world populations: systematic review

A. Kurtovic-Kozaric1,2, H. Komic2,3, L. Mehinovic2, M. Kozaric4, I. Eminovic2, Z. Halilovic5

1Laboratory of Human Genetics, Department of Clinical Pathology, Cytology and Human Genetics, Clinical Center of the University of Sarajevo,, Sarajevo, Bosnia and Herzegovina, 2Department of Genetics, Biology Section, Faculty of Science, University of Sarajevo, Sarajevo, Bosnia and Herzegovina, 3TIMM Laboratory at Sahlgrenska Cancer Center, University of Gothenburg, Gothenburg, Sweden, 4Clinic of Gynecology and Obstetrics, Clinical Center of the University of Sarajevo, Sarajevo, Bosnia and Herzegovina, 5Laboratory of Human Genetics, Department of Clinical Pathology, Cytology and Human Genetics, Clinical Center of the University of Sarajevo, Sarajevo, Bosnia and Herzegovina

Introduction: Y chromosome microdeletions in azoospermia factor (AZF) sub-regions of its long arm are the second most frequent genetic cause of male infertility. The aim of this study was to analyze data on Y chromosome microdeletion world-wide.

Materials and Methods: A recall search yielded 924 papers, 73 papers met the inclusion criteria. Risk of bias was the size of tested population (>100 men) and its application excluded 31 studies, yielding 42 papers for final analysis.

Results: The mean Y microdeletion frequency for all included studies was 8.35% (n=32996), and 6.4% (n=31552) for the studies that passed risk of bias criteria. The mean Y microdeletion frequencies per continent (Asian, American, European and African) were 9%, 6.83%, 4.45% and 3.97%, respectively. The highest frequencies were detected in South Korean (38%) and Syrian (28%) populations. The lowest frequencies were in Danish (0.7%) and Croatian (0.9%) populations. The frequencies among AZF sub-regions showed the same pattern in almost all studies, with AZFc being most frequent (90.5%), followed by AZFb+c.

Conclusion: Isolated AZFc microdeletion was the most frequent, except for Croatian, Swedish, Kuwaiti and Middle East populations. These results can serve as a basis for creating future guidelines for genetic diagnostics of male infertility.

A. Kurtovic-Kozaric: None. H. Komic: None. L. Mehinovic: None. M. Kozaric: None. I. Eminovic: None. Z. Halilovic: None.

P02 Sensory disorders (eye, ear, pain)

P02.01C PAX6 gene analysis for aniridia patients: single Lithuanian centre experience in 2017-2018 years period

R. Traberg, V. Asmoniene, D. Cereskevicius, M. Sukys

Hospital of Lithuanian University of Health Sciences Kauno klinikos, Kaunas, Lithuania

Introduction: Aniridia is a very rare congenital panocular disorder characterized by complete or partial iris hypoplasia. PAX6 gene mutations occur in around 90% of aniridia cases. Two-thirds of aniridia cases are inherited in an autosomal dominant pattern and 1/3 are sporadic cases.

Materials and Methods: PAX6 gene analysis was performed for patients who were diagnosed with isolated and syndromic aniridia. Their first line and/or affected family members also was analysed. The PAX6 gene was analysed by Sanger sequencing (2-13 exones). If no point mutations were found, analysis was extended to deletion/ duplication search with multiplex ligation-dependent probe amplification (MLPA) kit (MRC-Holland SALSA MLPA P219 PAX6 kit).

Results: 5 families and 3 single cases were tested for PAX6 gene mutations in 2 years period (2017 - 2018) (20 analyses altogether). Six different mutations were found in 15 (75%) cases: 1 missense mutation, 1 intragenic deletion, 4 nonsense mutations. The 2 truncating mutations (c.991C>T (p.Arg331Ter) and c.392_393delTA (p.Ile131Thrfs*15)) were newly described. De novo PAX6 gene variant c.991C>T (p.Arg331Ter) was found for patient, who diagnosed with aniridia and doubled kidney. PAX6 gene variant c.392_393delTA (p.Ile131Thrfs*15) was found for family members with isolated aniridia. Members of one family with various clinical presentation (unilateral aniridia and Peters anomaly, cataracts, optic nerve hypoplasia) shared missense variant of unknown significance c.422T>C (p.Leu141Pro).

Conclusions: We would like to suggest that PAX6 gene variants c.991C>T (p.Arg331Ter) and c.392_393delTA (p.Ile131Thrfs*15) are cause of aniridia for our patients.

R. Traberg: None. V. Asmoniene: None. D. Cereskevicius: None. M. Sukys: None.

P02.02D The role of next-generation sequencing in the differential diagnosis of congenital iris anomalies

F. Blanco-Kelly, M. Tarilonte, C. Villaverde, S. T. Swafiri, A. Arteche, A. Avila-Fernandez, J. Moya, I. Lorda, M. Trujillo-Tiebas, C. Ayuso, M. Corton

Genetics Department, IIS-Fundación Jiménez Díaz-CIBERER, UAM, Madrid, Spain

Abnormal development of the iris is a feature of several congenital ocular malformations. Classical aniridia, the most common form, is a panocular disease characterized by iris and foveal hypoplasia, keratopathy, cataracts and glaucoma, caused by PAX6 haploinsufficiency. The presence of variable expressivity and phenotypic overlapping with other types of anterior segment dysgenesis (ASD) hinders the genetic characterization of atypical presentations of aniridia. Here, we evaluate the usefulness of NGS to improve the clinical diagnosis of these pathologies. A cohort of 106 families was studied, including cases with classical aniridia or atypical forms (iris hypoplasia, atrophy or coloboma). The entire PAX6 gene and other 260 genes associated with ocular malformations were analyzed using custom targeted NGS and aCGH approaches, as well as clinical or whole exome and/or Sanger sequencing. About 82% of the cases presented likely pathogenic variants or CNVs in 5 different genes. Defects in PAX6 explain 75% of cases, including not only all characterized cases with classical aniridia, but also 30% of patients with more atypical presentations, in whom LOF or atypical missense and non-coding variants were identified, respectively. Other aniridia-like patients presented pathogenic variants in ASD-related genes or variants of uncertain significance in candidate genes. Molecular analysis of iris malformations presents high diagnostic yield not only in cases with classical aniridia (95%) but also in patients with aniridia-like phenotypes (>50%). Our work evidences the clinical utility of differential genetic studies based on NGS in patients with congenital iris anomalies.

Grants: ISCIII (PI17_01164, CPII17_00006), FEDER and Conchita Rabago Foundation.

F. Blanco-Kelly: None. M. Tarilonte: None. C. Villaverde: None. S.T. Swafiri: None. A. Arteche: None. A. Avila-Fernandez: None. J. Moya: None. I. Lorda: None. M. Trujillo-Tiebas: None. C. Ayuso: None. M. Corton: None.

P02.03A Genotype-phenotype correlations in congenital PAX6-associated aniridia

T. A. Vasilyeva1, V. V. Kadyshev1, A. A. Voskresenskaya2, N. V. Petrova1, A. V. Marakhonov1,3, R. A. Zinchenko1,4

1Research Centre for Medical Genetics, Moscow, Russian Federation, 2Cheboksary branch of S. Fyodorov Eye Microsurgery Federal State Institution, Cheboksary, Russian Federation, 3Far Eastern Federal University, Vladivostok, Russian Federation, 4Pirogov Russian National Research Medical University, Moscow, Russian Federation

Objectives: Aniridia is an autosomal dominant severe panocular disorder caused by pathogenic variants in the PAX6 gene or large chromosomal aberrations affecting 11p13 region. Aniridia phenotype varies greatly. Based on the occurrence of several aniridia characteristics in groups of patients with the same type of PAX6 mutation, genotype—phenotype correlations were analyzed.

Methods: 155 patients from 129 unrelated families were examined in the course of the earlier study, 118 patients were found to have intragenic pathogenic variants while 37 others have large chromosomal deletions. Patients were allocated into 6 groups according to the type of PAX6 mutation: missense, nonsense, frame shifting, splicing, 3′-cis-regulatory region deletions, and all other 11p13 chromosome deletions. Data on six frequently observed aniridic eye anomalies were collected: complete or partial aniridia, cataract, glaucoma, kerathopathy, fovea hypoplasia, and nystagmus. Fisher’s exact test with Benjamini—Hochberg correction for multiple testing was applied for 2×2 contingency tables created to compare occurrence of each ocular sign in the group of patients with one type of mutations versus the corresponding occurrence in the group with all other mutations.

Results: Only patients with 3′-cis-regulatory region deletions significantly more often develop a distinct and milder aniridia phenotype without kerathopathy, fovea hypoplasia and nystagmus. Identified missense and splicing mutations were associated with severe aniridia phenotype and in their consequences could not be distinguished from the loss-of-function mutations.

Conclusions: Revealed genotype-phenotype correlations in PAX6-associated aniridia suggests common disease mechanism for all mutations except for 3′-cis-regulatory region deletions.

Supported by grant RSF №17-15-01051.

T.A. Vasilyeva: None. V.V. Kadyshev: None. A.A. Voskresenskaya: None. N.V. Petrova: None. A.V. Marakhonov: None. R.A. Zinchenko: None.

P02.04B Congenital aniridia caused by new PAX6 variants involving exonic cryptic-splicing sites

M. Tarilonte, P. Ramos, J. Moya, C. Villaverde, G. Sanz, S. T. Swafiri, B. Gener, F. Blanco-Kelly, C. Ayuso, M. Corton

IIS - University Hospital Fundación Jiménez Díaz, Madrid, Spain

Congenital aniridia is a panocular disease, with a worldwide incidence of 1:50.000-100.000 births, characterized by iris and foveal hypoplasia and additional anomalies in the cornea, anterior segment and lens, leading to low visual acuity. Up to 90% of the aniridia cases carried monoallelic loss-of-function variants causing haploinsufficiency of PAX6, a highly-conserved transcriptional regulator that is critical for normal ocular development. Here, we aimed to assess functionally the implication on splicing of different exonic and intronic variants of uncertain significance (VUS), mainly located in the PAX6 homeodomain. First, novel VUS including synonymous, missense and non-canonical splicing variants were identified in our cohort of 87 Spanish families with aniridia after classical genotyping and/or targeted next generation sequencing (NGS) of the entire PAX6 locus. No additional pathogenic variants were found in other 250 eye developmental genes. Functional splicing assays were performed using in silico predictors, in vitro minigenes and, when available, patients-derived lymphocytes cell lines. We described new spliceogenic mechanisms for PAX6 variants through activating different exonic cryptic donors as the most plausible cause for congenital aniridia in several families. These included an additional case of parental PAX6 mosaicism, as confirmed by digital-droplet PCR, for a synonymous variant in an asymptomatic mother with two affected siblings. In conclusion, clinical interpretation of silent and non-canonical splicing variants represents a real challenge for the genetic counseling. Our study revealed the importance of functional studies to elucidate the role of non-coding PAX6 variants underlying ocular development.

Grants: ISCIII (PI17_01164, CPII17_00006), FEDER and Conchita Rabago Foundation

M. Tarilonte: None. P. Ramos: None. J. Moya: None. C. Villaverde: None. G. Sanz: None. S.T. Swafiri: None. B. Gener: None. F. Blanco-Kelly: None. C. Ayuso: None. M. Corton: None.

P02.05C Preliminary results from a new GWAS meta-analysis point at new loci for age-related hearing loss (ARHL)

M. Brumat1, A. Morgan1, M. Cocca2, M. Mezzavilla2, G. Van Camp3, E. Fransen4, G. Biino5, U. Ambrosetti6, D. Toniolo7, S. Ghiselli2, 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, 3Center of Medical Genetics, University of Antwerp and Antwerp University Hospital, Antwerp, Belgium, 4Department of Biomedical Sciences, University of Antwerp, Antwerp, Belgium, 5Institute of Molecular Genetics, National Research Council of Italy, Pavia, Italy, 6Audiologia, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, University of Milan, Milan, Italy, 7Center for Translational Genomics and Bioinformatics, S. Raffaele Scientific Institute, Milan, Italy

Introduction: With the progressive ageing of world population, there is a need to further investigate the genetic base of late-onset disorders such as ARHL. With this study, we aim to increase our knowledge of this complex disease.

Materials and Methods: We performed a case-control GWAS analysis on 2663 subjects (aged >=50) coming from Italy, Northern Europe and Caucasus clinically characterized including the audiometric phenotype. Cases and controls were classified as previously described (DOI:10.3109/21695717.2014.911472). Genotype data were imputed using 1000Gph3 reference panel. Results were pooled together with METAL. Replication of results was performed with UK-Biobank data (ie. speech-in-noise phenotype). Analyses on natural selection in European populations were performed and integrated haplotype scores (iHS) on significant GWAS results were collected.

Results: Meta-analysis identified some strongly significant loci. To prioritize these genes, signals of selection were checked to evaluate the presence of genetic variants that could have some evolutionary role and a stronger effect. A significant hit on chr5 was detected in a gene/protein (|iHS|score>2) catalysing the conversion of homocysteine to other amino-acids. Signal of selection was present in North European populations, but absent in South European ones. Interestingly, literature showed a relationship between hyperhomocysteinemia induced by folate deficiency and premature hearing loss, prompting further investigation and discussion on these results.

Conclusions: As the association signal is mainly driven by Northern Italian and European populations, a population-specific haplotype involved in folate metabolism should also implicated directly or indirectly in ARHL.

M. Brumat: None. A. Morgan: None. M. Cocca: None. M. Mezzavilla: None. G. Van Camp: None. E. Fransen: None. G. Biino: None. U. Ambrosetti: None. D. Toniolo: None. S. Ghiselli: None. P. Gasparini: None. G. Girotto: None.

P02.06D Multi-level evidence of an allelic hierarchy of USH2A variants; hearing loss, auditory processing and speech/language outcomes

P. A. Perrino1, L. Nedevska2, R. Reader3,4, A. Hill5, WGS500 Consortium, A. R. Rendall1, H. S. Mountford2, A. N. Buscarello1, N. Lahiri6, A. Saggar6, R. H. Fitch1, D. F. Newbury2

1Department of Psychological Science/Behavioral Neuroscience, University of Connecticut, Storrs, CT, United States, 2Faculty of Health and Life Sciences, Oxford Brookes University, Oxford, United Kingdom, 3Wellcome Trust Centre for Human Genetics, Oxford, United Kingdom, 4School of Veterinary Medicine and Science, University of Nottingham, Sutton Bonington, United Kingdom, 5Centre for Child & Adolescent Health, University of Bristol, Bristol, United Kingdom, 6Institute of Molecular and Clinical Sciences, University of London & St George’s University Hospitals NHS Foundation Trust, London, United Kingdom

Introduction: Hearing and auditory processing are fundamental to language development. Genetic mechanisms that alter these processes may therefore have secondary effects on emergent language. In this work, we consider variations across the USH2A gene and characterise an allelic hierarchy associated with alternate and distinct clinical manifestations including Auditory Processing Disorder (APD), Developmental Language Disorder (DLD) and Usher syndrome.

Materials and Methods: This investigation combines whole genome sequencing data from a family affected by a severe form of APD and speech dysarthria with genetic characterisation of two large population cohorts; the Avon Longitudinal Study of Parents and Children (ALSPAC) and UK10K. To compliment human findings, a mouse model with a genetic knockout of the rodent homolog Ush2a was assessed for auditory processing and vocalizations.

Results: Combined human and mouse results show: (1) heterozygous USH2A disruptions lead to impaired low-frequency sound perception; (2) low-level hearing impairments caused by heterozygous disruption of Ush2a associate with persistent higher-order acoustic processing deficits and altered vocalizations in mice; and (3) risk variants of USH2A combined with altered low-frequency hearing thresholds lead to significantly worse language outcomes in humans.

Conclusions: We identify a complex, interactive genetic mechanism under which variants in USH2A contribute to low-frequency hearing loss and alter higher-order auditory processing increasing the risk of language disorder. Since these variants are found in 0.85% of the population, we recommend comprehensive genetic screening to enable the early identification of carrier individuals alongside specific assessment of low-frequency hearing in individuals at risk.

P.A. Perrino: None. L. Nedevska: None. R. Reader: None. A. Hill: None. A.R. Rendall: None. H.S. Mountford: None. A.N. Buscarello: None. N. Lahiri: None. A. Saggar: None. R.H. Fitch: None. D.F. Newbury: None.

P02.07A Expanding the mechanisms underlying CEP290 pleiotropy: about 4 cases

I. Barny1, I. Perrault1, S. Thomas1, T. Attié-Bitach1, M. Rio2, C. Hamel3, H. Dollfus4, S. Defoort-Dhellemmes5, J. Kaplan1, J. Rozet1, X. Gerard1

1Institut Imagine, Paris, France, 2IHU Necker Enfants Malades, Paris, France, 3Institut des Neurosciences de Montpellier, Montpellier, France, 4Centre de référence pour les affections génétiques ophtalmologiques CARGO, Strasbourg, France, 5CHRU de Lille, Lille, France

Introduction: CEP290 is pivotal for the assembly/maintenance of primary and motile cilia in a wide range of cells. Consistently, CEP290 mutations cause a spectrum of ciliopathies ranging from Leber congenital amaurosis (LCA10) to embryo-lethal Meckel syndrome (MKS). LCA10 manifests invariably as a congenital and dramatically severe cone-dominant disease with visual function reduced to light perception. Here, we report 4 individuals with some preserved vision despite biallelism for presumably severe CEP290 mutations and functional analysis in fibroblasts we performed to better understand the molecular bases of CEP290 pleiotropy.

Materials and Methods: in silico, mRNA, protein and ciliation analyses were performed in P1 (p.Ile556Phefs*17, homozygous), P2 (p.Lys170*/p.Glu1364*), P3.1 and P3.2 (c.1824+3A>G/ p.Asn2290Lysfs*6) and controls.

Results: mRNA analysis of P1 and P2 mRNA revealed spontaneous skipping of exons encompassing the premature stop codons by mechanisms involving basal exon-skipping (BES) and nonsense-associated altered splicing (NAS) individually or in combination. Protein analysis detected small amounts of a CEP290 protein and unaltered ciliation ability compared to controls. In contrast, we detected no BES or NAS which could have enabled the production of PTC-free CEP290 isoforms from the mutant allele carrying the c.6869dup in the fibroblasts from P3.1 and P3.2. However, we detected some correctly spliced CEP290 mRNAs transcribed from the second allele carrying the c.1824+3A>G mutation and small amounts of the protein by Western blot analysis.

Conclusion: Here, we report that CEP290 pleiotropy involves splicing regulation through different mechanisms that can act individually or in combination and include NAS, BES and hypomorphic consensus splice site mutations.

I. Barny: None. I. Perrault: None. S. Thomas: None. T. Attié-Bitach: None. M. Rio: None. C. Hamel: None. H. Dollfus: None. S. Defoort-Dhellemmes: None. J. Kaplan: None. J. Rozet: None. X. Gerard: None.

P02.08B The role of HCN1 variation in circadian chronotypes

S. Smieszek1, C. P. S. Polymeropoulos2, M. Polymeropoulos2

1Vanda Pharmaceuticals, WASHINGTON, DC, United States, 2Vanda Pharmaceuticals, DC, OH, United States

To ascertain the genetic risk factors for morningness/eveningness phenotypes we conducted a genome-wide association analysis using 316 whole genome sequencing samples. We have directly tested the association between SNPs and MEQ (morning-evening questionnaire). We computed association test results by linear regression assuming additive allelic effects (covariates: age, gender, and the 3 PCs). We detect a large region on chromosome 5 (more than ~400 adjacent SNPs in LD, spanning ~2mb) centered within HCN1, Brain Cyclic Nucleotide-Gated Channel 1. Regional enrichment yields a p-value of < 10-99. It is highly expressed in the brain and potentially modulates excitability in the brain and responding to regulation by cyclic nucleotides, playing a critical role in shaping the autonomous activity of single neurons and the periodicity of network oscillations. It has been shown in a double-mutant mouse model that lack of HNC1 mediated feedback in rod photoreceptor cells prolongs rod responses and saturates the downstream retinal network during bright light stimulation. The risk allele is effectively correlated with lower MEQ score hence evening phenotype. The locus has been shown to be a significant (1.6e-09) eQTL for HCN1 in GTEX. HCN1 channel is responsible for the feedback on the rods regulating the dynamic range of light reactivity under dim or intermediate light conditions. We hypothesize that if this feedback is not functioning properly an individual may get saturated with even dim light resulting in misperception of the light conditions resulting in a circadian delay. This would suggest that HCN1 variations may directly impact the ME phenotype.

S. Smieszek: None. C.P.S. Polymeropoulos: A. Employment (full or part-time); Modest; Vanda Pharmaceuticals. M. Polymeropoulos: A. Employment (full or part-time); Modest; Vanda Pharmaceuticals.

P02.09C Clinical utility of panel-based genetic testing in infants and young children with inherited eye disease

E. Lenassi1, S. Ramsden2, J. Clayton-Smith2, S. Douzgou2, G. Hall2, V. Sharma3, I. Lloyd4, J. L. Ashworth3, S. Biswas3, G. C. Black1, P. Sergouniotis1

1University of Manchester, Manchester, United Kingdom, 2Manchester Centre for Genomic Medicine, St Mary’s Hospital, Manchester, United Kingdom, 3Manchester Royal Eye Hospital, Manchester, United Kingdom, 4Great Ormond Street Hospital for Children, London, United Kingdom

Introduction: Advances in ocular genetics have transformed our understanding of inherited eye diseases (IEDs) and have led to the development of powerful diagnostic tests. However, integration of these tests into routine healthcare is frequently ineffective. Providing robust evidence of benefit can accelerate implementation; for example, the adoption of a genetic test is much more likely when its clinical utility (i.e. its ability to influence management and health outcomes) has been clearly demonstrated. Here we assess the current clinical utility of genetic testing in paediatric IED.

Materials and Methods: Unrelated children (0-5 years old) with IED were retrospectively ascertained through the database of the UK North West Genomic Laboratory Hub. The cohort was consecutively collected and included individuals presenting between 09/2011 and 08/2018. All study participants were examined in tertiary paediatric ophthalmic genetic clinics and underwent panel-based genetic testing.

Results: 229 children were evaluated including 33 with albinism, 83 with bilateral paediatric cataracts, 19 with congenital glaucoma, 7 with ectopia lentis and 57 with inherited retinal disorders. The diagnostic yield was highest for albinism and ectopia lentis and lowest for congenital glaucoma. Genetic testing altered management (e.g. prevented additional investigations or led to the introduction of personalised surveillance measures or determined eligibility for treatment trials) in significant subsets of children with ectopia lentis, albinism, bilateral cataracts and inherited retinal disorders.

Conclusions: Genetic testing helped identify an aetiological diagnosis in the majority of children with IED. This prevented additional unnecessary testing and provided the opportunity for anticipatory guidance in a subset of patients that could be precisely defined.

E. Lenassi: None. S. Ramsden: None. J. Clayton-Smith: None. S. Douzgou: None. G. Hall: None. V. Sharma: None. I. Lloyd: None. J.L. Ashworth: None. S. Biswas: None. G.C. Black: None. P. Sergouniotis: None.

P02.11A Founder haplotype bearing mutation c.1621C>T (p.Gln541*) in the FYCO1 gene causing of autosomal recessive cataract (CTRCT18) in the Sakha Republic of Russia

N. A. Barashkov1,2, L. S. Vychuzhina1, A. V. Solovyev1,2, F. M. Teryutin1, V. G. Pshennikova1,2, T. E. Burtseva1,2, M. I. Tomsky1, F. A. Platonov2, G. P. Romanov2, N. N. Gotovtsev1, E. K. Khusnutdinova3,4, O. L. Posukh5,6, S. A. Fedororva1,2

1Yakut Scientific Centre of Complex Medical Problems, Siberian Branch of the Russian Academy of Medical Sciences, Yakutsk, Russian Federation, 2M.K. Ammosov North-Eastern Federal University, Yakutsk, Russian Federation, 3Ufa Federal Research Center of Russian Academy Sciences, Institute of Biochemistry and Genetics, Ufa, Russian Federation, 4Bashkir State University, Ufa, Russian Federation, 5Federal Research Center Institute of Cytology and Genetics, Novosibirsk, Russian Federation, 6Novosibirsk State University, Novosibirsk, Russian Federation

Novel nonsense mutation c.1621C>T (p.Gln541*) in the FYCO1 gene was previously revealed by whole exome sequencing as the main genetic cause of congenital autosomal recessive cataract (CTRCT18) in patients from the Sakha Republic (Eastern Siberia). In this report we present the results of the c.1621C>T (p.Gln541*) carrier frequency analysis in 424 adult individuals without of visual impairments from 7 populations of Eastern Siberia (Russians, Yakuts, Evenks, Evens, Dolgans, Yukaghirs and Chukchi). The highest carrier frequency of this mutation was found in Yakut population (7.9%), the lowest in Evenks (1.7%) and Evens (2.0%), and c.1621C>T (p.Gln541*) was absent in Russians, Yukaghirs, Dolgans and Chukchi. Common haplotypes for c.1621C>T (p.Gln541*) were reconstructed as a result of genotyping of 6 STR markers flanking the FYCO1 gene in 25 patients homozygous for c.1621C>T (p.Gln541*) and in 114 patients without this mutation. Common haplotypes bearing c.1621C>T (p.Gln541*) indicate the role of founder effect in the spread of this mutation in the Sakha Republic. The highest diversity of the c.1621C>T-haplotypes was revealed in the Central Yakuts ethno-territorial group. The mutant haplotypes of the Vilyui and Northern Yakuts groups are probably derived from the c.1621C>T-haplotypes found in the Central Yakuts group. Our results suggest that mutation c.1621C>T (p.Gln541*) in the FYCO1 gene causing CTRCT18 arose about 260±65 years ago (in the middle of the XVIII century) and spread among Yakut population as a result of founder effect. This study was supported by the Ministry of Education and Science of the Russia (#6.1766.2017) and the RFBR (18-05-600035_Arctica).

N.A. Barashkov: None. L.S. Vychuzhina: None. A.V. Solovyev: None. F.M. Teryutin: None. V.G. Pshennikova: None. T.E. Burtseva: None. M.I. Tomsky: None. F.A. Platonov: None. G.P. Romanov: None. N.N. Gotovtsev: None. E.K. Khusnutdinova: None. O.L. Posukh: None. S.A. Fedororva: None.

P02.12B Clinical Exome Sequencing reveals the genetic cause in a family with congenital cataract

E. Louizou, S. Rapti, A. Vasiageorgi, G. Kapetsis, E. Tsitsopoulos, C. Yfanti

Molecular Genetics Department, Bioiatriki S.A., Athens, Greece

Introduction: Cataracts are the principal cause of treatable blindness worldwide. Inherited congenital cataract shows all types of inheritance patterns in a syndromic and non-syndromic form. Congenital Cataract is an opacification of the lens, usually diagnosed at birth and without proper treatment, the vision never develops normally. In this study we present a family from Central Greece with six affected members born with Congenital Cataract, five males and one female, all of which had bilateral nuclear cataracts as newborns. Despite efforts to medically improve their condition, many members had a gradual deterioration of their vision which led to blindness, before the age of thirty years old.

Materials and Methods: Clinical Exome Sequencing (CES) was performed with the Sophia Genetics Protocol on a MiSeq Illumina platform, in one of the affected probands. In order to confirm our finding and to investigate the carrier status of most family members, Sanger sequencing was performed for the region of interest.

Results: The analysis revealed a possibly pathologic mutation -c.3808C>T(p.Gln1270*) -on the NHS gene, which is located on the X chromosome. The mutation has not been previously recorded in any database but the NHS gene is known to be the cause of X-linked Congenital Cataract - Nans Hooran Syndrome.

Conclusion: CES of related subjects from an isolated family with Congenital Cataract identified a causal NHS mutation and confirmed the mode of inheritance in the family as X-linked recessive. This finding will aid dramatically the clinical management of this whole family.

E. Louizou: None. S. Rapti: None. A. Vasiageorgi: None. G. Kapetsis: None. E. Tsitsopoulos: None. C. Yfanti: None.

P02.13C A heterozygous likely pathogenic variant in MAF transactivation domain as a cause of bilateral congenital cataract without intellectual disability or seizures

L. Lovrecic, A. Maver, I. Vrečar, B. Peterlin

University Medical Center Ljubljana, Clinical Institute of Medical Genetics, Ljubljana, Slovenia

Introduction: Congenital cataract is a developmental disorder of the lens resulting in lens cloudiness or opacities. The hereditary congenital cataracts are genetically heterogeneous and can be transmitted under an autosomal dominant, recessive or X-linked inheritance model. The MAF gene (OMIM*177075) pathogenic sequence variants in the c-terminal DNA binding domain have been shown to cause autosomal dominant isolated congenital cataract phenotype. On the other hand, MAF N-terminal transactivation domain genetic variants are associated with Ayme-Gripp syndrome (OMIM#601088), in which the eye anomalies are accompanied by intellectual disability, sensorineural hearing loss, seizures, distinctive facial appearance, reduced growth and skeletal anomalies.

Materials and Methods: A female patient, law graduate by profession, was referred to genetic consultation in her first pregnancy for her history of bilateral congenital cataract. Other medical history was unremarkable, except for mild delay in achieving developmental milestones and mild unilateral sensorineural hearing loss. She never needed hearing aid. General examination at 30 years showed normal height and distinctive flat facial appearance. Clinical exome sequencing was used to test for causative variants in 110 known genes associated with isolated and syndromic cataract.

Results: A heterozygous likely pathogenic de novo missense variant c.188C>G (Pro63Arg) in the N-terminal transactivation domain of the MAF gene was identified.

Conclusions: This is the first report of pathogenic variant in the N-terminal transactivation domain of the MAF gene in an individual without intellectual disability/learning difficulties, seizures or significant hearing loss.

L. Lovrecic: None. A. Maver: None. I. Vrečar: None. B. Peterlin: None.

P02.15A Important contribution of STRC copy number variations to the development of mild-to-moderate hearing loss

B. Harasimowicz1, D. Ozieblo1,2, 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/Kajetany, Poland

Background: Copy number variations (CNVs) are common cause of genetically determined hearing loss (HL). CNVs are frequently found in the STRC gene which is related to autosomal recessive HL. Due to presence of STRC pseudogene and technological limitation of CNVs detection, large deletions and duplications encompassing STRC are still an underestimated contributor to HL development.

Material and Methods: A group of 50 patients with mild-to-moderate HL diagnosed before the age of 10 and with no DFNB1 pathogenic was recruited for the study. All patients were tested for CNVs in STRC gene using quantitative comparative fluorescent PCR (QF-PCR) with primers specific to the STRC gene and its pseudogene followed by multiplex-ligation probe amplification (MLPA).

Results: Genetic prescreen with QF-PCR revealed a complete deletion of the STRC tested regions in 24% (24/100) and partial deletion in 5% of the studied alleles (5/100). This method allowed us also to identify two complete and two partial deletion of tested regions in the STRC pseudogene. Complete deletions of the STRC gene regions were fully confirmed by MLPA. One additional STRC partial deletion and one duplication were also detected. Considering the genotypes, in 26% (13/50) of HL patients homozygous or compound heterozygous CNVs were detected. In 10% (5/50) of patients only simple heterozygous CNVs was found.

Conclusions: This study shows that CNVs in STRC are a frequent cause of mild-to-moderate hearing loss in Polish patients. There is a strong need to include CNVs analysis of STRC gene in the standard HL diagnostic workflow.

B. Harasimowicz: None. D. Ozieblo: None. H. Skarzynski: None. M. Oldak: None.

P02.16B Genetic analysis of 320 Iranian patients affected by deafness using Sanger sequencing and Next Generation Sequencing (NGS) methods

S. Morovvati1, M. Ajallooeian2, N. Ayoubzadeh3

1Human Genetic Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran, Islamic Republic of, 2Baqiyatallah University of Medical Sciences, Tehran, Iran, Islamic Republic of, 3Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran, Islamic Republic of

Introduction: Hearing loss (HL) is one of the most common congenital impairments which occurs one in every thousand children. More than half of these cases are caused by genetic factors. At the present time, more than 150 genes have been identified to be associated with hearing loss.

Materials and Methods: In this study, for more than 600 patients affected by deafness, genetic counseling was done. 320 patients selected for genetic analysis. In first step, for all selected patients, analysis of GJB2 gene was done. Then, for patients who had normal result for GJB2 gene, hearing loss genetic panel (including 127 genes involved in deafness) was performed using NGS method. Finally, for patients for whom no pathogenic mutation was detected in their genetic panel, Whole Exome Sequencing (WES) was done by NGS method.

Results: 19 different pathogenic mutations were found GJB2 gene in 92 out of 320 patients (28.75%). Deafness genetic panel and WES were performed for 65 and 57 patients with normal GJB2 test respectively. Pathogenic and likely pathogenic mutations were detected in 75 subjects including 25 novel mutations.

Discussion: Due to the prevalence of consanguine marriages in Iran, autosomal recessive hearing loss is common in our country. Genetic analysis, especially WES can be very helpful in preventing the birth of deaf children.

S. Morovvati: None. M. Ajallooeian: None. N. Ayoubzadeh: None.

P02.17C Gene-wise burden analysis on painful diabetic neuropathy

E. Salvi1, M. Marchi1, M. Gerrits2, R. Almomani2, I. D’Amato1, R. A. Malik3,4, D. Ziegler5,6, G. J. Bönhof6, I. S. J. Merkies2,7, C. G. Faber2, G. Lauria Pinter1,8, PROPANE study group

1Neuroalgology Unit - Fondazione IRCCS Istituto Neurologico “Carlo Besta, Milano, Italy, 2Clinical Genetics and Department of Neurology, Maastricht University Medical Center, Maastricht, Netherlands, 3Institute of Human Development, Centre for Endocrinology and Diabetes, University of Manchester and Central Manchester NHS Foundation Trust, Manchester Academic Health Science Center, Manchester, United Kingdom, 4Department of Medicine, Weill Cornell Medicine, Doha, Qatar, 5Institute for Clinical Diabetology, German Diabetes Center, Leibniz Center for Diabetes Research, Heinrich Heine University, Düsseldorf, Germany, 6Department of Endocrinology and Diabetology, Medical Faculty, Heinrich Heine University, Düsseldorf, Germany, 7Department of Neurology, St. Elisabeth Hospital, Willemstad, Curaçao, 8Department of Biomedical and Clinical Sciences "Luigi Sacco", University of Milan, Milano, Italy

Introduction: Neuropathic pain is one of the most common complication of diabetic patients. It is a complex trait influenced by multiple genes and environmental factors. A debated hypothesis is that many rare variants might contribute to the missing heritability unexplained by common variants. Since frequencies of rare variants are very low, even with high penetrance, it is challenging to detect association. Burden tests collapse the rare variants within the gene as a single genetic variable. This study proposes a gene-wise burden test to look for genes with an overrepresentation of variations in painful (PDN) versus painless (PLDN) diabetic neuropathy patients.

Material and Methods: 107 candidate genes were sequenced by single molecule Molecular Probes-Next Generation Sequencing (MIP-NGS) in 61 PDN and 125 PLDN patients. To test differences in the mutational burden between PDN and PLDN, we applied SKAT-O test as implemented in EPACTS on all non-synonymous variants with a maximum minor allele frequency of 5%.

Results: Comparing the excess of rare variants in PDN respect to PLDN, P2RX7 and SCN11A showed the lowest p-values (p=0.01 and p=0.03, respectively) even if they did not reach the significant Bonferroni threshold.

Conclusions: The burden approach provided biologically plausible signals that would have remained indistinguishable from random noise with the traditional single-variant analysis, because of high allelic heterogeneity. Our results could leave the possibility that a combination of protein-altering variants in P2RX7 and SCN11A contribute to the risk of PDN. The work has been supported by FP7 PROPANE (602273) and the H2020-MSCA PAIN-Net (721841) Grants.

E. Salvi: None. M. Marchi: None. M. Gerrits: None. R. Almomani: None. I. DAmato: None. R.A. Malik: None. D. Ziegler: None. G.J. Bönhof: None. I.S.J. Merkies: None. C.G. Faber: None. G. Lauria Pinter: None.

P02.18D Genetic causes of non-syndromic bilateral deafness in North-Eastern Slovenia

A. erjavec skerget, B. zagradisnik, D. krgović, A. golub, J. rebol, A. kravos, N. kokalj vokač

university Medical Centre Maribor, maribor, Slovenia

Introduction: Mutations in the gene encoding protein connexin 26 (GJB2) have been shown as a major contributor to prelingual, sensorineural, nonsyndromic, recessive deafness. One specific mutation, 35delG, has accounted for the majority of the mutations detected in the GJB2 gene in Caucasian populations. The purpose of our study was to evaluate the characterisation and the prevalence of all, GJB2, GJB6 and other known mutations among affected individuals from North Eastern Slovenia.

Materials and Methods: There were 33 individuals diagnosed with hereditary deafness (DF) enrolled in the study. The allele specific PCR, MLPA technique and the Sanger sequencing was used to screen the GJB2 coding region. NGS sequencing with Oto-GeneSGKit® was used in 10 probands with bilateral, pre-lingual non syndromic DF with a negative medical history related to potential causes of acquired DF.

Results: The overall diagnostic yield of the DF cohort was 8/33 (24.2%). The homozygous mutation c.35delG was identified in four out of 33 patients (12.1%). By five patients mono-allelic mutation c.35delG was detected (15.15%; 5/33). Three among them were compound heterozygotes: c.35delG variant compounded with variants: c.109G>A; WTIVS 1+1: G>A; and c.235delC. NGS detected one homozygous disease-causing variant c.2464C>T in OTOF gene in one patient (10%), which is also heterozygote for the c.35delG.

Conclusion: The relative low ratio of individuals homozygous (12.1%) and heterozygous (15.15%) for the c.35delG mutation suggest that there are other genes responsible for nonsyndromic deafness in the North-Eastern Slovenian population. In one out of 10 tested probands with NGS this was confirmed.

A. erjavec skerget: None. B. zagradisnik: None. D. krgović: None. A. golub: None. J. rebol: None. A. kravos: None. N. kokalj vokač: None.

P02.19A Investigating the genetic architecture of fuchs endothelial corneal dystrophy (FECD) in a genetically refined cohort of patients

A. N. Sadan1, N. J. Hafford-Tear1, K. Muthusamy1,2, L. Dudakova3, P. Skalicka3,4, P. Liskova3,4, A. J. Hardcastle1, S. J. Tuft1,2, N. Pontikos1, A. E. Davidson1

1UCL Institute of Ophthalmology, London, United Kingdom, 2Moorfields Eye Hospital, London, United Kingdom, 3Department of Ophthalmology, First Faculty of Medicine, Charles University and General University Hospital in Prague, Prague, Czech Republic, 4Research Unit for Rare Diseases; First Faculty of Medicine, Charles University and General University Hospital in Prague, Prague, Czech Republic

Introduction: Fuchs endothelial corneal dystrophy (FECD) is a degenerative condition affecting up to 4.5% of the population over the age of 50 years. Symptoms consequently result in a reduction in vision. An intronic CTG triplet-repeat expansion (termed CTG18.1) situated in TCF4 has been significantly associated with disease.

Material and methods: DNA samples were obtained from British and Czech FECD patients (n=679). Samples were genotyped for CTG18.1 using a short tandem repeat (STR) assay and triplet-primed-PCR. Individuals harbouring bi-allelic non-expanded CTG18.1 alleles (<50 repeats) were analysed by whole exome sequencing (WES) and when possible followed up for familial history and deep phenotyping. WES data was interrogated for rare deleterious variants (minor allele frequency [MAF] <0.5% and CADD>20) in genes previously associated with corneal disease and genes with a defined functional role within the cornea. Samples without an identifiable rare causal variant, are currently being analysed by a gene burden analysis using an internal WES dataset as the control group.

Results: 20% (138/679) of the FECD patients had non-expanded copies of CTG18.1. Interrogation of WES data identified several candidate disease-associated variants, including COL8A2 and ZEB1 variants in atypical early-onset cases. Results of the gene burden analysis presented a number of potentially novel genes within the refined patient cohort.

Conclusion: This study reveals the incidence of non CTG18.1-mediated FECD in our cohort and aims to provide insight into the genetic causes of disease within this genetically refined patient group. This project is funded by the National Eye Research Centre and Rosetrees Trust.

A.N. Sadan: None. N.J. Hafford-Tear: None. K. Muthusamy: None. L. Dudakova: None. P. Skalicka: None. P. Liskova: None. A.J. Hardcastle: None. S.J. Tuft: None. N. Pontikos: None. A.E. Davidson: None.

P02.20B Clinical manifestations and novel SOX10 pathogenic variants in Danish probands with profound hearing impairment and malformations of the semicircular canals

M. Fjellberg Moldenæs1, N. Dahl Rendtorff2, L. Sandbjerg Hindbæk2, Ø. Nilssen1,3, L. Tranebjærg2,4

1Department of Clinical Medicine, University of Tromsø, Tromsø, Norway, 2The Kennedy Centre, Department of Clinical Genetics, Copenhagen University hospital, Copenhagen, Denmark, 3Department of Medical Genetics, University Hospital of North-Norway, Tromsø, Norway, 4Institute of Clinical Medicine, University of Copenhagen, Copenhagen, Denmark

Introduction: Waardenburg- and CHARGE syndromes are associated with overlapping clinical manifestations such as hearing impairment and malformations of the semicircular canals. The syndromes are heterogeneous, both with respect to the genes involved and the clinical abnormalities found.

Materials and Methods: Eleven patients from eight families were shown to have bilateral profound hearing impairment with variable additional clinical features. Complete or partial agenesis of the semicircular canals gave suspicion of either CHARGE or Waardenburg syndrome.

Results: Genetic testing was carried out which included genes involved in these two syndromes. Here we present eight families harboring SOX10 pathogenic variants. The patients demonstrated a spectrum of SOX10 mutations of which six were novel, six occurred de novo and two were found to be dominantly inherited. Both familial cases presented with novel SOX10 mutations.

Conclusions: The results presented here add six novel pathogenic variants, one missense and four frameshift variants, to the spectrum of SOX10 mutations and associated clinical features involved in Waardenburg syndrome are described. Grant references: University of Tromsø - The Arctic University of Norway.

M. Fjellberg Moldenæs: None. N. Dahl Rendtorff: None. L. Sandbjerg Hindbæk: None. Ø. Nilssen: None. L. Tranebjærg: None.

P02.21C Targeted Next-Generation Sequencing (NGS) gene panel testing in 200 Danish individuals with primary non-syndromic hearing impairment

N. D. Rendtorff1, H. G. Karstensen1, L. S. Hindbæk1, L. Tranebjærg1,2

1The Kennedy Centre, Copenhagen, Denmark, 2Institute of Clinical Medicine, Copenhagen, Denmark

Introduction: Hearing impairment is the most common sensory impairment and is genetically heterogeneous. Identification of the causative variants underlying hearing impairment is challenging, since >100 different genes for non-syndromic hearing impairment have so far been reported.

Materials and Methods: In this study, 200 index patients, mostly of Danish origin, with hearing impairment underwent targeted Next-Generation Sequencing (NGS) using hearing impairment gene panels for Illumina platforms. Causative variants were confirmed by Sanger sequencing and segregation analysis was performed when relevant samples were available. Prior to NGS sequencing, DFNB1 and SLC26A4-related hearing impairment had been excluded by Sanger sequencing in most patients.

Result: Of the 200 patients sequenced, we were able to detect causative variants in 73 cases (37%). Furthermore, in an additional 12 cases (6%) only one pathogenic variant was identified so far. The study emphasizes the genetic heterogeneity of hearing impairment since causative variants were found in 41 different genes. In total, 113 different likely pathogenic or pathogenic variants were detected. Forty-four variants (38%) were novel (absent from the literature).

Conclusions: Targeted Next-Generation Sequencing allowed us to detect causative variants in 73 of tested cases. In this cohort, the diagnostic yield was slightly higher in cases with presumed autosomal recessive hearing impairment and in patients with an early onset of hearing impairment. Copy number variation detection studies are ongoing. Regarding the non-solved cases, selected families will be analyzed by whole exome sequencing.

N.D. Rendtorff: None. H.G. Karstensen: None. L.S. Hindbæk: None. L. Tranebjærg: None.

P02.22D First reported Northern European with CABP2-related non-syndromic hearing loss

A. T. Højland1,2,3, H. Okkels1,4, M. B. Petersen1,3,2

1Research and Knowledge Center in Sensory Genetics, Aalborg University Hospital, Aalborg, Denmark, 2Department of Clinical Medicine, Aalborg University, Aalborg, Denmark, 3Department of Clinical Genetics, Aalborg University Hospital, Aalborg, Denmark, 4Section of Molecular Diagnostics, Department of Clinical Biochemistry, Aalborg University Hospital, Aalborg, Denmark

Introduction: Autosomal Recessive Deafness 93 is caused by homozygous mutations in the CABP2 gene located at 11q13.2. Five families with CABP2-related Autosomal Recessive Deafness 93 have been reported in PubMed. Four families (three Iranian and one Pakistani) are homozygous for the NM_016366.2:c.637+1G>T mutation and one Italian family is homozygous for the c.466G>T, p.(Glu156*) mutation.

Materials and Methods: We report an eight-year-old boy of non-consanguineous Danish Caucasian parents with prelingual bilateral moderate sensorineural non-syndromic hearing loss. Both parents had adult onset non-syndromic hearing loss and several members of the mother’s family also had early-onset hearing loss. Diagnostic whole-exome sequencing was performed using DNA from the index on the NextSeq 500 (Illumina Inc., San Diego, CA) using SureSelect CRE Exome Kit v2 (Agilent Technologies Inc., Santa Clara, CA). Data analysis was performed by an in-house pipeline limited to 123 known hearing loss genes using VarSeq version 2.0.2 (Golden Helix Inc., Bozeman, MT). The mutation was verified by direct sequencing.

Results: NM_016366.2:c.637+1G>T was found in a homozygous state in the index. No other causative variants were identified.

Conclusion: To our knowledge, this is the first time the NM_016366.2:c.637+1G>T mutation is identified in a patient of Caucasian descend and the first time CABP2-related non-syndromic hearing loss is reported in a patient of Northern European descend. Grants: None

A.T. Højland: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Modest; The Foundation for Promotion of Medical Science, The Hede Nielsen Family Foundation. B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Significant; The Obel Family Foundation, Jacob Madsen & wife Olga Madsen’s Foundation, Doctor Sofus Carl Emil Friis & wife Olga Doris Friis’s Foundation, Aase & Ejnar Danielsen’s Foundation, Axel Muusfeldt’s Foundation, L. F. Foght’s Foundation, The Augustinus Foundation. H. Okkels: None. M.B. Petersen: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Significant; Jacob Madsen & wife Olga Madsen’s Foundation, The Augustinus Foundation, Doctor Sofus Carl Emil Friis & wife Olga Doris Friis’s Foundation, Axel Muusfeldt’s Foundation, L.F. Foght’s Foundation.

P02.23A Presumed, unknown second mutation in GJB2 monoallelic patients is not a CNV in the DFNB1 (GJB2/GJB6) region, but it must exist

D. Safka Brozkova, P. Lassuthova, A. Uhrova Meszarosova, P. Seeman

DNA laboratory, Department of Pediatric Neurology, 2nd Medical School and University Hospital Motol, Prague, Czech Republic, Prague 5, Czech Republic

Introduction: In almost 40% of patients with autosomal recessive hearing loss, biallelic pathogenic mutations in the GJB2 gene are detected. For 6% patients with only one pathogenic GJB2 mutation (monoallelic) the second is unknown. Moreover, there is a significant excess of monoallelic among hearing loss population (6%) compared to the normal hearing population (3%). Therefore we assume the second causal mutation, yet unknown, in part of monallelic hearing loss patients which explain their hearing loss. Because of the character of GJB2 mutations, we expected to find the CNV, most likely one deletion within the critical region.

Materials and Methods: The CNV analysis was performed with SureSelect custom panel covering continuous chr13:20.735.021-21.102.144. Altogether 33 monoallelic patients were examined with use of three biallelic GJB2 patients as controls. The CNV analysis was performed with NextGene sw.

Results: The shared 1.6kb large deletion was found in four monoallelic. Nevertheless, the deletion was not possible to confirm with use of another independent method as Sanger sequencing or PCR. Even the haplotype analysis did not reveal any common haplotype in monoallelic. Moreover, the NGS of a gene panel of 71 recessive hearing loss genes did not reveal causal mutation in other gene in group of 15 monoallelic.

Conclusion: Therefore we believe the second unknown and undiscovered mutation which contribute and cause hearing loss in the substantial part of GJB2 monoallelic patients exist, but is probably not the CNV in the critical DFNB1 region.

Supported by: Ministry of Health of the CZ - nr 16-31921A

D. Safka Brozkova: None. P. Lassuthova: None. A. Uhrova Meszarosova: None. P. Seeman: None.

P02.24B First missense variant in N-terminal cytoplasmic region of KCNQ4: analysis of the genotype-phenotype correlation

M. Oldak1, A. Madejska1, D. Ozieblo1,2, M. Leja1,2, H. Skarzynski3

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

Introduction: Pathogenic variants in KCNQ4 are a well-known cause of autosomal dominant hearing loss (ADHL). There is a phenotypic variability observed in patients with different type and localization of KCNQ4 pathogenic variants. To date, only three frameshift mutations were described in the N-terminal cytoplasmic domain of KCNQ4 in ADHL families with late-onset and pure high frequency HL.

Materials and Methods: DNA was collected from a five-generation family with progressive high frequency ADHL. High throughput sequencing on proband’s DNA was performed. In family members (n=15) segregation analysis of the identified variants with HL was conducted using Sanger sequencing. Deep genotype-phenotype correlation analysis was performed using cross-sectional linear regression testing of pure tone audiometry results.

Results: Genetic testing revealed a novel, probably pathogenic c.274G>A (p.Glu92Lys) variant in KCNQ4, which fully segregated with HL in the studied family. Detected variant has not been reported in population databases and was classified as pathogenic. The p.Glu92Lys is the first missense variant identified in the N-terminal cytoplasmic region of KCNQ4. HL observed in the analyzed family was more severe at mid frequencies as compared to the previously published families with truncating variants located in this domain.

Conclusions: Identification of KCNQ4 p.Glu92Lys in a ADHL family confirms the association between missense KCNQ4 pathogenic variants and a high frequency HL with similar annual progression at mid and high frequencies. The data suggest that the type of KCNQ4 detected variants provides a better prognostic factor than their topological localization.

Supported by: NCN Research Grant no. 2016/22/E/NZ5/00470 SONATA BIS6.

M. Oldak: None. A. Madejska: None. D. Ozieblo: None. M. Leja: None. H. Skarzynski: None.

P02.25C Genetic basis of autosomal dominant hearing loss in pediatric patients

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

1Department of Genetics, Institute of Physiology and Pathology of Hearing, Warsaw/Kajetany, 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/Kajetany, Poland

Introduction: Autosomal Dominant Hearing Loss (ADHL) is the second most common form of inherited hearing loss with an onset after the second decade of life. Current knowledge on the genetic aspects of ADHL in Polish patients is limited, which significantly affects the diagnosis, genetic counselling and prevents prediction of disease progression.

Materials and Methods: Thirteen families with ADHL diagnosed before 18 years of age were enrolled in the study. DNA was isolated from peripheral blood or oral cavity swab samples from probands and family members. High-throughput genetic analysis using the TruSight One panel (Illumina Inc.) and the MiSeq sequencer was carried out for the probands. To confirm the presence of identified genetic variants and their segregation with ADHL in individual families Sanger’s sequencing was performed.

Results: Genetic cause of ADHL was identified in approximately 60% (8/13) of the families. The identified variants were located in ACTG1, COCH, DIAPH1, EYA4, KCNQ4, PTPRQ, TBC1D24 and TMC1. Among the identified variants approximately 60% (6/8) were novel. In the remaining families the selected variants did not segregate with ADHL.

Conclusions:Our results show high genetic heterogeneity of ADHL in Polish pediatric patients. Considering frequent identification of new genetic variants, it is necessary to perform thorough clinical examination and segregation analysis of the selected variants with ADHL in the largest possible number of family members. In patients with no genetic cause identified, the study area should be extended and include all protein coding regions or whole genome.

Supported by: NCN SonataBIS6 grant no. 2016/22/E/NZ5/00470

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

P02.27A Novel variants in known genes - results of genetic testing in families with autosomal dominant hearing loss

D. Ozieblo1,2, M. L. Leja1,2, A. Sarosiak1,2, 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/Kajetany, 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 usually characterized by postlingual age of onset and progression. To date 63 loci with 45 different genes were causally involved in the pathogenesis of ADHL.

Materials and Methods: Multigenerational families with ADHL were recruited in the study (n=19). Genomic DNA was isolated from whole blood and buccal swabs samples. In probands’ DNA samples a multigene high throughput sequencing was performed. Family segregation analysis of the identified variants was conducted using Sanger sequencing. All detected variants were analyzed in the context of population databases and literature. Pathogenicity of identified variants was predicted by different computational approaches.

Results: Genetic testing revealed probably pathogenic variants in almost 63% (12/19) of the analyzed families. The majority of identified variants were novel, previously not reported and hitherto not linked to the disease (7/12). Novel variants were missense changes and all of them (except for two genetic changes located in MYO6) were found in different genes.

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.

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

P02.28B Cochlear implantation outcome in patients with DFNB1 locus pathogenic variants - implications for precision medicine

D. Ozieblo1,2, A. Obrycka3, A. Lorens3, H. Skarzynski4, 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, 3Department of Implants and Auditory Perception, Institute of Physiology and Pathology of Hearing, Warsaw, Poland, 4Oto-Rhino-Laryngology Surgery Clinic, Institute of Physiology and Pathology of Hearing, Warsaw, Poland

Introduction: Almost 80% of children with profound prelingual hearing loss (HL) have a genetic cause of deafness; most often two GJB2/GJB6 (DFNB1 locus) recessive pathogenic variants. Cochlear implantation (CI) is a treatment of choice in profound HL patients but only few studies combine the etiology of HL with CI outcome.

Materials and Methods: Patients with profound prelingual HL who received CI before the age of 2 years and had a completed DFNB1 genetic testing were enrolled in the study (n=159). LittlEARS questionnaire and relative auditory development delay (RADD) at 6th month after CI activation were used to assess auditory development.

Results: Statistically significant differences were observed in RADD between patients implanted early (before 12 months of age) vs. late (after 12 months of age) and between patients with a short (≤6 months) vs. long (≥ 6 months) hearing aids (HAs) experience. Interestingly, in the most genetically homogenous patient group with two GJB2 c.35delG pathogenic variants there was no statistically significant difference in RADD between patients implanted early and late and between patients with a short and long HAs experience.

Conclusions: In children with homozygous GJB2 c.35delG pathogenic variants, application of CI before 12 or 24 months of age brings similar outcome. Children with an unknown genetic cause of HL should be implanted before 12 months of age to achieve better results in auditory development. Further studies in the non-DFNB1 group are planned to determine the link between genetic background of HL and the CI outcome.

Supported by: NCN Grant 2017/27/N/NZ5/02369

D. Ozieblo: None. A. Obrycka: None. A. Lorens: None. H. Skarzynski: None. M. Oldak: None.

P02.31A Hypermethylated genes and pathways in Polish children with high myopia

M. Gajecka1,2, S. Vishweswaraiah3, J. A. Karolak2,1, M. Mrugacz4, U. Ratnamala5, N. K. Mishra6, C. Guda6, S. S. Chettiar7, K. R. Johar7, U. Radhakrishna3, J. Swierkowska1

1Institute of Human Genetics, Polish Academy of Sciences, Poznan, Poland, 2Department of Genetics and Pharmaceutical Microbiology, Poznan University of Medical Sciences, Poznan, Poland, 3Department of Obstetrics and Gynecology, Oakland University William Beaumont School of Medicine, Royal Oak, MI, United States, 4Department of Ophthalmology and Eye Rehabilitation, Medical University of Bialystok, Bialystok, Poland, 5Department of Pharmacology, Creighton University, Omaha, NE, United States, 6Department of Genetics, Cell Biology & Anatomy College of Medicine, University of Nebraska Medical Center, Omaha, NE, United States, 7Department of Zoology, School of Sciences, Gujarat University, Ahmedabad, India

Introduction: High myopia (HM) is an eye disorder characterized by refractive error (RE) greater than -6.0 diopters (D) with both environmental and genetic factors involved. Although a number of loci, candidate genes, and sequence variants have been identified in HM, the general genetic factor explaining the causes of HM has not been specified. The aim of this study was to unravel the role of epigenetic changes in HM which could explain the relation between HM and environmental factors.

Materials and Methods: In order to verify if the dysregulated methylation of genes could contribute to the HM development, using a genome-wide DNA methylation array, we studied 18 Polish cases (aged 4-12 years, RE between -6.0 and -15.0 D) and 18 matched controls. CpGs with the highest fold change or the highest difference in the methylation level between cases and controls were analyzed. Pathway overrepresentation analyses among hypermethylated genes were performed using ConsensusPathDB.

Results: In total, 1,541 CpGs, representing 1,745 genes, were hypermethylated (≥2.0-fold change, FDR p≤0.05, ROC-AUC ≥0.75) in HM subjects compared to controls. The most hypermethylated CpGs with more than 20% difference in the methylation level between cases and controls were located in TIMM50, RAB3C, MICAL3, RWDD4A, XRCC2, and FARP2. Enrichment analysis of hypermethylated myopia-related genes set (>5 genes shared, p≤0.01) revealed overrepresentation of genes from several pathways, including signal transduction, transcription, and signaling pathways as receptor tyrosine kinases, JAK-STAT, TGF-beta, EGF-EGFR.

Conclusions: Methylation of genes and disruption of identified pathways could contribute to HM phenotype.

M. Gajecka: None. S. Vishweswaraiah: None. J.A. Karolak: None. M. Mrugacz: None. U. Ratnamala: None. N.K. Mishra: None. C. Guda: None. S.S. Chettiar: None. K.R. Johar: None. U. Radhakrishna: None. J. Swierkowska: None.

P02.32B Diagnostic yield of whole exome sequencing-based genetic testing for patients with inherited eye diseases

K. Wells, K. Kämpjärvi, E. Mårtensson, M. Mehine, J. Känsäkoski, L. Sarantaus, H. Västinsalo, J. Schleit, I. Saarinen, M. Muona, S. Myllykangas, T. Alastalo, J. W. Koskenvuo, S. Tuupanen

Blueprint Genetics, Helsinki, Finland

Genetic testing is essential in the diagnosis and management of inherited eye diseases (IEDs). However, IED diagnostics can be compromised by poorly validated and curated tests. We aimed to develop and validate a high quality whole exome sequencing (WES) based platform and to assess diagnostic yield in a large IED cohort. Performance of the WES assay with bespoke clinical content, was tested through next generation sequencing (NGS) of reference samples using the Illumina NovaSeq platform. The assay was designed to cover clinically-relevant intronic and difficult-to-sequence regions, and detect copy number variants (CNVs). Based on this assay, 23 ophthalmology gene panels were curated. The assay showed high average sequencing depth (183x) and coverage (99.7% regions covered >20x), and uniform coverage over difficult-to-sequence regions, including RPGR ORF15. Detection sensitivity was high for SNVs (0.998), INDELs (0.97), 1-exon CNVs (0.93) and 5-exon CNVs (0.99). The Usher Syndrome Panel gave the highest diagnostic yield (>80%). The most frequently ordered 266-gene Retinal Dystrophy Panel yielded a diagnosis in 58% (of 1587) cases. Diagnoses were made in a total of 111 genes, the most common genes being ABCA4, USH2A, RPGR, RHO and PRPH2, as well as in newly implicated IEDs genes, and in custom targeted deep intronic regions. Approximately 3% of patients had a diagnostic CNV. A WES assay with boosted clinical content provides high diagnostic yields for IED patients. The genetic variability of diagnoses in our cohort supports the use of comprehensive NGS panels in IED diagnostics, to optimize diagnostic yield and clinical care.

K. Wells: A. Employment (full or part-time); Significant; Blueprint genetics. K. Kämpjärvi: A. Employment (full or part-time); Significant; Blueprint genetics. E. Mårtensson: A. Employment (full or part-time); Significant; Blueprint genetics. M. Mehine: A. Employment (full or part-time); Significant; Blueprint genetics. J. Känsäkoski: A. Employment (full or part-time); Significant; Blueprint genetics. L. Sarantaus: A. Employment (full or part-time); Significant; Blueprint genetics. H. Västinsalo: A. Employment (full or part-time); Significant; Blueprint genetics. J. Schleit: A. Employment (full or part-time); Significant; Blueprint genetics. I. Saarinen: A. Employment (full or part-time); Significant; Blueprint genetics. M. Muona: A. Employment (full or part-time); Significant; Blueprint genetics. S. Myllykangas: E. Ownership Interest (stock, stock options, patent or other intellectual property); Significant; Blueprint genetics. T. Alastalo: E. Ownership Interest (stock, stock options, patent or other intellectual property); Significant; Blueprint genetics. J.W. Koskenvuo: E. Ownership Interest (stock, stock options, patent or other intellectual property); Significant; Blueprint genetics. S. Tuupanen: A. Employment (full or part-time); Significant; Blueprint genetics.

P02.33C New insights into the phenotypic spectrum of PROM1-associated retinopathy

M. del Pozo-Valero1, I. Martín-Mérida1,2, B. Jiménez-Rolando3, A. Arteche1, A. Ávila-Fernández1,2, F. Blanco-Kelly1, R. Riveiro-Álvarez1, C. Van Cauwenbergh4, E. De Baere4, C. Rivolta5, B. García-Sandoval3, M. Cortón1,2, C. Ayuso1,2

1Department of Genetics, Instituto de Investigación Sanitaria–Fundación Jiménez Díaz University Hospital (IIS-FJD-UAM), Madrid, Spain, 2Center for Biomedical Network Research on Rare Diseases (CIBERER), ISCIII, Madrid, Spain, 3Department of Ophthalmology, Instituto de Investigación Sanitaria–Fundación Jiménez Díaz University Hospital (IIS-FJD-UAM), Madrid, Spain, 4Center for Medical Genetics Ghent, Ghent University and Ghent University Hospital, Ghent, Belgium, 5Department of Computational Biology, Unit of Medical Genetics, University of Lausanne, Laussane, Switzerland

Purpose: Inherited retinal dystrophy (IRD) refers to a group of progressive and degenerative diseases that affect the photoreceptor cells and lead to visual impairment. One of the genes associated with IRD is PROM1. Disease-causing PROM1 variants have been associated with different phenotypes. The aim was to provide a detailed analysis of the genetic and phenotypic characteristics of the PROM1-associated retinopathy in a large cohort of patients.

Methods: PROM1 screening was performed using classical molecular techniques and/or targeted Next-Generation-Sequencing in a cohort of 2216 IRD families. Copy number variation analysis was carried out in unsolved monoallelic cases. Detailed ophthalmic evaluation was performed in 25 patients.

Results: Thirty-two families presented PROM1 variants, including likely pathogenic and unknown significance variants, 10 of which are novel. Causative variants were identified in 21 families segregating in autosomal recessive (17) or dominant (4) pattern, 3 of them with founder effect. Phenotypic analysis in 25 patients carrying disease-causing PROM1 variants revealed clinical heterogeneity regardless of genotype. Most of the patients suffered from cone-rod dystrophy and some patients presented with macular dystrophy or retinitis pigmentosa, all presenting macular damage.

Conclusions: Our study reports the largest and comprehensive description of genetic and clinical findings in PROM1-retinopathy. The prevalence of PROM1 variants that seems to reliably explain the IRD phenotype in our cohort is about 1%. This study also highlights the great heterogeneity of PROM1-associated phenotypes. Regardless of the initial diagnosis of primary cone or rod loss, all patients developed a common macular involvement, a characteristic phenotypic finding of PROM1.

M. del Pozo-Valero: None. I. Martín-Mérida: None. B. Jiménez-Rolando: None. A. Arteche: None. A. Ávila-Fernández: None. F. Blanco-Kelly: None. R. Riveiro-Álvarez: None. C. Van Cauwenbergh: None. E. De Baere: None. C. Rivolta: None. B. García-Sandoval: None. M. Cortón: None. C. Ayuso: None.

P02.35A Contribution of DRAM2 gene in inherited retinal dystrophies with early macular involvement

V. Abad-Morales1,2, S. Ruiz-Nogales1,2, R. Navarro1,3, P. Méndez1,2, M. Riera1,2, A. Burés-Jelstrup1,3, B. Corcóstegui1,3, E. Pomares1,2

1Fundació de Recerca de l’Institut de Microcirurgia Ocular, Barcelona, Spain, 2Department of Genetics, Institut de Microcirurgia Ocular (IMO), Barcelona, Spain, 3Department of Retina, Institut de Microcirurgia Ocular (IMO), Barcelona, Spain

Introduction: Macular Dystrophies (MD) are a group of Inherited Retinal Dystrophies (IRD) characterized by the degeneration of the central retina or macula, principally formed by cone photoreceptors. Although there are more than 55 MD genes, a considerable percentage of these cases remain unsolved.

Methods: Whole exome sequencing of a cohort of IRD families was performed, analyzing a panel of 300 IRD genes in order to determine the genetic pathogenic cause. For some new mutations, functional analyzes were ruled out from patients blood samples.

Results: Three new DRAM2 homozygous mutations were identified in three independent families: one frameshift and two splicing variants. All patients shared a singular phenotype, with an early macular involvement followed by a peripheral degeneration. However, the age of onset and the evolution of the pathology were different in each case. At a molecular level, functional studies revealed alterations in gene expression differing between patients.

Conclusions: DRAM2 was firstly associated to IRD in 2015, and only scanty cases and 10 different pathogenic mutations have been reported to date. Thus, its function in the retina and the associated phenotypes are still much unknown. In this context, the results obtained significantly contribute to the comprehension of the role of this gene in the molecular bases of IRD, increasing the number and type of reported pathogenic variants and their functional effects, and strengthening the DRAM2 genotype-phenotype correlations in the retina.

Grants: This study was supported by Fundació Privada Cellex (Fi-201501) and Fundació de Recerca de l’Institut de Microcirurgia Ocular (Fi-201401).

V. Abad-Morales: None. S. Ruiz-Nogales: None. R. Navarro: None. P. Méndez: None. M. Riera: None. A. Burés-Jelstrup: None. B. Corcóstegui: None. E. Pomares: None.

P02.36B Next generation sequencing in patients with bilateral optic atrophy: Leber hereditary optic neuropathy and beyond

M. Volk1, A. Maver1, A. Fakin2, N. Teran1, M. Jarc Vidmar2, S. Petrović2, M. Hawlina2, B. Peterlin1

1Clinical institute of medical genetics, UMC Ljubljana, Ljubljana, Slovenia, 2Eye hospital, UMC Ljubljana, Ljubljana, Slovenia

Leber hereditary optic neuropathy (LHON) is caused by missense mutations in the mitochondrial genes (mtDNA) encoding complex I subunits of the respiratory chain. However, mutations affecting genes in the nuclear genome may strikingly resemble the clinical presentation of LHON. We conducted a study to define best testing strategy using next generation sequencing in bilateral optic atrophy and inconclusive family history. Here we report on 30 unrelated individuals referred to our institution for genetic testing because of the optic nerve atrophy and clinically suspected LHON. None of the patients had lesions suggestive for multiple sclerosis on brain MRI. Genetic analysis consisted of mtDNA sequencing followed by clinical exome sequencing. In two unrelated patients mtDNA sequencing identified typical LHON pathogenic variants (m.11778G>A, m.3700G>A). Exome sequencing in three unrelated patients revealed OPA1 mutation (c.2489G>A, likely pathogenic variant), compound heterozygous mutations in ACO2 (c.2253dupC, VUS and c.719G>C, pathogenic variant) and a de novo mutation in WFS1 (c.2480C>T, likely pathogenic variant). Upon genetically guided clinical review, the last patient was found to exhibit signs on macular OCT, typical for Wolfram syndrome. We found possible causative variants in 5 of 30 patients with bilateral optic atrophy and clinically suspected LHON. In addition to typical LHON mitochondrial variants we found causative variants associated with autosomal dominant, autosomal recessive and syndromic forms of optic neuropathy and reclassified clinical diagnoses. Our results provide evidence that a combined approach with mtDNA and clinical exome sequencing is recommended in cases of clinically suspected LHON and negative family history.

M. Volk: None. A. Maver: None. A. Fakin: None. N. Teran: None. M. Jarc Vidmar: None. S. Petrović: None. M. Hawlina: None. B. Peterlin: None.

P02.37C Targeted next generation sequencing reveals homozygous PDE6C mutation in a pedigree with macular aplasia

K. Kamenarova1, S. Cherninkova2, K. Mihova1, F. Shakola1, V. Mitev1, I. Tournev2, R. Kaneva1

1Molecular Medicine Centre, Medical University – Sofia, Sofia, Bulgaria, 2Department of Neurology, University Hospital Alexandrovska, Medical University – Sofia, Sofia, Bulgaria

Introduction: Macular, or foveal aplasia refers to the lack of foveal depression with continuity of all neurosensory layers in the presumed location of the fovea. It has been reported in cases of aniridia, albinism, microphthalmia and achromatopsia. Inherited retinal dystrophies (IRD), among which macular aplasia (MA), constitute a group of diseases characterized by clinical variability and pronounced genetic heterogeneity. By using IRD-panel next-generation sequencing, we aimed to identify the disease-causing mutation in a large Bulgarian pedigree with MA inherited in autosomal-recessive pattern.

Materials and Methods: A large pedigree with MA, most likely achromatopsia, accompanied by nystagmus and decreased visual acuity presented from birth was recruited and DNA from the proband was sequenced using the Illumina® platform and TruSight One sequencing panel. Protein coding and splice-site variants were filtered in a panel with 341 genes related to retinal dystrophies.

Results: Genetic analysis identified a homozygous missense variant c.2141T>A (p.Ile714Asn) in a conserved amino acid within the gene encoding the catalytic subunit of the cone photoreceptor phosphodiesterase, PDE6C. The commonly used prediction tools classified the PDE6C-c.2141T>A variant to be putatively damaging/deleterious. There is only one PDE6C-c.2141A allele found in the Non-Finnish European population suggesting an extremely low frequency for this change. Segregation study is in progress.

Conclusions: We have identified a family affected by autosomal-recessive dystrophy of the central photoreceptor system and found homozygous possibly pathogenic variant in PDE6C, mutations in which have been previously reported in patients with autosomal-recessive inherited achromatopsia and early-onset cone photoreceptor dysfunction. Grant references: DUNK01/2/2009 and Grant-D-109/03.05.20018.

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

P02.38D Burden of missense variants in hearing loss genes in sporadic Meniere disease

A. Gallego-Martinez1, T. Requena1, P. Román-Naranjo1, J. Lopez-Escamez1,2

1Centro Pfizer-Universidad de Granada-Junta de Andalucía de Genómica e Investigación Oncológica (GENYO), Granada, Spain, 2Department of Otolaryngology, Instituto de Investigación Biosanitaria Ibs.GRANADA, Hospital Universitario Virgen de las Nieves, Granada, Spain

Introduction: Meniere disease (MD) is a rare inner ear disorder defined by episodes of vertigo, sensorineural hearing loss (SNHL) and tinnitus. MD has been described mostly in sporadic cases, being familial cases around 10% of total observed cases. Between 20-45% of the cases have bilateral hearing loss. The aim of this study is to investigate the burden of rare variants in SNHL genes in sporadic cases of MD.

Methods: We designed a targeted-sequencing panel including SNHL genes in supporting cells and sequenced 890 Spanish MD patients. The frequency of rare variants (with a MAF inferior to 0.1) in the gene panel was compared with three independent datasets as controls for the gene burden analysis. Interaction model between rare and common variants is proposed for most relevant genes in MD cases.

Results: Patients with sporadic MD showed a significant enrichment of missense variants in SNHL genes that was not found in the controls. The list of genes includes GJB2, USH1G, SLC26A4, ESRRB and CLDN14. A rare synonymous variant with unknown significance was found in the MARVELD2 gene in several unrelated patients with MD.

Conclusions: There is a burden of rare variation in certain SNHL genes in sporadic MD. Furthermore, the interaction of common and rare variants in SNHL genes may have and additive effect on MD phenotype.

Funding: FPS-PI0496-2014, Spain; EF-0247-2017 grant from Consejeria de Salud, Spain; Luxembourg National Research Fund (INTER/Mobility 17/11772209), Luxembourg, and 2016-MeniereSociety Grant, UK.

A. Gallego-Martinez: None. T. Requena: None. P. Román-Naranjo: None. J. Lopez-Escamez: None.

P02.39A Burden of rare variants in OTOF gene in familial Meniere disease

P. Roman-Naranjo1, C. A. Jimenez-Ruiz1, M. C. Moleon2, A. Gallego-Martinez1, J. A. Lopez-Escamez1,2

1Centre for Genomics and Oncological Research (GENYO), Granada, Spain, 2Department of Otolaryngology, Instituto de Investigación Biosanitaria ibs.GRANADA, Hospital Universitario Virgen de las Nieves, Granada, Spain

Introduction: Meniere disease (MD) is a rare inner ear disorder characterized by vertigo, sensorineural hearing loss (SNHL) and tinnitus. Familial aggregation is found in 10% of MD cases with autosomal dominant inheritance and several genes already described, such as DTNA, FAM136A and SEMA3D genes. Thus, the goal of this study is to define new candidate genes for familial MD (FMD).

Materials & Methods: We recruited 62 FMD cases to perform whole-exome sequencing. Variant calling was performed with GATK. Candidate genes were prioritized based on pathogenicity using PhenIX. Single rare variant analysis (SRVA) focused on SNHL genes was performed using MAF<0.001. Likewise, a gene burden analysis (GBA) with MAF<0.05 was performed to analyze the interaction of common and rare variants. We studied the relationship between rare and common variants which modulate gene expression.

Results: Forty-percent of FMD cases carried one novel or ultrarare likely pathogenic (CADD>15) variant in SNHL genes. Ninety-four rare variants were selected from SRVA. A total of 222 rare variants were retrieved with GBA resulting in a high enrichment of variants in OTOF gene (corrected p-value: 3x10-4). Four rare variants in our cohort showed a significant association with three common variants regulating the expression of OTOF gene.

Conclusions: There is a burden of rare variants in SNHL genes. Particularly, the interaction of rare and common variants in OTOF gene may play an important role in FMD.

Funding: Supported by the Luxembourg National Research Fund INTER/Mobility/17/11772209 and EF-0247-2017 from Andalusian Health Government to JALE.

P. Roman-Naranjo: None. C.A. Jimenez-Ruiz: None. M.C. Moleon: None. A. Gallego-Martinez: None. J.A. Lopez-Escamez: None.

P02.41C Differential expression analysis suggests lipid metabolism plays a role in North Carolina Macular Dystrophy

E. Tavares1, C. Tang1, A. Paterson1, S. Li1, M. Liang1, M. Wilson1, E. Campos1, A. Vincent1,2, E. Héon1,2

1The Hospital for Sick Children Research Institute, Toronto, ON, Canada, 2Department of Ophthalmology and Vision Sciences, The Hospital for Sick Children, University of Toronto, Toronto, ON, Canada

Introduction: North Carolina Macular Dystrophy (NCMD) is a high penetrance autosomal dominant inherited disease. The non-coding SNPs in the linkage region on chromosome 6 fall within a DNase I hypersensitive site upstream of PRDM13 and which regulates PRDM13 expression during development. This study aimed to understand the molecular basis of NCMD through differential expression analysis.

Methods: PSSM scanning was used to predict changes in binding of known transcription factors in retina for the non-coding variants on chromosome 6. RNA-seq from skin-derived RNA for three sibling pairs (affected/unaffected each) within a family was done for differential gene expression analysis.

Results: These variants introduce a gain of binding sites for two important retinal transcription factors (PAX6 and OTX2) upstream of PRDM13. This can potentially alter the PRDM13 regulation in retinal tissue. RNA-seq revealed ~50 differently expressed genes between the affected and unaffected. Although we did not find a direct association of these genes to the known genes involved in macular dystrophies, GO enrichment analysis showed ~30 statistically significant processes with lipid metabolism being most significant and it was previously linked to age-related macular dystrophy.

Conclusions: The introduction of PAX6 and OTX2 binding sites upstream of PRDM13 can potentially cause PRDM13 overexpression and may interfere with lipid metabolism and oxidative stress by increasing the amount of lipids in the retina.

Funding: Mira Godard, FFB, McLaughlin Centre.

E. Tavares: None. C. Tang: None. A. Paterson: None. S. Li: None. M. Liang: None. M. Wilson: None. E. Campos: None. A. Vincent: None. E. Héon: None.

P02.43A The most common forms of non-GJB2-related nonsyndromic (NSHL) and disguised syndromic (SHL) hearing impartment in Russian patients

O. L. Mironovich1, E. A. Bliznetz1, T. G. Markova2, M. R. Latayants2, T. V. Markova1, L. A. Bessonova1, M. S. Petukhova1, O. N. Makienko1, D. M. Guseva1, I. V. Anisimova1, O. P. Ryzhkova1, A. V. Polyakov1

1Federal State Budgetary Institution «Research Centre for Medical Genetics», Moscow, Russian Federation, 2Federal State Budgetary Institution «National Research Center for Audiology and Hearing Rehabilitation», Moscow, Russian Federation

Introduction: Hearing loss is a common disorder, and according to the audiological screening in Russia, congenital hearing impairment affects nearly 3 in every 1000 live births. Apart from the GJB2 gene, more than 100 genes responsible for NSHL and more than 400 genes associated with SHL are known. Currently, massive parallel sequencing (MPS) is the optimal diagnostic method for non-GJB2-related forms of hearing impairment.

Materials and Methods: DNA samples of 205 Russian patients with NSHL (without mutations in GJB2 gene) and 10 patients with the incoming diagnosis of Pendred, Branchiootorenal, Usher and Alstrom syndromes were tested by using custom targeted MPS panel which includes 33 genes of NSHL and SHL. 25 DNA samples were searched for abnormal copy numbers with MLPA.

Results: Among 205 patients with NSHL, pathogenic and probably pathogenic variants have been identified in 41 cases (20%) in following genes: STRC (10), USH2A (6), SLC26A4 (4), MYO7A (4), OTOF (4), POU3F4 (2), TECTA (2), TMPRSS3 (2), PTPRQ (1), ADGRV1 (1), TMC1 (1), LOXHD1 (1), ACTG1 (1), MYO15A (1), OTOA (1). Diagnostic efficiency in patients with SHL was 80% (8/10). In patients with NSHL, syndromic forms have been identified in 9% of total cases (18/205). The majority of causative mutations in STRC and OTOA were large CNVs (80% and 50%). Mutations c.11864G>A, c.2171_2174delTTTG, c.107A>C in USH2A, STRC, SLC26A4 genes are assumed to be frequent in Russian patients.

Conclusions: The proportion of detected genetic forms of hearing loss in Russia is about 12% of all non-syndromic hearing loss.

O.L. Mironovich: None. E.A. Bliznetz: None. T.G. Markova: None. M.R. Latayants: None. T.V. Markova: None. L.A. Bessonova: None. M.S. Petukhova: None. O.N. Makienko: None. D.M. Guseva: None. I.V. Anisimova: None. O.P. Ryzhkova: None. A.V. Polyakov: None.

P02.44B Prenatal diagnosis of Norrie disease after exome sequencing of affected proband during ongoing pregnancy

A. V. Marakhonov1,2, S. A. Repina1, I. A. Akimova1, M. V. Shurygina3, T. A. Vasilyeva1, S. I. Kutsev1,4, V. V. Kadyshev1, R. A. Zinchenko1,5

1Research Centre for Medical Genetics, Moscow, Russian Federation, 2Far Eastern Federal University, Vladivostok, Russian Federation, 3S. Fyodorov Eye Microsurgery Federal State Institution, Moscow, Russian Federation, 4Pirogov Russian National Research Medical University, Moscow, Russian Federation, 5Moscow Regional Research and Clinical Institute, Moscow, Russian Federation

Objectives: Hereditary ophthalmic pathology is a genetically heterogeneous group of diseases that occur either as an isolated eye disorder or as a symptom of hereditary syndromes (chromosomal and monogenic). That is why diagnostic search in some cases of ophthalmic pathology could be time- and cost-consuming. The most challenging situation could arise when prenatal diagnosis is needed during ongoing pregnancy.

Materials and Methods: A family is referred to the RCMG for affected child birth risk prognosis at 7-8 week of gestation because the previous child, six-years-old boy, has congenital aniridia, glaucoma, retinal detachment, severe psychomotor delay, lack of speech, several ophthalmic surgeries. The affected child had been previously tested for PAX6 mutations and 11p13 copy number variation which revealed no changes.

Results: Considering ongoing pregnancy, lack of pathogenic changes and precise diagnosis in an affected boy, NGS sequencing of clinically relevant genes was performed which revealed a novel hemizygous substitution NM_000266.3(NDP_v001):c.385G>T, p.(Glu129*), in NDP gene associated with Norrie disease (OMIM#310600). Subsequent Sanger validation of the affected boy and his mother confirmed identified substitution inherited in X-linked recessive mode. Amniotic fluid testing revealed the fetus is hemizygous for the variant. Complications were developed during subsequent interruption of pregnancy driven by medical necessity.

Conclusions: Clinical polymorphism of hereditary ophthalmic pathology could severely complicate the establishment of exact diagnosis and make it time- and cost-consuming. NGS appears to be the method-of-choice in complicated cases which could substantially hasten the establishment of diagnosis and genetic risk estimation.

Supported by RFBR grant № 19-015-00122.

A.V. Marakhonov: None. S.A. Repina: None. I.A. Akimova: None. M.V. Shurygina: None. T.A. Vasilyeva: None. S.I. Kutsev: None. V.V. Kadyshev: None. R.A. Zinchenko: None.

P02.45C Screening TYR gene variations in Turkish oculocutaneuse albinism patients

O. HATIRNAZ NG1, E. Yılmaz1, Z. Parlakgüneş2, K. Yararbaş3, S. Ziylan2, Y. Alanay3, U. Ozbek3

1Acibadem Mehmet Ali Aydınlar University, School of Medicine, Department of Medical Biology, ISTANBUL, Turkey, 2Yeditepe University, Medical Faculty, Department of Eye Diseases, ISTANBUL, Turkey, 3Acibadem Mehmet Ali Aydınlar University, School of Medicine, Department of Medical Genetics, ISTANBUL, Turkey

Introduction: Oculocutaneous albinism (OCA) is characterised by a generalized reduction in pigmentation of hair, skin and eyes and variable ocular findings including nystagmus, reduced visual acuity and photophobia. The most common mutated gene in OCA is TYROSINASE (TYR). Currently, no data available on the frequency or molecular background of Albinism in Turkey.

Materials and Methods: Forty OCA patients (23 male and 17 female, 3 pairs of siblings) with a median age 19.2 years (min: 6mo-max 70.1 years) were enrolled. Following DNA isolation, the coding regions of TYR genes were analysed by PCR and direct sequencing.

Results: We have detected 28 different TYR variations in 31(77.5%, 17 homozygous, 16 compound heterozygous and 1 heterozygous) of the OCA patients (Table1). The most frequent variants were c.815G>A, p.Trp272* and c.741C>A, p.Cyc247* with an allele frequency of 0.06. Among 28 variations five of them were novel (Table 1). The allele frequencies of two common TYR polymorphisms S192Y and R402Q were 34% and 14.6% respectfully.

Conclusion: TYR gene is commonly mutated in OCA patients in Turkey. The patient with only heterozygous TYR variant, addition to other 8 patients with no variation should be evaluated for other related genes. Further studies with new patients and investigation of other melanin related genes in TYR negative patients are ongoing.

Table 1: TYR variations and their allele frequencies detected in our patients.




coding effect

alle freq



NM_000372.4 c.815G>A


stop gain




NM_000372.4 c.741C>A


stop gain




NM_000372.4 c.996G>A






NM_000372.4c.1217 C>T






NM_000372.4 c.1204C>T


stop gain




NM_000372.4 c.1 A>G






NM_000372.4 c.573delA


stop gain




NM_000372.4 c.613C>A






NM_000372.4 c.230G>A






NM_000372.4 c.1118C>A






NM_000372.4 c.816G>C






NM_000372.4 c.446A>C






NM_000372.4 c.919_925delTCCAGAA


frame shift




NM_000372.4 c.616G>A






NM_000372.4 c.140G>A






NM_000372.4 c.61C>T






NM_000372.4 c.661G>T


stop gain




NM_000372.4 c.1058G>A






NM_000372.4 c.225T>A


stop gain




NM_000372.4 c.139G>T






NM_000372.4 c.1430G>A


stop gain




NM_000372.4 c.763C>T


stop gain




NM_000372.4 c.715C>T






NM_000372.4 c.1193A>G






NM_000372.4 c.1255_1256insTTG






NM_000372.4 c.1036-9_1041delTAATGAACAGGATTT


splice site




NM_000372.4 c.1036+3A>C


splice site




NM_000372.4 c.308G>C





O. Hatirnaz ng: None. E. Yılmaz: None. Z. Parlakgüneş: None. K. Yararbaş: None. S. Ziylan: None. Y. Alanay: None. U. Ozbek: None.

P02.46D Copy number analysis of the OPN1LW and OPN1MW genes by MLPA

A. S. Hoekstra1, L. Haer-Wigman2, M. Tjon-Pon-Fong2, M. Zegers1, M. Ketema1, R. Vijzelaar1

1MRC-Holland, Amsterdam, Netherlands, 2Radboud University Medical Center, Nijmegen, Netherlands

Introduction: X-linked cone dysfunction disorders such as Blue Cone Monochromacy and Cone Dystrophy are caused by pathogenic variants and rearrangements in OPN1LW and OPN1MW which share more than 98% sequence similarity. Located in tandem, they are susceptible to meiotic mispairing, unequal homologous and nonhomologous recombination. This might lead to different copy numbers of OPN1LW and/or OPN1MW or the formation of hybrid opsin genes. Because of the frequent exchange, X chromosomes have around one to five OPN1MW copies, whereas they rarely have more than one OPN1LW copy. Using the current diagnostic tests, it remains difficult to determine the copy number and presence of gene rearrangements in OPN1LW and OPN1MW.

Methods: A novel multiplex ligation-dependent probe amplification (MLPA) assay was developed and tested on 14 patient samples with a known copy number status of OPN1LW and OPN1MW. Copy number and rearrangements in OPN1LW and OPN1MW were analysed in more than 100 DNA samples.

Results: The copy number of OPN1LW and OPN1MW was confirmed in the 14 patients and could be easily determined in 75 male and female DNA samples and in 45 clinical samples consisting of affected individuals and carriers. Ten patients with hybrid opsin genes were identified, including one patient with two hybrid genes.

Conclusion: The newly developed MLPA assay enables determination of the copy number status and identification of gene rearrangements in OPN1LW and OPN1MW. The addition of MLPA to routine sequencing of OPN1LW and OPN1MW will improve detection of the cause of X-linked cone dysfunction disorders.

A.S. Hoekstra: A. Employment (full or part-time); Significant; MRC-Holland. L. Haer-Wigman: None. M. Tjon-Pon-Fong: None. M. Zegers: A. Employment (full or part-time); Significant; MRC-Holland. M. Ketema: A. Employment (full or part-time); Significant; MRC-Holland. R. Vijzelaar: A. Employment (full or part-time); Significant; MRC-Holland.

P02.47A Novel mutations in TMEM126A causing non-syndromic autosomal recessive optic atrophy

N. Weisschuh1, M. Synofzik2,3, C. Kernstock1, S. Schimpf-Linzenbold4, F. Schuettauf5, A. Neu6, K. Kloth7

1Institute for Ophthalmic Research, Tuebingen, Germany, 2Hertie Institute for Clinical Brain Research, Tuebingen, Germany, 3German Center for Neurodegenerative Diseases, Tuebingen, Germany, 4CeGaT GmbH and Praxis für Humangenetik, Tuebingen, Germany, 5Department of Ophthalmology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany, 6Department of Pediatrics, University Medical Center Hamburg-Eppendorf, Hamburg, Germany, 7Institute of Human Genetics, University Medical Center Hamburg-Eppendorf, Hamburg, Germany

Reports on autosomal recessive optic atrophy (arOA) are sparse and so far, only one gene has been specifically associated with non-syndromic arOA, namely TMEM126A. To date, all reports of TMEM126A mutations are from patients of Maghrebian origin, who all carry an identical nonsense mutation. We report two novel variants in the TMEM126A gene from two non-Maghreb patients/families, both resulting in an arOA phenotype.

The proband of family A was diagnosed with visual loss in early childhood but a diagnosis of optic atrophy was only made at 14 years. A diagnostic gene panel revealed a splice donor variant (c.86+2T>C) in the TMEM126A gene. Analysis of this variant based on RNA from whole blood revealed a single aberrant transcript lacking exon 2, presumably representing a functional null allele. Two siblings from family B were diagnosed with optic atrophy in early childhood. A missense variant (p.S36L) in the TMEM126A gene was identified in a gene panel-based diagnostic setting in both siblings. This missense variant is ultra rare in the general population, affects a highly evolutionarily conserved amino acid and segregates with the disease within the family. The three probands reported in this study had a relatively mild clinical course without any evidence of a syndromic (e.g. neurological) comorbidity, which is in line with previous studies.

We provide additional evidence for the implication of biallelic TMEM126A mutations in arOA. Our findings extend both the mutational spectrum and geographic presence of TMEM126A in arOA.

N. Weisschuh: None. M. Synofzik: None. C. Kernstock: None. S. Schimpf-Linzenbold: None. F. Schuettauf: None. A. Neu: None. K. Kloth: None.

P02.48B A familial optic atrophy associated with mild neurodevelopmental disorder caused by SOX5 haploinsufficiency

A. Gouronc1, Y. Perdomo2, L. Maurin2, A. Schalk1, S. El Chehadeh3, E. Schaefer3, H. Dollfus2,3, S. Scheidecker1

1Laboratoires de Diagnostic Génétique, Hôpitaux Universitaires de Strasbourg, Strasbourg, France, 2Centre de Référence pour les Affections Rares en Génétique Ophtalmologique (CARGO), Hôpitaux Universitaires de Strasbourg, Strasbourg, France, 3Service de Génétique Médicale, Hôpitaux Universitaires de Strasbourg, Strasbourg, France

Introduction: Inherited optic neuropathies (ION) are a clinically and genetically heterogeneous group of disorders caused by nuclear or mitochondrial DNA variants. Isolated and syndromic forms are described. The most frequent ION are autosomal dominant optic atrophy (ADOA), due to OPA1 variant, and Leber hereditary optic neuropathy (LHON), caused by mitochondrial DNA variant.

Materials and Methods: Herein we report on three patients from a family suffering from bilateral optic neuropathy and suggesting an autosomal dominant inheritance. Two of them show additional features such as speech delay, mild learning disabilities and behavior disorders. The sequencing of a panel of ION’s genes including the OPA1 gene is normal. The SNP array analysis reveals an 82 kb intragenic heterozygous deletion of the SOX5 gene that segregates with the disease.

Discussion: The SOX5 haploinsufficiency leads to Lamb-Shaffer syndrome classically associating global developmental delay, with predominant speech impairment, mild to severe intellectual disability and mild dysmorphic features. Some patients have additional features such as brain abnormalities or musculoskeletal anomalies. Optic atrophy is described in only two patients carrying SOX5 heterozygous de novo variants: a 53 kb intragenic deletion and a point variant resulting in a premature stop codon. These two patients displayed a moderate to severe neurodevelopmental disorder.

Conclusion: Here we report on the first family of inherited autosomal dominant optic atrophy due to SOX5 heterozygous deletion associated with mild neurodevelopmental features. This observation suggests that a SOX5 gene analysis could be performed in patients presenting with optic atrophy associated with developmental disorder even mild.

A. Gouronc: None. Y. Perdomo: None. L. Maurin: None. A. Schalk: None. S. El Chehadeh: None. E. Schaefer: None. H. Dollfus: None. S. Scheidecker: None.

P02.49C PAX6 non-coding sequence variants cause congenital aniridia

A. Filatova1, T. Vasilyeva1, A. Marakhonov1,2, R. Zinchenko1,3, M. Skoblov1,2

1Research Centre for Medical Genetics, Moscow, Russian Federation, 2School of Biomedicine, Far Eastern Federal University, Vladivostok, Russian Federation, 3Pirogov Russian National Research Medical University, Moscow, Russian Federation

Introduction: Congenital aniridia (AN) is a rare autosomal dominant panocular disorder caused by mutations in the PAX6 gene. A previously conducted molecular genetic study of a large cohort of Russian patients with AN revealed several groups of PAX6 nucleotide variants which pathogenicity is not obvious, including variants out of canonical splicing site dinucleotides, possibly affecting splicing, and 5ʹ-UTR variants. These variants were classified as VUS or likely pathogenic according to the ACMG recommendations. Thus, to validate the pathogenicity of such variants functional studies are required.

Materials and Methods: To determine the effect of PAX6 variants on splicing we used a minigene assay. 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 assay, and the RNA expression level was evaluated by qPCR.

Results: Using a minigene assay, seven out of eight investigated probably affecting slicing variants (six deep intronic and two exonic) were showed to disrupt normal splicing patterns and result in frame shifting followed by mRNA degradation by NMD mechanism. Besides, five 5ʹ-UTR variants were found to lead to a significant decrease in the translation efficiency, including three of them, which also lead to aberrant splicing. Our further analysis allows us to suggest the mechanism of 5ʹ-UTR variants pathogenicity through disruption of upstream ORF which possibly exists in PAX6 5ʹ-UTR.

Conclusions: Using functional analysis we have confirmed the pathogenicity of 12 PAX6 noncoding mutations. Moreover, our results suggest the exact mechanism of their pathogenic action.

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

P02.50D Mutations in PLS1, encoding fimbrin, cause autosomal dominant non-syndromic hearing loss (ADNSHL)

A. Morgan1, D. Koboldt2,3, E. Barrie2, E. Crist2,3,4, M. Mezzavilla5, F. Faletra5, T. Mosher2,3,4, R. Wilson2,3, K. Manickam3,4, P. Gasparini1,5, D. Dell’Orco6, G. Girotto1,5

1University of Trieste, Trieste, Italy, 2Institute for Genomic Medicine at Nationwide Children’s Hospital, Columbus, OH, United States, 3Department of Pediatrics at The Ohio State University, Columbus, OH, United States, 4Division of Genetic and Genomic Medicine at Nationwide Children’s Hospita, Columbus, OH, United States, 5IRCCS Burlo Garofolo, Trieste, Italy, 6Department of Neurosciences, Biomedicine and Movement Sciences, Section of Biological Chemistry, University of Verona, Verona, Italy

Introduction: NSHL is a common sensory disorder characterized by high genetic heterogeneity. Despite huge efforts in genes discovery, almost half of patients still fail to receive a molecular diagnosis.

Methods: Two unrelated ADNSHL-families of European ancestry negative for mutations in known deafness-genes underwent next-generation sequencing. Data were filtered according to frequency, pathogenicity and pattern of inheritance. To prioritize genes a population-based analysis was performed (including statistics on natural selection in Europeans and gene constrains from gnomAD database) followed by in silico protein modelling.

Results: Among the genes found mutated in the two analysed families, interestingly, PLS1 showed signatures of natural selection and low observed/expected ratio of both missense and loss of function mutations. Thus, these findings suggested a level of conservation and allowed to prioritize PLS1 in which two novel likely-pathogenic missense variants were identified. PLS1 encodes fimbrin, one of the most abundant actin-bundling proteins of the stereocilia. Recently, it was demonstrated that Pls1-/- mice show a moderate/progressive form of HL across all frequencies (PMID: 25124451). In silico protein modelling displayed that both variants affect the actin-binding domain-1, and suggested an overall destabilization of the protein structure, reducing the protein’s ability to bind F-actin.

Conclusion: Present results (genomic data of two independent ADNSHL-families, literature updates, evidence of evolutionary constrains and protein modelling results) provide evidence that PLS1 is required for normal hearing and once mutated might cause ADNSHL. The identification of PLS1 as a new ADNSHL-gene provides a new possible target for the development of therapeutic approaches.

A. Morgan: None. D. Koboldt: None. E. Barrie: None. E. Crist: None. M. Mezzavilla: None. F. Faletra: None. T. Mosher: None. R. Wilson: None. K. Manickam: None. P. Gasparini: None. D. DellOrco: None. G. Girotto: None.

P02.51A Coincidental occurrence of Schnyder corneal dystrophy and posterior polymorphous corneal dystrophy type 3

P. Liskova1, L. Dudakova1, P. Skalicka1,2, A. E. Davidson3

1First Faculty of Medicine, Charles University, Prague, Czech Republic, 2General University Hospital in Prague, Prague, Czech Republic, 3UCL Institute of Ophthalmology, London, United Kingdom

Purpose: To report the simultaneous occurrence of two rare corneal dystrophies.

Methods: Case report of a 30-year-old male with a family history of posterior polymorphous corneal dystrophy type 3 (PPCD3) was invited for ophthalmic examination. Sanger sequencing of the coding regions and intron/exon boundaries of disease-associated genes, ZEB1 and UBIAD1 was performed.

Results: The clinical findings suggested co-occurrence of PPCD3 and Schnyder corneal dystrophy (SCD) in the proband. This dual diagnosis was supported by genetic findings. He was identified to carry a previously reported heterozygous nonsense mutation in ZEB1; c.2157C>G, p.(Tyr719*), and a novel heterozygous missense mutation in UBIAD1; c.569T>C; p.(Ile190Thr). The mother of the proband only carried the c.2157C>G ZEB1 variant and slit-lamp examination of her corneas showed endothelial lesions characteristic of PPCD3. The sister of the proband carried the c.569T>C in UBIAD1 and had corneal crystal deposition in her anterior stroma consistent with the diagnosis of SCD.

Conclusion: This case illustrates the coincidental occurrence of two rare and genetically distinct corneal dystrophies in a single patient. Furthermore, it highlights the need to perform comprehensive phenotyping in combination with appropriate genetic diagnostic testing to achieve an accurate diagnosis. This work was supported by GACR 17-12355S.

P. Liskova: None. L. Dudakova: None. P. Skalicka: None. A.E. Davidson: None.

P02.52B Molecular genetic study of primary congenital glaucoma: the mutational analysis of CYP1B1and LTBP2 genes

V. Szabó1, K. Knézy1, M. Csidey1, A. Szigeti1, E. Maka1, M. Bausz1, K. Sényi1, Z. Nagy1, G. Holló1, P. Kövy2, T. Krähling2, A. Tordai3, H. Andrikovics2, A. Bors2

1Semmelweis University, Dept. of Ophthalmology, Budapest, Hungary, 2Central Hospital of Southern Pest - National Institute of Hematology and Infectious Diseases, Budapest, Hungary, 3Semmelweis University, Dept. of Pathophysiology, Budapest, Hungary

Introduction: Purpose of our study was to examine the mutations of CYP1B1 and LTBP2 genes in patients and their relatives with primary congenital glaucoma, (PCG) and to evaluate genotype-phenotype correlations.

Patients and Methods: molecular analysis of 26 children of 21 families with PCG was performed. First we examined the common mutation p.Glu387Lys of CYP1B1 gene using RFLP technique. In cases where wild-type or heterozygous p.Glu387Lys variant was detected, Sanger-sequencing of CYP1B1 was performed. Common p.Arg299Ter (c.895C>T) mutation in LTBP2 gene was also genotyped. We carried out a retrospective evaluation, using clinical data as follows: age at diagnosis, ophthalmological status, pre- and postoperative visual acuity and intraocular pressure and pedigree.

Results: we identified two mutations in 20 patients (76,92%) in our study group. The p.Arg299Ter (c.895C>T) mutation in LTBP2 gene was found in one family in homozygous form. We identified the homozygous p.Glu387Lys mutation of CYP1B1 gene in 12 members of 8 families, in two siblings of one family we found two deletions causing frameshift (p.Arg355Hisfs*69/p.His401Leufs*24), compound heterozygous mutations described in the literature before were identified in further 5 families. No causative mutation was detected in 6 families (28,57%) neither in CYP1B1 gene, nor in examined position of LTBP2 gene.

Conclusion: No genotype-phenotype correlations were found in our study group with PCG, one could not conclude on the grounds of genotype of clinical course and prognosis. This is the first study investigating the mutations of CYP1B1 gene and the p.Arg299Ter (c.895C>T) in LTBP2 gene of PCG in Hungary.

V. Szabó: None. K. Knézy: None. M. Csidey: None. A. Szigeti: None. E. Maka: None. M. Bausz: None. K. Sényi: None. Z. Nagy: None. G. Holló: None. P. Kövy: None. T. Krähling: None. A. Tordai: None. H. Andrikovics: None. A. Bors: None.

P02.53C Expanding the CPAMD8-associated eye disease spectrum to primary congenital glaucoma: lessons learned from a large consanguineous family with pseudodominance

H. Verdin1, I. Balikova1,2,3, J. Van De Velde1, P. G. Kestelyn2, B. P. Leroy1,2, E. De Baere1

1Center for Medical Genetics, Ghent University, Ghent, Belgium, 2Department of Ophthalmology, Ghent University and Ghent University Hospital, Ghent, Belgium, 3Department of Ophthalmology, Children Hospital Queen Fabiola, Brussels, Belgium

Primary congenital glaucoma (PCG) is defined as an isolated trabeculodysgenesis that occurs in the first three years of life and is most often caused by biallelic mutations in CYP1B1. Other early-onset, developmental glaucomas may arise secondary to developmental malformations of the anterior segment of the eye, and occur as part of an anterior segment dysgenesis (ASD) disorder. Consequently, these overlapping phenotypes may challenge the diagnosis of PCG.

Here we studied a highly consanguineous Jordanian pedigree with over 17 affected individuals in three generations, displaying a clinical diagnosis of PCG. In addition to PCG, the proband also presented with iris atrophy, ectropion uveae and corectopia. Moreover, the familial history revealed the presence of other ocular manifestations in the family including cataracts, lens luxation and iris abnormalities. Using homozygosity-based filtering of exome data, we identified a homozygous frameshift variant in CPAMD8: c.785_788del, p.Tyr262Serfs*17. This variant is not present in population databases and segregates with the phenotype in the family. Recently, CPAMD8 was reported as the cause for a unique autosomal recessive ASD that is characterized by bilateral iris hypoplasia, ectopia lentis, corectopia, ectropion uveae, and cataracts, clinical features also observed here. Interestingly, two affected siblings who were initially diagnosed with PCG received a clinical diagnosis of megalocornea in retrospect. In conclusion, we identified the molecular cause underlying a PCG phenotype in a large consanguineous family using combined homozygosity-mapping and WES. Based on this family and the previously reported families, CPAMD8-associated ASD should be taken into consideration as a differential diagnosis for PCG.

H. Verdin: None. I. Balikova: None. J. Van De Velde: None. P.G. Kestelyn: None. B.P. Leroy: None. E. De Baere: None.

P02.54D Whole exome sequencing and linkage analysis of extended pedigrees to identify glaucoma susceptibility genes

P. Graham1, J. Peralta1,2, N. Blackburn1,2, J. Blangero1,2, M. Wirtz3, A. Hewitt1, D. Mackey4, K. Burdon1, J. Charlesworth1

1Menzies Institute for Medical Research, Hobart, Australia, 2South Texas Diabetes and Obesity Institute, Brownsville, TX, United States, 3Casey Eye Institute, Portland, OR, United States, 4Lions Eye Institute, Perth, Australia

The use of next generation sequencing in extended pedigrees has significant potential for identifying functional variants linked with complex disease. We are using whole exome sequencing (WES) of five large, complex families from Tasmania (Australia) and Oregon (USA) to identify susceptibility genes for primary open-angle glaucoma (POAG), the leading cause of irreversible blindness worldwide. Extended pedigrees, enriched for POAG, provide a powerful tool to search for rare and private genetic variants influencing the disease, where enrichment of rare variants occurs as a function of segregation from the founders. The families in this study range in size from 48 to 201 individuals (28 to 91 sequenced) and span 5 to 7 generations. These families are being used to locate quantitative trait loci (QTLs) for intraocular pressure (IOP), an important glaucoma endophenotype. Variance components linkage analysis of IOP was conducted on the WES data from 249 individuals and we identified QTLs on chromosomes 2,3,6,7 and 15. The chromosome 2 locus (2q22.2-24.3) spans a 20 million base pair region. Three of the five families independently contribute linkage information to this peak, which is a novel locus for both IOP and POAG. Further analysis is being undertaken to identify genes with family specific, potentially deleterious variants; which will be validated in large POAG case/control cohorts. Finding genes involved with POAG susceptibility will increase our understanding of the biological pathways involved with the disease process and from that, diagnostic tools and more effective treatments can be developed.

P. Graham: None. J. Peralta: None. N. Blackburn: None. J. Blangero: None. M. Wirtz: None. A. Hewitt: None. D. Mackey: None. K. Burdon: None. J. Charlesworth: None.

P02.55A PRPS1loss-of-function variants, from isolated hearing loss to severe congenital encephalopathy

S. marlin1,2, O. Mercati1, M. Abi Warde3, G. Lina-Granade4, M. Rio5, S. Heide6, P. de Lonlay7, I. Ceballos-Picot8, M. Robert9, V. Couloigner10, J. Beltrand11, N. Boddaert12, D. Rodriguez13, A. Rötig14, H. Prokisch15, S. Lyonnet2, N. Loundon10, J. Kaplan16, J. Bonnefont17, A. Munnich18, C. Besmond18, L. Jonard1,17

1Centre de Référence des Surdités Génétiques, Institut Imagine, Hôpital Necker-Enfants Malades, APHP, Paris, France, 2Laboratoire d’Embryologie et de Génétique des Malformations Congénitales, INSERM UMR 1163, Institut Imagine, Université Paris Descartes, Paris, France, 3Département de NeuroPédiatrie, CHU, Strasbourg, France, 4Service d’Otorhinolaryngologie et chirurgie cervico-faciale Pédiatrique, CHU, Lyon, France, 5Département de Génétique, Hôpital Necker-Enfants Malades, APHP, Paris, France, 6Département de Génétique, Groupe Hospitalier Pitié-Salpêtrière, AP-HP, Paris, France, 7Centre Référence des Maladies héréditaires du Métabolisme, Hôpital Necker-Enfants Malades, AP-HP, Paris, France, 8Laboratoire de Biochimie Métabolomique et Protéomique, Hôpital Necker-Enfants Malades, AP-HP, Paris, France, 9Service d’Ophtalmologie pédiatrique, Hôpital Necker-Enfants Malades, AP-HP, Paris, France, 10Service d’Otorhinolaryngologie et chirurgie cervico-faciale Pédiatrique, Hôpital Necker-Enfants Malades, AP-HP, Paris, France, 11Service Endocrinologie, Gynécologie et Diabétologie Pédiatrique, Hôpital Necker-Enfants Malades, AP-HP, Paris, France, 12Département de Radiologie pédiatrique, Hôpital Necker-Enfants-Malades, AP-HP; UMR 1163, Institut Imagine, Université Paris Descartes, Paris, France, 13Service de NeuroPédiatrie, Hôpital Trousseau, AP-HP, Paris, France, 14Laboratoire de Génétique des maladies Mitochondriales, INSERM UMR1163, Institute Imagine, Université Paris Descartes, Paris, France, 15Institute of Human Genetics, Helmholtz Zentrum München, Neuherberg, Germany, 16Laboratoire de Génétique Ophtalmologique INSERM UMR1163, Institute Imagine, Université Paris Descartes, Paris, France, 17Laboratoire de Génétique Moléculaire, Hôpital Necker-Enfants Malades, AP-HP, Paris, France, 18Université Paris Descartes, Institut Imagine, Paris, France

We describe two sporadic and two familial cases with loss-of-function variations in PRPS1, which is located on the X chromosome and encodes phosphoribosyl pyrophosphate synthetase 1 (PRS-1). We illustrate the clinical variability associated with decreased PRS-1 activity, ranging from mild isolated hearing loss to severe encephalopathy. One of the variants we identified has already been reported with a phenotype similar to our patient’s, whereas the other three were unknown. The clinical and biochemical information we provide will hopefully contribute to gain insight into the correlation between genotype and phenotype in this rare condition, in females as well as in males. Moreover, our observation of a new family in which hemizygous males display hearing loss without any neurological or ophthalmological symptoms prompts us to suggest analysing PRPS1in cases of isolated hearing loss.

S. marlin: None. O. Mercati: None. M. Abi Warde: None. G. Lina-Granade: None. M. Rio: None. S. Heide: None. P. de Lonlay: None. I. Ceballos-Picot: None. M. Robert: None. V. Couloigner: None. J. Beltrand: None. N. Boddaert: None. D. Rodriguez: None. A. Rötig: None. H. Prokisch: None. S. Lyonnet: None. N. Loundon: None. J. Kaplan: None. J. Bonnefont: None. A. Munnich: None. C. Besmond: None. L. Jonard: None.

P02.56B Biallelic sequence and structural variants in RAX2 are a novel cause for autosomal recessive inherited rod-dominated retinal disease

S. Van de Sompele1, C. Smith2, M. Karali3,4, M. Corton5,6, K. Van Schil1, F. Peelman7, T. Cherry8, T. Rosseel1, H. Verdin1, J. Derolez1, T. Van Laethem1, K. N. Khan9, M. McKibbin9, C. Toomes2, M. Ali2, A. Torella3, F. Testa10, B. Jimenez11, F. Simonelli10, J. De Zaeytijd12, J. Van den Ende13, B. P. Leroy1,12,14, F. Coppieters1, C. Ayuso5,6, C. F. Inglehearn2, S. Banfi3,4, E. De Baere1

1Center for Medical Genetics, Ghent University and Ghent University Hospital, Ghent, Belgium, 2Section of Ophthalmology and Neuroscience, School of Medicine, University of Leeds, St James’s University Hospital, Leeds, United Kingdom, 3Medical Genetics, Department of Precision Medicine, Università degli Studi della Campania “Luigi Vanvitelli”, Naples, Italy, 4Telethon Institute of Genetics and Medicine, Pozzuoli, Italy, 5Genetics Department, Instituto de Investigación Sanitaria-Fundación Jimenez Diaz University Hospital, Madrid, Spain, 6Center of Biomedical Network Research on Rare Diseases, Madrid, Spain, 7Flanders Institute for Biotechnology (VIB), Department of Medical Protein Research, Faculty of Medicine and Health Sciences, Ghent University, Ghent, Belgium, 8Center for Developmental Biology and Regenerative Medicine, Seattle Children’s Research Institute, Seattle, WA, United States, 9Department of Ophthalmology, St. James’s University Hospital, Leeds, United Kingdom, 10Eye Clinic, Multidisciplinary Department of Medical, Surgical and Dental Sciences, Università degli Studi della Campania “Luigi Vanvitelli”, Naples, Italy, 11Department of Ophthalmology, Fundación Jimenez Diaz University Hospital, Madrid, Spain, 12Department of Ophthalmology, Ghent University and Ghent University Hospital, Ghent, Belgium, 13Center for Medical Genetics, Antwerp University Hospital, Antwerp, Belgium, 14Division of Ophthalmology, The Children’s Hospital of Philadelphia, Philadelphia, PA, United States

RAX2 encodes a homeobox-containing transcription factor, in which four monoallelic pathogenic variants have been previously described in autosomal dominant cone-dominated retinal disease. Here, exome sequencing in a European cohort with inherited retinal disease (IRD) (n=2086) revealed biallelic RAX2 sequence and structural variants in five unrelated European index cases, displaying non-syndromic autosomal recessive retinitis pigmentosa (ARRP) with an age of onset ranging from childhood to the mid 40s (average mid 30s). Protein structure modeling of the novel recessive missense variants points to loss-of-function while a dominant-negative effect is predicted for the previously reported dominant RAX2 alleles. Structural variants were fine-mapped to disentangle their underlying mechanisms and haplotyping of c.335dup in two cases suggests a common Belgian ancestry. One additional unrelated Belgian ARRP patient carried the same c.335dup allele in the absence of a second RAX2 coding or structural variant, suggesting a role for non-coding variants in RAX2 associated ARRP. To conclude, we found biallelic pathogenic variants in RAX2 to be associated with ARRP, revealing RAX2 as a novel gene for recessively inherited rod-dominated retinal diseases. The identification of RAX2 biallelic pathogenic variants in five families of European origin indicates that this gene may underlie a non-negligible fraction of ARRP cases of other populations that still lack a molecular diagnosis. The RAX2 mutational spectrum was broadened from sequence to structural variants (SVs). Finally, the identification of pathogenic structural variants in RAX2 stresses the importance of SV assessment in WES and WGS data in IRD.

Funding: BOF15/GOA/011; BOF/01D04716; FWO/1145719N; FWO/1802215N; Italian Telethon Foundation

S. Van de Sompele: None. C. Smith: None. M. Karali: None. M. Corton: None. K. Van Schil: None. F. Peelman: None. T. Cherry: None. T. Rosseel: None. H. Verdin: None. J. Derolez: None. T. Van Laethem: None. K.N. Khan: None. M. McKibbin: None. C. Toomes: None. M. Ali: None. A. Torella: None. F. Testa: None. B. Jimenez: None. F. Simonelli: None. J. De Zaeytijd: None. J. Van den Ende: None. B.P. Leroy: None. F. Coppieters: None. C. Ayuso: None. C.F. Inglehearn: None. S. Banfi: None. E. De Baere: None.

P02.57C Investigation of the role of a homozygous mutation in BBS10 in non syndromic retinal degeneration

A. V. Vig1,2, E. Tavares2, O. Kehelwathugoda1,2, A. Mollica1,2, J. Maynes2,1, A. Vincent2,1, E. Heon2,1

1University of Toronto, Toronto, ON, Canada, 2The Hospital for Sick Children, Toronto, ON, Canada

Introduction: Bardet-Biedl syndrome (BBS) is rare autosomal recessive disorder, characterized by retinal degeneration, obesity, digital anomalies, genito-urinary defects, and variable cognitive impairment. It is part of a broader pleotropic class of diseases called ciliopathies. Ciliopathies are caused by mutations in genes that play important roles in the function of cellular signaling organelles called cilia. BBS10 encodes a chaperonin–like protein that mediates assembly of the BBSome, which transports vesicles to cilia. BBS10 is one of the most frequently mutated BBS genes, accounting for approximately 16% of cases.

Methods: A 29 yo female from a consanguineous family had non-syndromic cone-rod dystrophy (CRD) and did not have any mutation identified using standard of care clinical genetic testing. Whole exome sequencing was performed. Candidate variants were assessed based on family segregation, predicted effect on protein structure and function, amino acid conservation, and population frequency.

Results: Whole genome sequencing revealed a novel homozygous mutation in BBS10. The variant is rare and segregated in the family. It occurs at a residue which is highly conserved among chaperonins. Based on modelling of BBS10 secondary structure, it is predicted that the variant could 1) affect multimer assembly or 2) alter how ATP hydrolysis induces the protein conformation changes that are vital to chaperone function.

Conclusions: We have identified a rare homozygous variant we presume significant in BBS10 in a patient with non-syndromic CRD. We are exploring the functional consequences of the variant through phenotyping a patient-derived fibroblast cell line.

Funding: FFB, Mira Godard, McLaughlin Centre, ORF

A.V. Vig: None. E. Tavares: None. O. Kehelwathugoda: None. A. Mollica: None. J. Maynes: None. A. Vincent: None. E. Heon: None.

P02.58D Clinical utility of a custom NGS panel in routine evaluation of retinal dystrophies

I. Hernan, E. Borràs, M. Gamundi, B. Mañé, V. García-Prieto, B. Delàs, M. Carballo

Hospital de Terrassa (CST), Terrassa, Spain

Introduction: Retinal dystrophies (RD) are a broad group of clinically and genetically heterogeneous disorders affecting the retina. The non-syndromic forms of RD can be attributed to mutations in more than 100 genes. Consequently, next generation sequencing (NGS) technologies are among the most promising approaches to identify mutations in RD patients.

Materials and Methods: A cohort of patients including independent cases with various forms of RD, especially Retinitis Pigmentosa and Stargardt disease were analysed. To identify the RD causative gene, a custom SureSelect capture panel (Agilent) targeting 104 genes associated with non-syndromic retinal disease was developed. NGS libraries were run on Illumina MiSeq or HiSeq Sequencer. Analyses, annotation, filtration and variant curation were done using GeneSystems software (Sistemas Genómicos). When it was possible, segregation analysis of the candidate variant was performed in additional family members by Sanger sequencing.

Results: A pathogenic variant was identified in 25 RD patients (58%), including novel mutations in ABCA4, CACNA1F, EYS and PRCD gene. In 7 patients, a variant of unknown significance was detected; a functional test would be necessary to assess their clinical significance.

Conclusions: Five novel variants have been identified as a causative mutation for RD using the custom capture panel. The designed NGS assay achieves a detection rate of almost 60% and provides an adequate routine assay for genetic analysis of patients with retinal disease.

I. Hernan: None. E. Borràs: None. M. Gamundi: None. B. Mañé: None. V. García-Prieto: None. B. Delàs: None. M. Carballo: None.

P02.59A Copy number variants (CNVs) identified by comprehensive genetic testing of inherited retinal dystrophies

L. Guidugli1, S. Tuupanen1, M. Mehine1, K. Kämpjärvi1, L. Koskinen1, K. Wells1, J. Känsäkoski1, M. Valori1, I. Saarinen1, M. Muona1, E. Sankila2, S. Myllykangas1, J. W. Koskenvuo1, T. Alastalo1

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

Retinal dystrophies (RD) are disorders that damage the photoreceptors in the retina and cause visual impairment. Prompt, comprehensive genetic diagnosis of RD can assist in risk assessment, symptom management and selection of the appropriate targeted treatment. The genetic testing needs to take into account sequence alterations and copy number variants (CNVs). We evaluated the rates and characteristics of CNVs in 2754 patients tested using a comprehensive RD panel.

DNA from patients was sequenced by targeted OS-Seq using the Illumina NextSeq500 sequencing platform or the IDT xGEN Exome Research Panel using the Illumina NovaSeq platform. CNVs were detected by CNVkit and an in-house developed deletion caller.

CNVs in 47 genes matching the patient’s phenotype were reported in 4.6% of the cases. Of the CNVs, 71.1% were partial gene deletions, 13.3% whole gene deletions, 3.1% one exon deletions, and 0.8% partial exon deletions. 11.7% of the identified CNVs were duplications (partial or whole gene). 88.3% of CNVs were likely pathogenic or pathogenic, 11.7% were variants of uncertain significance. Of the likely pathogenic and pathogenic CNVs, 73.4% were diagnostic. USH2A and PRPF31 were enriched in CNVs compared to other genes. Notably, CNVs were identified also in genes in which CNVs are not commonly reported, e.g. ABCA4 and RPE65.

These results highlight the importance of comprehensive genetic testing for the diagnosis of retinal dystrophies. We identified CNVs ranging from one exon to whole gene deletions in multiple genes. In addition, we detected a relative high percentage of copy number duplications that warrant further investigation.

L. Guidugli: A. Employment (full or part-time); Significant; Blueprint Genetics. S. Tuupanen: A. Employment (full or part-time); Significant; Blueprint Genetics. M. Mehine: A. Employment (full or part-time); Significant; Blueprint Genetics. K. Kämpjärvi: A. Employment (full or part-time); Significant; Blueprint Genetics. L. Koskinen: A. Employment (full or part-time); Significant; Blueprint Genetics. K. Wells: A. Employment (full or part-time); Significant; Blueprint Genetics. J. Känsäkoski: A. Employment (full or part-time); Significant; Blueprint Genetics. M. Valori: A. Employment (full or part-time); Significant; Blueprint Genetics. I. Saarinen: A. Employment (full or part-time); Significant; Blueprint Genetics. M. Muona: A. Employment (full or part-time); Significant; Blueprint Genetics. E. Sankila: A. Employment (full or part-time); Modest; 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. 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. 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.

P02.60B Prevalence and characteristics of RPGR ORF15 variants in patients with inherited retinal dystrophies

J. Sistonen1, S. Tuupanen1, K. Kämpjärvi1, P. Siivonen1, M. Mehine1, J. Känsäkoski1, K. Wells1, J. Schleit1, M. Valori1, P. Salmenperä1, E. Sankila2, E. Salminen1, T. Alastalo1, J. Koskenvuo1, S. Myllykangas1

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

Introduction: The exon ORF15 in RPGR is a mutational hotspot for X-linked retinitis pigmentosa (XLRP). However, it generally performs poorly in standard sequencing-based assays due to a highly repetitive purine-rich sequence. To address the clinical importance of RPGR ORF15 and the lack of high-quality next generation sequencing (NGS) -based diagnostics, we aimed to develop a comprehensive clinical test for inherited retinal dystrophies.

Materials and Methods: We optimized a whole exome sequencing workflow with the Illumina NovaSeq 6000 platform to cover 266 retinal dystrophy-associated genes, including the difficult-to-sequence region in RPGR ORF15. We evaluated the performance of RPGR sequencing in 1587 unselected patient samples.

Results: In our clinical cohort, the overall diagnostic yield was 58%. A molecular diagnosis in RPGR was identified in 5.7% (90/1587) of the patients. The 90 pathogenic/likely pathogenic variants consisted of 63 frameshift (70.0%), 21 nonsense (23.3%), three missense (3.3%), and two consensus splice site (2.2%) variants, and one gross deletion (1.1%). Seventy-one out of 90 (79%) pathogenic/likely pathogenic variants were detected in the ORF15, of which 28 (39%) were in the difficult-to-sequence central region between residues p.824 and p.1077. Female patients accounted for 24% of the diagnostic cases.

Conclusions: Our results highlight the importance of RPGR ORF15 sequencing in retinal dystrophy patients. The high-quality NGS-based assay enables rapid and reliable molecular diagnostics of RPGR ORF15, and enhances the identification of patients for ongoing gene therapy trials.

J. Sistonen: A. Employment (full or part-time); Significant; Blueprint Genetics. S. Tuupanen: A. Employment (full or part-time); Significant; Blueprint Genetics. K. Kämpjärvi: A. Employment (full or part-time); Significant; Blueprint Genetics. P. Siivonen: A. Employment (full or part-time); Significant; Blueprint Genetics. M. Mehine: A. Employment (full or part-time); Significant; Blueprint Genetics. J. Känsäkoski: A. Employment (full or part-time); Significant; Blueprint Genetics. K. Wells: A. Employment (full or part-time); Significant; Blueprint Genetics. J. Schleit: A. Employment (full or part-time); Significant; Blueprint Genetics. M. Valori: A. Employment (full or part-time); Significant; Blueprint Genetics. P. Salmenperä: A. Employment (full or part-time); Significant; Blueprint Genetics. E. Ownership Interest (stock, stock options, patent or other intellectual property); Significant; Blueprint Genetics. E. Sankila: A. Employment (full or part-time); Modest; Blueprint Genetics. E. Salminen: A. Employment (full or part-time); 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. J. 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.

P02.61C Prevalence and genetic characteristics of RPE65-associated retinal disease

J. Tommiska1, T. Alastalo2, K. Kämpjärvi1, L. Guidugli2, J. Känsäkoski1, K. Wells1, H. Västinsalo1, M. Kaare1, L. Sarantaus1, P. Salmenperä1, M. Gentile1, S. Bruce1, E. Sankila3, J. W. Koskenvuo1, S. Myllykangas1, S. Tuupanen1

1Blueprint Genetics, Helsinki, Finland, 2Blueprint Genetics, San Francisco, CA, United States, 3Helsinki University Eye Hospital, Helsinki, Finland

RPE65 variants are associated with severe retinal diseases including Leber congenital amaurosis (LCA) and retinitis pigmentosa (RP). Recent advancements in targeted therapy have incentivised genetic diagnostics and increased the efforts to identify RPE65 patients eligible for therapy. We evaluated the prevalence of RPE65 variants in 2240 retinal dystrophy patients by using next-generation sequencing (NGS).

Methods: Patients with LCA, RP, rod-cone dystrophy, or early onset retinal dystrophy were tested at a CLIA certified laboratory 2016 - 2018. Analysis was done using in-house developed and validated NGS, bioinformatics and clinical interpretation.

Results: Of 18 patients (0.8%) with RPE65-related disease, 7 (38.8%) had LCA, 4 (22.2%) had RP, 6 (33.3%) were affected with severe early onset retinal dystrophy, and one (5.5%) had congenital stationary night blindness (CSNB). The median age at molecular diagnosis was 13 years (range 1 - 60 years). Of the 35 disease-associated RPE65 variants 18 (51%) were missense, 10 (29%) protein truncating, 5 (13%) splice site variants, and 2 (5.7%) copy number variants (CNVs). Two (11.1%) patients carried a CNV: a single exon deletion and a deletion of the whole RPE65 gene, respectively. Twelve (67%) patients had at least one loss-of-function variant. One patient had the c.1430A>G, p.(Asp477Gly) variant associated with autosomal dominant disease and displayed an atypical form of RP.

Conclusions: RPE65 has a significant role in LCA and is important in differential diagnostics of retinal dystrophies. Our results also highlight the importance of high-quality genetic diagnostics covering both sequence variants and CNVs for optimized diagnosis and clinical care.

J. Tommiska: A. Employment (full or part-time); 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. K. Kämpjärvi: A. Employment (full or part-time); Significant; Blueprint Genetics. L. Guidugli: A. Employment (full or part-time); Significant; Blueprint Genetics. J. Känsäkoski: A. Employment (full or part-time); Significant; Blueprint Genetics. K. Wells: A. Employment (full or part-time); Significant; Blueprint Genetics. H. Västinsalo: A. Employment (full or part-time); Significant; Blueprint Genetics. M. Kaare: A. Employment (full or part-time); Significant; Blueprint Genetics. L. Sarantaus: A. Employment (full or part-time); Significant; Blueprint Genetics. P. Salmenperä: A. Employment (full or part-time); Significant; Blueprint Genetics. E. Ownership Interest (stock, stock options, patent or other intellectual property); Significant; Blueprint Genetics. M. Gentile: A. Employment (full or part-time); Significant; Blueprint Genetics. E. Ownership Interest (stock, stock options, patent or other intellectual property); Significant; Blueprint Genetics. S. Bruce: 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. S. Tuupanen: A. Employment (full or part-time); Significant; Blueprint Genetics.

P02.62D Identification of pathogenic mutations in genes involved in non syndromic hearing loss and Usher syndrome

F. Cesca1,2, E. Bettella1,2, R. Polli1,2, E. Leonardi1,2, M. C. Aspromonte1,2, M. Bellini1,2, A. Sensi3, S. Bigoni4, P. Scimemi5,6, R. Santarelli5,6, A. Murgia1,2

1Laboratory of Molecular Genetics of Neurodevelopment, Department of Women’s and Children’s Health, University of Padua, Padua, Italy, 2Fondazione Istituto di Ricerca Pediatrica (IRP), Città della Speranza, Padua, Italy, 3U.O. Medical Genetics Romagna, M. Bufalini Hospital, Cesena, Italy, 4Medical Genetics Unit, Ferrara University Hospital, Ferrara, Italy, 5Audiology and Phoniatric Service, Department of Neurosciences, University of Padua, Padua, Italy, 6Santi Giovanni e Paolo Hospital, ULSS3 Serenissima, Venice, Italy

Non-syndromic hearing loss is characterized by a vast genetic heterogeneity; some syndromic forms have onset as isolated deafness and then evolve later in life, as Usher syndrome. We developed an NGS targeted gene-panel of 59 genes using the Ion Torrent PGMTM platform combined with a customized bioinformatic pipeline for the analysis of DNA samples from clinically highly selected subjects with sensorineural hearing loss, negative for GJB2 mutations/GJB6 deletions. Among the 158 subjects tested, 64 were found to carry pathogenic variants (41%) that in 18 cases (28%) altered genes involved both in NSHL and Usher syndrome (ADGRV1, CDH23, MYO7A, PCDH15, USH1C, USH2A); 13 of these subjects were under 15 years of age and were largely referred for NSHL. 7/18 positive-subjects carried mutation in CDH23, the most frequently mutated gene in our cohort. We achieved a diagnosis of Usher syndrome type I in three subjects (3 y.o.; 6 y.o.; 16 y.o.) with congenital profound hearing loss, retinal anomalies/retinitis pigmentosa and/or history of motor delay, who carried mutations in CDH23, MYO7A and PCDH15. Usher syndrome type II was diagnosed in a 12 y.o. boy referred for congenital bilateral mild hearing loss and subsequently found to have early signs of retinal alteration. 13 novel likely pathogenic mutations were identified in NSHL/Usher genes; 1 splice-site mutation has been further characterized at the RNA level. We demonstrate the importance and efficacy of integrating the powerful NGS technology with a comprehensive careful clinical evaluation, to reach an earlier diagnosis and provide important prognostic and follow-up information.

F. Cesca: None. E. Bettella: None. R. Polli: None. E. Leonardi: None. M.C. Aspromonte: None. M. Bellini: None. A. Sensi: None. S. Bigoni: None. P. Scimemi: None. R. Santarelli: None. A. Murgia: None.

P03 Internal organs & endocrinology (lung, kidney, liver, gastrointestinal)

P03.01A Visceral myopathy due to a novel deletion of the ACTG2 gene: a case report

M. Kraatari1,2,3, H. Kokkonen2,4, M. Mäkinen2,5, S. Turunen6, J. Moilanen1,2,3, O. Kuismin1,2,3

1Department of Clinical Genetics, Oulu University Hospital, Oulu, Finland, 2Medical Research Center, Oulu University Hospital, University of Oulu, Oulu, Finland, 3PEDEGO Research Unit, University of Oulu, Oulu, Finland, 4Northern Finland Laboratory Centre NordLab, Oulu, Finland, 5Cancer and Translational Medicine Research Unit, Department of Pathology, University of Oulu, Oulu, Finland, 6Department of Children and Adolescents, Oulu University Hospital, Oulu, Finland

Introduction: ACTG2-related disorders are a part of visceral myopathy, a rare hereditary myopathic degeneration of gastrointestinal and urinary tracts causing chronic intestinal pseudo-obstruction. Visceral myopathy is characterized by impaired intestinal function and motility resulting in severe abdominal pain, malnutrition and even death. Inter- and intrafamilial variability is present. Diagnostic criteria include absence of mechanical obstruction and histological examination of intestinal biopsies.

Materials and Methods: We report a 10-year-old boy referred to Oulu university hospital in March 2016 due to pseudo-obstruction of the small intestine. Previously, persistent ductus arteriosus was closed operatively and diagnosis of functional growth hormone disturbance was placed. He had suffered from periodic abdominal pain, vomiting and slow weight gain from infancy. His clinical picture was severe and he was completely dependent on parental nutrition. Clinical suspicion of visceral myopathy was aroused.

Results: Deletion/duplication analysis of ACTG2 identified a heterozygous likely pathogenic deletion encompassing the entire ACTG2. Chromosomal microarray showed a heterozygous microdeletion 2p13.1 of 84 kb including the exons 2-9 of ACTG2 and entire DGUOK. The deletion was not identified in the parents and thus, was considered de novo.

Conclusions: We identified a novel heterozygous deletion of ACTG2 explaining the patient’s phenotype. Mutations in ACTG2 cause visceral myopathy. The microdeletion also included DGUOK. Mutations in DGUOK lead to autosomal recessive deoxyguanosine kinase deficiency causing neurological symptoms and liver dysfunction. No other pathogenic mutations were identified in DGUOK. To the authors’ knowledge, no deletions in ACTG2 have previously been reported causing visceral myopathy.

M. Kraatari: None. H. Kokkonen: None. M. Mäkinen: C. Other Research Support (supplies, equipment, receipt of drugs or other in-kind support); Modest; Amgen. S. Turunen: D. Speakers Bureau/Honoraria (speakers bureau, symposia, and expert witness); Modest; Abbvie. J. Moilanen: None. O. Kuismin: None.

P03.02B The genetic background in a group of 56 Polish patients with suspected Alport syndrome

P. Halat-Wolska1, E. Ciara1, L. Obrycki2, J. Antoniewicz2, K. Gadomska-Prokop2, J. Kosińska3, M. Rydzanicz3, D. Siestrzykowska1, P. Stawiński3,4, B. Chałupczyńska1, D. Jurkiewicz1, P. Kowalski1, M. Pelc1, D. Piekutowska-Abramczuk1, K. Iwanicka-Pronicka1,5, P. Iwanowski1, J. Lesiak2, A. Łuba2, A. Niemirska2, A. Rogowska6, D. Wicher1, M. Krajewska-Walasek1, R. Grenda2, K. Chrzanowska1, R. Płoski3, M. Litwin2

1Department of Medical Genetics, The Children’s Memorial Health Institute, Warsaw, Poland, 2Department of Nephrology, The Children’s Memorial Health Institute, Warsaw, Poland, 3Department of Medical Genetics, Warsaw Medical University, Warsaw, Poland, 4Department of Genetics, Institute of Physiology and Pathology of Hearing, Warsaw, Poland, 5Department of Audiology and Phoniatrics, The Children’s Memorial Health Institute, Warsaw, Poland, 6Department of Ophthalmology, The Children’s Memorial Health Institute, Warsaw, Poland

Introduction: Alport syndrome (AS) is a clinically and genetically heterogeneous nephropathy caused by pathogenic variants in COL4A3-COL4A5. While most (80%) AS cases are X-linked, approximately 15% and 5% are autosomal recessive or difficult to differentiate from thin basement membrane nephropathy (TBMN) dominant forms. Digenic inheritance among COL4A3-COL4A5 or their concomitance with other glomerulopathy or chronic kidney disease (CKD) related genes, has been recently proposed to explain the variable phenotypic expression and incomplete penetrance frequently observed in AS patients.

Materials and Methods: NGS analysis of 55 glomerulopathy and CKD related genes was performed in a group of 56 unrelated Polish patients with suspected AS.

Results: In all patients clinical diagnosis was confirmed at the molecular level. Overall, 17 known and 32 novel, likely pathogenic alterations in COL4A3-COL4A5 were identified. The inheritance was X-linked in 73% of cases, remaining 9% autosomal recessive and 18% were dominant AS/TBMN. Changes were randomly distributed across all COL4A3-COL4A5 coding regions, however we revealed a recurrent COL4A5 variant c.1871G>A in twelve patients. Additionally, four patients with this alteration had likely pathogenic variant in COL4A3, HNF1B or MYH9, which may modify disease’s severity.

Conclusions: The results of this study broaden the genotypic spectrum of AS, which will facilitate future research on the genotype-phenotype correlations. Multiple-gene sequencing is an effective approach to obtain genetic information in AS, particularly about the mode of inheritance which is important for counselling and may help to predict the clinical course, especially for those patients with mild, non-specific or atypical phenotype.

Partially supported: CMHI-M29/18

P. Halat-Wolska: None. E. Ciara: None. L. Obrycki: None. J. Antoniewicz: None. K. Gadomska-Prokop: None. J. Kosińska: None. M. Rydzanicz: None. D. Siestrzykowska: None. P. Stawiński: None. B. Chałupczyńska: None. D. Jurkiewicz: None. P. Kowalski: None. M. Pelc: None. D. Piekutowska-Abramczuk: None. K. Iwanicka-Pronicka: None. P. Iwanowski: None. J. Lesiak: None. A. Łuba: None. A. Niemirska: None. A. Rogowska: None. D. Wicher: None. M. Krajewska-Walasek: None. R. Grenda: None. K. Chrzanowska: None. R. Płoski: None. M. Litwin: None.

P03.03C Genotype/phenotype correlations in carriers of a single autosomal COL44A3 and COL4A4 mutation

A. Cianflone1,2,3, H. Storey4, F. Flinter1, F. Forzano1

1Clinical Genetics department, Guy’s & St Thomas’ NHS Foundation Trust, London, United Kingdom, 2Department of Neurosciences, Rehabilitation, Ophthalmology, Genetic and Maternal and Infantile Sciences (DINOGMI), University of Genova, Genova, Italy, 3Ospedale Policlinico San Martino IRCCS-Medical Genetic Unit, Largo R. Benzi 10, Genova, Italy, 4Molecular Genetics, Viapath, Guy’s Hospital, London, United Kingdom

Alport’s syndrome is complex: 85% cases show X linked inheritance with pathogenic mutations in COL4A5 and 15% autosomal recessive inheritance with pathogenic mutations in COL4A3 and COL4A4. All affected individuals develop renal failure, usually as young adults.

Carriers of a single autosomal mutation develop Thin Basement Membrane Nephropathy (TBMN) associated with ‘benign familial haematuria’. We analysed a cohort of 119 individuals (29 probands and 85 relatives identified by cascade testing) with heterozygous mutations in COL4A3 or COL4A4 identified by Next Generation Sequencing and assessed genotype/phenotype correlations. 70% had haematuria, 38% developed proteinuria, 21% had chronic kidney disease and 10% reached end stage renal disease. 36% of our cohort had a single mutation in COL4A4 c.2906C>G p.(Ser969Ter), representing 18% of cases of proteinuria. 50% of these patients developed proteinuria compared with 31% of patients with other mutations. The risk of eGFR falling below 60ml/min increased from 6% to 8% in patients with the COL4A4c.2906C>G p.(Ser969Ter) mutation. Single COL4A3/4 mutations are recognised susceptibility factors for developing proteinuria, hypertension and renal disease in later life, but these risks have previously not been quantified. It is recommended that all patients with a single COL4A3/4 mutation should have annual checks of blood pressure and urine, with a low threshold for prescribing an ACE inhibitor, and this is important for those with the high-risk genotype.

In conclusion, this is the largest study ever performed of the implications of carrying a single autosomal COL4A3/4 mutation and our findings clarify the risk of renal disease in this population.

A. Cianflone: None. H. Storey: None. F. Flinter: None. F. Forzano: None.

P03.04D Molecular genetic analysis in patients with clinically suspected autosomal recessive polycystic kidney disease

L. Obeidova1, V. Elisakova1, T. Seeman2, J. Reiterova3, J. Vcelak4, J. Stekrova1

1Institute of Biology and Medical Genetics, First Faculty of Medicine, Charles University and General University Hospital in Prague, Prague, Czech Republic, 2Department of Paediatrics, 2nd Faculty of Medicine, Charles University in Prague and Motol University Hospital in Prague, Prague, Czech Republic, 3Department of Nephrology, First Faculty of Medicine, Charles University in Prague and General University Hospital in Prague, Prague, Czech Republic, 4Institute of Endocrinology, Prague, Czech Republic

Introduction: Autosomal recessive polycystic kidney disease (ARPKD) is a severe form of chronic kidney disease, frequently diagnosed prenatally or in an early age. ARPKD is primaly caused by mutations in the PKHD1 gene, nevertheless, phenotype of polycystic kidneys clinically resembling ARPKD can be caused by mutations in number of other genes, such as HNF1β, PKD1, PKD2, NPHP etc. Thus, the molecular genetic analysis can be very useful in differential diagnosis of ARPKD in patient. The results of molecular genetic analysis in eleven patients with clinically suspected ARPKD, who did harbor mutations in other genes than PKHD1, are presented.

Materials and Methods: The molecular analysis was carried out using next-generation sequencing method with enrichment capture-based and amplicon-based library preparation. The panel of approximately 80 genes associated with the formation of polycystic kidneys was analyzed.

Results: The most frequent mutations found in our group of patients were variants in the TMEM67 gene (5 patients). In three patients, mutation in PKD1 was detected (in one patient in combination with PKHD1 mutation in trans). Two patients harbored combination of two mutations: (1) mutation in PKHD1 and TMEM237, (2) PKHD1 mutation and deletion of 3 exons in the NPHP3 gene. In one patient, deletion of whole HNF1β gene was identified.

Conclusions: Because of an etiologic heterogeneity of polycystic kidney disease phenotype, the complex mutational analysis, encompassing analysis in other genes (especially TMEM67), should be used for reliable differential diagnosis. Supported by the grant projects GAUK 1015, PROGRES- Q25/LF1 and RVO VFN64165

L. Obeidova: None. V. Elisakova: None. T. Seeman: None. J. Reiterova: None. J. Vcelak: None. J. Stekrova: None.

P03.05A Gastrointestinal dysfunction in autism spectrum disorder: New insights from the Foxp1+/-mouse with altered gut motility and achalasia

H. Fröhlich1, M. Kollmeyer1, M. Stuhlinger1, V. Linz1, D. Groneberg2, A. Reigl2, E. Zizer3, A. Friebe2, B. Niesler1, G. Rappold1

1Department of Human Molecular Genetics, Institute of Human Genetics, University of Heidelberg, Heidelberg, Germany, 2Institute of Physiology, University of Würzburg, Würzburg, Germany, 3Department of Internal Medicine I, University of Ulm, Ulm, Germany

Gastrointestinal (GI) dysfunction is common in individuals with autism spectrum disorder (ASD), but these symptoms are often overlooked and it is still not clear how GI dysfunction relates to the core features of ASD. FOXP1 syndrome is characterized by autistic traits, such as social deficits, language impairment, and intellectual disability. But feeding difficulties, constipation, and other GI problems were also reported. Whether these symptoms are due to primary impairment or a secondary effect of altered behaviour or side effects of psychotropic medication remains unclear.

We investigated the GI tract of patient-relevant Foxp1+/-mice. These mice have a lower body weight than wild type animals and show altered feeding behaviour. Foxp1 was expressed in all GI sections and was reduced in Foxp1+/-mice compared with WT. A pronounced atrophy of the tunica muscularis was detected in the oesophagus and colon, caused by reduced muscle cell proliferation. Nitric oxide-induced relaxation of the lower oesophagus sphincter was impaired and achalasia confirmed in vivo by manometry. Several Foxp1 targets previously identified by microarray analysis in the brain were significantly deregulated in the oesophagus of Foxp1+/- mice. The total gut transit was significantly prolonged. Spatiotemporal maps depicting the colonic contraction patterns revealed strongly disturbed contractility and peristalsis. Overall, our findings provide the first evidence that GI disturbances in patients with FOXP1 autism spectrum disorder may be caused by impaired gut motility and achalasia, driven by FOXP1-dependent deregulation of genes. Furthermore, this is the first report of achalasia being caused by a heterozygous gene deletion.

H. Fröhlich: None. M. Kollmeyer: None. M. Stuhlinger: None. V. Linz: None. D. Groneberg: None. A. Reigl: None. E. Zizer: None. A. Friebe: None. B. Niesler: None. G. Rappold: None.

P03.06B HLA and non-HLA susceptibility genes to childhood steroid-sensitive nephrotic syndrome in the Japanese population

X. Jia1, T. Horinouchi2, Y. Hitomi1, Y. Kawai1, K. Nozu2, C. Nagano2, T. Yamamura2, M. Nagasaki3, K. Tokunaga1, K. Iijima2

1Dept Human Genetics, Grad Sch Medicine, Univ Tokyo, Tokyo, Japan, 2Dept Pediatrics, Grad Sch Medicine, Kobe Univ, Kobe, Japan, 3Dept Integrative Genomics, Tohoku Medical Megabank, Tohoku Univ, Sendai, Japan

Idiopathic nephrotic syndrome (INS) is the most common cause of kidney disease in children, about 80% of pediatric patients respond to steroid treatment and are classified as steroid-sensitive NS (SSNS). Previous genome-wide association studies (GWASs) have identified HLA-DR/DQ as the predominant risk factors of childhood SSNS. Our group reported the first GWAS for childhood SSNS in Japanese. Genome-wide significant associations were identified in HLA-DR/DQ region and disease-associated HLA alleles and haplotypes were further clarified (J Am Soc Nephrol, 2018). To identify other loci contributing to susceptibility to childhood SSNS, especially in non-HLA regions, we performed an extended GWAS with a larger number of samples. Discovery stage including 897 patients with childhood-onset SSNS and 2,807 adult healthy controls was carried out in the Japanese population, genotyped using Affymetrix ‘Japonica Array’. Whole-genome imputation was conducted using a phased reference panel of 2,049 healthy Japanese individuals (2KJPN panel). Quality control was performed to exclude the samples with low calling rate (<97%) and the variants with low genotyping rate (<97%), minor allele frequency (MAF) <0.5% and Handy-Weinberg equilibrium (HWE) test p-value <1×10-5. Association analysis was conducted using logistic regression with the adjustment of gender and principal components. In this discovery GWAS, the most significant association was detected in HLA-DR/DQ region as we reported before (P=2.98×10-32, odds ratio (OR)=0.34). Furthermore, two regions on chromosome 19 and 18 showed genome-wide significant associations (p=3.28×10-18, OR=1.95; p=5.38×10-9, OR=1.65). Replication studies were successfully performed in other Asian sample sets.

X. Jia: None. T. Horinouchi: None. Y. Hitomi: None. Y. Kawai: None. K. Nozu: None. C. Nagano: None. T. Yamamura: None. M. Nagasaki: None. K. Tokunaga: None. K. Iijima: None.

P03.07C Two genetic disease in one family: Cockaine Syndrome (ERCC8) and Fanconi Bickels (SLC2A2)

M. Doco-Fenzy1, N. Calmels2, H. Thorn1, C. Poirsier1, M. Spodenkiewiscz1, E. Gouy1, L. Le Collen1, R. Santer3, B. Digeon4, A. Doe5, A. Lebre1, A. Lehmann6, C. Obringer7, V. Laugel7, G. Thiefin8

1Service de Génétique, CHU-Reims, Reims, France, 2Service de génétique, strasbourg, France, 3genetic, Hambourg, Germany, 4Service de Pédiatrie, CHU-Reims, Reims, France, 5Service de Neurologie, CHU-Reims, Reims, France, 6Genome center Sussex university, Cambridge, United Kingdom, 7Service de Génétique, CHU-Strasbourg, strasbourg, France, 8Service de Gastroentérologie, CHU-Reims, Reims, France

We report a rare consanguinous family suffering from 2 congenital autosomic recessive genetic disease. The parents are cousins and have 7 children. 3 boys and 1 girl show Cockaine syndrome (OMIM #216400) and 3 children (1 girl and 2 boys) show Fanconi-Bickels symptoms (OMIM #227810) linked to GLUT2 deficiency. Two boys are affected by both Cockaine and GLUT2 deficiency.

Fanconi-Bickels syndrome is related to SLC2A2 mutations (3q26) and glucose transporter protein-2 (GLUT2) defect. The typical clinical picture is characterized by hepatorenal glycogen accumulation resulting in hepato- and nephromegaly. In this family the patients with SLC2A2 mutation show growth retardation, major hepatomegaly and renal failure. The homozygous mutation is intronic: lVS9-1 g>a /IVS9-1g>a.

In Cockaine syndrome, 2 genes are affected ERCC6 and ERCC8. Here a new homozygous pathogenic variation has been identified in ERCC8 (5q12.1): c.730C>T (exon 9) (Calmel N et al. Orphanet J Rare Dis. 2016). ERCC8 encodes the CSA protein involved in DNA repair. The patients aging 15 to 28 years in the family show neonatal photosensibility, growth retardation, dysmorphic features, lypoatrophy, intellectual deficiency, late onset deafness and tremor. The RRS test (Recovery RNA synthesis after DNA damage) was altered and UDS test (unscheduled DNA synthesis) was normal. ERCC8 is involved in Cockaine Syndrome and UV-Sensitive syndrome, both very rare in Europe (1/200000 and 1/1000000 respectively).

The occurrence of several genetic pathology in the same family is now more frequently presented. We report here the association of two very rare syndromes affecting most children in a unique family.

M. Doco-Fenzy: None. N. Calmels: None. H. Thorn: None. C. Poirsier: None. M. Spodenkiewiscz: None. E. Gouy: None. L. Le Collen: None. R. Santer: None. B. Digeon: None. A. Doe: None. A. Lebre: None. A. Lehmann: None. C. Obringer: None. V. Laugel: None. G. Thiefin: None.

P03.08D Apparently isolated congenital hyperinsulinism due to KDM6A mosaic pathogenic variant

M. Yacobi Bach1, E. Elkon Tamir2, S. Ben Shachar1, O. Eyal3

1Genetics institute, Tel Aviv, Israel, 2Pediatric Endocrinology institute, Tel Aviv, Israel, 3Pediatric Endocrinology institute, Tel Aviv, Israel

Introduction: Kabuki Syndrome- KS is a syndrome with multiple congenital anomalies. It is characterized by dysmorphic facial features, craniofacial and skeletal anomalies, dermatoglyphic abnormalities, mild to moderate cognitive decline, and postnatal growth deficiency. Congenital hyperinsulinism as the presenting feature of KS, was described in 10 affected individuals. The gene KDM6A is responsible for KS in approximately 5% of patients. KDM6A mosaicism was reported in at least one patient with typical KS manifestations.

Case report: A 20-month-old girl diagnosed with isolated, persistent congenital hyperinsulinism responsive only to Diazoxide treatment. She has no dysmorphic features, normal development, and no structural anomalies. Molecular Analysis of the coding regions and exon/intron boundaries of 16 genes responsible for congenital hyperinsulinism was performed on a clinical basis by targeted next generation sequencing. A mosaic pathogenic variant in the gene KDM6A c.514C>T p.Arg172Ter was detected in at least 20%. of the cells. No other variants were identified.

Discussion: It has been suggested previously that female patients with KDM6A mutations have milder phenotypes than males. This is the first report of KDM6A pathogenic variant causing isolated congenital hyperinsulinism in an apparently healthy girl. It is likely that the combination of a mosaic state of variant and female gender resulted in congenital hyperinsulinism. We suggest the gene KDM6A will be included in the molecular studies of isolated congenital hyperinsulinism.

M. Yacobi Bach: None. E. Elkon Tamir: None. S. Ben Shachar: None. O. Eyal: None.

P03.10B Genetic Drivers of Congenital Chylothoraces

S. Schneider1,2, A. C. Hilger1,2, H. Thiele3, J. Altmüller3, A. Müller2, H. Reutter1,2

1Institute of Human Genetics, University of Bonn, Bonn, Germany, 2Department of Pediatrics, Children’s Hospital, University of Bonn, Bonn, Germany, 3Cologne Center for Genomics, University of Cologne, Cologne, Germany

Introduction: Congenital chylothoraces (CCT) are rare fetal conditions, occurring in 1/10,000 pregnancies. Its clinical course varies from small thoracic effusions to life‐threatening thoracic compression and secondary hydrops fetalis. Primary CCT result from malformations of the thoracic lymphoid system, secondary CCT result from cardiac anomalies or inflammatory disease. Previous studies suggest underlying genetic causes and found several genes to be mutated in some patients with lymphatic disorders. The aim of this study is to investigate new genetic drivers for primary CCT.

Methods: We applied whole-exome sequencing (WES) in two affected sibling pairs and their healthy parents. To identify disease causing variants we applied standardized filtering of WES-data. First, we filtered for autosomal-recessive and autosomal-dominant novel variants. Second, the predicted deleteriousness and the conservation of the mutations was controlled. Third, further research on the candidate genes was done to specify the functional effects of the mutations. The variants found in potential candidate genes were validated and segregated by re-sequencing. After prioritisation of the best candidate gene, re-sequencing of this candidate gene in a cohort of 30 sporadic cases is warranted.

Results: The filtering of WES-data identified four rare homozygous variants in ZNF512 (p.His90Arg), SMC6 (p.Thr679Met), BRE (p.Ser11Cys) and GCKR (p.Arg149Lys).

Conclusions: We suggest novel recessive candidate genes for congenital chylothoraces. Re-sequencing in a cohort of 30 sporadic cases with congenital chylothoraces and functional studies in zebrafish of the most promising candidate genes is pending.

S. Schneider: None. A.C. Hilger: None. H. Thiele: None. J. Altmüller: None. A. Müller: None. H. Reutter: None.

P03.11C A novel homozygous nonsense mutation (p.R516X) in the SLC5A5 gene causing congenital hypothyroidism

F. B. Isik1, M. D. Sozuguzel1, B. K. Aydin2, C. Parlayan1, M. Yildiz2, H. Cangul1

1Istanbul Medipol University, istanbul, Turkey, 2Kanuni Sultan Süleyman Training and Research Hospital, istanbul, Turkey

Congenital hypothyroidism (CH) is the most common neonatal endocrine disorder with an incidence of 1 in 3500 live births and 2% of CH cases have familial origin. Unless a timely treatment is introduced, CH causes mental retardation and growth delay. More than 10 causative genes have been described for the pathogenesis of inherited CH to date. In this study we investigated a Mendelian cause of CH in a consanguineous family with 2 affected children. Both cases showed short stature, mental retardation and bone age retardation. Next generation sequencing analysis of 16 candidate CH genes identified a homozygous nonsense change (p.R516*, c.1546C>T) in the SLC5A5 gene (NM_000453.2) in both cases. We confirmed the presence of this variant by Sanger sequencing and both parents and the unaffected sibling carried this variant at heterozygous state, underlying the co-segregation of the variant with the disease status in the family. Moreover, this variant was not present in 400 ethnically matched control chromosomes. p.R516* variant in the SLC5A5 gene is not currently listed as a mutation in clinical databases. Since (i) it introduces a premature stop codon in the gene; (ii) co-segregates with the disease status in the family and (iii) is not present in 400 ethnically matched control chromosomes, here we report it as a novel mutation causing congenital hypothyroidism.

F.B. Isik: None. M.D. Sozuguzel: None. B.K. Aydin: None. C. Parlayan: None. M. Yildiz: None. H. Cangul: None.

P03.12D Molecular analysis of CYP21A2 gene in 100 patients with Congenital Adrenal Hyperplasia (CAH) due to 21-hydroxylase deficiency

E. Fylaktou1, A. Sertedaki1, E. Charmandari1,2

1Division of Endocrinology, Metabolism and Diabetes, First Department of Pediatrics, National and Kapodistrian University of Athens, Medical School, ‘Aghia Sophia’ Children’s Hospital, Athens, Greece, 2Division of Endocrinology and Metabolism, Biomedical Research Foundation of the Academy of Athens, Athens, Greece

Introduction: Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency is an autosomal recessive condition in which mutations of the cytochrome P450 21-hydroxylase gene (CYP21A2) result in decreased synthesis of glucocorticoids and often mineralocorticoids. The clinical spectrum of the disease ranges from most severe, to mild forms depending on the degree of 21-hydroxylase activity.

Materials and Methods: Seventy-one cases (69 referred for CAH and 2 for prenatal diagnosis), as well as 29 siblings of carriers/patients with CAH, were tested for CYP21A2 gene mutations. Long range PCR was employed to selectively amplify the CYP21A2 gene against its pseudogene, followed by Sanger sequencing. MLPA analysis was undertaken for samples with an indication of duplication/deletion of the gene.

Results: Thirty patients with CAH were compound heterozygotes or homozygotes, while forty were heterozygotes for CYP21A2 gene mutations. One of the prenatal cases was compound heterozygote, while the other was simple heterozygote for CYP21A2 gene mutations. The most common mutations detected were as follows: p.V281L (25%), p.P30L (14%), I2 Splice Site (10%), p.I172N (4%), p.Q318X (2%), p.P482S (3%), p.P453S (13%) and *13G>A 3' UTR (7%). Four parents with no clinical symptoms were found to be compound heterozygotes for the non-classic form of CAH. Eight samples harbored genomic rearrangements in one or both alleles of the gene.

Conclusion: Molecular analysis of the CYP21A2 gene is essential for proper management of patients with CAH due to 21-hydroxylase deficiency, as well as for genetic counseling and prenatal diagnosis.

E. Fylaktou: None. A. Sertedaki: None. E. Charmandari: None.

P03.13A Application of next-generation sequencing in the search of genetic causes for Taiwanese patients with syndromic disorders of sex development

M. Tsai

Department of Pediatrics, National Cheng Kung University Hospital, Tainan, Taiwan

Objective: This study aimed primarily to establish a rapid and high-throughput genetic test for syndromic disorders of sex development (DSD). Meanwhile, the applicability and effectiveness of next-generation sequencing were evaluated in searching for the remaining unknown causative genes of this inherited disease.

Methods: Patients with clinically diagnosis of DSD with additional dysmorphic features were recruited. We applied the filter-based hybridization method as the main strategy for whole exome sequence (WES) enrichment. The amplified exomes were hybridized against the DNA libraries and sequenced subsequently. We finally used the integrated computing programs to call the genetic variants for DSD.

Results: We enrolled a total of 10 syndromic DSD patients. Targeted amplicons were smoothly generated with adequate capture efficiency (coverage 98% of exons and 100% >30 read depths). Among them, we found 2 patients compatible with CHARGE syndrome (one is c.1480C>T, p.Arg494X and the other is c.6571G>A, p.Glu2191Lys in CHD7 gene), one with Robinow syndrome (c.1571delCGGGTGGGGCAGCGfs in DVL1 gene), one with OPHN1 syndrome (c.1171T>A, p.Arg391Trp in OPHN1 gene), and one with FG syndrome (c.1864C>T, p.Glu622Lys in FLNA gene).

Conclusion: We identified a number of genetic variants accounting for syndromic DSD in Taiwanese population. With identification of these causative genes, it extended our current understanding of sex development and related congenital disorders. Further functional verification of these variants may be needed on the cell line models.

Funding: This research was funded by the research grants awarded by the National Cheng Kung University Hospital (NCKUH-10702001 and NCKUH-10307006).

M. Tsai: None.

P03.14B Splice site mutation in GRHPR gene in Iranian family with ESRD and nephrocalcinosis

R. Miri Moosavi1, S. Saber1, A. Ebrahimi1, T. Malakoutian2

1Jordan medical and genetic laboratory, Tehran, Iran, Islamic Republic of, 2Department of Nephrology, Hasheminejad Kidney Center, Iran University of Medical Sciences, Tehran, Iran, Islamic Republic of

Introduction: End-stage renal disease (ESRD) occurs when your kidneys clearly begin to shut down. Both genetic and epigenetic increase risk of ESRD. A three-to nine-fold greater risk of ESRD is observed in individuals with a family history of ESRD, so role of genetic study is important nowadays.

Material and Method: A 43-year-old, Iranian man were detected ESRD with nephrocalcinosis (multiple stones). According to nephrologist decision, primary hyperoxaluria was considered so that was candidate for kidney and liver transplant. So, genetic analysis was requested to approve mentioned diagnosis. Whole exome sequencing (WES) test was performed to find variants responsible for renal disorder.

His parents had consanguineous marriage. His father and two sisters suffered from nephrocalcinosis. According to results of WES test and analysis all genes responsible to renal disorder, one likely pathogenic/pathogenic variant (c.735-1G>A) in splice site region was found in GRHPR gene. This gene is responsible for primary hyperoxaluria type 2 with an autosomal recessive inheritance pattern.

Clinical manifestations in patients were same as Mentioned disease, after examining of clinical data. Familial segregation was done and his father and two sisters are carrier of this variant. Liver transplant might be considered within kidney transplant in this case.

Conclusion: Genetic analysis should be considered in renal failure to do the best management in therapy.

R. Miri Moosavi: None. S. Saber: None. A. Ebrahimi: None. T. Malakoutian: None.

P03.15C GCK mutations in Croatian MODY patients

A. Merkler1, A. Špehar Uroić1, N. Krnić1, H. Ljubić1, D. Caban1, A. Acman Barišić1, D. Kaštelan1,2, J. Sertić1,2

1University Hospital Centre Zagreb, Zagreb, Croatia, 2University of Zagreb School of Medicine, Zagreb, Croatia

Introduction: Maturity onset diabetes of the young (MODY) is clinically and genetically heterogeneous group of diabetes inherited in autosomal dominant manner. It usually occurs in adolescence or young adulthood and accounts for at least 1-3% of all diabetes. GCK-MODY is one of four most common type of MODY with estimated prevalence of 1:1000. It is characterized by mild, stable fasting hyperglycemia which is often discovered incidentally during routine medical screening.

Materials and Methods: After clinical examination, 56 patients with stable hyperglycemia, small 2 hour increment in OGTT, positive family history of type 2 or gestational diabetes and negative pancreatic antibodies were tested for GCK-MODY. The promoter, whole coding region and flanking intronic regions of the GCK gene were analyzed by Sanger sequencing. Pathogenicity of identified mutations was verified in reference databases for mutations related with GCK-MODY.

Results: 17 different mutations in GCK gene were found in 32 patients. Most of the mutations were in exon 7 (six mutations in 16 patients) and in exon 9 (five mutations in 5 patients). The most common mutation was p.Thr228Met in exon 7 found in 8 patients from 4 different famillies. One patient was apparently homozygous for mutation p.Gly170Asp, but its true homozygosity is not yet confirmed, it can be a result of an allele dropout due to SNP in the primer region. In one patient we detected a novel variant c.806T>G, p.Phe269Cys in exon 7.

Conclusions: GCK-MODY is frequently underdiagnosed and inadequately treated. Treatment is rarely necessary if the mild hyperglycemia remains stable.

A. Merkler: None. A. Špehar Uroić: None. N. Krnić: None. H. Ljubić: None. D. Caban: None. A. Acman Barišić: None. D. Kaštelan: None. J. Sertić: None.

P03.16D Novel variants in DHH gene identified with 46,XY gonadal dysgenesis

A. Aghayev1, G. Toksoy1, S. Poyrazoglu2, B. Karaman1, S. Avcı1, Z. Yavas Abalı2, U. Altunoglu1, F. Bas2, F. Darendeliler2, S. Basaran1, Z. Uyguner1

1Dept. Med. Genet., Istanbul Med. Faculty, Istanbul University, Istanbul, Turkey, 2Dept. Ped. Endocrinology, Istanbul Med. Faculty, Istanbul University, Istanbul, Turkey

Introduction: Desert Hedgehog (DHH) a member of the hedgehog family, is located in 12q13.1 and acts on early testicular development, testis cord formation and differentiation of fetal Leydig cells. To date, six homozygous mutations have been described in DHH in 46,XY patients conferring phenotypes ranging from partial to complete gonadal dysgenesis, associated with of without polyneuropathy.

Materials and Methods: We investigated three patients from two families with 46,XY gonadal dysgenesis, for pathogenic sequence alterations in 31 associated genes, with in-house-designed next generation sequencing (NGS) targeted gene panel, using an Ion Torrent platform. Ultrasound and histopathological examination of the gonads with electrophysiological examination of peripheral nerves were performed. Protein modeling was done to predict the effect of the missense mutations.

Results: We have identified three different homozygous mutations, one in two siblings, c.[1146G>A];[1146G>A], (p.[Trp382*];[Trp382*]), and two in singleton case, c. [71G>C;1063C>T];[71G>C;1063C>T], (p.[Gly24Ala;Arg355Cys];[Gly24Ala;Arg355Cys]). One of the siblings presented with penoscrotal hypospadias, bilateral inguinal testes, Mullerian structure evident on biopsy, no response to HCG at one-year and raised as female initially. Second sibling presented micropenis, bilateral inguinal testes, no Mullerian structure, response was normal to HCG at age 14 days and raised as male. In addition, both patients suffered from polyneuropathy. Third patient presented at age 19 days with penoscrotal hypospadias, bilateral inguinal testes, no Mullerian structure, low AMH and raised as male.

Conclusions: DHH mutation should be analyzed in patients with 46,XY gonadal dysgenesis for diagnosis and the presence of potential neuropathy and gonadal tumors.

A. Aghayev: None. G. Toksoy: None. S. Poyrazoglu: None. B. Karaman: None. S. Avcı: None. Z. Yavas Abalı: None. U. Altunoglu: None. F. Bas: None. F. Darendeliler: None. S. Basaran: None. Z. Uyguner: None.

P03.17A HBx destabilizes TIP60 in HBV-induced hepatocellular carcinoma

S. Hora1,2, N. Kumari1, D. Rajagopalan1, L. Hooi1, T. B. Toh1, K. K. Lee1, W. S. Teo1, T. Tan2, P. L. Chen1, E. K. Chow1, H. P. Koeffler1, S. Jha1,3

1Cancer Science Institute of Singapore, Singapore, Singapore, 2Department of Biochemistry, National University of Singapore, Singapore, Singapore, 3Department of Biochemistry, National University of Singapore, Sinagpore, Singapore

TIP60(TAT-interactive protein of 60 kDa),is a lysine acetyltransferase (KAT5) and acts as a haplo-insufficient tumor suppressor in multiple cancer-types. TIP60 is known to be destabilized by different onco-viruses and this intrigued us to inspect its role in another onco-virus pathogenesis, hepatitis B virus (HBV). HBV, the leading cause of hepatocellular carcinoma (HCC) is the 6th most common cancer in the world. Among the four overlapping open reading frames of HBV, the HBV X gene (HBx) is considered the causative agent for malignant transformation associated with HBV infection. In this conference I am going to discuss data that show TIP60 being targeted by HBx, a proto-oncogene of HBV proteasomally, by utilizing an E3 ubiquitin ligase, EDD1 in the cell. By utilizing various cell culture models, we demonstrate that HBx and TIP60 form a complex with EDD1. Interestingly, this hacking of TIP60 protein, enables increased proliferation of hepatocytes, as confirmed by various growth assays and mechanistically by increased expression of TERT and decreased levels of TIP60 protein. Additionally, tissue microarray analysis(TMA)of HBV-positive malignant tumor samples demonstrate a strong correlation between TIP60 and EDD1 levels, further supporting our hypothesis.We therefore propose TIP60 as one of the cellular targets in HBx mediated viral infection and to utilize small molecule inhibitors against the identified ubiquitin-ligase in HBx-mediated HBV-carcinogenesis. In conclusion, our study has identified a relatively unknown role of HBx, its destabilization of an epigenetic writer and the mechanism involved in this phenomenon. This work was supported by MOE grants(MOE AcRF Tier 1 T1-2012 Oct-04 and T1-2016 Apr-01)and CSI(R-713-006-014-271). SH is supported by NUS-Research Scholarship awarded by NUS Yong Loo Lin School of Medicine.

S. Hora: None. N. Kumari: None. D. Rajagopalan: None. L. Hooi: None. T.B. Toh: None. K.K. Lee: None. W.S. Teo: None. T. Tan: None. P.L. Chen: None. E.K. Chow: None. H.P. Koeffler: None. S. Jha: None.

P03.18B Next generation sequencing approach for determining the molecular basis of familial hematuria

O. Beltcheva1, K. Kamenarova1, K. Mihova1, A. Boueva2, D. Roussinov3, G. Zlatanova3, V. Lazarov4, P. Miteva3, M. Gaydarova3, B. Deliyska4, V. Mitev1, R. Kaneva1

1Molecular Medicine Center, Dept. of Medical Chemistry and Biochemistry, Medical University-Sofia, Sofia, Bulgaria, 2Nephrology and Dialysis Clinics, SBALDB “Dr. Lisichkova”, Varna, Bulgaria, 3SBAL Pediatric Diseases, Nephrology and Hemodialysis Clinic, Department of Pediatrics, Medical University -Sofia, Sofia, Bulgaria, 4Nephrology Clinic, University Hospital “Queen Giovanna”, Medical University - Sofia, Sofia, Bulgaria

Introduction: Hematuria is a non-specific symptom of kidney or urinary tract pathology. The presence of blood in the urine could be due to trauma, infections, systemic disease, disorders of the coagulation or genetic mutations affecting the development and function of particular cell and tissue types. The presence of family history is indicative of a hereditary condition such as disorders of the basement membrane, complement or podocytes. Differential diagnosis of hematuria is crucial for successful treatment of the underlying condition.

Materials and Methods: Illumina TruSight One Sequencing Panel was used for mutation screening in 8 families with multiple members with hematuria. The index patients were referred with initial diagnoses of glomerulopathy, thin basement membrane nephropathy, uric acid nephropathy, focal segmental glomerulosclerosis, tubulointerstitial nephritis and/or chronic kidney disease.

Results: Using next generation screening we determined the genetic cause of hematuria in Bulgarian families. Pathogenic variants were found in the type IV collagen (autosomal recessive and dominant variants in COL4A4, X-linked in COL4A5) and complement genes. Both missense and splice mutations, known pathogenic and novel variants were found. A potential role of heterozygous C1QC mutations in the pathogenesis of G3 glomerulopathy was observed.

Conclusion: Clinical diagnosis of hereditary hematuria is often hindered by phenotype variability, lack of information for disease progression in older family members, difficulties in obtaining biopsy samples, etc. Addition of massive parallel sequencing of large gene to the diagnostic procedure would allow timely and precise determination of the molecular cause of the disease.

Grant references: D-73/2018; DUNK 01/2/2009

O. Beltcheva: None. K. Kamenarova: None. K. Mihova: None. A. Boueva: None. D. Roussinov: None. G. Zlatanova: None. V. Lazarov: None. P. Miteva: None. M. Gaydarova: None. B. Deliyska: None. V. Mitev: None. R. Kaneva: None.

P03.19C A rare diagnosis: Hermansky-Pudlak syndrome in a patient with pulmonary fibrosis, oculocutaneous albinism and thrombocytopathy

J. Trizuljak1,2, M. Doubková3, A. Hrazdírová3, Z. Vrzalová2, I. Blaháková2, L. Radová2, Š. Pospíšilová1,2, M. Doubek1,2

1Department of Internal Medicine, Hematology and Oncology, University Hospital and Faculty of Medicine, Brno, Czech Republic, 2Central European Institute of Technology, Brno, Czech Republic, 3Department of Pulmonary Diseases and Tuberculosis, University Hospital and Faculty of Medicine, Brno, Czech Republic

Introduction: Hermansky-Pudlak Syndrome (HPS) is an autosomal recessive disorder associated with oculocutaneous albinism, bleeding diathesis, granulomatous colitis, and highly penetrant pulmonary fibrosis in some subtypes, Homozygous or compound heterozygous mutations in HPS1, HPS3, HPS4 and several other genes lead to clinical manifestation of the disease.

Materials and Methods: A 57-year-old patient with congenital oculocutaneous albinism, thrombocytopathy and late onset accelerated pulmonary fibrosis was referred to our clinic. Negative family history of these symptoms suggested autosomal-recessive mode of inheritance. We performed NGS analysis of proband-parents trio. Whole-exome libraries were prepared according to the Nimblegen SeqCap EZ Exome v3 protocol and sequencing was performed on NextSeq 500 for all of them. Furthermore, we performed in silico analysis of a virtual gene panel, including HPS1, AP3B1, HPS3, HPS4, HPS5, HPS6, DTNBP1, BLOC1S3, and PLDN.

Results: Whole-exome sequencing identified a compound heterozygous genotype in HPS1 gene in the proband. We identified a pathogenic frameshift variant c.1189delC (p.Gln397Serfs*2), resulting in a premature stop codon, associated with HPS. Furthermore, we identified a rare, previously undescribed nonsense variant, c.1507C>T (p.Gln503*), resulting in a premature stop and mRNA degradation. Presence of both variants was verified by Sanger sequencing. The following molecular-genetic analysis of parents confirmed their heterozygous carrier status.

Conclusions: Compound heterozygous mutations in HPS1 in the proband lead to disruption of HPS1 gene and clinical manifestation of Hermansky-Pudlak syndrome with severe pulmonary fibrosis leading to respiratory failure and death. This study was supported by Czech Ministry of Health (grant AZV 16-29447A) and Masaryk University (grant MUNI/A/1105/2018).

J. Trizuljak: None. M. Doubková: None. A. Hrazdírová: None. Z. Vrzalová: None. I. Blaháková: None. L. Radová: None. Š. Pospíšilová: None. M. Doubek: None.

P03.20D Genetic testing for hereditary hemorrhagic telangiectasia diagnosis: identification of new ENG and ACVRL1 mutations in Italian families

F. Cro’1, C. Lapucci1, E. Buscarini2

1Synlab Italia - Laboratory of Medical Genetics, Castenedolo, Italy, 2Hospital "Maggiore" ASST - European Reference Network (ERN) HHT, Crema, Italy

Introduction: Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant disease characterized by mucocutaneous telangiectasias, epistaxis and visceral arteriovenous malformations (AVMs). HHT-type-1 is caused by ENG gene mutations and it is characterized by a high incidence of pulmonary and cerebral AVMs. Type-2 is caused by ACVRL1 gene mutations and it is associated with hepatic AVMs. Mutations in SMAD4 gene cause Juvenile Polyposis/HHT syndrome. Here we report two cases with suspected HHT-1 and HHT-2 in which new ENG and ACVRL1 mutations where found.

Materials and Methods: DNA was extracted from blood samples of patients with suspected HHT. ENG, ACVRL1 and SMAD4 genes were analyzed by Next Generation Sequencing and results were confirmed by Sanger Sequencing.

Results: Case-1: mutation c.780_781insCCTACG (p.Ser260_Trp261insProTh) on ENG gene was identified in a 16-year-old boy with epistaxis, PAVM and HHT-1 family history. This mutation hasn’t been reported yet. Genetic testing was also performed in patient’s three brothers and two cousins. The mutation was identified in one brother (4-years-old, experiencing bleeding episodes) and both cousins (9 and 7 years-old). Two brothers (15 and 9 years-old, without any HHT symptoms) resulted negative. Case-2: mutation c.1327T>G (p.Cys443Gly) on ACVRL1 gene was identified in a 45-years-old woman with severe liver AVMs and enlisted for transplantation, rare epistaxis and no HHT family history. The mutation, consistent with clinical suspect of HHT-2, hasn’t been described yet.

Conclusions: Here we report new ENG and ACVRL1 mutations thought to be causative of hereditary hemorrhagic telangiectasia. Further clinical evaluation on carrier and not-carrier relatives will be performed.

F. Cro: A. Employment (full or part-time); Significant; Synlab Italia. C. Lapucci: A. Employment (full or part-time); Significant; Synlab Italia. E. Buscarini: None.

P03.21A Enrichment for large deletions encompassing genes expressed in the enteric nervous system in Hirschsprung disease patients with additional associated anomalies

K. C. MacKenzie1, C. S. Tang2, J. D. Windster1,3, T. Le4,5, 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. Wijnen3, S. Lyonnet4,6,5, M. Garcia-Barcelo2, P. K. H. Tam2, M. Alves1, A. Brooks1, R. M. W. Hofstra1, E. Brosens1

1Clinical Genetics, 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, 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, 6Service de Génétique, Necker Enfants Malades University Hospital,, Paris, France

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, including autism and intellectual disability. A subset of these patients have a known genetic syndrome in which HSCR has a variable expression or penetrance. In others, the genetic etiology is unknown and we hypothesize that rare Copy Number Variation (CNV) impacts their disease development. Indeed, rare Copy Number (CN) losses were significantly enriched in patients with HSCR and additional anomalies without a known causal variant (n=23, p=3.64E-7), and not in isolated HSCR (n=20, p=0.700) or in HSCR patients with a known RET or other causal variant (n=15, p=0.705). Of the HSCR patients with additional anomalies tested, at least five (three males and two females) had a large de novo CNV and one male inherited an X-linked CN loss. Patients with a known causal variant had a significant lower burden of the known HSCR predisposing risk haplotypes (P=0.0232), and isolated HSCR patients a higher burden (P=0.0277) compared to patients with HSCR and additional anomalies without a known causal variant. The rare CN losses identified are enriched for dosage sensitive genes, expressed in the developing mouse enteric nervous system (ENS; p=1.760E-10): SLC8A1, DDR1, GNL1, GABBR1, MAPK8, UFD1L, FHIT, AKT3, TUBB, TBX2, BCAS3 and USP32. Additionally, the latter four, had rare putative deleterious variants in HSCR patients without a deleterious CNV, confirming our hypothesis that rare CNV contributes to syndromic HSCR with unknown genetic etiology.

K.C. MacKenzie: None. C.S. Tang: None. J.D. Windster: None. T. Le: 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. S. Lyonnet: None. M. Garcia-Barcelo: None. P.K.H. Tam: None. M. Alves: None. A. Brooks: None. R.M.W. Hofstra: None. E. Brosens: None.

P03.22B The impact ofNRG1expressions and methylation on multifactorial Hirschsprung disease in Indonesia

Gunadi1, A. Kalim2, N. Budi2, N. Arfian2, E. Purnomo3, K. Iskandar3

1Faculty of Medicine, Public Health and Nursing, Universitas Gadjah Mada/Dr. Sardjito Hospital, Yogyakarta, Indonesia, 2Faculty of Medicine, Public Health and Nursing, Universitas Gadjah Mada, Yogyakarta, Indonesia, 3Faculty of Medicine, Public Health and Nursing, Universitas Gadjah Mada/UGM Academic Hospital, Yogyakarta, Indonesia

Background: Hirschsprung disease (HSCR) is a complex genetic disorder characterized by absence of ganglion cells in the gastrointestinal tract. Our previous study revealed that NRG1 rare variant frequency in Indonesian HSCR patients is <1%. Therefore, we wished to investigate NRG1 expression and methylation effect on HSCR pathogenesis in Indonesia.

Methods: NRG1 type I (HRGα, HRGβ1, HRGβ2, HRGβ3, HRGγ and NDF43 isoforms), type II and type III expressions in both ganglionic and aganglionic colon of HSCR patients and control colons were analysed by real-time polymerase chain reaction (RT-PCR). Next, we treated the extracted gDNA from HSCR patients’ and control colons with sodium bisulfate and analyzed the methylation pattern of NRG1 exon 1 with methylation-specific PCR.

Results: NRG1 expressions were up-regulated in HSCR patients colon compared with control (type I: 13.81±1.79 vs.15.22±1.22 vs. 17.78±1.27; type II: 13.93±1.52 vs. 15.67±1.52 vs. 16.57±1.02; type III: 11.98±2.86 vs.16.24±2.28 vs. 17.51±1.48, respectively), with p-value of 0.02, 0.03 and 0.01, respectively. Furthermore, HRGβ1/HRGβ2 expressions almost reached a significant difference between ganglionic and control colons (13.07±1.32 vs. 14.75±1.26, p=0.09). Most HSCR patients (80%) and controls (75%) revealed partially methylated NRG1. NRG1 methylation levels were lower in the ganglionic and aganglionic than control colons, however did not reach a significant level (p=0.31 and 0.13, respectively).

Conclusions: We shows the aberrant NRG1 expression in Indonesian HSCR patients and might not be due to DNA methylation. Moreover, our study provides further insights into the contribution of aberrant NRG1 expression in the HSCR pathogenesis.

Gunadi: None. A. Kalim: None. N. Budi: None. N. Arfian: None. E. Purnomo: None. K. Iskandar: None.

P03.23C The circadian aspect of PNPLA3 p.I148M and TM6SF2 p.E167K in hypercholesterolemia

U. Kovac1, J. Kovac2, E. Malicev3, E. A. Jasper4, K. Trebušak Podkrajšek1,2, T. Battelino2,1, K. K. Ryckman4, D. Rozman1

1University of Ljubljana, Faculty of Medicine, Ljubljana, Slovenia, 2University Medical Centre Ljubljana, University Children´s Hospital, Ljubljana, Slovenia, 3University of Ljubljana, Blood Transfusion Centre of Slovenia, Ljubljana, Slovenia, 4University of Iowa, Department of Epidemiology, Iowa, IA, United States

Background and aims: Lipid metabolism is under circadian control and long-term disturbance can lead to metabolic syndrome. Herein we test the hypothesis that variants rs738409 (p.I148M) in PNPLA3 and rs58542926 (p.E167K) in TM6SF2 associated with pathogenesis of Non-Alcoholic Fatty Liver Disease also represent novel hypercholesterolemia-associated variants. We propose that both non-synonymous variants associate with the change of circadian behavior in hepatic cells and contribute to the development of the hypercholesterolemia.

Materials and Methods: DNA samples from a Slovenian general screening programme of preschool children, age of 5, with known total cholesterol and LDL-cholesterol diagnosed with FH were obtained (N=121). The frequency of variants rs738409 and rs58542926 were compared to European individuals from 1000 Genomes and Exome variant server databases. With CRISPR/Cas9 technology we generated HepG2 cells with different PNPLA3 and/or TM6SF2 genotypes and assessed the metabolic, expression and proliferation aspects.

Results: An association between the PNPLA3 rs738409 and TM6SF2 rs58542926 minor allele with hypercholesterolemia has been identified if compared to European males from 1000 Genomes (OR=1.451, 95%CI=1.093-1.926, p<0.01; OR=0.3583, 95%CI=0.1536-0.8358, p<0,05) and European Americans from EVS database (OR=1.491, 95%CI=1.075-2.097, p<0.05; OR=0.3621, 95%CI=0.1603- 0.8181, p<0,01). HepG2 cells were found homozygous for rs738409 minor allele and rs58542926 major allele. We currently evaluate the circadian expression of both genes and the effect of cholesterol on genotypes of genetically modified cell lines.

Conclusions: We propose both polymorphisms as a novel hypercholesterolemia-associated genetic variant that disrupts the circadian rhythm and contribute to elevated plasma lipids.

Acknowledgement: Slovenian Research Agency program grant P1-0390 and graduate fellowship.

U. Kovac: None. J. Kovac: None. E. Malicev: None. E.A. Jasper: None. K. Trebušak Podkrajšek: None. T. Battelino: None. K.K. Ryckman: None. D. Rozman: None.

P03.24D Large deletions are an underappreciated cause of hyperinsulinism

T. W. Laver1, M. N. Wakeling1, R. Caswell1, B. Bunce2, J. A. L. Houghton2, K. A. Patel1, K. Hussain3, S. Ellard1, S. Flanagan1

1Institute of Biomedical and Clinical Science, Exeter, United Kingdom, 2Royal Devon & Exeter NHS Foundation Trust, Exeter, United Kingdom, 3Sidra Medicine, Doha, Qatar

Introduction: Hyperinsulinism is a disorder where dysregulated insulin secretion leads to hypoglycaemia. 50% of patients do not have a mutation in a known gene. Large contiguous gene deletions have been reported as an extremely rare cause of hyperinsulinism but are not routinely screened thus these may be an underappreciated cause of the disorder.

We aimed to assess the contribution of large deletions to the aetiology of hyperinsulinism.

Materials and Methods: Using off-target CNV (copy number variant) calling from targeted next generation sequencing data we screened 865 patients with hyperinsulinism for large deletions (>1Mb).

Results: We identified causative contiguous gene deletions in 16/865 patients in our cohort. 13 were previously reported to cause hyperinsulinism: X chromosome deletions (Turner syndrome) (n=3), 9p deletions (n=9) and a 16p deletion (n=1). We also identified 3 patients with overlapping de novo deletions on chromosome 20. These were the only novel large (>1Mb) de novo deletions within the cohort. They are a new cause of hyperinsulinism.

Conclusions: 2% of patients in our cohort had causative large contiguous gene deletions. This is likely to be an underestimate of the prevalence of large deletions in hyperinsulinism as some patients will have had cytogenetic testing prior to referral for hyperinsulinism genetic testing. Large deletions are a rare but significant cause of hyperinsulinism and should be screened for as part of genetic panel tests for the disease. We also highlight a novel cause of hyperinsulinism: 20p11.2 deletions.

Funding: Wellcome Trust and the Royal Society (105636/Z/14/Z and WT098395/Z/12/Z).

T.W. Laver: None. M.N. Wakeling: None. R. Caswell: None. B. Bunce: None. J.A.L. Houghton: None. K.A. Patel: None. K. Hussain: None. S. Ellard: None. S. Flanagan: None.

P03.25A Searching for genes related to idiopathic central precocious puberty (ICPP) in a Spanish cohort

N. V. Ortiz Cabrera1,2, R. Riveiro Álvarez2, M. A. López Martínez2, J. Cruz Rojo3, L. Garzón Lorenzo3, T. Gavela Pérez2, P. Pérez Segura2, I. Aragón Gómez2, L. Soriano Guillén2, M. J. Trujillo Tiebas2

1Hospital Infantil Universitario Niño Jesús, Madrid, Spain, 2Health Research Institute-Jiménez Díaz Foundation University Hospital, Madrid, Spain, 3Hospital Universitario 12 de Octubre, Madrid, Spain

In the last decade, the number of Idiopathic Central Precocious Puberty (ICPP) cases has decreased thanks to the discovery of mutations in different genes. The identification of loss of function of the maternal imprinted gene MKRN3 is the principal genetic cause of ICPP1-12. More recently DLK1 has also been discovered as another imprinted gene implicated in the pathogenesis of ICPP13.

Aims of the study: 1) To analyze the presence of pathogenic variants in 13 genes related to GnRH pathway; 2) To evaluate the diagnostic yield of the coding region analysis of MKRN3; 3) To determine the presence of copy number variation (CNV) of DLK1 and MKRN3 genes; 4) To exclude uniparental disomy (UPD) of chromosome14.

Patients and Methods: patients with ICPP were gathered from 2015 to 2018. In 20 patients we analyzed the clinical exome filtered by 13 genes that included MKRN3. Additionally, in another 11 patients we analyzed MKRN3 coding region by Sanger sequencing. In all patients we analyzed: a) DLK1 coding region by Sange sequencing; b) CNVs of MKRN3 and DLK1 by MLPA technique; c) UPD of chromosome 14 using STRs markers.

Results: from the 31 patients recruited we found two likely pathogenic variants in MKRN3 in one sporadic case and in one familial case.

Conclusion: MKRN3 gene is the most frequent genetic cause both sporadic and familial ICPP so we propose it as the first one to be screened in the genetic approach of patients with ICPP.

N.V. Ortiz Cabrera: None. R. Riveiro Álvarez: None. M.A. López Martínez: None. J. Cruz Rojo: None. L. Garzón Lorenzo: None. T. Gavela Pérez: None. P. Pérez Segura: None. I. Aragón Gómez: None. L. Soriano Guillén: None. M.J. Trujillo Tiebas: None.

P03.26B Complex indel variant calling in a repetitive genomic region

S. L. Wilkinson1, M. Edwards1, S. John1, F. Honti1, J. Mackintosh2, D. J. Morris-Rosendahl1,3

1Clinical Genetics and Genomics Laboratory, Royal Brompton and Harefield NHS Foundation Trust, London, United Kingdom, 2Respiratory Medicine, Royal Brompton and Harefield NHS Foundation Trust, London, United Kingdom, 3Genomic Medicine, National Heart and Lung Institute, Imperial College London, London, United Kingdom

Introduction: Interstitial Lung Disease (ILD) represents a collection of many different pulmonary disorders that interfere with the tissue and space surrounding the alveoli, causing irreversible damage. We performed genetic testing on a patient diagnosed with idiopathic pulmonary fibrosis who also had telomere lengths in the 1st centile.

Materials and Methods: Next-Generation sequencing was performed on patient DNA using our in-house Respigene™ panel of 172 genes. An in-house bioinformatics pipeline, configured to interpret genes associated with rare respiratory conditions, was used to classify SNVs and CNVs.

Results: Initial analysis did not detect any potentially pathogenic variants in genes associated with ILD in the patient. Another laboratory reported a RTEL1 splice variant: c.1266+3A>G. Further investigation using different variant callers detected a 78bp deletion at the RTEL1 exon 15/intron 15 boundary. Sanger sequencing and Tapestation analysis confirmed the patient to be heterozygous for the 78bp deletion. This deletion is classified as a VUS, but we consider it more likely pathogenic than benign. The c.1266+3A>G variant showed an allelic balance of less than 40%, and our results suggest it is an artefact caused by bioinformatic misalignment of the complex repeat region.

Conclusion: Complex and repetitive genomic regions still prove challenging for routinely-used variant calling software and Sanger sequencing. The error in variant calling may have resulted in a missed genetic diagnosis in this patient. Increasing intronic flanking regions for bioinformatic analysis when performing targeted sequencing and ‘training’ of bespoke bioinformatic pipelines will improve detection of complex variants in the exon/intron boundaries and beyond.

S.L. Wilkinson: None. M. Edwards: None. S. John: None. F. Honti: None. J. Mackintosh: None. D.J. Morris-Rosendahl: None.

P03.27C Changes in human physiology in response to three-week inactivity or how your training trains your microbiome

R. Šket1,2, T. Debevec3,4, S. Kublik5, M. Schloter5, N. Treichel5, B. Murovec6, D. Makuc7, K. Pečnik7, J. Plavec7, I. Mekjavić3, O. Eiken8, J. Kovač1, Z. Prevoršek9, B. Stres2,10,11

1University Medical Centre Ljubljana Slovenia, Division of Paediatrics, Unit of Special Laboratory Diagnostics, Ljubljana, Slovenia, 2Group for Microbiology and Microbial Biotechnology, Department of Animal Science, Biotechnical Faculty, University of Ljubljana, Ljubljana, Slovenia, 3Department of Automation, Biocybernetics and Robotics, Jozef Stefan Institute, Ljubljana, Slovenia, 4Faculty of Sport, University of Ljubljana, Ljubljana, Slovenia, 5Research Unit for Comparative Microbiome Analysis, Helmholtz Zentrum München - German Research Center for Environmental Health, Neuherberg, Germany, 6Laboratory for Machine Inteligence, Faculty of Electrical Engineering, University of Ljubljana, Ljubljana, Slovenia, 7Slovenian NMR Centre, National Institute of Chemistry, Ljubljana, Slovenia, 8Department of Environmental Physiology, Swedish Aerospace Physiology Centre, Royal Institute of Technology, Stockholm, Sweden, 9Group for Genetics, Animal Biotechnology and Immunology, Department of Animal Science, Biotechnical Faculty, University of Ljubljana, Ljubljana, Slovenia, 10Faculty of Civil and Geodetic Engineering, University of Ljubljana, Ljubljana, Slovenia, 11Center for Clinical Neurophysiology, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia

Introduction: We explored pathophysiological consequences of inactivity and hypoxia on human physiology and intestinal microbiota in healthy males during the randomized crossover design of run-in (5 day) and experimental phases [21-day normoxic bedrest (NBR), hypoxic bedrest (HBR) and hypoxic ambulation (HAmb) (hypoxic ~4000 m simulated altitude)] in a strictly controlled laboratory environment, with balanced fluid and dietary intakes and 24/7 medical surveillance.

Materials and Methods: Incorporating system medicine approach, intestinal transit spanning constipation, eosinophil-derived neurotoxin, bile acids, diet composition, intestinal electrical conductivity, indole etc., along with NMR metabolomics, were assessed. Furthermore, structure, diversity and function of intestinal microbiota using shot-gun metagenomics were investigated. Finally, all observations were integrated and analyzed in correlation to negative physiological symptoms, related to obesity and metabolic syndrome in same participants.

Results: Inactivity negatively affected fecal consistency and in combination with hypoxia aggravated the state of gut inflammation. On the other hand, many of the microbial parameters were shown to lag behind the changes in human physiology and intestinal environment, since significant changes in bacterial community were delayed until week four in HBR only, where members of the genus Bacteroides and proteins involved in iron acquisition and metabolism, cell wall, capsule, virulence and mucin degradation were enriched.

Conclusions: Our multi-omics approach suggest a time-dependent and complex interplay between the host physiology (including apparent constipation), immunity (inflammation), controlled diet, intestinal environment variables and microbiome physiology during the acute cessation of exercise. Funding: PlanHab project (Grant no. 284438) and Young Research Fellowship to RŠ (SRA#37426).

R. Šket: None. T. Debevec: None. S. Kublik: None. M. Schloter: None. N. Treichel: None. B. Murovec: None. D. Makuc: None. K. Pečnik: None. J. Plavec: None. I. Mekjavić: None. O. Eiken: None. J. Kovač: None. Z. Prevoršek: None. B. Stres: None.

P03.28D A novel homozygous CARMIL2 variant leads to infantile-onset colitis and gastrointestinal eosinophilic disease without recurrent infections

A. Kurolap1,2, O. Eshach Adiv1,2,3, L. Konnikova4,5,6, L. Werner7, C. Gonzaga-Jauregui8, M. Steinberg2, V. Mitsialis5,6, A. Mory2, M. Y. Nunberg7, S. Wall5, R. Shaoul1,3, J. D. Overton8, A. R. Shuldiner8, Y. Zohar1,9, T. Paperna2, S. B. Snapper5,6,10, D. S. Shouval7, H. Baris Feldman1,2

1The Bruce and Ruth Rappaport School of Medicine, Technion, Haifa, Israel, 2The Genetics Institute, Rambam Health Care Campus, Haifa, Israel, 3Pediatric Gastroenterology, Rambam Health Care Campus, Haifa, Israel, 4Devision of Newborn Medicine, Department of Pediatrics, Children’s Hospital of Pittsburgh of UPMC, Pittsburgh, PA, United States, 5Division of Gastroenterology, Hepatology and Nutrition, Boston Children’s Hospital, Boston, MA, United States, 6Harvard Medical School, Boston, MA, United States, 7Pediatric Gastroenterology Unit, Edmond and Lily Safra Children’s Hospital, Sheba Medical Center, Ramat Gan, Israel, 8Regeneron Genetics Center, Tarrytown, NY, United States, 9Institute of Pathology, Rambam Health Care Campus, Haifa, Israel, 10Division of Gastroenterology, Hepatology and Endoscopy, Brigham and Women’s Hospital, Boston, MA, United States

Introduction: Infantile-onset colitis refers to inflammatory gastrointestinal (GI) disorders manifesting before the two years of age. Nearly 100 genes have been implicated in syndromes presenting with early-onset colitis. We aimed to decipher the genetic basis of severe infantile colitis and eosinophilic GI disease without recurrent infections in a 10-years-old boy, and subsequently characterize his GI and immunological phenotypes.

Materials and Methods: We used trio whole exome sequencing (WES) for pathogenic variant discovery. Protein expression was studied using western blot (WB) analysis and immunohistochemical (IHC) staining. Immunological workup included in vitro T-cell studies, flow cytometry and CyTOF analyses.

Results: WES revealed a rare homozygous variant in CARMIL2: c.1590C>A; p.Asn530Lys. The variant was predicted pathogenic and co-segregated with the disease in the nuclear family. WB and IHC studies revealed markedly reduced CARMIL2 expression in patient cells compared to controls. Comprehensive in vitro immunological analyses demonstrated severely reduced regulatory T-cells (Treg) with impaired CD4+ T cell proliferation and Treg generation. CyTOF analysis revealed significant shifts in the innate and adaptive immune cells of the patient compared to ulcerative colitis patients and healthy controls.

Conclusions: Pathogenic bi-allelic CARMIL2 variants cause an immunodeficiency syndrome characterized by recurrent infections and skin lesions, occasionally with concurrent diarrhea. This study expands our knowledge on the immune landscape alterations caused by CARMIL2 defects and underscores the role of CARMIL2 as a candidate gene for early-onset inflammatory and eosinophilic GI disease. The predominant GI manifestations in the patient warrant further study of CARMIL2 function in the gut.

A. Kurolap: None. O. Eshach Adiv: None. L. Konnikova: None. L. Werner: None. C. Gonzaga-Jauregui: A. Employment (full or part-time); Significant; Regeneron Pharmaceuticals Inc.. M. Steinberg: None. V. Mitsialis: None. A. Mory: None. M.Y. Nunberg: None. S. Wall: None. R. Shaoul: None. J.D. Overton: A. Employment (full or part-time); Significant; Regeneron Pharmaceuticals Inc. A.R. Shuldiner: A. Employment (full or part-time); Significant; Regeneron Pharmaceuticals Inc.. Y. Zohar: None. T. Paperna: None. S.B. Snapper: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Significant; NIH grants HL59561, DK034854, and AI50950; the Helmsley Charitable Trust; and the Wolpow Family Chair in IBD Treatment and Research. D.S. Shouval: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Significant; The Israel Science Foundation and Jefferey Modell Foundation grants. H. Baris Feldman: None.

P03.29A Clinical utility of gene panel sequencing for diagnosis of early-onset chronic kidney disease

A. Domingo Gallego1, M. Pybus1, G. Bullich1, P. Ruiz1, L. Lorente-Grandoso1, M. Furlano1, G. Fraga2, G. Ariceta3, M. Borregán3, J. Piñero-Fernández4, L. Rodríguez-Peña4, I. Llano-Rivas5, R. Sáez6, L. Guirado1, R. Torra1, E. Ars1

1Fundació Puigvert, Barcelona, Spain, 2Hospital de la Santa Creu i Sant Pau, Barcelona, Spain, 3Hospital Vall d’Hebron, Barcelona, Spain, 4Hospital Universitario Virgen de la Arrixaca, Murcia, Spain, 5Hospital Universitario Cruces, Bizkaia, Spain, 6Hospital Donostia, San Sebastián, Spain

Introduction: Inherited kidney diseases (IKD) are the leading cause of chronic kidney disease (CKD) in the first three decades of life and encompass a broad range of nephropathies.

Methods: Targeted next generation sequencing with high sequencing depth of a large IKD-gene panel was performed in 426 patients with early-onset CKD (<30 years), including patients with: 1) congenital abnormalities of the kidney and urinary tract (CAKUT) (n=89) (with bilateral anomalies and/or extrarenal defects and/or familial CAKUT), 2) renal cystic ciliopathies (n=180), 3) suspected inherited glomerular disease (n=129) and 4) renal tubulopathies/nephrolithiasis/nephrocalcinosis (n=28).

Results: Causative mutations were found in 64% (271/426) of patients: 42% (37/89) with CAKUT, 76% (137/180) with renal cystic ciliopathies, 60% (77/129) with suspected inherited glomerular disease and 71% (20/28) with renal tubulopathy/nephrolithiasis/nephrocalcinosis. Copy number variants were detected in 11% of all genetically diagnosed patients (29/271). Pathogenic variants were identified in 57 different genes, 10 of which (COL4A3, COL4A4, COL4A5, HNF1B, NPHP3, PAX2, PKD1, PKD2, PKHD1 and TSC2) explained 75% (203/271) of patients.

Conclusion: Our IKD-gene panel allowed a precise molecular diagnosis in nearly two-thirds of patients with early-onset CKD. The high diagnosis yield of our approach can be explained by 1) the efficient detection of copy number variants and variants in complex genomic regions, such as PKD1 gene, due to the high sequencing depth and 2) the strict clinical inclusion criteria to favor genetic testing in patients with likely monogenic cause of nephropathy. Funding: Instituto de Salud Carlos III/FEDER(PI16/01998, PI18/00362).

A. Domingo Gallego: None. M. Pybus: None. G. Bullich: None. P. Ruiz: None. L. Lorente-Grandoso: None. M. Furlano: None. G. Fraga: None. G. Ariceta: None. M. Borregán: None. J. Piñero-Fernández: None. L. Rodríguez-Peña: None. I. Llano-Rivas: None. R. Sáez: None. L. Guirado: None. R. Torra: None. E. Ars: None.

P03.30B Large effect of rare variants with large effect sizes in (sporadic) IPF

J. Šelb, M. Rijavec, K. Osolnik, P. Korošec

University Clinic of Respiratory and Allergic Diseases Golnik, Golnik, Slovenia

Introduction: Rare variants in telomere metabolism associated genes have recently been shown to be important contributors to idiopathic pulmonary fibrosis (IPF), regardless of the family history. We aimed to validate the result in an independent cohort of IPF patients.

Methods: We calculated that in order to have 80% power to detect at least one IPF patient with a causative mutation in a telomere metabolism associated gene, if the prevalence of mutations is the same as in the above referenced study (13.2%), we had to sequence 11 patients for the presence of mutations in those genes. To get a random sample we included 11 consecutive IPF patients, regardless of their family history of IPF, at their control visit at our University Clinic. All patients had IPF diagnosis according to ATS/ERS/JRS/ALAT guidelines. Sequencing of telomere metabolism associated genes was performed on the Illumina platform.

Results: Demographic statistics of our sample were comparable to other IPF demographics with regard to age (mean age = 71.2 years), IPF family history (1/11 (9.1%)) and sample male predominance (8/11 (72.7%)). In 2 out of 11 (18.2%) patients, none of which had a family history of IPF and were therefore classified as sporadic IPF cases, the causative variant was found (NM_001283009(RTEL1):c.326_329del (p.I109fs); NM_001193376(TERT):c.1374delC (p.W459fs)). Despite sufficient coverage (mean coverage = 121X) we didn’t confirm the causative variant in the only patient with a family history of IPF in our cohort.

Conclusions: Causative variants in telomere metabolism associated genes play a significant role also in sporadic IPF.

J. Šelb: None. M. Rijavec: None. K. Osolnik: None. P. Korošec: None.

P03.31C A new form of hereditary iron overload unlinked to known hereditary hemochromatosis genes

D. Wallace, N. Subramaniam

IHBI, School of Biomedical Sciences, Queensland University of Technology, Brisbane, Australia

Hereditary hemochromatosis (HH) is normally caused by homozygosity for pathogenic mutations in the HFE (homeostatic iron regulator) gene. Other autosomal recessive forms of HH are associated with mutations in genes involved in iron homeostasis including hemojuvelin (HJV), hepcidin (HAMP) and transferrin receptor 2 (TFR2). Autosomal dominant forms of iron overload are associated with mutations in the cellular iron exporter ferroportin (SLC40A1), with a single case linked to a mutation in the 5’UTR of the H-ferritin gene (FTH1). We describe a family with apparent autosomal dominant iron overload with characteristics similar to the classical form of ferroportin disease. The condition is characterised by elevated serum ferritin levels with normal or mildly elevated transferrin saturation. Perls’ Prussian blue staining of liver biopsy sections in two affected individuals show prominent iron deposition in both Kupffer cells and hepatocytes. Sequencing of the coding sequences and splice sites of genes known to cause HH revealed no mutations in the proband. Whole genome SNP genotyping was performed on 15 members of the pedigree. Linkage analysis showed haplotypes on regions of chromosomes 1, 3, 12, 18 and 19 that were shared among the four most clearly affected members of the pedigree but not present in the three most clearly unaffected members. These regions encompass a total of 93 megabases, approximately 3% of the human genome and contain over 1500 canonical genes. Importantly, these genomic intervals do not contain the SLC40A1 gene indicating that affected members of this family do not have a variant form of ferroportin disease.

D. Wallace: None. N. Subramaniam: None.

P03.32D Transcriptome profiling in regenerating rat liver after partial hepatectomy, portal vein ligation, and associated liver partition and portal vein ligation for staged hepatectomy

D. Colak1, O. Al-Harazi1, I. H. Kaya2,3, O. M. Mustafa1, F. Meng4,5, A. M. Assiri5,6,2, D. K. Dhar4,5,7, D. C. Broering4,2

1Biostatistics, Epidemiology, and Scientific Computing Department, King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia, Riyadh, Saudi Arabia, 2College of Medicine, AlFaisal University, Riyadh, Saudi Arabia, 3Biostatistics, Epidemiology, and Scientific Computing Department, King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia, 4Department of Surgery and Organ Transplantation Center, King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia, 5Comparative Medicine Department, King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia, 6Institute for Research and Medical Consultations, Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia, 7Institute for Liver and Digestive Health, Regenerative Medicine & Fibrosis Group University College London, Royal Free Hospital, London, United Kingdom

Introduction: Liver has phenomenal capacity to regenerate, which has been shown clinically where diseased liver parts are removed to preserve and stimulate the growth of the remaining healthy tissues. Several surgical techniques have been developed for such purpose, including partial hepatectomy (PH), intraoperative portal vein ligation (PVL), and associated liver partition and portal vein ligation for staged hepatectomy (ALPPS). Indeed, while clinical outcomes are somewhat comparable, different procedures show clinically distinct recovery patterns. The observable clinical differences likely mirror some underlying variations in the patterns of gene activation and regeneration pathways.

Materials and Methods: We provided a comprehensive comparative analysis of the gene regulation in regenerating rat livers temporally spaced at 24h and 96h after PH, PVL, and ALPPS using the next-generation RNA sequencing transcriptomics analysis.

Results: The time-dependent factors appear to be the most important determinant of post-injury alterations of gene expression in liver regeneration. Early transcriptomic changes that were found in all three procedures included cell cycle associated genes and immune-response activation genes as well as transcription factors, DNA replication regulators, G1/S-transition regulators, and cytokinesis.

Conclusions: The functional pathway and gene network analyses revealed both unique and overlapping molecular mechanisms and pathways for each surgical procedure. Identification of molecular signatures, such as gene-to-gene interactions, regenerative signaling pathways, specific to each surgical procedures further our understanding of key regulators of liver regeneration as well as patient populations that are likely to benefit from each procedure. Funding: This study is funded by KFSHRC Research Grants (2110006 and 2180030 to DC).

D. Colak: None. O. Al-Harazi: None. I.H. Kaya: None. O.M. Mustafa: None. F. Meng: None. A.M. Assiri: None. D.K. Dhar: None. D.C. Broering: None.

P03.33A Biallelic LZTR1 mutation in a patient with non-classical Noonan phenotype

L. Tiberi1, A. La Barbera2, A. Provenzano2, P. Reho2, E. Bosi2, M. Bozzola3, S. Giglio2,4

1medical genetics unit Department of Experimental and Clinical Biomedical Sciences "Mario Serio" University of Florence, Florence, Italy, 2medical genetics unit Department of Experimental and Clinical Biomedical Sciences, Florence, Italy, 3Pediatrics Unit University of Pavia, Pavia, Italy, 4Medical Genetics Unit Meyer Children's Hospital, Florence, Italy

Noonan syndrome (NS) is an autosomal dominant, multisystemic disorder caused by dysregulation of the RAS/mitogen activated protein kinase (MAPK) pathway. Heterozygous variants in 11 known genes account for approximately 80% of cases. Whole exome sequencing (WES) studies recently identified the existence of a recessive form of Noonan syndrome caused by biallelic pathogenic LZTR1 variants, however information on the phenotypes of LZTR1 patients and functional properties of the mutations are limited. We report a 15-year-old boy with typical facial features of NS in the absence of congenital heart defects, cardiomyopathy, skin anomalies and intellectual disability. The patient had been treated with growth hormone (GH) between ages 11 and 15 without any effect on growth. WES revealed compound heterozygosity for a missense LZTR1 variant c.2102C>G; p.Pro701Arg inherited from his healthy father and a missense variant c.2062C>G: p.Arg688Gly NM_006767 inherited from his mother. We identified the variant inherited from his mother by exome reanalyses modifying pipelines parameters. Both variants were in the BTB domain of LTZR1. Mutations in LZTR1, already known to be causal in familial schwannomatosis type 2, have been recently involved in some patients with autosomal dominant and recessive Noonan syndrome in which clinical manifestations include cardiomyopathy and intellectual disability. This is the first case in which only facial features, short stature and no response to GH therapy are reported. Recently LZTR1 was associated to sporadic cerebral tumors so it is necessary a careful clinical observation in order to consider GH treatment in patients carrying LZTR1 variants.

L. Tiberi: None. A. La Barbera: None. A. Provenzano: None. P. Reho: None. E. Bosi: None. M. Bozzola: None. S. Giglio: None.

P03.34B Utility of MODY probability calculator among HNF1A- and GCK-MODY Polish patients (a retrospective analysis)

M. Szopa, D. Ucieklak, I. Solecka, I. Solecka, S. Mrozinska, J. Hohendorff, M. Sani, M. Malecki

JUMC, Krakow, Poland

Introduction: The most common form of monogenic diabetes is MODY. An easy-to-use MODY prediction model for identifying genetic-test indicated patient cases was developed in 2012: www.diabetesgenes.org/content/mody-probability-calculator. The aim of this study was to validate the utility of this tool with MODY patients in the Polish population.

Materials and Methods: Our MODY patient database was established 18 years ago at the Department of Metabolic Diseases, JUMC, Krakow, Poland and was based on typical clinical inclusion criteria. Patients in the database who provided all answers to the MODY calculator questionnaire --106 GCK-MODY and 92 HNF1A-MODY-- were included. The control group was established with 85 T1DM individuals and it does not include any T2DM patients due to insufficient number of sub-35-year-old T2DM cases in our outpatient clinic --as required in the calculator model.

Results: The mean predictive value using the calculator was 63.1% for GCK-MODY and 44.92% for HNF1A-MODY patients. This is in contrast with the mean calculator predictive value of 4.82% for T1DM patients. Only two T1DM patients out of 85 obtained a calculator predictive value higher than 25% -- the minimum suggested criteria for genetic testing referral. Only 11 GCK-MODY patients and 33 HNF1A-MODY patients received a sub-25% score. Sensitivity and specificity for both types of MODY were 77.78% and 97.65% respectively. GCK-MODY when compared to HNF1A-MODY obtained better sensitivity.

Conclusion: The model based on the Hattersley's group calculator reliably indicated genetic testing for GCK-MODY patients among our sub-population of Polish patients. The obtained results for HNF1A-MODY patients were also satisfactory.

M. Szopa: None. D. Ucieklak: None. I. Solecka: None. I. Solecka: None. S. Mrozinska: None. J. Hohendorff: None. M. Sani: None. M. Malecki: None.

P03.35C Assessment of genes known to be associated with MODY by next-generation sequencing

O. Cilingir1, B. Durak Aras1, E. Simsek2, D. Cınar1, M. A. Temena1, S. Arslan1, H. Bas1, E. Erzurumluoglu1, S. Artan1

1Eskisehir Osmangazi University, Faculty of Medicine, Department of Medical Genetics, Eskısehır, Turkey, 2Eskisehir Osmangazi University, Faculty of Medicine, Department of Pediatric Endocrinology, Eskısehır, Turkey

MODY (Maturity Onset Diabetes of the Young) is a autosomal dominant heterogeneous form of diabetes mellitus which mainly affects children and young adults. The defect in beta cell function is the cause of MODY. There are several known subtypes of MODY and each subtype occurs by pathogenic variants of different genes. Approximately 80% of MODY occurs by GCK and HNF1A pathogenic variants in European population, but different ethnics may have different rates of subtypes. This study aimed to explain the genetic architecture of MODY-suspected 132 Turkish patients. In this study, genes known to be associated with MODY (SLC161,KLF11,G6PC2,HADH,MOG,ZFP57,RFX6,GCK,PAX4,BLK,GLIS3,NEUROG3,GLUD1,INS,KCNJ11,ABCC8, HNF1A,PDX1,HNF1B,INSR,NKX2-2,HNF4A,FOXP3) were sequenced by using IonTorrent S5 and runs were then analyzed with bioinformatics pipeline. Of 132 patients, pathogencic, VUS(variant-unknown-significance), and DAP(disease-associated polymorphism) were detected in 39 patients. Additionally, a new variant identified in GCK is classified as pathogenic according to ACGM criteria and this variant was determined as de novo with the segragation analysis. The most common pathogenic variants were seen in GCK and the results are shown on the table. The novel variant detected in GCK results in premature stop codon because of frame shift mutation. The pathogenic variants detected in different genes revealed that NGS method is suitable to test genetic ethiology of multigenic diseases like MODY.

Table: Mutations and frequencies (%) in 39 patients.

O. Cilingir: None. B. Durak Aras: None. E. Simsek: None. D. Cınar: None. M.A. Temena: None. S. Arslan: None. H. Bas: None. E. Erzurumluoglu: None. S. Artan: None.

P03.36D Molecular characterization of a cohort of MODY patients from the North of Portugal

F. E. D. R. Laranjeira1, I. Ribeiro1,2, A. Amado3, E. Pinto1, J. Vilaverde3, A. R. Soares4, S. Rocha1, A. Carvalho3, S. Teixeira3, J. Dores3,2, C. Amaral3, I. Palma3, C. Reis4, M. T. Pereira3, C. Freitas3, C. Soares4, M. J. Oliveira3, R. Almeida3, G. Soares4, A. Fortuna4,2, D. Quelhas1,2, H. Cardoso3,2

1Unidade de Bioquímica Genética, Centro de Genética Médica Doutor Jacinto Magalhães, Centro Hospitalar Universitário do Porto, Porto, Portugal, 2Unidade Multidisciplinar de Investigação Biomédica (UMIB), Instituto de Ciências Biomédicas Abel Salazar, Universidade do Porto, Porto, Portugal, 3Serviço de Endocrinologia, Centro Hospitalar Universitário do Porto, Porto, Portugal, 4Serviço de Genética Médica, Centro de Genética Médica Jacinto de Magalhães, Centro Hospitalar Universitário do Porto, Porto, Portugal

Introduction: Maturity-onset diabetes of the young (MODY) is a group of monogenic disorders of autosomal dominant transmission resulting from a primary defect in insulin secretion, associated with pancreatic β-cell dysfunction. It is the cause in approximately 2% of diabetic patients, but it is frequently misdiagnosed as type 1 or type 2 diabetes. Although fourteen different genes have been implicated, mutations in the glucokinase gene, GCK, and genes coding for hepatocyte nuclear factor 1α, 1β and 4α (HNF1A, HNF1B and HNF4A, respectively), are the most common causes of MODY, accounting for up to 70% of mutated alleles.

Methodology: 112 patients with criteria for MODY type diabetes, from adults Endocrinology or Genetics consultation, underwent molecular genetics testing at UBG. Molecular genetics studies were performed by Sanger sequencing of one or more of the following genes, according to clinical suspicion: HNF1A, GCK, HNF4A and HNF1B.

Results: We present the molecular characterization of 24 patients where a definite or probable causing mutation was identified, thus 21%. Fourteen (58%) are HNF1A-MODY, eight (33%) are GCK-MODY and two (8%) are HNF1B-MODY. Eighteen different mutations were identified - 10 in HNF1A, 6 on GCK and 2 on HNF1B - including 3 novel mutations: c.1146_1156del and c.1422_1424delGCCinsCAG in HNF1A and c.863T>C on GCK.

Conclusions: The prevalence of HNF1A-MODY and GCK-MODY in our cohort is much lower than the values described. The MODY spectrum in Portugal is expected to be different from the one reported in other populations and will be revealed after the study of remainder MODY genes.

F.E.D.R. Laranjeira: None. I. Ribeiro: None. A. Amado: None. E. Pinto: None. J. Vilaverde: None. A.R. Soares: None. S. Rocha: None. A. Carvalho: None. S. Teixeira: None. J. Dores: None. C. Amaral: None. I. Palma: None. C. Reis: None. M.T. Pereira: None. C. Freitas: None. C. Soares: None. M.J. Oliveira: None. R. Almeida: None. G. Soares: None. A. Fortuna: None. D. Quelhas: None. H. Cardoso: None.

P03.37A Mutations in the Hepatocyte Nuclear Factor 1 Alpha (HNF1A), 4 Alpha (HNF4A) and 1 Beta (HNF1B) in Maturity-Onset Diabetes of the Young in Croatia

D. Caban1,2, A. Merkler1, H. Ljubić1, A. Špehar Uroić1, N. Krnić1, M. Čavlović Naglić3, L. Smirčić-Duvnjak3, D. Kaštelan1, J. Sertić1,4

1University Hospital Centre Zagreb, Zagreb, Croatia, 2University of Applied Health Sciences, Zagreb, Croatia, 3Merkur University Hospital, Zagreb, Croatia, 4University of Zagreb School of Medicine, Zagreb, Croatia

Introduction: Maturity-onset diabetes of the young (MODY) is a monogenic form of diabetes that is characterized by an early onset (usually before 25 years), autosomal dominant mode of inheritance and a primary defect in pancreatic β-cell function. MODY is a common form of monogenic diabetes and it may account for 1% to 2% of all diabetes cases in Europe. Many people with MODY are misdiagnosed with type 1 or type 2 diabetes.

Materials and Methods: All adult subjects with diabetes onset under age of 45 years and currently older than 18 years. Other inclusion criteria are: evidence of endogenous insulin secretion (fasting or random C-peptide ≥ 0.2 nmol/L) and negative glutamic acid decarboxylase antibodies (GADA), islet cell autoantibodies (ICA) and islet tyrosine phosphatase 2 (IA2). We analysed 44 patients for HNF1A-MODY, 14 patients for HNF4A-MODY and 8 patients for HNF1B-MODY. For identification of mutations in the coding and promoter region of analyzed genes, we used the Sanger sequencing method.

Results: We identified six different mutations in nine patients for subtype HNF1A-MODY. These mutations are located in exons 2, 3, 4 and 6, but the greatest number are found in exon 4, p.Gly292Argfs*25. For subtype HNF1B-MODY we identified one mutation, p.Val458Gly in exon 7. No HNF4A-MODY gene mutations were identified in any of patients analyzed.

Conclusions: Correctly identifying MODY has important implications for treatment, surveillance of complications and associated extra-pancreatic disorders, and identification of affected and at-risk family members.

D. Caban: None. A. Merkler: None. H. Ljubić: None. A. Špehar Uroić: None. N. Krnić: None. M. Čavlović Naglić: None. L. Smirčić-Duvnjak: None. D. Kaštelan: None. J. Sertić: None.

P03.38B MODY genes HNF1A, GCK and HNF4A pathogenic variant spectrum in pediatric patients

M. Šukys, V. Ašmonienė, R. Traberg, D. Čereškevičius

Hospital of Lithuanian University of Health Sciences, Kauno klinikos, Kaunas, Lithuania

Introduction: Maturity onset diabetes of the young (MODY) is a rare form of diabetes which is caused by pathogenic variant in one of the thirteen known responsible genes, but mostly in 3 genes: HNF1A, GCK, HNF4A. Correct diagnosis might allow change treatment, especially when it is mistaken for type 1 diabetes. Here we analyzed genetic variants in Lithuanian pediatric patients with suspected monogenic diabetes.

Materials and Methods: We performed GCK, HNF1A, HNF4A genes Sanger sequencing for pediatric patients (0-18 years old) for whom there was mild hyperglycemia or were diagnosed type 1 diabetes with negative antibody markers (glutamic acid decarboxylase, insulin and insulinoma-associated-2 antibodies) in the year 2017-2018.

Results: 32 patients were analyzed for all 3 genes, or one of them depending on clinical situation. 50% of them had at least second degree relative with anamnesis of diabetes or hyperglycemia. For 8 of the patients we found GCK nonsynonymous heterozygous variants which were previously reported as pathogenic. 2 patients had heterozygous variants in HNF1A: one with frameshift variant c.872dupC and another nonsynonymous variant c.809A>C which was not reported earlier. This variant changes amino acid in DNA binding region (p.Asn270Thr), cosegregates in patients family as patients father and grandmother (father‘s line) were diagnosed with diabetes. Also, as multiple in silico predictors showed as pathogenic variant, we ascertained variant as likely pathogenic.

Conclusion: In our study, only 1/3 of patients were proven for carrying a disease causing allele of all suspected MODY patients. One patient had novel variant with no previous report.

M. Šukys: None. V. Ašmonienė: None. R. Traberg: None. D. Čereškevičius: None.

P03.39C Functional characterization of HNF1A variants identified in the Norwegian MODY diabetes registry can implement precision medicine in diabetes clinics

I. Aukrust1,2, A. Kaci2,3, P. Svalastoga2, J. Molnes1,2, L. Bjørkhaug4, P. R. Njølstad2,3

1Department of Medical Genetics, Haukeland University Hospital, Bergen, Norway, 2Center for Diabetes Research, Department of Clinical Science, University of Bergen, Bergen, Norway, 3Department of Pediatrics and Adolescents, Haukeland University Hospital, Bergen, Norway, 4Department of Biomedical Laboratory Sciences and Chemical Engineering, Western Norway University of Applied Sciences, Bergen, Norway

Introduction: Variants in the hepatocyte nuclear factor-1alpha gene (HNF1A) can cause Maturity-Onset Diabetes of the Young (MODY3). MODY3 patients, who are often misdiagnosed as type1/type2 diabetes, are important to identify, since they can benefit from sulfonylurea treatment rather than insulin. The aim of this study was to functionally investigate possible pathogenic effects of 11 HNF1A variants from the Norwegian MODY registry, in order to provide a precise diagnosis and treatment.

Materials and Methods: The Norwegian MODY registry includes 2125 individuals with suspected MODY. HNF1A variants (Sanger identified) were investigated by transactivation (luciferase assay), protein expression (immunoblotting), DNA binding (electrophoretic mobility shift assay) and nuclear localization assays.

Results: All HNF1A variants investigated were classified as variants of uncertain significance. Variants p.(Ala116Thr), p.(Lys222del) and p.(Asn266Ser) showed low transcriptional activity (15-36%). p.(Ala116Thr) and p.(Lys222del) demonstrated low DNA binding (23% and 17%), and impaired nuclear localization. Moreover, p.(Lys222del) exhibited low protein expression (26%). Carriers of functionally impaired variants manifested with early diabetes-onset (<25 years). The autoantibody status was negative or not tested. Carriers of p.(Ala116Thr) and p.(Lys222del) had a family history of diabetes. The p.(Ala116Thr)-carrier had earlier been treated with sulfonylurea with no optimal effect. Her sister (also carrier), is treated successfully with sulfonylurea. The p.(Lys222del) and p.(Asn266Ser) patients are currently treated with insulin/metformin, and should be considered shifted to sulfonylurea. The remaining variants were either functionally normal or moderately impaired.

Conclusions: Functional investigation of HNF1A variants should support precision medicine in MODY3 patients.

Grants: Helse Vest, Norwegian Diabetes Association, foundation of K.G. Jebsen

I. Aukrust: None. A. Kaci: None. P. Svalastoga: None. J. Molnes: None. L. Bjørkhaug: None. P.R. Njølstad: None.

P03.40D New generation sequencing as an effective method of diagnosing patients with various form of monogenic diabetes

M. Borowiec1, K. Antosik1, P. Mludzik1, A. Zmyslowska2

1Department of Clinical Genetics, Medical University of Lodz, Lodz, Poland, 2Department of Pediatrics, Diabetology, Endocrinology and Nephrology, Medical University of Lodz, Lodz, Poland

Introduction: Monogenic forms of diabetes represent 5-7% of all diabetes types. They are a heterogeneous group of disorders caused by mutations in single genes, most of which regulate the pancreatic β cells function. The aim of the study was to determine the genetic background in patients referred to the Outpatient Genetics Clinic of the Centre for Monogenic Diabetes in Lodz, Poland.

Materials and Methods: The study group consisted of 379 patients with suspected monogenic diabetes aged from 3 months to 38 years diagnosed from February 2017 to January 2019. They were referred based on the age of diagnosis, family history, autoantibodies status, preserved insulin secretion, different clinical course of diabetes or coexistence of other symptoms. The next generation sequencing (NGS) method was performed using a designed panel of 35 genes (SureSelect, Agilent) and the following platforms and programs: Variant Studio (Illumina®), PhenIX (Charité Universitätsmedizin Berlin) and IGV (Broad Institute).

Results: Various forms of monogenic diabetes were confirmed in 75/379 (19.8%) of patients. 40 pathogenic variants of the GCK gene (MODY2), 8 variants of the HNF1A gene (MODY3), 7 variants of the HNF4A gene (MODY1), 7 variants of the BLK gene (MODY11), 7 variants of the KCNJ11 gene (PNDM - permanent neonatal diabetes mellitus), 5 variants of the HNF1B gene (RCAD - renal cysts and diabetes syndrome) and 1 variant of the ABCC8 gene (PNDM) were identified.

Conclusions: The NGS method seems to be effective diagnostic tool for patients with monogenic diabetes. Supported by the grants No 2015/19/B/NZ5/02243, 502-03/2-159-02/502-24-307 and 502-03/2-159-02/502-24-306.

M. Borowiec: None. K. Antosik: None. P. Mludzik: None. A. Zmyslowska: None.

P03.41A Analysis of genes associated with the development of nephronophthisis

J. Indrakova1, N. Havranova1, M. Dvorakova1, I. Uhliarikova1, J. Rydlova1, M. Zelinova2, R. Kremlikova Pourova2, J. Lastuvkova3, A. Boday1

1AGEL Laboratories, Novy Jicin, Czech Republic, 2Charles univerzity 2nd Faculty of Medicine and University Hospital Motol, Prague, Czech Republic, 3Masaryk's Hospital in Usti nad Labem, Usti nad Labem, Czech Republic

Objectives: Nephronophthisis is an autosomal recessive disease genetically very heterogenous. Currently is described 25 genes responsible for up to 70% of the disease from which 30-40% is caused by NPHP1 gene deletion. The main symptoms are kidney degeneration and fibrotic tissue formation, leading in renal failure often in childhood. Other less common symptoms include growth retardation, anemia and metabolic changes. The disease is caused by the presence of two causal mutations from parents, formation of disease de novo is rare. We describe three families their children have a defined clinical diagnosis of nephronophtisis.

Methods: We tested DNA isolated from peripheral blood and the molecular analysis of genes associated with nephronophthisis was performed by MLPA and custom designed NGS. Found causal mutations were verified by Sanger sequencing.

Results: By the analysis of 3 patients with clinical diagnosis of nephronophtisis we found homozygous causal mutations in two of them and two different heterozygous causal mutations (compound heterozygote) in one of them.

Conclusion: Molecular genetic testing confirmed a clinical diagnosis of nephronophtisis in all three patients. Follow-up segregation of causal variants in the patients’ families identified healthy carriers of these variants and in one family we confirmed the diagnosis in affected siblings. The knowledge of causal mutations is important not only for confirmation of the diagnosis, but also for healthy carriers in case of pregnancy planning and possible prenatal testing.

J. Indrakova: None. N. Havranova: None. M. Dvorakova: None. I. Uhliarikova: None. J. Rydlova: None. M. Zelinova: None. R. Kremlikova Pourova: None. J. Lastuvkova: None. A. Boday: None.

P03.43C PEMT, PCYT1 A and B polymorphisms in children with obesity

P. Tutac1, N. Andreescu1, A. Mihailescu1, D. Tiugan1, V. Serafim1, C. Zimbru1, C. Paul2, I. Velea2, M. Niculescu1, M. Puiu1

1University of Medicine and Pharmacy Victor Babes, Center of Genomic Medicine, Timisoara, Romania, 2University of Medicine and Pharmacy Victor Babes, Pediatrics Discipline, Timisoara, Romania

Introduction: Variations in PCTY1A and B genes associate with alterations of PCTY1 expression and of phosphatidylcholine (PC) synthesis. PC, the major glycerophospholipid in eukaryotic cells, can be synthesized by three pathways: the de-novo pathway, also known as Kennedy pathway, the Lands cycle and the phosphatidylethanolamine methyl transferase (PEMT) pathway. PC biosynthesis is required for normal very low-density lipoprotein secretion from hepatocytes and generation of arachidonic acid (ARA); its inhibition may lead to excess storage of lipids within liver, contributing to the development of obesity-associated hepatosteatosis.

Aim: Whether PCTY1A, PCTY1A B and PEMT gene polymorphisms in obese children associate with obesity-related somatometric and biochemical parameters.

Material and Method: 200 obese children, aged 7 to 18 years old; BMI>+2SD; abdominal circumference above the 90th percentile; +/- HTA and dyslipidemia or insulin resistance. PCTY1A (rs 1580820) and PCTY1B (rs 4898190), PEMT(rs1109859, rs12103822, rs16961845, rs4244593, rs4479310, rs7214988, rs7946, rs8068641, rs936108, rs13342397, rs6502603) were analysed using next generation sequencing.

Results: PCTY1A, PCTY1B and PEMT rs1109859 variations were positively correlated with glycemia, cholesterol, HDLc, total LA, ARA, BMI, cardiac frequency, and arterial and systolic blood pressure. PCTY1A and PCTY1B polymorphisms were correlated with plasma choline, and possibly with plasma betaine and 5-methyl tetrahydrofolate.

Conclusion: Genetic variations involved in choline metabolism were associated with alterations in choline, folate, and cholesterol metabolism in obese children, and could play important roles in the severity of associated metabolic alterations. Acknowledgement: This work was performed at The Center of Genomic Medicine, POSCCE Project, SMIS:48749, and funded by POC Project NutriGen, SMIS:104852.

P. Tutac: None. N. Andreescu: None. A. Mihailescu: None. D. Tiugan: None. V. Serafim: None. C. Zimbru: None. C. Paul: None. I. Velea: None. M. Niculescu: None. M. Puiu: None.

P03.44D Detection of adenoma derived circulating cell-free DNA of human pituitary adenoma using semiconductor sequencing

H. Niedra1, K. Megnis1, R. Peculis1, V. Rovite1, I. Balcere2, I. Konrade2, J. Stukens3, V. Pirags1,3,4, J. Klovins1

1Latvian Biomedical Research and Study Centre, Riga, Latvia, 2Riga East Clinical University Hospital, Riga, Latvia, 3Pauls Stradins Clinical University Hospital, Riga, Latvia, 4University of Latvia Faculty of Medicine, Riga, Latvia

Introduction: Circulating cell-free DNA (ccfDNA) analysis has been lately used as a minimally invasive method in cancer diagnostics. Currently, there is a scarce amount of information on adenoma derived ccfDNA. We examined the possibility to detect pituitary adenoma (PA) derived ccfDNA, by performing targeted NGS on ccfDNA using semiconductor sequencing.

Materials and Methods: Blood samples used were obtained from five NFPA patients before PA surgery. Sixteen tumor specific mutations each in unique gene (discovered in exome sequencing) were targeted in ccfDNA.

Results: Sequencing coverage was 6191X on average (range 444 - 13026). In two mutation sites (two patients) the alternate allele was in less than in 10% of reads: RYR1 (G/A, 2.27%), MPRIP (C/T, 3.65%). Five mutations (from two patients) had the alternate allele close to 50% of reads: VPS13D (C/T, 48%), LDLRAD2 (C/T, 47%), SPEN (A/G, 49%), GPATCH4 (C/T, 58%), G6PC2 (C/G, 46%). The alternate allele was in less than in 0.1% of reads in nine mutations, however, the estimated background alternate allele rate in non-mutation sites also was below 0.1%.

Conclusions: Results indicate that while it is possible to detect somatic mutations found in adenoma in ccfDNA using semiconductor sequencing the source of mutated ccfDNA molecules should be further researched. Mutation rate in 50% of reads could be due to somatic mosaicism or due to increased tumor cell apoptosis/necrosis in these patients.

Research was supported by the European Regional Development Fund project no. “Molecular markers of pituitary tumor development, progression and therapy response”.

H. Niedra: None. K. Megnis: None. R. Peculis: None. V. Rovite: None. I. Balcere: None. I. Konrade: None. J. Stukens: None. V. Pirags: None. J. Klovins: None.

P03.45A Exome sequencing of non-functioning pituitary adenomas reveal tumors with high amount of somatic mutations and previously reported genes

R. Pečulis1, V. Rovīte1, H. Niedra1, I. Balcere2, J. Nazarovs3, J. Stuķēns3, I. Konrāde2, V. Pīrāgs1,3,4, J. Kloviņš1

1Latvian Biomedical Research and Study Centre, Riga, Latvia, 2Riga East Clinical University Hospital, Riga, Latvia, 3Pauls Stradiņš Clinical University Hospital, Riga, Latvia, 4University of Latvia, Faculty of Medicine, Riga, Latvia

Introduction: The most common type of pituitary diseases is pituitary adenomas (PA). Although not undergoing metastasis, PAs are responsible for increased mortality and morbidity. Genetic causes of PAs are known in a minority of cases: several genes are implicated in familial PA. Somatic mutations in GNAS and USP8 are cause in subgroups of sporadic GH and ACTH secreting PA, respectively. Exome and genome sequencing is used to study PA genetics nowadays, but is hampered by heterogeneity of PAs.

Materials and Methods: Exomes (TruSeq Rapid Exome TargetedRegions v1.2) of normal - tumor DNA pairs of seven non-functioning PA patients were sequenced using Illumina NextSeq. Data were analysed using Isaac-Starling pipeline. Somatic mutations were reviewed with IGV2.2.4. Sanger sequencing validation was performed for a subset of mutations covering all PA patients.

Results: Various somatic mutation rate was observed in the exomes of sequenced tumors. Two PAs were hypermutated with 272 and 671 somatic mutations. Other five PA had a low somatic mutation rate between one to 12 somatic mutation per exome. VPS13D in hypermutated tumor and RYR1 had somatic mutations and had been reported containing somatic mutation previously in GH secreting adenomas. Somatic mutations in G6PC and SMARCAD1 previously have been detected in adenomas of different location.

Conclusions: Some PAs contain a significantly higher amount of somatic mutations than expected from non-metastasizing tumor. Somatic mutations in genes reported previously in connection with PA provide research targets for upcoming studies. The study was supported by ERDF grant

R. Pečulis: None. V. Rovīte: None. H. Niedra: None. I. Balcere: None. J. Nazarovs: None. J. Stuķēns: None. I. Konrāde: None. V. Pīrāgs: None. J. Kloviņš: None.

P03.46B Tracing origin of cultured pituitary adenoma cells using “omics" analysis

V. Rovite1, R. Peculis1, I. Mandrika1, R. Petrovska1, K. Megnis1, I. Balcere2, J. Stukens3, I. Konrade2, A. Breiksa3, J. Nazarovs3, V. Pirags1,3,4, J. Klovins1

1Latvian Biomedical Research and Study centre, Riga, Latvia, 2Riga Eastern Clinical University Hospital, Riga, Latvia, 3Pauls Stradins Clinical University Hospital, Riga, Latvia, 4University of Latvia Faculty of Medicine, Rigs, Latvia

Introduction: Pituitary adenomas (PA) are benign tumours of the anterior pituitary that cause increased mortality and morbidity. Cell cultures derived from PA tissue can form free floating aggregates called pituispheres (PS) or adherent mesenchymal stromal cells (MSC). We studied genetic relationship between patients’ germline DNA, tumour tissue somatic DNA, DNA of PS and MSC obtained from culture to trace origin of each type of cultured cells.

Materials and Methods: PA patients were enrolled to national biobank - Genome Database of Latvian Population from Pauls Stradins Clinical University Hospital where transsphenoidal surgery of PA was performed for all patients. Exomes (Illumina TruSeq_Rapid_Exome_TargetedRegions_v1.2) of germline, tumour somatic, PS and MSC were sequenced using Illumina NextSeq with 75bp paired end reads. Sequencing data were analyzed with Illumina Basespace Enrichment App (v3.0.0) aligning to human HG19 reference genome using Isaac Genome Alignment Software, variants called with Starling algorithm and variants annotated with Illumina Annotation Engine. Filtered variants were reviewed using IGV 2.3.14.

Results: PAs contain low amount of tissue specific mutations (median 4, range 2 - 6). Somatic mutations of the primary tumour can be detected in the respective PS, but not in the respective MSC. Genetic alterations of MSCs corresponded to mutations in PA patients’ germline DNA.

Conclusions: Using exome comparison we were able to trace origin of PS and MSC cell cultures, showing that genome of PS represents genome of PA while MSC most likely represent normal cells of pituitary or surrounding tissues.

V. Rovite: None. R. Peculis: None. I. Mandrika: None. R. Petrovska: None. K. Megnis: None. I. Balcere: None. J. Stukens: None. I. Konrade: None. A. Breiksa: None. J. Nazarovs: None. V. Pirags: None. J. Klovins: None.

P03.47C Improved mapping quality and coverage in highly homologous PKD1 gene enable high diagnostic yield in ADPKD

S. Valo1, J. Tallila1, M. Kaare1, H. Jalanko2, J. Sistonen1, A. Korppoo1, K. Gall3, M. Muona1, P. Salmenperä1, M. Gentile1, S. Myllykangas1, T. Alastalo3, J. Koskenvuo1

1Blueprint Genetics, Helsinki, Finland, 2Helsinki University Central Hospital, Helsinki, Finland, 3Blueprint Genetics, San Francisco, CA, United States

Introduction: Autosomal dominant polycystic kidney disease (ADPKD) is an adult-onset multisystem disorder characterized by cysts in the kidneys and liver. It is estimated that PKD1 explains 85% and PKD2 15% of ADPKD cases. Genetic testing has become an important factor in the management of ADPKD. However, analysis of PKD1 is technically challenging due to its large size, high GC-content and duplication of the first 33 exons with a high degree of homology to pseudogenes (PKD1P1-P6). We evaluated the diagnostic yield and performance of our In-house panels for cystic kidney disease.

Materials and Methods: Next-generation sequencing (NGS) was performed using the IDT xGEN Exome Research Panel with added custom probes and the Illumina NovaSeq 6000 platform. This assay provides improved mapping quality and coverage of PKD1 compared to other NGS methods assessed in our laboratory.

Results: PKD1 provided both high mean coverage (199x) and excellent mapping quality with 99.5% of the target nucleotides covered at least 20x. In the study cohort of 131 index patients, a genetic diagnosis was established in 52% of cases with disease causing variants detected in 7 different genes. In 63% of the diagnostic cases the disease causing variant was identified in PKD1. Interestingly, 84% (n=36) of the variants were located in the duplicated region of PKD1.

Conclusions: Our results demonstrate that our in-house NGS platform is well-suited for clinical diagnostics with comprehensive coverage in difficult-to-sequence regions of PKD1. The method provides a cost-effective diagnostic tool to simultaneously diagnose various types of mutations.

S. Valo: A. Employment (full or part-time); Significant; Blueprint Genetics. J. Tallila: A. Employment (full or part-time); Significant; Blueprint Genetics. M. Kaare: A. Employment (full or part-time); Significant; Blueprint Genetics. H. Jalanko: A. Employment (full or part-time); Modest; Blueprint Genetics. J. Sistonen: A. Employment (full or part-time); Significant; Blueprint Genetics. A. Korppoo: A. Employment (full or part-time); Significant; Blueprint Genetics. K. Gall: A. Employment (full or part-time); Significant; Blueprint Genetics. M. Muona: A. Employment (full or part-time); Significant; Blueprint Genetics. P. Salmenperä: A. Employment (full or part-time); Significant; Blueprint Genetics. E. Ownership Interest (stock, stock options, patent or other intellectual property); Significant; Blueprint Genetics. M. Gentile: 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. J. Koskenvuo: A. Employment (full or part-time); Significant; Blueprint Genetics. E. Ownership Interest (stock, stock options, patent or other intellectual property); Significant; Blueprint Genetics.

P03.48D Detection of DZIP1L mutations by whole exome sequencing in two consanguineous families with polycystic kidney disease

J. M. Hertz1, P. Svenningsen2, H. Dimke3, M. B. Engelund1, H. Norgaard4, A. Hansen5, N. Marcussen6, H. C. Thiesson7, B. L. Jensen3, M. J. Larsen1

1Department of Clinical Genetics, Odense University Hospital, Odense C, Denmark, 2Department of Cardiovascular and Renal Research, Institute of Molecular Medicine, University of Southern Denmark, Odense C, Denmark, 3Department of Cardiovascular and Renal Research, Institute of Molecular Medicine, University of Southern Denmark, Odense C, Denmark, Odense C, Denmark, 4Department of Pediatrics, Rigshospitalet, Copenhagen, Denmark, 5Vestergade 21, Koge, Denmark, 6Department of Clinical Pathology, Odense University Hospital, Odense C, Denmark, 7Department of Nephrology, Odense University Hospital, Odense C, Denmark

Autosomal recessive polycystic kidney disease (ARPKD) is an early onset cystic renal disease with an incidence of about 1 per 20,000 live births. It is most frequently caused by mutations in the PKHD1 gene, encoding fibrocystin, but mutations in other cilia-related disease genes may mimic the phenotype. In 2017, Lu et al. reported homozygous mutations in DZIP1L located at 3q22.1, encoding the basal body protein DAZ interacting protein 1-like protein (DZIP1L) in seven children with ARPKD from four unrelated and consanguineous families. DZIP1L is located at the basal body of the primary cilium, and impaired function of DZIP1L is associated with a defect in the ciliary trafficking of polycystin-1 and polycystin-2. So far, no other DZIP1L mutations have been reported, and ARPKD caused by DZIP1L mutations is a rare form of polycystic kidney disease.We performed whole exome sequencing in two consanguineous families with three children with polycystic kidney disease, and identified two different and not previously reported DZIP1L mutations in homozygous form: c.193T>C; p.(Cys65Arg), and c.216C>G; p.(Cys72Trp). All three children are phenotypically characterized by enlarged echogenic kidneys with poor cortico-medullary differentiation. No liver cysts were detected. Liver stiffness were evaluated in two of the children by transient elastography, and with normal result. Functional analyses of the p.Cys72Trp mutation indicate that this variant causes a disruption of a localization signaling or interaction domain of DZIP1L.

J.M. Hertz: None. P. Svenningsen: None. H. Dimke: None. M.B. Engelund: None. H. Norgaard: None. A. Hansen: None. N. Marcussen: None. H.C. Thiesson: None. B.L. Jensen: None. M.J. Larsen: None.

P03.49A Genetic analysis in pulmonary arterial hypertension Sicilian patients: preliminary results

I. Loddo, F. Barbera, M. Beretta, A. Callari, L. Martino, D. Di Carlo, P. Conaldi, P. Vitulo

ISMETT Mediterranean Institute for Transplantation and Advanced Specialized Therapies (ISMETT), Palermo, Italy

Background: Pulmonary arterial hypertension (PAH) is a progressive and fatal disorder associated with high pulmonary artery pressure. Most heritable PAH (75%) is caused by a pathogenic variant in BMPR2, pathogenic variants in other genes are considerably less common (1-3%).

Genetic testing enables early diagnosis and offers an opportunity for family screening.

Materials and Methods: We designed a target sequencing custom panel that includes 15 genes of interest. Genetic counselling was offered to every patients.

To identify genetic mutations and help make a precise diagnosis and family screening, we performed genetic testing by NGS in six patients with PAH.

Results: In three patients we identified variants classified according to ACMG Guidelines.

In a 47 years old female affected by familial PAH, we identified a heterozygous nonsense variant (c.2617C>T) p.Arg873* in BMPR2 gene, classified as pathogenic.

In a 31 years old female affected by PAH with Pulmonary Capillary Hemangiomatosis, we identified a homozygous frameshift pathogenic variant (c.2666_2667insAATC) p.Lys891Aspfs* in EIF2AK4 gene, classified as pathogenic and never described before.

In a 55 years old male affected by Pulmonary Arterial Hypertension, we identified a heterozygous missense variant (c.1151T>A) p.Val384Asp in SMAD9 gene, classified as likely pathogenic and never described before. All variants were confirmed by Sanger sequencing.

Genetic counselling and molecular analysis was offered to high-risk relatives.

Conclusions: Our study confirm that NGS Target resequencing represents a valuable tool in PAH genetic testing. Analyses of PAH casual genes have a great help to clinical diagnosis and deep implications in patients management and disease treatment.

I. Loddo: None. F. Barbera: None. M. Beretta: None. A. Callari: None. L. Martino: None. D. Di Carlo: None. P. Conaldi: None. P. Vitulo: None.

P03.50B Preparing for genomics: a retrospective audit of a metropolitan renal genetics clinic

E. I. Krzesinski1,2, Y. B. Prawer1,2, M. J. P. Regan1, A. Yeung1,2,3, M. F. Hunter1,2

1Monash Genetics, Monash Health, Melbourne, Australia, 2Department of Paediatrics, Monash University, Melbourne, Australia, 3Victorian Clinical Genetics Service, Melbourne, Australia

Introduction: Hereditary kidney conditions are rare but account for a significant burden on hospitals in both adults and children. Monash Health is a KidGen/ Australian Genomics Health Alliance/Melbourne Genomics Health Alliance flagship recruitment site, aiming to assess the impact of multidisciplinary renal-genetic clinics on patient care while providing access to whole exome sequencing (WES).

Materials and Method: This three year audit (2015-2017) of renal patients referred to the Monash Health genetics service provides a picture of the renal genetics landscape at the outset of renal flagship WES testing. The electronic medical record was searched using renal keywords; each file was manually assessed for inclusion and exclusion criteria. Data collection included referrer specialty, condition, family history, inheritance pattern, type and cost of genetic testing and impact of genetic testing on management and reproductive options.

Results: Through keyword matching 676 patients were identified, 86 met inclusion criteria; 59 (69%) were probands with a primary renal condition and 35 (40.1%) were referred by a nephrologist. Cystic renal disease was the commonest reason for referral. Genetic testing was arranged for 50 patients of whom 21 (42%) had informative results. Diagnosis rate improved from 2015 to 2017; cost per informative patient diagnosis was $2499.

Conclusion: Audit provides a snapshot of the utility of clinical genetics input in a renal genetics context at the outset of improved WES access. Fourteen referred patients were unaffected providing them with reproductive reassurance. Diagnosis rate improved from 2015 to 2017 as more molecular diagnoses were obtained, thus improving patient management.

E.I. Krzesinski: None. Y.B. Prawer: None. M.J.P. Regan: None. A. Yeung: None. M.F. Hunter: None.

P03.51C Molecular genetic analysis of Steroid Resistant Nephrotic Syndrome: Detection of a novel mutation

A. Shojaei1, N. Serajpour1, B. Karimi2, P. Khosravi2

1Department of Medical Genetics and Molecular Biology, Faculty of Medicine, Iran University of Medical Sciences, Tehran, Iran, Islamic Republic of, 2Medical Genetic laboratory, Shahid Akbarabad hospital, Iran University of Medical science, Tehran, Iran, Islamic Republic of

Background: Nephrotic syndrome is one of the most common kidney diseases in childhood. About 20% of children are steroid-resistant NS (SRNS) which progress to end-stage renal disease (ESRD). More than 53 genes are associated with SRNS which represent the genetic heterogeneity of SRNS. This study was aimed to screen disease causing mutations within NPHS1 and NPHS2 and evaluate new potential variants in other genes.

Method: In first phase of study, 25 patients with SRNS were analyzed for NPHS1 (exon 2, 26) and all exons of NPHS2 genes by Sanger sequencing. In the second phase, whole exome sequencing was performed on 10 patients with no mutations in NPHS1 and NPHS2.

Result: WES analysis revealed a novel mutation in FAT1 (c.10570C>A; Q3524K). We identified 4 pathogenic mutations, located in exon 4 and 5 of NPHS2 gene in 20% of patients (V180M, P118L, R168C and Leu156Phe). Also our study has contributed to the descriptions of previously known pathogenic mutations across WT1 (R205C) and SMARCAL1 (R764Q) and a novel polymorphism in CRB2.

Conclusion: Our study concludes that mutations of exon 4 and 5 NPHS2 gene are common in Iranian and some other ethnic groups. We suggest conducting WES after NPHS2 screening and further comprehensive studies to identify the most common genes in the development of SRNS, which might help in Clinical impact on management in patients with SRNS.

A. Shojaei: None. N. Serajpour: None. B. Karimi: None. P. Khosravi: None.

P03.52D TNF-alpha gene polymorphisms influence disease phenotype in inflammatory bowel disease

O. M. Popa1, C. Tieranu2, M. Bojinca1, I. Tieranu1, M. Diculescu1

1University of Medicine and Pharmacy 'Carol Davila', Bucharest, Romania, 2Elias University Emergency Hospital, Bucharest, Romania

Introduction: Tumor necrosis factor (TNF) alpha is a major proinflammatory cytokine involved in the immune response in inflammatory bowel disease (IBD). IBD is composed of two main subtypes, namely Crohn’s disease (CD) and Ulcerative Colitis (UC). There are multiple phenotypes of IBD in terms of disease extension, disease severity and extraintestinal manifestations (EIM). EIM in IBD are frequent and may significantly impact the quality of patients life. Our aim was to investigate two TNF-alpha gene SNPs for association with clinical manifestations in IBD patients.

Materials and Methods: The study included 142 IBD patients (78 CD, 43M/35F and 64 UC, 40M/24F), all of Romanian origin. EIM were documented in 34 patients (20 CD, 14 UC). The two SNPs of TNF-alpha gene rs361525 (-238G/A) and rs1800629 (-308G/A) were genotyped by TaqMan Allelic Discrimination Assay (7300 Real-Time PCR System, Applied Biosystems by Thermo Fisher Scientific, USA). Association tests for each polymorphism were performed with DeFinetti online software (http://ihg2.helmholtzmuenchen.de/cgi-bin/hw/hwa1.pl) and p values ≤0.05 were considered significant. Results. We found a significant association between -308A variant and the presence of EIM in both ulcerative colitis (p=0.0002, OR 8.51) and Crohn’s disease (p=0.002, OR 5.19). For UC, -238A allele and the GA genotype were more frequent in patients with EIM than in patients without EIM (p=0.01, OR 27.03 and p=0.0009, OR 30.13 respectively). No other associations with clinical phenotype were observed.

Conclusion: TNF-α polymorphisms influence clinical manifestations of IBD in Romanian patients. These results should be confirmed on larger patients cohorts. Grant support: IDEI 311/2007.

O.M. Popa: None. C. Tieranu: None. M. Bojinca: None. I. Tieranu: None. M. Diculescu: None.

P04 Skeletal, connective tissue, ectodermal and skin disorders

P04.01A Comprehensive re-assessment of causality of ABCC6 missense variants associated with pseudoxanthoma elasticum

S. Verschuere1,2, P. Coucke1,2, O. M. Vanakker1,2

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

Introduction: Pseudoxanthoma elasticum (PXE) is an autosomal recessive ectopic mineralization disorder caused by ABCC6 mutations. Inconsistencies in variant interpretation raised the question whether all variants reported in literature or variant databases are truly disease-causing. Especially for missense substitutions it is challenging to predict the impact on protein function. We therefore re-evaluated pathogenicity of all ABCC6 missense variants.

Materials and Methods: The score-based variant classification system Sherloc was used to analyze 234 variants from literature, ClinVar and in-house patient screenings. Clinical and functional evidence were scored according to the rules and guidelines of Nykamp et al. (2017).

Results: Comprehensive classification revealed 74% variants of uncertain significance (VUS), 12% likely pathogenic, 10% pathogenic, 3% benign and 1% likely benign variants. VUS classification was further refined into truly uncertain variants (69% of VUS) and those leaning towards likely benign (1% of VUS) or likely pathogenic (30% of VUS). Taking the latter into account, 44% of ABCC6 variants is (likely) pathogenic when considering all available population, clinical, experimental and in silico data. This is significantly different from ClinVar, where 87% of ABCC6 missense variants are allegedly pathogenic and only 11% VUS.

Conclusions: Our results underline that variant classification should be done systematically and with caution, as it has important consequences for patients and heterozygous carriers. The high number of VUS confirms the need for functional testing to prove or refute their causality before returning them to patients.

S. Verschuere: None. P. Coucke: None. O.M. Vanakker: None.

P04.02B De novo pathogenic ABL1 variants cluster in a myristoyl-binding pocket

A. J. M. Blakes1, E. Gaul2, W. Lam3, N. Shannon4, A. Chase2, DDD Study, A. G. L. Douglas1,2

1Wessex Clinical Genetics Service, University Hospital Southampton NHS Foundation Trust, Southampton, Southampton, United Kingdom, 2Human Development and Health, Faculty of Medicine, University of Southampton, Southampton, UK, Southampton, United Kingdom, 3South East of Scotland Clinical Genetics Service, Western General Hospital, Crewe Road, Edinburgh, UK, Edinburgh, United Kingdom, 4Clinical Genetics Service, Nottingham University Hospitals NHS Trust, Hucknall Road, Nottingham, UK, Nottingham, United Kingdom

Introduction: ABL1 is a proto-oncogene encoding a nonreceptor tyrosine kinase. It is best known in the somatic BCR-ABL fusion gene associated with chronic myeloid leukaemia. Recently, Wang et al. described two germline missense variants in ABL1 causing an autosomal dominant developmental syndrome (OMIM 617602) with congenital heart disease, skeletal malformations, and characteristic facies.

Materials and Methods: Patients with de novo heterozygous missense variants in ABL1 were identified via trio WES through the DDD Study with Sanger confirmation via UK regional genetics laboratories. Clinical assessments of patients were carried out via regional clinical genetics services. To investigate ABL1 kinase activity in vitro, HEK293T cells were transfected with plasmid constructs encoding wild-type or mutant ABL1 cDNA. Phosphorylation of ABL1-specific substrates was measured by immunoblotting and potential for reversal investigated by treatment with imatinib. Bio-informatic analysis of gene-wide conservation and germline/somatic variation was performed.

Results: We identified three unrelated patients with de novo pathogenic missense variants in ABL1, including two novel variants. All variants cluster in the myristoyl-binding pocket of ABL1 – a region critical for auto-inhibitory regulation of the kinase domain, which is subject to high missense constraint and evolutionary conservation. All patients recapitulate the phenotype of the ABL1 developmental syndrome. Functional studies of mutant ABL1 kinase activity are ongoing and will be presented.

Conclusions: We describe three new cases with pathogenic ABL1 missense variants causing skeletal malformations and congenital heart disease. Mutations cluster around the myristoyl binding-pocket of ABL1, suggesting a gain-of-function mechanism due to loss of auto-inhibition.

A.J.M. Blakes: None. E. Gaul: None. W. Lam: None. N. Shannon: None. A. Chase: None. A.G.L. Douglas: None.

P04.03C Rare loss-of-function variants in the Epidermal Differentiation Complex predispose individuals to Atopic Dermatitis

S. P. Smieszek, C. Polymeropoulos, G. Birznieks, M. Polymeropoulos

Vanda Pharmaceuticals, Washington, DC, United States

The epidermal differentiation complex (EDC) includes over fifty genes encoding proteins involved in keratinocyte development. Of these genes, filaggrin (FLG) located on chromosome 1 q21, is the most studied in the context of skin barrier dysfunction. We investigated the frequency and effect of rare loss-of-function (LOF) variants in patients of a clinical study, VP-VLY-686-2102 (randomized, double-blind, placebo-controlled, in patients with chronic pruritus associated with AD) with 117 whole genome sequencing (WGS) samples. We have shown that 45/117 AD patients carry significantly more, rare loss-of-function (LOF) mutations in the SFTP family of genes as compared to 55/316 in a control population (p-value = 0.000004). This group of EDC LOF (stopgain, frameshift) rare variants (EDC-LR) consists of 20 variants observed in the 45 AD patients resulting in a calculated Odds Ratio of 2.96 and a Relative Risk of 2.38. Among the detected LOF variants, there are 25 cases of FLG LOF mutations as defined by R501X (rs61816761), 2282del4 (rs558269137), other LOFs in FLG as well as LOFs in FLG2, HRNR, LCE4A, LCE5A, TCHH, TCHHL1 and other members of the EDC. We examine the regional accumulation of rare LOF variants in FLG region. For the entire EDC, we obtained a p-value of 4.7e-20, much lower than for FLG alone p-value of 4.5e-6, indicative of an even greater effect when analyzed jointly (entire family vs. FLG alone) in the AD context. The identified LOF variants within the region can serve as biomarkers as well as help delineate the genetic profile in AD patients.

S.P. Smieszek: A. Employment (full or part-time); Modest; Vanda Pharmaceuticals. C. Polymeropoulos: A. Employment (full or part-time); Modest; Vanda Pharmaceuticals. G. Birznieks: A. Employment (full or part-time); Modest; Vanda Pharmaceuticals. M. Polymeropoulos: A. Employment (full or part-time); Modest; Vanda Pharmaceuticals.

P04.04D A balanced translocation t(2;7)(p21;p15) in three generations: Genome sequencing offers an opportunity to understand molecular etiology of Saethre-Chotzen/Robinow-Sarouf syndromes

B. Turkgenc1, R. P. Aguilar2,3, B. Curral3,4, C. Lowther3,4, E. Schields Wilch3, M. Talkowski3,4,5, C. Morton2,3,5,6, S. G. Temel7

1Acibadem Diagnostic Center, Istanbul, Turkey, 2Brigham and Women's Hospital, Boston, MA, United States, 3Harvard Medical School, Boston, MA, United States, 4Massachusetts General Hospital, Boston, MA, United States, 5Broad Institute of MIT and Harvard, Cambridge, MA, United States, 6University of Manchester, Manchester, United Kingdom, 7University of Bursa, Faculty of Medicine, Department of Medical Genetics, Bursa, Turkey

Recent developments in genomic analyses have facilitated the precise mapping of translocation breakpoints associated with Mendelian disorders to decipher if the rearrangement is likely to contribute to the observed phenotype. We evaluated the phenotypic consequences of an apparently balanced translocation in a family affected by craniosynostosis and limb malformations presenting with a dominant inheritance pattern and variable expressivity. We applied large-insert jumping libraries to localize the breakpoints and confirmed these results with Sanger sequencing to characterize breakpoints of a t(2;7)(p21;p15), followed by gene expression studies to explore the functional impact of the rearrangement. Sequencing revealed five breakpoints, disrupting two genes, HDAC9 and MACC1. We discovered altered expression of TWIST1 located downstream of HDAC9. Our results suggest that an inversion in the HDAC9-TWIST region in 7p21.1 affects regulation of TWIST, thereby producing a skeletal dysplasia with intrafamilial variable expressivity. These analyses suggest that the mechanism of this rearrangement might involve alteration of regulatory sequences located at a distance in cis that dysregulate TWIST and insinuate HDAC9 as a new disease locus producing craniosynostosis.

B. Turkgenc: None. R.P. Aguilar: None. B. Curral: None. C. Lowther: None. E. Schields Wilch: None. M. Talkowski: None. C. Morton: None. S.G. Temel: None.

P04.05A Novel variant in PRKAR1A associated with brachydactyly type E without hormone resistance

A. Pereda1, K. Heath2,3,4, J. Wu5, I. Valenzuela-Palafoll6, S. S. Taylor5,7, G. Perez de Nanclares1

1Molecular (Epi)Genetics Laboratory, BioAraba National Research Institute, Vitoria-Gasteiz, Spain, 2Institute of Medical and Molecular Genetics (INGEMM), Hospital Universitario La Paz, Universidad Autonóma de Madrid, IdiPAZ, Madrid, Spain, 3Skeletal dysplasia Multidisciplinary Unit (UMDE), Hospital Universitario La Paz, Madrid, Spain, 4CIBERER, ISCIII, Madrid, Spain, 5Department of Pharmacology, University of California at San Diego, San Diego, CA, United States, 6Area de Genètica Clínica i Malalties Minoritàries. Hospital Vall d'Hebron, Barcelona, Spain, 7Department of Chemistry and Biochemistry, University of California at San Diego, San Diego, CA, United States

Introduction: Protein kinase A (PKA) is a tetramer containing a regulatory (R) subunit dimer locking 2 catalytic (C) subunits. The most abundantly expressed R-subunit is PRKAR1A, composed of a dimerization domain, an inhibitory site, and 2 cAMP-binding domains (CNB:A; CNB:B), each containing a phosphate binding cassette (PBC). Inactivating mutations in PRKAR1A cause acrodysostosis with hormonal resistance (ACRDYS1).

Patients and Methods: We present a case of a 15 year-old girl with clinical suspicion of pseudopseudohypoparathyroidism based on brachydactyly type E, severe scoliosis, and short stature without hormone resistance. Her mother presented a similar phenotype. After discarding alterations in GNAS, PTHLH, IHH and GDF5 (Sanger sequencing, MS-MLPA/MLPA), a skeletal dysplasia NGS panel was performed.

Results: A heterozygous novel variant in PRKAR1A [NM_002734.4:c.592G>C; p.(Gly198Arg)], bioinformatically predicted as likely pathogenic, was identified. Familial studies confirmed cosegregation (mother harbored; healthy grandparents did not harbor). Gly198 precedes the PBC of CNB:A and its substitution to Arg is predicted to affect function because: (1) it might affect cAMP binding to CNB:A, as the long side-chain of arginine will crash with its neighboring residues (inactivating mutation); (2) the introduction of the longer side-chain into the tight PBC space could change its conformation leading to an altered binding between the C and the R (activating/inactivating mutation).

Conclusions: PRKAR1A variants can lead to phenotypes milder than ACRDYS1 depending on the affected protein domain. Incomplete or partial PKA inactivation could lead to an isolated non-severe brachydactyly without hormone resistance.

Funding: Instituto de Salud Carlos III (PI16/00073); Basque Department of Health (GV2016/111105).

A. Pereda: None. K. Heath: None. J. Wu: None. I. Valenzuela-Palafoll: None. S.S. Taylor: None. G. Perez de Nanclares: None.

P04.06B KIAA0753 mutations in skeletal ciliopathies: unveiling disease mechanisms

R. Vaz1, A. Hammarsjö2, F. Taylan1, D. Chitayat3, G. Grigelioniene2, A. Lindstrand2

1Department of Molecular Medicine and Surgery, Center for Molecular Medicine, Karolinska Institutet, Stockholm, Sweden, 2Department of Molecular Medicine and Surgery, Center for Molecular Medicine, Karolinska Institutet; Clinical Genetics, Karolinska University Hospital, Stockholm, Sweden, 3Division of Clinical and Metabolic Genetics, Department of Pediatrics, The Hospital for Sick Children, University of Toronto, Toronto, ON, Canada

Skeletal ciliopathies are a heterogeneous group of disorders caused by mutations in more than 25 genes, such as KIAA0753. Mutations in KIAA0753 have been associated with Joubert syndrome, orofaciodigital syndrome, and moderate to fetal lethal skeletal dysplasia with narrow thorax and abnormal metaphyses.

We have previously reported patients with biallelic frameshift and nonsense mutations in this gene. We have also shown that KIAA0753 is expressed in normal fetal human growth plate and immunohistochemistry of the growth plate of an affected fetus revealed an abnormal proliferative zone and a broad hypertrophic zone. To confirm the role of Kiaa0753 in skeletal ciliopathies, we used the zebrafish model. Phenotyping of zebrafish larvae carrying a nonsense mutation in homozygosity showed that mutant larvae presented with curved body, a typical ciliopathy phenotype, as well as abnormal cartilage patterning. kiaa0753-null zebrafish do not survive beyond the first week of development, suggesting that loss-of-function (LoF) mutations in kiaa0753 are embryonic lethal. Interestingly, these kiaa0753-null larvae show loss of cilia and abnormal cell rearrangements, helping us understand how LoF mutations in kiaa0753 affect cell organisation and embryonic development. In aggregate, our results show that Kiaa0753 is important for the early embryo development in zebrafish, in accordance with the phenotypes seen in patients. Future plans involve using this disease model to develop treatments for patients with KIAA0753 mutations.

R. Vaz: None. A. Hammarsjö: None. F. Taylan: None. D. Chitayat: None. G. Grigelioniene: None. A. Lindstrand: None.

P04.07C p63 establishes epithelial enhancers at craniofacial development genes involved in orofacial clefting

E. Lin-Shiao1,2, J. Welzenbach3, Y. Lan1,2, K. A. Alexander1,2, Z. Zhang1,2, M. Knapp4, E. Mangold3, M. Sammons1,2, S. L. Berger1,2, K. U. Ludwig3

1Department of Cell and Developmental Biology, University of Pennsylvania, Philadelphia, PA, United States, 2Epigenetics Institute, University of Pennsylvania, Philadelphia, PA, United States, 3Insitute of Human Genetics, School of Medicine, University of Bonn, Bonn, Germany, 4Insitute of Medical Biometry, Informatics and Epidemiology, School of Medicine, University of Bonn, Bonn, Germany

Transcription factor p63 is a key mediator of epidermal commitment, development, and differentiation. In humans, point mutations in the p63 coding region lead to developmental defects, including syndromic orofacial clefting. Notably, common risk variants located intronically in p63 contribute also to the risk of nonsyndromic cleft lip with/without cleft palate (nsCL/P), the most frequent isolated form of orofacial clefting. To date, our knowledge is limited about the role of p63 in human craniofacial development, due in part to a lack of tractable models. Here we investigated the role of p63 in human craniofacial development, and in the pathogenesis of nsCL/P. Using an inducible trans-differentiation model and systematic epigenomic sequencing (including ATAC-seq together with ChIP-seq of p63 and H3K27ac) we show that p63 establishes enhancers at craniofacial development genes to modulate their transcription. Specific substitution mutations in the DNA binding or SAM protein interaction domain of p63 respectively eliminate or reduce establishment of these enhancers. Furthermore, using large-scale GWAS data we show that enhancers established by p63 are highly enriched for SNPs associated with nsCL/P. These orthogonal approaches indicate a strong molecular link between p63 enhancer function and nsCL/P, illuminating molecular mechanisms underlying this developmental defect and revealing vital regulatory elements and new candidate causative genes.

E. Lin-Shiao: None. J. Welzenbach: None. Y. Lan: None. K.A. Alexander: None. Z. Zhang: None. M. Knapp: None. E. Mangold: None. M. Sammons: None. S.L. Berger: None. K.U. Ludwig: None.

P04.08D Cleidocranial dysplasia: natural history of bone manifestations

A. LAVILLAUREIX1,2, C. Michot2, G. Baujat2, G. Maruani3, M. Polak4, M. De la Dure-Molla5, J. Souberbielle6, E. Koumakis7, V. Cormier-Daire2

1Service de Génétique Clinique, Centre de référence "Maladies Rares" CLAD-Ouest, CHU Rennes - Université Rennes 1, Rennes, France, 2Service de Génétique Clinique, Centre de Référence Maladies Osseuses Constitutionnelles, APHP - Hôpital Necker-Enfants Malades - Institut Imagine - INSERM UMR 1163 - Université Paris Descartes Sorbonne Paris Cité, Paris, France, 3Service d'explorations fonctionnelles, AP-HP - Hôpital européen Georges-Pompidou, Paris, France, 4Service d'endocrinologie, gynécologie et diabétologie pédiatrique, APHP - Hôpital Necker-Enfants Malades, Paris, France, 5Service d'Odontologie, AP-HP - Hôpital Rothschild, Paris, France, 6Laboratoire d'explorations fonctionnelles, APHP - Hôpital Necker-Enfants Malades, Paris, France, 7Service de Rhumatologie, APHP, Hôpital Cochin, Paris, France

Cleidocranial dysplasia (CCD) is a rare autosomal dominant skeletal dysplasia whose most characteristic signs are the delayed closure of cranial sutures with wide fontanelle at birth, hypoplastic or aplastic clavicles, a short height and multiple dental anomalies. The natural history of CCD is poorly known. Isolated cases of patients with multiple fractures or osteoporosis have been described, without evaluating the other risk factors for bone fragility. The purpose of this study is to evaluate bone mineralization and the occurrence of fractures inpatients with CCD, children and adults. In an observational study, we collected clinico-radiological and genetics data, phosphocalcic balance, markers of bone remodeling and bone densitometry data of 45 CCD patients aged 9 months to 60.8 years (median = 15.4 years). 7% of patients evaluated had at least one fracture without trauma (3/45). 32% (13/41) patients have secondary hyperparathyroidism, a recognized risk factor for osteopenia due to increased bone resorption induced by parathyroid hormone and 20% (8/41) patients have vitamin D deficiency (25-OH D <20ng / mL). The analysis of BMD shows that the Z-score at the lumbar level of 38.7% of patients (12/31) is less than -2DS. These results indicate the need for active prevention of osteoporosis and fractures in patients with CCD, ensuring at an early age of sufficient intakes calcium and vitamin D, but also regular physical activity to allow optimal / maximal bone mass acquisition in adulthood.

A. Lavillaureix: None. C. Michot: None. G. Baujat: None. G. Maruani: None. M. Polak: None. M. De la Dure-Molla: None. J. Souberbielle: None. E. Koumakis: None. V. Cormier-Daire: None.

P04.09A A novel mutation in COL2A1 leading to spondyloepiphyseal dysplasia congenita

Y. Kendir Demirkol1, Ö. Akgün Doğan1, M. Say2, T. Kızılboğa Akgün3,4, L. Doğanay4

1Department of Pediatric Genetics, Health Sciences University, Umraniye Education and Research Hospital, İstanbul, Turkey, 2Bioinformatic Team, Gen-Era Diagnostic, İstanbul, Turkey, 3Department of Molecular Biology and Genetics, İstanbul Technical University, İstanbul, Turkey, 4GLAB (Genomic Laboratory) Health Sciences University, Umraniye Education and Research Hospital, İstanbul, Turkey

Introduction: Spondyloepiphyseal dysplasia congenita (SEDC, OMIM# 183900) is a rare autosomal dominant inherited chondrodysplasia. SEDC is characterised by dwarfism and skeletal abnormalities caused by mutations of the COL2A1 gene, which prevent bone growth. The most common features of SEDC are skeletal deformities such as short-trunk dwarfism, odontoid hypoplasia, cervical spine subluxation, scoliosis, kyphosis, lumbar lordosis, coxa vara, genu valgum, clubfoot, pes planus and metaphyseal changes. Cervical cord compression is the most hazardous skeletal deformity in patients with SEDC which requires special attention and management. Here, we present a mother and a son with SEDC due to a novel heterozygous mutation in SEDC.

Material and Methods: The patient was the first child of non consanguineous parents, born at 33 gestation week a birth length of 39 cm (3-10th centile). Postnatal he was hospitalized in intensive care unit due to respiratory insufficiency. Physical examination revealed facial dysmorphic features and short neck. The mother has disproportionate short stature, facial dysmorphic features and operation scar due to hip dislocation. Her X ray showed platyspondyly, kyphosis, scoliosis, coxa vara and flattened epiphyses.

Results: Next-generation sequencing was performed on Illumina MiSeq (v1.9) platform using the virtual panel for skeletal dysplasia consisting of 130 genes. The novel heterozygous variant in COL2A1 (c.2006G>C) was detected both son and mother. Segregation within the mothers family showed the mutation was denova.

Conclusions: SEDC is a rare skeletal dysplasia. Targeted exome analysis has great importance in the fast and accurate setting in the diagnosis and avoid serious neurological deficits and or mortality.

Y. Kendir Demirkol: None. Ö. Akgün Doğan: None. M. Say: None. T. Kızılboğa Akgün: None. L. Doğanay: None.

P04.10B Novel variant in COL2A1 gene causing spondyloepimetaphyseal dysplasia Strudwick type (SEMD-S)

M. Kero, A. Meašić, A. Bobinec, I. Sansović, I. Barišić

Children´s Hospital Zagreb, Zagreb, Croatia

Introduction: Collagenopathies type II are rare autosomal-dominant conditions characterized by skeletal dysplasia, short stature and sensorial defects. More than 400 heterozygous mutations in type II collagen gene COL2A1 have been identified, but only 5 of them are found in patients with spondyloepimetaphyseal dysplasia Strudwick type (SEMD-S), a rare phenotype of intermediate severity. We present a female patient with SEMD-S phenotype and a novel mutation in the COL2A1 gene.

Patients and Methods: The patient presented at birth with severe short trunk-short limb dwarfism, short chest, coxa vara, clubfoot, cleft palate, conductive hearing loss and mild external hydrocephalus. Metaphyseal irregularities permitted clinical differentiation from SED congenita. Now, at the age of 7 years, her stature is significantly reduced and a waddling gait, genu valgum and lumbar lordosis developed. Hands and feet are normal. The genomic DNA from blood leukocytes of the patient was isolated and Next Generation Sequencing (NGS) analysis was done focusing on about 200 genes associated with skeletal dysplasias.

Results: NGS revealed a novel heterozygous missense variant (c.2177G>A, p.Gly726Asp) in exon 33 of the COL2A1 gene, categorised as likely pathogenic according to ACMG/AMP classification. As glycine substitutions are typical for SEMD-S patients, it is highly probable that the identified variant is causative of the SEMD-S phenotype.

Conclusion: To our knowledge, this missense variant in COL2A1 has not been reported before. It extends the mutational spectrum allowing for better genotype/phenotype correlation in patients with SEMD-S. Molecular genetic analysis is helpful for the diagnosis of skeletal dysplasias with overlapping phenotypes.

M. Kero: None. A. Meašić: None. A. Bobinec: None. I. Sansović: None. I. Barišić: None.

P04.12D Defining the clinical features associated with variants in COL4A2: case report and review of the literature

A. Hanson-Kahn1, K. Vlessis1, E. J. Smith2, M. Manning1

1Stanford University, Stanford, CA, United States, 2Stanford Children's Health, Palo Alto, CA, United States

COL4A2 encodes the α2 chain of type IV collagen, which assembles into heterotrimers with COL4A1 chains to form a component of the basal membrane. Heterozygous variants in COL4A2 have been described in fewer than 20 families in association with familial cerebrovascular disease manifesting as porencephaly and intracerebral hemorrhage. Seizures, cortical malformations, lens opacities, hematuria and elevated creatine kinase have been reported. We present a 4-1/2 year old male with ischemic stroke and deep vein thrombosis, intracranial hemorrhage, systemic arterial involvement (tapering of the abdominal aorta, occlusion of the left common iliac artery, small right iliac artery and collaterals of the upper extremities) and mildly dilated aortic root and ascending aorta. Whole exome sequencing reported a maternally inherited variant in COL4A2 (c.1396G>A, p.G466S). This variant is not reported in ClinVar but is reported in gnomAD with an allele frequency of 5.0x10-5. In silico models predict this variant to be damaging. The patient’s mother is asymptomatic; brain MRI, echocardiogram and ophthalmology examinations are being coordinated. While intracranial hemorrhage has also been seen in other patients with COL4A2 variants, systemic arteriopathy and mild enlargement of the aortic root have not been previously described. We review the literature and further define the clinical features associated with variants in COL4A2 to include arteriopathy and aortic root dilatation.

A. Hanson-Kahn: None. K. Vlessis: None. E.J. Smith: None. M. Manning: None.

P04.13A Next generation sequencing in neonates presenting with collodion baby syndrome

A. Bobinec1, A. Meašić1, M. Kero1, I. Sansović1, S. Ožanić Bulić2, N. Pustišek2, S. Murat-Sušić3, I. Barišić1,2

1Department of Medical Genetics and Reproductive Health, Children's Hospital Zagreb, Scientific Centre of Excellence for Reproductive and Regenerative Medicine (CERRM), University of Zagreb School of Medicine, Zagreb, Croatia, 2Division of Dermatology, Children's Hospital Zagreb, University of Zagreb School of Medicine, Zagreb, Croatia, 3Department of Dermatology and Venerology, University Hospital Centre Zagreb, University of Zagreb School of Medicine, Zagreb, Croatia

Introduction: Collodion baby (CB) is a rare condition characterized by the presence of parchment or-cellophane-like collodion membrane encompassing the whole body. CB is a genetically heterogeneous disorder that usually presents with lamellar ichthyosis (LI) or congenital ichthyosiform erythroderma (CIE). Other less common disorders as self-improving collodion ichthyosis, Netherton syndrome, Gaucher disease type 2, Chanarin-Dorfman syndrome, ectodermal dysplasia or Sjögren Larsson syndrome may also be responsible for this phenotype.

Materials and Methods: Clinical exome sequencing (CES) was performed in 4 patients with referral diagnosis of CB using Illumina TruSight One Kit.

Results: CES analysis revealed mutations in ALOX12B, SPINK5, NIPAL4 and TGM1 genes. In patient 1 (P1), we found a homozygous mutation in the ALOX12B gene, known to cause self-improving collodion ichthyosis. This was consistent with a rapid resolution of almost all skin lesions. P2 and P3 developed Netherton syndrome. In P2 compound heterozygous mutations in the SPINK5 gene confirmed the diagnosis. In P3 no causative genetic variations were found. In P4, presenting with CB evolving to LI, we detected two heterozygous variants, one in NIPAL4 gene (likely pathogenic) and other in TGM1 (uncertain significance).

Conclusion: Clinical evolution of CB is hard to predict as clinical presentation and severity may vary from mild to life-threatening. CES provided a timely and precise diagnosis in most of our patients which is important for treatment planning, disease prognosis and genetic counselling of affected families.

A. Bobinec: None. A. Meašić: None. M. Kero: None. I. Sansović: None. S. Ožanić Bulić: None. N. Pustišek: None. S. Murat-Sušić: None. I. Barišić: None.

P04.14B IL11RA and pansynostosis

A. Topa1,2, A. Rohlin2, L. Lovmar2, G. Stenman1, L. Kölby3

1Department of Pathology and Genetics, University of Gothenburg, The Sahlgrenska Academy, Gothenburg, Sweden, 2Department of Clinical Genetics, Sahlgrenska University Hospital, Gothenburg, Sweden, 3Department of Plastic Surgery, University of Gothenburg, The Sahlgrenska Academy, Gothenburg, Sweden

The genetic background of craniosynostosis is complex and implicates multiple signaling pathways. The role of interleukin 11 receptor, alpha (IL11RA), a cell-derived cytokine, has been evidenced in the pathogenesis of multiple synostosis with autosomal recessive inheritance pattern (Craniosynostosis and dental anomalies, OMIM#614188). A Crouzon-like appearance with different types of craniosynostosis has been observed. Several causal missense and truncating variants have been reported. We present three additional cases with homozygous novel pathogenic and likely pathogenic variants in IL11RA (NM_001142784.2: c.598C>A, p.Pro200Thr; c.696C>A, p.Tyr232*; c.866A>G, p.His289Arg). All three patients presented with high intracranial pressure, a Crouzon-like phenotype and early closure of all cranial sutures (pansynostosis). Our cases, together with previous reports of IL11RA-related pansynostosis, suggest a key regulator role for IL11RA in the morphogenesis and maintenance of patency of all cranial sutures.

A. Topa: None. A. Rohlin: None. L. Lovmar: None. G. Stenman: None. L. Kölby: None.

P04.15C Diagnostic value of targeted next-generation sequencing in craniosynostosis

E. M. Olech1, A. Sowińska-Seidler1, D. Popiel2, G. Koczyk2,3, M. Socha1, J. Walczak-Sztulpa1, A. Materna-Kiryluk1,2, A. Latos-Bieleńska1,2, R. Posmyk4, R. Śmigiel5, A. Dawidziuk1, A. Jamsheer1

1Department of Medical Genetics, Poznan University of Medical Sciences, Poznan, Poland, 2Centers for Medical Genetics GENESIS, Poznan, Poland, 3Institute of Plant Genetics (PAS), Department of Biometry and Bioinformatics, Poznan, Poland, 4Podlaskie Center of Clinical Genetics, Bialystok, Poland, 5Department of Pediatric Propedeutics and Rare Diseases, Wroclaw Medical University, Wroclaw, Poland

Background: Craniosynostosis (CS), the premature fusion of one or more cranial sutures, occurs either as an isolated malformation or in a syndromic form, representing a genetically heterogeneous and clinically variable group of disorders. Routine diagnostic screening of common craniosynostosis-associated genes enables to establish genetic aetiology in 21% to 62% depending on the size of the study, ethnicity of the population, and range of the molecular analysis.

Materials and Methods: We performed targeted next-generation sequencing in a group of 60 patients manifesting CS with negative results of molecular screening. We have designed a custom hybridisation-based panel, encompassing 61 genes and 11 SNPs chosen based on current knowledge about variants involved in craniofacial development using online databases. We sequenced captured and indexed libraries on Ion Torrent S5 sequencing system.

Results: This approach revealed pathogenic variants or probably pathogenic variants among patients presenting with different forms of CS. We found pathogenic and probably pathogenic variants or variants of unknown significance within the ALX4, BMP4, CYP26B1, EDN3, EFNB1, EFTUD2, FGFR1, FGFR2, FGFR3, IFT122, IFT140, IHH, MEGF8, RECQL4, RUNX2, SKI, TCF12 genes.

Conclusion: We present an NGS targeted gene panel approach as a valuable diagnostic tool in the genetic testing of patients presenting with craniosynostosis.

This work was supported by the grants from the Poznan University of Medical Sciences, Poland 502-14-11261860-41259 and the Polish National Science Centre, Poland UMO-2016/23/N/NZ5/02577 to Ewelina M. Olech, and UMO-2016/22/E/NZ5/00270 to Aleksander Jamsheer

E.M. Olech: None. A. Sowińska-Seidler: None. D. Popiel: None. G. Koczyk: None. M. Socha: None. J. Walczak-Sztulpa: None. A. Materna-Kiryluk: None. A. Latos-Bieleńska: None. R. Posmyk: None. R. Śmigiel: None. A. Dawidziuk: None. A. Jamsheer: None.

P04.16D A genome-wide association study implicates BMP7 as a risk factor for nonsyndromic metopic craniosynostosis

C. M. Justice1, A. Cuellar2, K. Bala2, J. A. Sabourin1, M. L. Cunningham3, A. O. M. Wilkie4, J. M. Phipps4, Y. Zhou4, K. Crawford4, D. Cilliers5, J. E. V. Morton6, A. Weber7, L. C. Wilson8, E. Simeonov9, R. Kaneva10, N. Yaneva11, K. Georgiev12, A. Busarski12, C. Senders13, M. Zwienenberg14, T. Roscioli15, M. Barba16, W. Lattanzi16, K. Conway17, V. Sheffield18, L. Brody19, J. Mills20, D. Kay21, R. Sicko21, R. Tittle22, A. F. Wilson1, P. A. Romitti17, S. A. Boyadjiev2, the National Birth Defects Prevention Study

1Genometrics Section, Computational and Statistical Genomics Branch, Division of Intramural Research, NHGRI, NIH, Baltimore, MD, United States, 2Department of Pediatrics, University of California Davis, Sacramento, CA, United States, 3Department of Pediatrics, Seattle Children's Craniofacial Center, University of Washington, Seattle, WA, United States, 4MRC Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford, United Kingdom, 5Clinical Genetics Service, Oxford Centre for Genomic Medicine, Oxford University Hospitals NHS Foundation Trust, Oxford, United Kingdom, 6West Midlands Regional Clinical Genetics Service and Birmingham Health Partners, Birmingham Women’s and Children’s Hospitals NHS Foundation Trust, Birmingham, United Kingdom, 7Department of Clinical Genetics, Liverpool Women's NHS Foundation Trust,, Liverpool, United Kingdom, 8Clinical Genetics Service, Great Ormond Street Hospital, London, United Kingdom, 9National Institute of Pediatrics, Sofia Medical University, Sofia, Bulgaria, 10Molecular Medicine Center, Department of Medical Chemistry and Biochemistry, Medical Faculty, Medical University of Sofia, Sofia, Bulgaria, 11National Genetic Laboratory, University Hospital of Obstetrics and Gynecology “Maichin Dom”, Medical University of Sofia, Sofia, Bulgaria, 12Department of Neurosurgery, University Hospital 'St. Ivan Rilski', Medical University of Sofia, Sofia, Bulgaria, 13Department of Otolaryngology, Head and Neck Surgery, University of California Davis,, Sacramento, CA, United States, 14Department of Neurosurgery, University of California Davis, Sacramento, CA, United States, 15Neuroscience Research Australia, University of New South Wales, Sydney, Australia, 16Institute of Anatomy and Cell Biology, The Catholic University Sacred Heart, Rome, Italy, 17Department of Epidemiology, College of Public Health, The University of Iowa, Iowa City, IA, United States, 18Department of Pediatrics, Stead Family Children’s Hospital, The University of Iowa, Iowa City, IA, United States, 19Gene and Environment Interaction Section, NHGRI, NIH, Bethesda, MD, United States, 20Epidemiology Branch, Eunice Kennedy Shriver NICHD, NIH, Bethesda, MD, United States, 21Division of Genetics, Wadsworth Center, NYS Department of Health, Albany, NY, United States, 22Department of Nutritional Sciences, University of Texas at Austin, Austin, TX, United States

Craniosynostosis (CS) arises from premature closure of one or more cranial sutures. Previously, we observed that BMP2 (rs1884302) and BBS9 (rs10262453) were independently associated with sagittal nonsyndromic CS (sNCS). In this study, we conducted a genome-wide association study (GWAS) for metopic NCS (mNCS). In an international sample of 228 non-Hispanic white case-parent trios, three variants were genome-wide significant: rs781712 (P=4.788x10-9; odds ratio (OR)=2.407) intronic to SPRY3; rs6127972 (P=5.611x10-9; OR=2.257) intronic to BMP7; and rs62590971 (P=4.046x10-8; OR=0.380) located ~155 kb upstream from TGIF2LX. The association for rs6127972 was replicated in an independent sample (194 unrelated mNCS cases, 333 controls, P=0.0042, OR=1.45) and in a meta-analysis, P = 3.11x10-9 (OR = 1.45). Rs1884302 and rs10262453, identified as risk factors from our previous sNCS GWAS, were also genotyped in our mNCS samples. Rs10262453 was borderline significant in the mNCS discovery and replications cohorts (P=9.216x10-5, OR=1.716), (P=0.0003, OR=1.62) respectively, and had a meta-analysis P=1.330x10-7(OR=1.62). Notably, the C allele for this variant was over-transmitted in mNCS probands, whereas the A allele was over-transmitted in sNCS probands. Functional assessment of rs6127972 using western blot and ELISA showed no difference in BMP7 expression or protein levels in synostotic metopic versus open sutures. Data from luciferase studies suggested that the locus may act as a repressor element with the risk allele exerting stronger repression. Zebrafish transgenic analysis produced inconclusive results. Our findings implicate the BMP/TGFβ signaling pathway in midline NCS and support the role of BBS9 locus in the etiology of metopic craniosynostosis. [Grants NIH X01HG008936 and R01DE016886]

C.M. Justice: None. A. Cuellar: None. K. Bala: None. J.A. Sabourin: None. M.L. Cunningham: None. A.O.M. Wilkie: None. J.M. Phipps: None. Y. Zhou: None. K. Crawford: None. D. Cilliers: None. J.E.V. Morton: None. A. Weber: None. L.C. Wilson: None. E. Simeonov: None. R. Kaneva: None. N. Yaneva: None. K. Georgiev: None. A. Busarski: None. C. Senders: None. M. Zwienenberg: None. T. Roscioli: None. M. Barba: None. W. Lattanzi: None. K. Conway: None. V. Sheffield: None. L. Brody: None. J. Mills: None. D. Kay: None. R. Sicko: None. R. Tittle: None. A.F. Wilson: None. P.A. Romitti: None. S.A. Boyadjiev: None.

P04.17A BMP2 or not BMP2? A SMAD6-related question in craniosynostosis

E. Calpena1, A. Cuellar2, K. Bala2, S. M. A. Swagemakers3, N. Koelling1, S. J. McGowan1, M. Balasubramanian4, J. E. V. Morton5, A. Weber6, L. C. Wilson7, D. Johnson8, S. A. Wall8, S. R. F. Twigg1, I. M. J. Mathijssen9, E. Simeonov10, W. Lattanzi11, F. Boardman-Pretty12,13, S. A. Boyadjiev2, A. O. M. Wilkie1,8

1MRC Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, United Kingdom, 2Department of Pediatrics, University of California Davis, Sacramento, CA, United States, 3Departments of Pathology and Bioinformatics, Erasmus MC, University Medical Center Rotterdam, Rotterdam, Netherlands, 4Sheffield Clinical Genetics Service, Sheffield Children's NHS Foundation Trust, Sheffield, United Kingdom, 5West Midlands Regional Clinical Genetics Service and Birmingham Health Partners, Birmingham Women’s and Children’s Hospitals NHS Foundation Trust, Birmingham, United Kingdom, 6Department of Clinical Genetics, Liverpool Women's NHS Foundation Trust, Liverpool, United Kingdom, 7Clinical Genetics Service, Great Ormond Street Hospital, London, United Kingdom, 8Craniofacial Unit, Oxford University Hospitals NHS Trust, John Radcliffe Hospital, Oxford, United Kingdom, 9Department of Plastic and Reconstructive Surgery, Erasmus MC, University Medical Center Rotterdam, Rotterdam, Netherlands, 10National Institute of Pediatrics, Sofia Medical University, Sofia, Bulgaria, 11Institute of Anatomy and Cell Biology, The Catholic University Sacred Heart, Rome, Italy, 12Genomics England, London, United Kingdom, 13William Harvey Research Institute, Queen Mary University of London, London, United Kingdom

Incomplete penetrance is a complicating factor in diagnosis and makes genetic counselling very challenging. Usually the mechanisms explaining the non-penetrance are not understood, with few epistatically-interacting genetic factors described in humans. One recent example of such a “two-locus” model is the proposal by Timberlake et al (eLife, 2016) of an interaction of SMAD6 variants in midline craniosynostosis (premature fusion of the metopic and/or sagittal cranial sutures of the skull) with a common polymorphism near BMP2, to explain frequent non-penetrance in the parent transmitting the SMAD6 variant. We aimed (i) to validate the findings in an independent, larger cohort, and (ii) to delineate the phenotype associated with SMAD6 variants. We performed NGS-based resequencing of multiplexed PCR products of SMAD6 in 800 unsolved patients with any type of craniosynostosis, and genotyped the rs1884302 BMP2 polymorphism in SMAD6-positive individuals. We identified 18 different rare damaging SMAD6 variants (2.25% overall), with the highest prevalence in metopic synostosis (~6%) and a ~23-fold enrichment of loss-of-function variants compared to gnomAD data (P<0.00001). This confirms that SMAD6 variants increase the risk of craniosynostosis (especially metopic). Combining these data with a further 6 independently identified variants, 17/24 were transmitted from an unaffected parent but the BMP2 genotype did not correlate with presence/absence of craniosynostosis. Hence further work is needed to identify the factors accounting for reduced penetrance of SMAD6 variants - both in craniosynostosis, and in the panoply of other phenotypes (including congenital heart disease, thoracic aortic aneurysm and intellectual disability), with which SMAD6 variants have been associated.

E. Calpena: None. A. Cuellar: None. K. Bala: None. S.M.A. Swagemakers: None. N. Koelling: None. S.J. McGowan: None. M. Balasubramanian: None. J.E.V. Morton: None. A. Weber: None. L.C. Wilson: None. D. Johnson: None. S.A. Wall: None. S.R.F. Twigg: None. I.M.J. Mathijssen: None. E. Simeonov: None. W. Lattanzi: None. F. Boardman-Pretty: None. S.A. Boyadjiev: None. A.O.M. Wilkie: None.

P04.18B Chromosomal rearrangements and CNVs identified in craniosynostosis cases

E. König1, E. Kunstmann2, A. Quattländer3, T. Schweitzer4, E. Klopocki1

1Institute of Human Genetics, Würzburg, Germany, 2Praxis für Humangenetik, Würzburg, Germany, 3Kinderarztpraxis Volkach, Volkach, Germany, 4Department of Neurosurgery, Section of Pediatric Neurosurgery, University Hospital of Würzburg, Würzburg, Germany

Craniosynostosis, the premature fusion of cranial sutures, is a common birth defect (~1:2500 newborns) occurring either as an isolated or syndromic form. Single nucleotide variants (SNVs) in known craniosynostosis genes i.e. FGFR2, FGFR3 and TWIST1 account for only 20% of cases. Besides these SNVs, a significant subgroup (15%) of patients with syndromic craniosynostosis show chromosomal rearrangements. Due to the small number of patients with identical chromosomal aberrations reported in the literature, it is often difficult to determine the critical region/gene which contributes to the phenotype and perform a specific diagnostic analysis. Thus, aCGH was chosen as genome-wide CNV screening approach for identification of CNVs in a cohort of 33 craniosynostosis patients.

In total we detected likely pathogenic CNVs in 18% of our cohort. These CNVs encompass known craniosynostosis genes like MSX2 and TCF12 and novel craniosynostosis candidate genes, respectively. Three patients carried unbalanced translocations and in additional three cases we detected interstitial gains and losses. Clinically these patients presented with isolated as well as syndromic forms of craniosynostosis.

Our results emphasize the importance of chromosomal rearrangements and CNVs as a genetic cause of craniosynostosis, particularly in case of unclear syndrome classification due to clinical variability. Furthermore, genes which reside in or are disrupted by rare CNVs represent candidate genes for craniosynostosis.

In addition to sequencing approaches CNV screening methods like aCGH should be included in the genetic testing algorithm for craniosynostosis patients. This will enable diagnosis in a significant portion of yet unsolved syndromic as well as non-syndromic cases.

E. König: None. E. Kunstmann: None. A. Quattländer: None. T. Schweitzer: None. E. Klopocki: None.

P04.19C Biallelic loss of function mutations in CSGALNACT1 cause a mild skeletal dysplasia with joint laxity

R. Meyer1, M. Begemann1, A. Schulze2, A. Kochs3, I. Kurth1, M. Elbracht1

1Institute of Human Genetics, RWTH Aachen University, Aachen, Germany, 2Department of Orthopedic Surgery, RWTH Aachen University, Aachen, Germany, 3Praxis für Orthopädie und Unfallchirurgie, Aachen, Germany

Biallelic loss of function mutations in CSGALNACT1 were recently proposed to cause a prenatal onset mild skeletal dysplasia with joint laxity in a single 3.5 years old female patient. The phenotype in this patient reflects the skeletal dysplasia of Csgalnact1-/- mice and resembles the clinical and radiographic picture of a milder spectrum of Desbuquois dysplasia (Vodopiutz et al., 2017). CSGALNACT1 encodes chondroitin sulfate N-acetylgalactosaminyltransferase-1 (CSGalNAcT-1, ChGn-1) which has a critical role in the biosynthesis of chondroitin sulfate and dermatan sulfate. Here we report on a 12 years old boy born from consanguineous parents with a mild skeletal dysplasia, short stature (-3SD), and joint laxity. Psychomotor development was normal. Whole exome sequencing revealed a homozygous 1bp-deletion in CSGALNACT1 (c.372del, p.(His125Thrfs*9)). Segregation analysis confirmed both parents as heterozygous carriers. The identified variant is neither reported in gnomAD and dbSNP nor in the literature. It leads to a frameshift and premature stop codon and, thus, with high probability to loss of function of the corresponding protein. This is the second report of a patient with biallelic loss of function mutations in CSGALNACT1 which underlines its role in mild skeletal dysplasia, short stature, and joint laxity without obvious additional organ manifestations and normal psychomotor development.

R. Meyer: None. M. Begemann: None. A. Schulze: None. A. Kochs: None. I. Kurth: None. M. Elbracht: None.

P04.20D Prenatal diagnosis of Desbuquois dysplasia by whole exome sequencing before the occurrence of specific ultrasound signs

C. Houdayer1, F. Boussion2, A. Ziegler1, S. Blesson3, C. Bris1, A. Toutain3, F. Biquard2, A. Guichet1, D. Bonneau1,4, E. Colin1,4

1Department of Biochemistry and Genetics, Angers University Hospital, ANGERS, France, 2Department of Obstetrics and Gynecology, Angers University Hospital, ANGERS, France, 3Department of Genetics, Tours University Hospital, TOURS, France, 4MitovascLab, Team, Institut MitoVasc, UMR CNRS 6015, INSERM U1083, Angers University, Angers, France

Osteochondrodysplasia is a group of hereditary disorders of the connective tissue, bones or cartilage that impair skeletal development with a prevalence of about 2 per 10 000 births. The genetic and phenotypic heterogeneity of this group of disorders is tremendous involving more than 300 genes that are responsible for more than 450 distinct conditions. One of them, Desbuquois syndrome dysplasia (DBQD) is a rare skeletal osteochondrodysplasia characterized by severe micromelic dwarfism, joint laxity with multiple joint dislocations, specific radiographic findings features and facial dysmorphism. DBQD is a very severe and often sometime lethal form of micromelic dwarfism. We report here a case for which whole exome sequencing allowed to perform an early prenatal diagnosis before the occurrence of characteristic ultrasound signs. Indeed, at 21 WG, the ultrasound scan confirmed that the fetus was very likely affected with an osteochondrodysplasia because all biometric parameters were below -4 SD associated with bilateral vertical talus. However, it was impossible, at that time, to pinpoint the exact diagnosis. Using exome sequencing, we evidenced two variants in CANT1, the gene responsible for DBDQ. The parents were informed that the fetus was very likely affected with DBQD at a gestational age of 24 weeks. The present case, is to our knowledge, the first one in whom a molecular prenatal diagnosis of DBQD is performed in the absence of relevant family history and before the appearance of evocative sonographic features. It highlights the utility of prenatal exome sequencing in performing a precocious diagnosis for severe fetal conditions.

C. Houdayer: None. F. Boussion: None. A. Ziegler: None. S. Blesson: None. C. Bris: None. A. Toutain: None. F. Biquard: None. A. Guichet: None. D. Bonneau: None. E. Colin: None.

P04.21A Vascular ehlers-danlos syndrome

N. Agaoglu1, Y. Kendir Demirkol2, O. Akgun Dogan2, M. Say3, T. Kizilboga Akgun1, H. Doganay1

1GLAB (Genomic Laboratory) Health Sciences University, Umraniye Education and Research Hospital, Istanbul, Turkey, 2Department of Pediatric Genetics, Health Sciences University, Umraniye Education and Research Hospital, Istanbul, Turkey, 3Bioinformatic Team, Gen-Era Diagnostic, Istanbul, Turkey

Introduction: Vascular Ehlers-Danlos syndrome is an autosomal dominant connective tissue disorder caused by mutations in COL3A1 and COL1A1. It is considered as the most severe form of Ehlers-Danlos syndrome (EDS) due to the high mortality rate associated with the fragility of arteries and internal organs. Common symptoms include thin, translucent skin; easy bruising; characteristic facial appearance; and fragile arteries, muscles and internal organs. Treatment and management options are limited and focused on preventing serious complications. Here we present a 5 years old female with vascular type EDS due to a novel heterozygous mutation COL3A1.

Materials and Methods: The patient was the first child of non-consanguineous healthy parents, born at term with a birth weight of 2600gr (3-10th centile). Easly bruising even with small traumas have been noticed from the infancy period. She also had difficulty in wound healing. Physical examination revealed wide forehead, deep-set eyes, thin translucent skin with multiple bruising and atrophic scars, joint laxity, and profound superficial veins.

Results: Next-generation sequencing was performed on Illumina MiSeq(v1.9) platform using the virtual panel for collagenopathy consisting of 51 genes. The heterozygous variant in COL3A1(c.3563G>A) was detected. Segregation within the family showed that the variant is de-novo.

Conclusions: Although vascular type EDS is a rare genetic disease, it should be kept in mind in patients with easy bruisable skin and atrophic scar. Accurate and early diagnosis have great importance in preventing serious life threating complications.

N. Agaoglu: None. Y. Kendir demirkol: None. O. Akgun dogan: None. M. Say: None. T. Kizilboga akgun: None. H. Doganay: None.

P04.23C Homozygous mutations of EGFR gene as a cause of lethal syndrome with progeroid features in two Roma families

J. Laštuvková1, P. Hitka2, S. Mazurová3, M. Magner3, M. Tesařová3, V. Stránecký3, V. Čejnová1

1Department of Medical Genetics, Masaryk Hospital in Usti nad Labem, Krajska zdravotni, a.s., Usti nad Labem, Czech Republic, 2Clinic of Neonatology Faculty of Health Care Studies Jan Evangelista Purkyně University in Usti nad Labem and Krajska zdravotni, a.s., Masaryk Hospital in Usti nad Labem, Usti nad Labem, Czech Republic, 3Department of Paediatrics and Adolescent Medicine First Medical Faculty, Charles University in Prague and General University Hospital in Prague, Czech Republic, Prague, Czech Republic

EGFR gene encodes an epidermal growth factor receptor which is important in cell proliferation and differentiation. So far, only rare reports of germline mutations in the EGFR gene as a cause of monogenic disease in children are available. Our patient was a young pregnant woman of Roma origin. Her previous pregnancy was complicated by polyhydramnios and IUGR and her daughter was born in the 31st week of pregnancy, birth weight 660g. Child had no organ malformation but very fragile skin and died after 46 days because of multiorgan failure. The current pregnancy was also complicated by severe polyhydramnios, there were hyperechogenic kidneys and bowels and IUGR. The boy was born in the 30th week with birth weight of 760g. The boy had progeroid features with lack of subcutaneous fat and very thin and fragile skin. He died in 34 days because of metabolic dysbalance. Whole exome sequencing revealed presence of homozygous mutation c.1283G>A (p.Gly428Asp) in the EGFR gene in both deceased siblings, both parents are heterozygous carriers. Thirteen years ago, we examined Roma family in which 3 children died in early infancy with very similar clinical features. The diagnosis of Netherton syndrome was considered, but wasn't confirmed. With our new experience we tested mutation of EGFR gene in one of these deceased children. Homozygous mutation c.1283G>A was confirmed by Sanger sequencing. Mutations of EGFR gene should be considered in a differential diagnosis of progeroid syndrome especially in Roma families. Our report describes features of these children. Dedication: RVO-VFN 64165/2012

J. Laštuvková: None. P. Hitka: None. S. Mazurová: None. M. Magner: None. M. Tesařová: None. V. Stránecký: None. V. Čejnová: None.

P04.24D Tessier no. 4 facial cleft leading to the diagnosis of familial vascular Ehlers-Danlos syndrome

C. R. Fagerberg1, R. M. Schmidt2, S. Farholt3, K. P. Sørensen1

1Department of Clinical Genetics, Odense University Hospital, Odense C, Denmark, 2H C Andersen Childrens Hospital, Odense University Hospital, Odense C, Denmark, 3Center for Rare Diseases, Aarhus University Hospital, Aarhus C, Denmark

The parents of a girl born with Tessier no. 4 facial clefting sought genetic counselling concerning recurrence risk in the next pregnancy. The pattern of the facial clefting and a constriction ring on the right leg raised the suspicion of amniotic band to be the cause. Trio exome analysis was performed to best possible rule out other causes. A maternally inherited substitution of glycin to serin in the triple helix domaine (COL3A1:c.2689G>A;p.Gly897Ser) was identified in the COL3A1-gene, raising the suspicion of vascular Ehlers-Danlos syndrome. The family history was without vascular catastrophes, but one affected had severe bleeding after a gynecological surgery. Some carriers of the variant had easy bruising, whereas others did not. It is well known that substitutions of glycin to serin in the triple helix domaine lead to a better prognosis and a milder phenotype than other glycin substitutions in vascular Ehlers-Danlos Syndrome, which most likely explains the mild vascular phenotype in this family. This case reminds us that some collagen diseases increase the risk of amniotic banding, the risk being estimated to approximately 1 %. The other way round, the risk of a collagen disease in case of amniotic banding is to the best of our knowledge unknown. We suggest that genetic analysis for selected collagen diseases should be done in case of amniotic banding.

C.R. Fagerberg: None. R.M. Schmidt: None. S. Farholt: None. K.P. Sørensen: None.

P04.26B Natural exon skipping reveals that antisense oligonucleotide-mediated exon skipping should be directed at the recessive type of dystrophic epidermolysis bullosa

J. Bremer1, E. H. van der Heijden1, D. S. Eichhorn2, R. Meijer3, H. H. Lemmink1, H. Scheffer3, R. S. Sinke1, M. F. Jonkman2, A. M. G. Pasmooij2, P. C. Van den Akker1

1University of Groningen, University Medical Center Groningen, Department of Genetics, Groningen, Netherlands, 2University of Groningen, University Medical Center Groningen, Department of Dermatology, Groningen, Netherlands, 3Department of Human Genetics, Radboud University Nijmegen Medical Centre, Nijmegen, Netherlands

Introduction: Dystrophic epidermolysis bullosa (DEB) is a devastating genetic blistering disease affecting skin and mucous membranes. DEB is caused by pathogenic variants in the COL7A1 gene encoding type VII collagen, and can be inherited dominantly or recessively. The structure of COL7A1 makes it an attractive candidate for exon-skipping therapy. Recently, we demonstrated promising proof-of-principle for antisense oligonucleotide (AON)-mediated exon-skipping as a systemic therapeutic approach for DEB. However, it is unclear what phenotypic effect may be expected from exon-skipping and which patient groups may benefit the most.

Patients and Methods: To answer these questions, we studied new clinical and molecular data on seven patients from the Dutch EB registry and reviewed the literature on pathogenic COL7A1 variants inducing ‘natural exon-skipping’.

Results: We found that the natural skipping of certain exons led to disease in a heterozygous state, while the skipping of other exons only led to disease if combined with a pathogenic variant on the other COL7A1 allele. The dominant DEB phenotypes associated with heterozygous exon-skipping could not be distinguished from dominant phenotypes caused by heterozygous COL7A1 variants not inducing exon-skipping. Phenotypes associated with recessive exon-skipping mutations were, however, on average relatively mild in the spectrum of recessive DEB.

Conclusions: For dominant DEB, AON-mediated exon-skipping is unlikely to make a clinical difference. In contrast, we anticipate that exon-skipping has the potential to induce a clinically relevant improvement of the devastating recessive DEB phenotype, especially the types caused by bi-allelic null variants.

Grants and Fellowships: Clinical Fellowship ZonMW (90715614, PvdA)

J. Bremer: None. E.H. van der Heijden: None. D.S. Eichhorn: None. R. Meijer: None. H.H. Lemmink: None. H. Scheffer: None. R.S. Sinke: None. M.F. Jonkman: None. A.M.G. Pasmooij: None. P.C. Van den Akker: None.

P04.27C Targeted next-generation sequencing in the diagnosis of facial dysostoses

E. M. Olech1, D. Popiel2, G. Koczyk1,3, A. Materna-Kiryluk1,2, M. Badura-Stronka1,2, M. Wiśniewska1,2, A. Latos-Bieleńska1,2, A. Jamsheer1,2

1Department of Medical Genetics, Poznan University of Medical Sciences, Poznan, Poland, 2Centers for Medical Genetics GENESIS, Poznan, Poland, 3Institute of Plant Genetics (PAS), Department of Biometry and Bioinformatics, Poznan, Poland

Background: Facial dysostoses (FD) encompass rare and heterogeneous congenital craniofacial anomalies subdivided into mandibulofacial dysostoses (MFDs) and acrofacial dysostoses (AFDs). Both MFDs and AFDs result from impaired migration of neural crest cells to the pharyngeal arches and the face during embryogenesis. The main clinical features observed in affected patients are maxillary, malar and mandibular hypoplasia, cleft palate, and/or ear defects. In AFDs, limb defects are present as an additional feature.

Materials and Methods: To determine the molecular aetiology of FD, we performed a custom amplification-based panel of 37 genes (145.5 kb, 761 amplicons) by next-generation sequencing on Ion Torrent S5 sequencing system. We analysed a group of 25 patients affected by variable forms of FD and validated clinically relevant variants by conventional sequencing in both affected individuals and their parents.

Results: Testing with targeted next-generation gene panel revealed pathogenic or probably pathogenic variants in 14 out of 25 patients presenting with different forms of FDs. We found either novel or known variants within the ALDH1A1, ALX1, DHODH, EFNB1, EFTUD2, FGFR1, SF3B4, SRCAP, TCOF1, and ZSWIM6 genes.

Conclusion: We proved the efficiency and clinical utility of the designed gene panel. The targeted strategy presented here is a suitable and helpful approach in the genetic diagnostics of variable forms of FD.

This work was supported by the grant from the Poznan University of Medical Sciences, Poland 502-14-11261860-41259 to Ewelina M. Olech. and grant from the Polish National Science Centre, Poland UMO-2016/22/E/NZ5/00270 to Aleksander Jamsheer

E.M. Olech: None. D. Popiel: None. G. Koczyk: None. A. Materna-Kiryluk: None. M. Badura-Stronka: None. M. Wiśniewska: None. A. Latos-Bieleńska: None. A. Jamsheer: None.

P04.29A Novel mutations in autosomal dominant inherited syndromes with cutaneous manifestations

E. Borràs, I. Hernan, M. A. Pol, F. Corella, M. J. Gamundi, B. Mañé, M. Carballo

Hospital de Terrassa (CST), Terrassa, Spain

Introduction: Molecular diagnosis provides useful information for disease prognosis and genetic counseling, especially when cutaneous changes are result from an underlying hereditary tumor syndrome. We present four solved cases of autosomal dominant hereditary disorders displaying skin manifestations.

Materials and Methods: The cases consist on a 28 yo woman with multiple trichoepitheliomas (Brooke-Spiegler syndrome); a 10 yo girl with connective tissue nevi and osteopoikilosis (Buschke-Ollendorff syndrome); and two patients with multiple cutaneous leiomyomas (Reed syndrome), a 31 yo woman with cutaneous/uterine leiomyomas and renal cancer and a 38 yo man with cutaneous leiomyomas whose mother and sister had uterine lesions. All had a family history of the condition compatible with an autosomal dominant inheritance pattern. Disease-associated genes were analysed using PCR and direct sequencing and deletions/duplications were detected by MLPA.

Results: Pathogenic mutations were identified in all cases, three of them being novel. Patients with Brooke-Spiegler and Buschke-Ollendorff syndrome had non-sense mutations in respectively CYLD (p.Gly282Ter) and LEMD3 (p.Trp581Ter), while Reed syndrome patients carried complete or partial FH deletions. This novel partial deletion extends 1.9 kb and its breakpoints are located within exon 2 and intron 2, disrupting the FH gene. Genetic counseling was performed and relatives at risk were offered direct molecular testing. Positive cases were further examined by imaging techniques to assess the presence of other lesions or malignancies.

Conclusions: Molecular testing confirmed the hereditary syndromes initially diagnosed and allowed to adopt adequate prevention and treatment measures in those positive cases with an increased risk of internal cancer.

E. Borràs: None. I. Hernan: None. M.A. Pol: None. F. Corella: None. M.J. Gamundi: None. B. Mañé: None. M. Carballo: None.

P04.30B Two cases Ehlers-Danlos syndrome, kyphoscoliotic type, 2 caused by FKBP14 mutation

V. Rumyantseva1, I. Girba1, N. Scherbakova1, N. Samoilova1, E. Zaklyazminskaya1,2

1Petrovsky Russian Research Centre of Surgery, Moscow, Russian Federation, 2Pirogov Russian Research Medical University, Moscow, Russian Federation

Introduction: FKBP14-related Ehlers-Danlos syndrome (EDS) is an extremely rare recessive connective tissue disorder. The poor prognosis is anticipated due to typical features of the syndrome and possible potentially life-threatening vascular complication in childhood and atlantoaxial instability.

Materials and Methods: We have observed clinical and genealogical examination of two patients (14 month-old boy and 11 month-old girl) with unusual phenotypes: the evaluation confirmed following symptoms: generalized muscle hypotonia, progressive kyphoscoliosis, and joint hypermobility. Echocardiogram and hearing was normal. Boy’s ultrasound scan showed expansion of the axillary vein and girl’s one showed ectasia of the internal jugular vein. Patients underwent genetic testing via high-throughput sequencing followed by capillary Sanger sequencing.

Results: In 2 patients from 2 unrelated Russian families identified homozygosity for a 1-bp insertion within a 5C-nucleotide repeat in exon 3 (c.362dupC, NM_017946.2) of the FKBP14 gene, causing a frameshift predicted to result in a premature termination codon (Glu122ArgfsTer7). Their unaffected parents were heterozygous of the mutation.

Conclusion: Clinicians should give more consideration to rare genetic syndromes, especially in the case of symptoms from different clinical areas. Genotype/phenotype association studies will be necessary to elucidate further the cause of the variability of the disease severity. As it has early clinical manifestations, FKBP14-related EDS is still a challenge and the key issue for its effective follow-up that includes cardiovascular monitoring that is cerebral, thoracic, abdominal MRA, and cervical dynamic radiograph. The 362dupC mutation was linked to the same haplotype in all individuals despite their geographically diverse origins, suggesting a possible founder event

V. Rumyantseva: None. I. Girba: None. N. Scherbakova: None. N. Samoilova: None. E. Zaklyazminskaya: None.

P04.31C Bi-allelic mutations in LSS, encoding lanosterol synthase, cause autosomal-recessive hypotrichosis simplex

M. Romano1, A. Tafazzoli1, M. Mattern1, S. Sivalingam1, S. Wolf1, A. Rupp2, H. Thiele3, J. Altmüller4, P. Nürnberg4, J. Ellwanger5, R. Gambon6, A. Baumer7, N. Kohlschmidt8, D. Metze9, S. Holdenrieder2, R. Paus10, D. Lütjohann11, J. Frank12, M. Geyer13, M. Bertolini14, P. Kokordelis1, R. C. Betz1

1Institute of Human Genetics, Bonn, Germany, 2Institute of Laboratory Medicine, Technical University Munich, Munich, Germany, 3Cologne Center for Genomics, University of Cologne, Cologne, Germany, 4Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany, 5Dermatological Practice, Munich, Germany, 6Pediatric Practice Feldstrasse, Thusis, Switzerland, 7Institute for Genetic Medicine, University of Zürich, Zürich, Switzerland, 8Institute of Clinical Genetics, Bonn, Germany, 9Department of Dermatology, University of Münster, Münster, Germany, 10Centre for Dermatology Research, University of ManchesterInstitute of Human Genetics, ManchesterInstitute, United Kingdom, 11Institute for Clinical Chemistry and Clinical Pharmacology, University of Bonn, Bonn, Germany, 12Department of Dermatology, Venereology and Allergology, University Medical Center Göttingen, Göttingen, Germany, 13Institute of Innate Immunity, Department of Structural Immunology, University of Bonn, Bonn, Germany, 14Monasterium Laboratory - Skin and Hair Research Solutions GmbH, Münster, Germany

Hypotrichosis simplex (HS) is a rare form of hereditary alopecia characterized by childhood-onset of diffuse and progressive scalp and body hair loss. Although research has identified a number of causal genes, genetic etiology in about 50% of HS cases remains unknown. The present report describes the identification via whole exome sequencing of five different mutations in the gene LSS in three unrelated families with unexplained, potentially autosomal recessive HS. Affected individuals showed sparse to absent, lanugo-like scalp hair, sparse and brittle eyebrows, sparse eyelashes and body hair. The LSS gene encodes lanosterol synthase (LSS), which is a key enzyme in the cholesterol biosynthetic pathway. This pathway plays an important role in hair follicle biology. After localizing LSS protein expression in the hair shaft and bulb of the hair follicle, the impact of the mutations on keratinocytes was analyzed using immunoblotting and immunofluorescence. Interestingly, wild-type LSS was localized in the endoplasmic reticulum (ER), whereas mutant LSS proteins were localized in part outside of the ER. A plausible hypothesis is that this mislocalization has potential deleterious implications for hair follicle cells. Immunoblotting revealed no differences in the overall level of wild-type and mutant protein. Analyses of blood cholesterol levels revealed no decrease in cholesterol or cholesterol intermediates, thus supporting the previously proposed hypothesis of an alternative cholesterol pathway. The identification of LSS as causal gene for autosomal recessive HS highlights the importance of the cholesterol pathway in hair follicle biology, and may facilitate novel therapeutic approaches for hair loss disorders in general.

M. Romano: None. A. Tafazzoli: None. M. Mattern: None. S. Sivalingam: None. S. Wolf: None. A. Rupp: None. H. Thiele: None. J. Altmüller: None. P. Nürnberg: E. Ownership Interest (stock, stock options, patent or other intellectual property); Modest; ATLAS Biolabs GmbH. J. Ellwanger: None. R. Gambon: None. A. Baumer: None. N. Kohlschmidt: None. D. Metze: None. S. Holdenrieder: None. R. Paus: None. D. Lütjohann: None. J. Frank: None. M. Geyer: None. M. Bertolini: None. P. Kokordelis: None. R.C. Betz: None.

P04.32D Phenotypic presentations of Hajdu-Cheney syndrome according to age - 5 distinct clinical presentations

L. Graversen1, M. Handrup2, M. Irving3, H. Hove4, B. Diness5, L. Risom5, D. Svaneby6, M. Aagaard6, I. Vogel1, H. Gjørup7, M. Davidsen8, M. Hellfritzsch9, P. Gregersen1

1Department of clinical genetics, Aarhus University Hospital, Aarhus, Denmark, 2Pediatrics and Adolescent Medicine, Centre for Rare Diseases, Aarhus University Hospital, Aarhus, Denmark, 3Guy’s and St Thomas NHS Trust, London, United Kingdom, 4Centre for Rare Diseases, Copenhagen University Hospital, Copenhagen, Denmark, 5Department of clinical genetics, Copenhagen University Hospital, Copenhagen, Denmark, 6Department of clinical genetics, Hospital Lillebaelt, Vejle, Denmark, 7Center for Oral Health in Rare Diseases, Department of Maxillofacial Surgery, Aarhus University Hospital, Aarhus, Denmark, 8Department of Orthopaedic Surgery, Aarhus University Hospital, Aarhus, Denmark, 9Department of Radiology, Aarhus University Hospital, Aarhus, Denmark

We present five Danish individuals with Hajdu-Cheney syndrome (HJCYS) (OMIM #102500), a rare multisystem skeletal disorder with distinctive facies, generalised osteoporosis and progressive focal bone destruction. In four cases positive genetic screening of exon 34 of NOTCH2 supported the clinical diagnosis; in one of these cases, mosaicism was demonstrated, which, to our knowledge, has not previously been reported. In one case no genetic testing was performed since the phenotype was definite, and the diagnosis in the mother was genetically confirmed. The age of the patients differs widely from seven to 56 years, allowing a natural history description of the phenotype associated with this ultra-rare condition. The evolution of the condition is most apparent in the incremental bone loss leading to osteoporosis and the acro-osteolysis, both of which contribute significantly to disease burden.

L. Graversen: None. M. Handrup: None. M. Irving: None. H. Hove: None. B. Diness: None. L. Risom: None. D. Svaneby: None. M. Aagaard: None. I. Vogel: None. H. Gjørup: None. M. Davidsen: None. M. Hellfritzsch: None. P. Gregersen: None.

P04.33A Design of a candidate smMIPS sequencing study in patients with hereditary angioedema of unknown cause (U-HAE)

C. Mathey1, C. Stieber1,2, K. U. Ludwig1, A. Maaser1, S. Heilmann-Heimbach1, M. M. Nöthen1,2

1Institute of Human Genetics, University of Bonn School of Medicine & University Hospital Bonn, Bonn, Germany, 2Center for Rare Diseases Bonn, University of Bonn, Bonn, Germany

Hereditary angioedema (HAE) is a rare, potentially life-threatening disease with an estimated prevalence of 1:50,000 characterized by recurrent episodes of subcutaneous or submucosal swellings. Transmitted as an autosomal dominant trait, there are five types of HAE with known genetic defects. While HAE with decreased C1-Inhibitor can be explained by mutations in SERPING1, only about 20-25% of the cases of HAE with normal C1-Inhibitor can be explained by mutations in FXII. But two recently discovered mutations in ANGPT1 and PLG have been linked to two further forms of HAE with normal C1-Inhibitor. Despite these successes in identifying causes of HAE, there still remains a significant fraction of patients with the diagnosis of so called HAE with unknown cause (U-HAE). Aiming to identify potential new disease-causing genes that may play a role in the development or clinical heterogeneity of HAE, we performed multiplex targeted sequencing using the single-molecule molecular inversion probes (smMIPs). Our smMIPs panel comprises 29 genes that were prioritized by systematic literature research and pathway analysis and are mainly related to known disease-causing genes and pathways in HAE. The panel was established on a MiSeq platform where we achieved a coverage ≥100x for 95% of all exons in 27 out of 29 selected genes. Sequencing in our patient cohort of 125 HAE patients with U-HAE is currently ongoing on a Illumina HiSeq platform and results will be presented at the upcoming conference.

C. Mathey: None. C. Stieber: None. K.U. Ludwig: None. A. Maaser: None. S. Heilmann-Heimbach: None. M.M. Nöthen: None.

P04.34B Expanding the phenotype of Pseudoxanthoma elasticum with hereditary spastic paraplegia

A. LEGRAND1,2, A. Mesnil3, C. Durand4, C. Tesson5, S. Zuily6, X. Jeunemaitre3,7,2, C. Goizet8, J. Albuisson3,7,2

1INSERM U970 Paris Cardiovascular Research Centre, PARIS, France, 2Université Paris Descartes, Sorbonne Paris Cité, faculté de Médecine, Paris, France, 3Assistance Publique-Hôpitaux de Paris, Hôpital Européen Georges Pompidou, Centre de Référence des Maladies Vasculaires Rares, PARIS, France, 4INSERM U1211, Laboratoire Maladies Rares: Génétique et Métabolisme, Université de Bordeaux, Bordeaux, France, 5INSERM U1127, CNRS UMR 7225, UPMC Université Paris 06 UMR S1127, Sorbonne Université Institut du Cerveau et de la Moelle épinière, ICM F-75013, PARIS, France, 6Université de Lorraine, Inserm, DCAC, Vascular Medicine Division and Regional Competence Center for Rare Vascular and Systemic Autoimmune Diseases, Centre Hospitalier Regional Universitaire de Nancy, Nancy, France, 7INSERM U970 Paris Cardiovascular Research Centre, Paris, France, 8Service de Génétique Médicale, CHU de Bordeaux et Laboratoire MRGM, INSERM U1211, Université de Bordeaux, Centre de Référence Neurogénétique, Service de Génétique Médicale, CHU de Bordeaux, France, Bordeaux, France

Background: Pseudoxanthoma elasticum (PXE) is an autosomal recessive disorder characterized by ectopic mineralization and fragmentation of the elastic fibers of connective tissues. ABCC6 has been the only identified gene, but up to 18% of cases remain genetically unsolved with one or no pathogenic variant in ABCC6 (Legrand et al, 2017). The corresponding negative French PXE cases were genetically explored for new PXE gene(s).

Methods: Exome sequencing was performed in one ABCC6 negative PXE family with additional features, to search for pathogenic variants. We identified a candidate gene (CG) in a congruent metabolic pathway, and sequenced 46 additional ABCC6 negative PXE index cases (IC). An in vitro biochemical assay was carried out to determine the pathogenicity of identified missense variants and a clinical description of positive cases was performed to refine their phenotypic spectrum.

Results: Out of 47 PXE IC, 3 harbored 2 pathogenic variants in our CG. Histologically-confirmed skin lesions, and diminished visual acuity due to maculopathy evoked the diagnosis of PXE. The association with neurological symptoms was strikingly present in the 3 cases, evoking a phenotype of PXE “plus” neurological symptoms. Biochemical analyses confirmed loss of activity of the corresponding mutated enzyme.

Conclusion: Our CG is mutated in 6.4% of our unsolved PXE cases and should be systematically sequenced in suspected PXE cases with neurological findings: spastic paraplegia, dystonia, cognitive impairment, peripheral neuropathy and brain MRI abnormalities. The mechanism leading to overlapping phenotypes for these 2 genes remains to be explored to explain their role and link in mineralization.

A. Legrand: None. A. Mesnil: None. C. Durand: None. C. Tesson: None. S. Zuily: None. X. Jeunemaitre: None. C. Goizet: None. J. Albuisson: None.

P04.35C Post-zygotic MTOR mutations and Ito hypomelanosis: phenotypic spectrum and ultrastructural characterization

V. Carmignac1,2, A. Sorlin3,2, V. E. R. Parker4, E. Blanchard-Laumonnier5,6, C. Mignot7,8, M. Aubriot-Lorton9, J. Courcet2, Y. Duffourd2, P. Kuentz10, D. Rodriguez11, R. G. Knox12, A. Boland13, R. Olaso13, V. Darmency14, C. Quelin15, S. Odent15, D. Amram16, M. Chevarin2, c. Vincent-Delorme17, B. Catteau18, L. Guibaud19, A. Arzimanoglou20, D. Bessis21, D. Geneviève22, J. Deleuze13, R. Semple23, C. Philippe2,24, L. Faivre2, J. Rivière25, P. Vabres1

1MAGEC-Mosaique Dijon, CHU Dijon-Bourgogne, Dijon, France, 2INSERM UMR1231-GAD team, Dijon, France, 3Genetic Department - CHU Dijon-Bourgogne, Dijon, France, 4University of Cambridge Metabolic Research Laboratories, Cambridge, United Kingdom, 5Service d’Anatomie et Cytologie Pathologiques, UF de Biologie Cellulaire et Microscopie Electronique, Tours, France, 6INSERM U1259 « Morphogenèse et Antigénicité du VIH et des virus des hépatites », Tours, France, 7Service de neuropédiatrie et pathologie du développement, Hôpital Trousseau,, Paris, France, 8Département de Génétique et Centre de Référence Déficiences Intellectuelles de Causes Rares, Groupe Hospitalier Pitié-Salpêtrière, Paris, France, 9Service de Pathologie, CHU Dijon, Dijon, France, 10Genetic Department - CHU Besançon, Besançon, France, 11Service de neuropédiatrie et pathologie du développement, Hôpital Trousseau, Paris, France, 12University of Cambridge Metabolic Research Laboratories, Institute of Metabolic Science, Cambridge, France, 13Centre National de Génotypage, Institut de Génomique, CEA, Evry, France, 14Pediatric and medical genetics, Dijon, France, 15clinical genetics, CHU Rennes, Rennes, France, 16clinical genetics, CH Créteil, Créteil, France, 17Medical genetics- CHU Lille, Lille, France, 18Dermatology departement-CHU Lille, Lille, France, 19pediatric and foetal imagery department, HCL lyon, Lyon, France, 20ESEFNP, HCL Lyon, Lyon, France, 21Dermatology departement-CHU Montpellier, Montpellier, France, 22Medical genetics department, CHU Montpellier, Montpellier, France, 23University of Cambridge Metabolic Research Laboratories, Institute of Metabolic Science, Cambridge, United Kingdom, 24UF6254-CHU Dijon, Dijon, France, 25Mc Gill University, Montréal, QC, Canada

Germline MTOR mutations are associated with Smith-Kingsmore syndrome (SKS), whereas somatic mutations are reported in patients with hemimegalencephaly/focal cortical dysplasia; only few presented with linear depigmentation. Here, we report clinical manifestations in 12 sporadic cases with Ito hypomelanosis and post-zygotic mutation of MTOR.

We performed exome analysis or ultra-deep targeted sequencing of MTOR gene on hypopigmented skin biopsy, and performed precise clinical reappraisal. In two patients, we analyzed the phenotype histologically and ultrastructurally.

We identified 9 MTOR post-zygotic mutations including six novel. Two were recurrent. Clinically, patients had blaschkolinear hypopigmentation (11/12), epilepsy (9/12), macrocephaly (8/12), hemi-hypertrophy (7/12), and severe intellectual disability (7/12), 2 have iria heterochromia, and one nephromegaly. Primary fibroblasts (p.Glu2419Lys) showed activation of PIK3K-AKT pathway. Skin biopsies showed a decreased number of melanocytes melanosome immaturity in melanocytes and a decreased number of melanosomes in keratinocytes in hypopigmented areas.

Although somatic MTOR mutations have already been reported in Ito's hypomelanosis, this is the first clinical-genetic characterization of this association. Neurologically, there are analogies with SKS and hemimegalencephaly. Reduction of intrakeratinocyte melanosomes had already been reported in Ito's hypomelanosis, but without genetic study results as well as in hypochromic spots of tuberous sclerosis (TS), which is due to mutations of mTOR repressor TSC1/2. We found that tuberous sclerosis and MTOR-related hypomelanosis shared common pathophysiology process, with a lack of maturation of melanosomes. Importantly, no renal involvement known in TS has been identified in cases with MTOR mutations. Therapeutic use of rapamycin, a MTOR inhibitor, is therefore possible.

V. carmignac: None. A. Sorlin: None. V.E.R. Parker: None. E. Blanchard-Laumonnier: None. C. Mignot: None. M. Aubriot-Lorton: None. J. Courcet: None. Y. Duffourd: None. P. Kuentz: None. D. Rodriguez: None. R.G. Knox: None. A. Boland: None. R. Olaso: None. V. Darmency: None. C. Quelin: None. S. Odent: None. D. Amram: None. M. Chevarin: None. C. Vincent-Delorme: None. B. Catteau: None. L. Guibaud: None. A. Arzimanoglou: None. D. Bessis: None. D. Geneviève: None. J. Deleuze: None. R. Semple: None. C. Philippe: None. L. Faivre: None. J. Rivière: None. P. Vabres: None.

P04.36D Molecular analysis of ALPL gene in Russian cohort of patients with suspected hypophosphatasia

M. Fedyakov1, Y. Eismont1, T. Ivaschenko2, I. Sosnina3, E. Snegova3, A. Sarana1,4, S. Scherbak1,4, O. Glotov1,2

1City Hospital 40, Saint-Petersburg, Russian Federation, 2D.O.Ott Research Institute of Obstetrics, Gynecology and Reproductology, Saint-Petersburg, Russian Federation, 3Consultative and diagnostic center for children, Saint-Petersburg, Russian Federation, 4Saint-Petersburg State University, Saint-Petersburg, Russian Federation

Introduction: Hypophosphatasia (HPP) is a rare heritable metabolic disorder characterized by defective mineralization of bone and/or teeth in the presence of reduced activity of unfractionated serum alkaline phosphatase (ALP). The overall prevalence of severe HPP is range from 1/100 000 to 1/300 000. Mild forms of HPP are more frequent than severe forms - expected prevalence can reach 1/6000 in Western populations. Russian prevalence of mild and severe HPP is still unknown. Genetic analysis provides determining of diagnosis in cases with suspected HPP.

Materials and Methods: We analyzed genomic DNA samples from 112 unrelated individuals with signs of HPP (low and/or recurrent low levels of ALP, low growth, recurrent fractures and others). Primers’ system for Sanger sequencing was designed and validated for 2-12 exons of ALPL gene. First exon of this gene was excluded because it’s non-coding and GC-rich region.

Results: Studied group included 66 males and 46 females (mean age 9yo, range from 1 month to 66yo). Low ALP (age- and sex-dependent reference range) was founded in 79% (89/112). We detected 15 pathogenic mutations (13% detection rate): 13 in heterozygous and 2 in compound-heterozygous. 3 novel missense variants were founded. Most frequent variant was p.E191K in exon 6. The prevalence of this mutation was 6,25% (7/112) in our study whereas gnomAD prevalence is 0,25%.

Conclusions: We presented the data of prevalence mild HPP in Russian cohort of patients with suspected HPP. Mild HPP was founded in 87% positive cases (13/15). Variant p.E191K in ALPL is common for Russian population.

M. Fedyakov: None. Y. Eismont: None. T. Ivaschenko: None. I. Sosnina: None. E. Snegova: None. A. Sarana: None. S. Scherbak: None. O. Glotov: None.

P04.37A Recurrent somatic IDH1 mutation in an adult with Maffucci syndrome

N. J. Brown1,2,3, Z. Ye4, C. Stutterd2,3, A. Schneider4, S. Mullen4,5, S. Mandelstam3,5,6, I. E. Scheffer3,4,7, M. S. Hildebrand4

1Victorian Clinical Genetics Services, MCRI, Parkville, Australia, 2Department of Clinical Genetics, Austin Health, Heidelberg, Australia, 3Royal Children’s Hospital Department of Paediatrics, University of Melbourne, Parkville, Australia, 4Department of Medicine, Austin Hospital, University of Melbourne, Heidelberg, Australia, 5Florey Institute of Neuroscience and Mental Health, Heidelberg, Australia, 6Department of Medical Imaging, Royal Children's Hospital, Parkville, Australia, 7Florey Institute of Neuroscience and Mental Health, Parkville, Australia

Background: Maffucci Syndrome is a rare, highly variable, somatic mosaic condition, caused by recurrent mutations in either IDH1 or IDH2. Features include benign enchondroma and spindle cell haemangioma, with a recognized increased risk of various malignancies. Fewer than 200 cases have been reported, therefore accurate estimates of malignancy risk are difficult to quantify and recommended surveillance guidelines are not available. IDH1 and IDH2 mutations are also implicated in a variety of other benign and malignant tumours. Recently, the FDA approved specific IDH1 and IDH2 inhibitors for use in individuals with relapsed acute myeloid leukaemia.

Methods: An adult male was assessed via the Austin Health Clinical Genetics Unit, after he presented with soft palpable lesions on the left upper limb. Imaging and histopathology raised the possibility of Maffucci syndrome. DNA was extracted from peripheral blood lymphocytes and from surgically resected tissue. Sanger sequencing and Droplet-digital PCR analysis of the IDH1 gene was performed.

Results: Imaging and histopathology results were virtually pathognomonic of Maffucci syndrome. We identified a recurrent, somatic mosaic c.394C>T (p.R132C) mutation in exon 5 of IDH1, in DNA derived from haemangioma tissue (~ 17% mutant allele frequency) that was absent in DNA derived from blood. This mutation is a well-established cause of Maffucci syndrome.

Conclusion: We present an instructive case of a rare condition, and explore the potential therapeutic benefits and risks of IDH1 and IDH2 inhibitors in this disorder.

Funding: NHMRC Program Grant (1091593):I.E.S.,Project Grant (1079058):M.S.H., Practitioner Fellowship (1006110):I.E.S., R.D Wright Career Development Fellowship (1063799):M.S.H.

N.J. Brown: None. Z. Ye: None. C. Stutterd: None. A. Schneider: None. S. Mullen: None. S. Mandelstam: None. I.E. Scheffer: None. M.S. Hildebrand: None.

P04.38B FBN1 gene mutations in 26 Hungarian patients with suspected Marfan syndrome or related fibrillinopathies

L. Madar1, K. Szakszon2, G. Pfliegler3, G. P. Szabó2, B. Brúgós3, N. Ronen1, J. Papp4, K. Zahuczky4, E. Szakos4, G. Fekete5, É. Oláh2, K. Koczok1, I. Balogh1

1Division of Clinical Genetics, Department of Laboratory Medicine, Faculty of Medicine, University of Debrecen, Debrecen, Hungary, 2Department of Pediatrics, Faculty of Medicine, University of Debrecen, Debrecen, Hungary, 3Division of Rare Diseases, Department of Internal Medicine, Faculty of Medicine, University of Debrecen, Debrecen, Hungary, 4Department of Pediatrics, Borsod-Abaúj-Zemplén County University Hospital, Miskolc, Hungary, 52nd Department of Pediatrics, Semmelweis University, Budapest, Hungary

Introduction: Marfan syndrome (MFS) is an autosomal dominant connective tissue disorder with cardiovascular, ocular and musculo-skeletal system involvement. FBN1 gene mutations lead to MFS and related fibrillinopathies. In this work we describe clinical and molecular data of 26 unrelated individuals with suspected MFS who were referred to our laboratory for FBN1 gene mutation analysis.

Materials and Methods: Genomic DNA samples and in one case RNA sample isolated from dermal fibroblasts were analyzed by next generation sequencing (NGS) and Sanger sequencing methods.

Results: We identified 23 causative or potentially causative (including variants of uncertain significance) FBN1 variants, seven of them was novel (~30%). About 30% of the cases were sporadic. Missense mutations were detected in 69.6% (16/23), the majority of them were located in one of the cbEGF motifs and ~70% of them substituted conserved cystein residues. Small deletions/duplications accounted for 13% of the cases (3/23), while splice site variants were identified in 17.4% (4/23). In three unrelated patients a low frequency (<1%) recurrent silent variant (c.3294C>T (p.Asp1098=) was identified. FBN1 mRNA analysis showed that the mutation does not lead to aberrant splicing, based on available data the variant was classified as benign.

Conclusions: FBN1 mutations were associated with MFS in the majority of the patients, in two cases with severe and early onset manifestation of the syndrome.

Funding: this study was supported by the Ministry of National Economy, Hungary, Grant/Award Number: GINOP‐2.3.2‐15‐2016‐00039.

L. Madar: None. K. Szakszon: None. G. Pfliegler: None. G.P. Szabó: None. B. Brúgós: None. N. Ronen: None. J. Papp: None. K. Zahuczky: None. E. Szakos: None. G. Fekete: None. É. Oláh: None. K. Koczok: None. I. Balogh: None.

P04.39C Identification of a novel two-exon deletion of FBN1gene in a patient with Marfan syndrome and homocystinuria

G. Buki1,2, K. Hadzsiev1,2, L. Pinter1,2, B. Melegh1,2, J. Bene1,2

1University of Pecs, Medical School, Clinical Center, Department of Medical Genetics, Pecs, Hungary, 2Szentagothai Research Center, University of Pecs, Pecs, Hungary

Introduction: Marfan syndrome (MFS) is an autosomal dominant multi-system disorder of connective tissue with high clinical heterogeneity. Some characteristic features of the syndrome, such as ectopia lentis, long-bone overgrowth, a high arched palate, a crowded dentition, scoliosis, kyphosis and arachnodactyly overlap with those seen in homocystinuria. Co-occurrence of these disorders can be observed in a number of patients. MFS is mainly caused by mutations in the fibrillin-1 gene (FBN1), mapped to chromosome 15q21.1. Majority of classic MFS patients bear small alterations, however, a smaller number of patients have larger genomic rearrangements in FBN1 gene.

Materials and Methods: After negative results of systematic sequencing of FBN1, TGFBR1 and TGFBR2 genes, an MLPA (P065-P066 MRC-Holland) analysis was performed in a 21 years old female patient suffering from Marfan syndrome based on Ghent criteria and homocystinuria.

Results: In contrast to the generally detected large deletions, a novel two-exon deletion (exon 46-47) has been observed in our patient. This results in the loss of 31-32nd calcium binding EGF-like domain which is responsible for the development of classic Marfan phenotype.

Conclusions: Less than 10% of the disease causing mutations are copy number alterations, in which single or multiple exon deletions can be detected by MLPA in a cost-effective manner even in the NGS era. Our case points out the importance of testing the MFS patients for homocystinuria, since an elevated level of homocysteine is associated with the risk of severe cardiovascular manifestations, though at present only a few guidelines recommend testing for homocystinuria.

G. Buki: None. K. Hadzsiev: None. L. Pinter: None. B. Melegh: None. J. Bene: None.

P04.40D A fibrillinopathy the marfanoid-progeroid-lipodystrophy syndrome - in mother and daughter

A. Kutkowska-Kaźmierczak, M. Gos, E. Obersztyn

Department of Medical Genetic, Institute of the Mother and Child, Warsaw, Poland

Introduction: Marfanoid-progeroid-lipodystrophy syndrome (MPLS) is a very rare genetic disorder with clinical features overlapping those of congenital Marfan syndrome - ocular, cardiovascular and skeletal manifestations, progeroid syndromes - progeroid appearance of the face and body not associated with other manifestation of early aging and lipodystrophy with the extreme congenital lack of subcutaneous fat tissues not associated with metabolic disturbances. To our knowledge only seven patients with this syndrome were reported so far. In all patients mutation in exon 64 of the FBN1 gene was detected.

Patients and Methods: We present clinical evaluation of mother and daughter in whom the same mutation Trp2756Ter in exon 64 of the FBN1 gene was detected using next generation sequencing method (NimbleGen SeqCap Target Enrichment Roche) with panel of genes associated with craniosynostosis genes (developed in the Department of Medical Genetics of the Institute of the Mother and Child in Warsaw, Poland).

Results: Both patients have marfanoid habitus with overgrowth, myopia, archnodactyly, pectus excavatum, hyperextensible joints, scoliosis, dysmorphic features with progeroid facial appearance associated with congenital lipodystrophy. Scaphocephalic head’s shape observed especially in daughter was probably a result of wide fontanelles and disturbances in cranial sutures fusion in childhood. Mental and motor development in both were within normal limits.

Conclusions: This is the second report presenting the patient with MPLS and cranial suture development disturbances. This syndrome should be included in the differential diagnosis of Marfan syndrome and progeroid-lipodystrophy syndromes coexisting with disturbances in cranial suture development.

Supported from IM&Ch intramural grant no. OPK-510-18-48

A. Kutkowska-Kaźmierczak: None. M. Gos: None. E. Obersztyn: None.

P04.41A Identification and characterization of microRNA-149, a candidate for orofacial clefting

R. Hollstein1, L. G. Stüssel1, M. Laugsch2,3, F. Haeberlein4, L. M. Hochfeld1, J. Welzenbach1, J. Schröder1, F. Thieme1, A. Heimbach1, T. Hess1,5, J. Gehlen1,5, S. Heilmann-Heimbach1, E. Mangold1, A. Rada-Iglesias2,6, B. Odermatt4, K. U. Ludwig1

1Institute of Human Genetics, University of Bonn, School of Medicine & University Hospital Bonn, Bonn, Germany, 2Center for Molecular Medicine Cologne (CMMC), University of Cologne, Cologne, Germany, 3Institute of Human Genetics, University of Cologne, Cologne, Germany, 4Institute of Anatomy, University of Bonn, Bonn, Germany, 5Institute of Human Genetics, Philipps University Marburg, Marburg, Germany, 6Cologne Excellence Cluster for Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany

Introduction: Nonsyndromic cleft lip with/without cleft palate (nsCL/P) is a common facial malformation with multifactorial etiology. The majority of genetic risk loci map to non-coding regions of the genome, suggesting that the underlying pathomechanisms act through regulatory effects on gene expression. One potential mechanism might be posttranscriptional gene regulation by micro RNAs (miRNA). For nsCL/P, a suitable cellular model are human neural crest cells (hNCCs), the mesenchymal precursor cells that give rise to the majority of cranial cartilage and bones.

Methods and Results: We combined array-based miRNA profiling in hNCC with in-house GWAS data to identify candidate miRNAs for nsCL/P. This analysis revealed miR-149-3p as strong candidate for nsCL/P involvement, based on consistent hNCC-expression across replicates and the presence of associated risk variants within its genomic region. Next, we aimed at characterizing the molecular mechanisms of miR-149-3p in the context of craniofacial development. In vitro, we modified miRNA149 abundance levels in cultured hNCC by overexpression and inhibition assays and monitored the migration behavior of hNCC using scratch assays. We found that inhibition of miR-149-3p significantly increases cellular migration of hNCC compared to untreated hNCCs. RNA-Seq data at different time points revealed differential expression of GPC1 and BMP7. To follow up these findings, in vivo analyses are currently performed in the zebrafish.

Conclusion: Through integration of large-scale genetic data and expression patterns in relevant cell types we here identified a novel regulatory mechanism that is involved in craniofacial development and might be related to the etiology of nsCL/P.

R. Hollstein: None. L.G. Stüssel: None. M. Laugsch: None. F. Haeberlein: None. L.M. Hochfeld: None. J. Welzenbach: None. J. Schröder: None. F. Thieme: None. A. Heimbach: None. T. Hess: None. J. Gehlen: None. S. Heilmann-Heimbach: None. E. Mangold: None. A. Rada-Iglesias: None. B. Odermatt: None. K.U. Ludwig: None.

P04.43C Detection of a new AluY insertion in the NF1 gene

C. Schmidt1, W. Brechan1, K. Tveten2, T. I. Nordtveit3, R. Østern1, O. Bojovic3

1Department of Medical Genetics, St. Olavs Hospital, Trondheim, Norway, 2Department of Medical Genetics, Telemark Hospital Trust, Skien, Norway, 3Department of Medical Genetics, Haukeland University Hospital, Bergen, Norway

Introduction: Neurofibromatosis type I (OMIM# 162200) is a common autosomal dominant disorder associated with variants in the NF1 gene. The spectrum of pathogenic variants is large, up to 30% affect splicing. Our diagnostic procedure combines analysis of genomic DNA and cDNA. By cDNA analysis we were able to detect a previously undescribed Alu insertion that was not detected by next generation sequencing (NGS).

Patient and Methods: A 44-year-old patient presented with café-au-lait spots, multiple subcutaneous nodules, vertebral neurofibroma, multiple iris hamartoma and choroidal nevi. DNA was first analyzed by a targeted capture-based NGS-panel (NF1, NF2 and SPRED1) and MLPA analysis (NF1 and NF2) with normal results. Heparin blood was used for cDNA analysis. Genetic analysis was performed on RNA extracted from short term lymphocyte culture; puromycine was used to prevent mRNA decay.

Result: cDNA sequencing revealed a deletion of exon 26 (r.3315_3496del, p.(Tyr1106Leufs*28); NM_001042492.2). Extended DNA-analysis of exon 26 and flanking regions by Sanger sequencing showed insertion of a truncated AluY transposable element in exon 26: c.3337_3338insAluY,3327_3337dupATTTATGAACC. The insertion leads to disruption of the 3’ acceptor splice site in exon 26, deletion of exon 26 results in reading frame shift and premature stop codon. Retrospective analysis of NGS data indicated a loss of coverage in this specific region.

Conclusion: While NGS is a powerful method it has limitations in detection of more uncommon variants. In NF1 genetic diagnostics, cDNA sequencing remains the gold standard for mutation detection, specifically for variants affecting splicing.

C. Schmidt: None. W. Brechan: None. K. Tveten: None. T.I. Nordtveit: None. R. Østern: None. O. Bojovic: None.

P04.46B Whole-exome sequencing and large-scale re-sequencing in nonsyndromic cleft lip with/without cleft palate identify novel susceptibility genes

N. Ishorst1, L. Henschel1, F. Thieme1, D. Drichel2, S. Sivalingam3,4,5, S. L. Mehrem1, A. C. Fechtner1, J. Fazaal1, J. Welzenbach1, A. Heimbach1, C. Maj4, J. Hausen3,4,5, R. Raff1, A. Hoischen6,7,8, M. Dixon9, A. Rada-Iglesias10,11, M. Bartusel10,11, A. Rojas-Martinez12, K. Aldhorae13, B. Braumann14, T. Kruse14, C. Kirschneck15, H. Reutter16,1, S. Nowak1, L. Gölz17,18, M. Knapp5, P. Krawitz4, M. M. Nöthen1, M. Nothnagel2, T. Becker19, K. U. Ludwig1, E. Mangold1

1Institute of Human Genetics, University of Bonn, School of Medicine & University Hospital Bonn, Bonn, Germany, 2Cologne Center for Genomics, Cologne, Germany, 3Core Unit for Bioinformatics Analysis, University of Bonn, Bonn, Germany, 4Institute for Genomic Statistics and Bioinformatics, University of Bonn, School of Medicine & University Hospital Bonn, Bonn, Germany, 5Institute of Medical Biometry, Informatics and Epidemiology, University of Bonn, Bonn, Germany, 6Department of Human Genetics, Radboud University Medical Center, Nijmegen, Netherlands, 7Department of Internal Medicine, Radboud University Medical Center, Nijmegen, Netherlands, 8Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, Nijmegen, Netherlands, 9Manchester Academic Health Sciences Centre, University of Manchester, Manchester, United Kingdom, 10Center for Molecular Medicine Cologne (CMMC), University of Cologne, Cologne, Germany, 11Cologne Excellence Cluster for Cellular Stress Responses in Aging‐Associated Diseases (CECAD), University of Cologne, Cologne, Germany, 12Tecnologico de Monterrey, School of Medicine, and Universidad Autonoma de Nuevo Leon, Centro de Investigación y Desarrollo en Ciencias de la Salud, Monterrey, Mexico, 13Orthodontic Department, College of Dentistry, Thamar University, Thamar, Yemen, 14Department of Orthodontics, University of Cologne, Cologne, Germany, 15Department of Orthodontics, University of Regensburg, Regensburg, Germany, 16Department of Neonatology, Children's Hospital, University of Bonn, Bonn, Germany, 17Department of Orthodontics, University of Erlangen, Erlangen, Germany, 18Department of Orthodontics, University of Bonn, Bonn, Germany, 19Institute for Community Medicine, University of Greifswald, Greifswald, Germany

Non-syndromic cleft lip with or without cleft palate (nsCL/P) is a common congenital malformation and has a multifactorial etiology. To date, 40 genome-wide significant risk loci for nsCL/P have been identified, but these explain less than 40% of the genetic liability. Epidemiological observations suggest that a fraction of the unidentified heritability might be explained by rare dominant de novo mutations (DNMs) in genes involved in craniofacial development. If highly penetrant, such DNMs may be of relevance in a diagnostic setting and for genetic counselling.

We performed whole-exome sequencing in 50 trios (discovery cohort) and identified 33 rare protein-altering DNMs in 33 “candidate genes”, two of them previously found associated with nsCL/P (CDH1, TRMO). Of note, this is the first study in nsCL/P trios using DNMs for candidate gene identification.

The “candidate genes” were subjected to resequencing with single molecule molecular inversion probes in 1,010 nsCL/P patients from European, Arabian, and Mexican ancestry and 1,574 population-matched controls (replication cohort). Of the total callset of 2,956 variants, 373 were absent from controls and showed CADD≥15 and MAF≤0.1% in public reference datasets. As a first step, we performed segregation analysis in the European cohort. Here, we could identify further DNMs in four of the genes (CSMD1, MDN1, ANK1, PAXIP1). In those genes we also found co-segregating variants (MDN1, CSMD1) and one compound heterozygous index (MDN1). RNA sequencing datasets confirmed expression of MDN1, ANK1 and PAXIP1 in relevant embryonic mouse tissues and human neural crest cells, making those genes promising candidates for functional follow-up.

N. Ishorst: None. L. Henschel: None. F. Thieme: None. D. Drichel: None. S. Sivalingam: None. S.L. Mehrem: None. A.C. Fechtner: None. J. Fazaal: None. J. Welzenbach: None. A. Heimbach: None. C. Maj: None. J. Hausen: None. R. Raff: None. A. Hoischen: None. M. Dixon: None. A. Rada-Iglesias: None. M. Bartusel: None. A. Rojas-Martinez: None. K. Aldhorae: None. B. Braumann: None. T. Kruse: None. C. Kirschneck: None. H. Reutter: None. S. Nowak: None. L. Gölz: None. M. Knapp: None. P. Krawitz: None. M.M. Nöthen: None. M. Nothnagel: None. T. Becker: None. K.U. Ludwig: None. E. Mangold: None.

P04.47C Genetic distribution of early onset osteoarthritis in a series of 40 patients

V. Ruault1, D. Genevieve1, F. Blotman1, P. Blanchet1, A. Fabre1, M. Fradin2, B. Isidor3, C. Jorgensen1, D. Lacombe4, M. Le Merrer5, S. Moutton6, S. Odent2, G. Plessis7, E. Sanchez1, S. Sigaudy8, F. Tran Mau Them6, M. Willems1, I. Touitou1, M. Barat-Houari1

1CHU, Montpellier, MONTPELLIER, France, 2CHU, Rennes, RENNES, France, 3CHU, Nantes, NANTES, France, 4CHU, Bordeaux-GH Pellegrin, BORDEAUX, France, 5CHU Paris - Hôpital Necker-Enfants Malades, PARIS, France, 6CHU, Dijon, DIJON, France, 7CHU, Caen, CAEN, France, 8CHU, Marseille, MARSEILLE, France

Introduction: Osteoarthritis (OA) is the most common joint disease worldwide. OA is a highly heterogeneous condition, with a wide range age of onset. Non-syndromic early-onset OA (EO-OA) is very rare and mainly familial. Strict inclusion criteria for EO-OA are based on: XRays evidence, body mass index (BMI) ≤ 30, age of onset ≤ 40y, ≥ 1 joint site involved and positive familial history. Although multifactorial mode of inheritance is common in OA, EO-OA are mainly monogenic conditions. Based on these data, we aimed to study the monogenic causes of non-syndromic EO-OA in French patients.

Materials and Methods: From 2013 to 2018, clinician experts in the field of skeletal dysplasia referred EO-OA patients to our Montpellier competence center, which assessed the diagnostic and excluded chondrodysplasia, based on clinical and radiological data. For genetic analysis, we used either Sanger sequencing or an NGS custom panel approach.

Results: We recruited 40 EO-OA patients, with a mean age of onset of articular pain at 23 years, ranging from 7 to 60 years. Pathogenic or a likely pathogenic heterozygous mutations were identified in 14/40 cases (35%), with 11/14 COL2A1 mutation (78.6%). We also observed a genetic heterogeneity involving COL11A2, COL9A3, and surprisingly a homozygous pathogenic variation in SLC26A2. Familial segregation of COL2A1 pathogenic variants was observed for 6 cases.

Conclusions: We confirmed that COL2A1 is the most common genetic cause of familial EO-OA. However, at least three other genes are involve in EO-OA. Therefore, we recommend to screen genes involved in cartilage matrix and homeostasis.

V. Ruault: None. D. Genevieve: None. F. Blotman: None. P. Blanchet: None. A. Fabre: None. M. Fradin: None. B. Isidor: None. C. Jorgensen: None. D. Lacombe: None. M. Le Merrer: None. S. Moutton: None. S. Odent: None. G. Plessis: None. E. Sanchez: None. S. Sigaudy: None. F. Tran Mau Them: None. M. Willems: None. I. Touitou: None. M. Barat-Houari: None.

P04.48D Mutations in osteoarthritis susceptibility genes cause joint shape variation detectable during ontogeny in zebrafish

E. Kague1, F. Turci2, Y. Yang2, S. Cross3, E. Lawrence1, L. McGowan1, J. Moss1, P. Royal2, C. L. Hammond1

1University of Bristol, The School of Physiology, Pharmacology and Neuroscience, Biomedical Sciences, Bristol, United Kingdom, 2University of Bristol, School of Physics, Bristol, United Kingdom, 3University of Bristol, Wolfson Bioimaging Facility, Biomedical Sciences, Bristol, United Kingdom

Osteoarthritis (OA) is a joint degenerative disease and leading cause of pain and disability worldwide. Joint shape is commonly used to predict OA. GWAs have rapidly increased the number of OA associated genes. Despite few genes were linked to shape variation, evidence for causality and functionality or their effect on joint shape are still lacking. Therefore, there is an unmet need to develop rapid and alternative screening platforms to test OA genes. We use the zebrafish jaw joint (JJ) to investigate the impact of OA genes and ageing on joint shape variation. 3D morphometrics showed shape abnormalities in aged JJ, accompanied by OA histopathological signs. Interestingly, such modifications were prematurely found in OA mutants (chsy1, col9a1, col11a2, and wnt16). We investigated if shape changes would be detected during JJ ontogeny by testing a broader list of OA mutants (chsy1, col9a1, col11a2, gdf5, barx1, mcf2l, dot1l, wnt16 and ncoa3) using confocal imaging of larvae immunostained for collagen type 2 followed by 2D and 3D analysis. Distinct classes of OA proteins leaded to significant shape variation in larvae, mostly explained by abnormal cell behaviour and collagen distribution. Dramatic shape variation caused joint movement impairment. We tested whether the use of mosaics, CRISPR G0s, could be sufficient to detect shape and cell changes. Surprisingly, wnt16 mosaics and wnt16-/- displayed similar cellular and shape changes in larvae and adults. By developing computational tools to facilitate 3D analysis we delivered a powerful and rapid screening platform to test OA genes. Versus Arthritis (grants 19497, 21161)

E. Kague: None. F. Turci: None. Y. Yang: None. S. Cross: None. E. Lawrence: None. L. McGowan: None. J. Moss: None. P. Royal: None. C.L. Hammond: None.

P04.49A Genetic heterogeneity in Polish patients with Osteogenesis Imperfecta

K. Sałacińska1, L. Rutkowska1, I. Pinkier1, D. Salachna1, A. Rusińska2, E. Jakubowska-Pietkiewicz2, A. Jamsheer3, L. Jakubowski1, A. Gach1

1Polish Mother's Memorial Hospital Research Institute, Łódź, Poland, 2Department of Propaedeutics of Paediatrics and Metabolic Diseases, University Teaching Hospital, Łódź, Poland, 3Department of Medical Genetics, University of Medical Science, Poznań, Poland

Introduction: Osteogenesis Imperfecta is a rare genetic disorder of connective tissue characterized by numerous fractures, blue sclera, tooth abnormalities, short stature and skeletal deformity. The diversity of clinical features and its severity varies between patients, even within a single family, ranging from severe perinatal lethal to a mild form. The aim of this project was to determine genetic heterogeneity versus clinical variability observed in patients affected by OI.

Materials and Methods: NGS using custom panel of 34 genes with confirmed and probable significance in OI pathogenesis was performed. The study included 118 patients, aged 1-44 years, presenting a broad spectrum of clinical manifestation. Based on bioinformatic analysis, selected variants were verified with Sanger sequencing in a group of 77 patients.

Results: We have identified COL1A1 mutations in 47 patients, COL1A2 in 24 patients and mutations in 3 non-collagenous genes in 6 patients. A total of 41 different point mutations in COL1A1 and 20 in COL1A2 were found. Respectively 15 and 11 have not been reported in dedicated Osteogenesis Imperfecta Variant Database. We also reported patient with a deletion spanning 25 exons in COL1A1 gene. Thus far expected lethality of particular domains in collagen type I genes revealed to be dubious, as we identified 11 patients aged 1,5 to 38 years with pathogenic mutations situated in these regions.

Conclusions: Inter- and intrafamilial phenotype variability in OI makes genetic counseling based on clinical symptoms challenging and proves panel targeted resequencing is a powerful and useful diagnostic tool. Young Scientist Grant 2016/IV/57-MN

K. Sałacińska: None. L. Rutkowska: None. I. Pinkier: None. D. Salachna: None. A. Rusińska: None. E. Jakubowska-Pietkiewicz: None. A. Jamsheer: None. L. Jakubowski: None. A. Gach: None.

P04.50B Genotype and phenotype in 201 Portuguese patients with osteogenesis imperfecta - unusual molecular results challenge genetic counseling

A. M. Travessa1, P. Dias1, M. Aza-Carmona2, J. Rosmaninho-Salgado3, T. Saraiva4, A. Grangeia5, M. Amorim6, M. Gonçalves-Rocha7, G. Araújo8, H. Santos1, M. Rodrigues1, A. Medeira1, I. Cordeiro1, J. Dupont1, O. Moldovan1, A. Beleza3, J. Sá3, J. M. Saraiva3, L. Ramos3, M. Venâncio3, S. Maia3, S. Fernandes3, G. Soares4, J. P. Freixo6, F. Díaz2, C. Barreiros9, C. de la Torre2, A. Bandeira10, J. Campagnolo11, F. Godinho9, M. Cassiano-Neves12, V. Tavares13, F. Teixeira8, T. Kay6, R. Oliveira5, A. Fortuna4, S. B. Sousa3, K. E. Heath2, A. B. Sousa1

1Serviço de Genética Médica, Departamento de Pediatria, Hospital de Santa Maria, Centro Hospitalar Universitário Lisboa Norte, Centro Académico de Medicina de Lisboa, Lisbon, Portugal, 2Institute of Medical & Molecular Genetics (INGEMM) and Skeletal dysplasia Multidisciplinary Unit, IdiPAZ, Hospital Universitario La Paz, UAM, & CIBERER, ISCIII, Madrid, Spain, 3Medical Genetics Unit, Hospital Pediátrico, Centro Hospitalar e Universitário de Coimbra, Coimbra, Portugal, 4Centro de Genética Médica Doutor Jacinto Magalhães, Centro Hospitalar Universitário do Porto, Oporto, Portugal, 5Serviço de Genética Médica, Centro Hospitalar de São João, Oporto, Portugal, 6Unidade de Genética Médica, Hospital Dona Estefânia, Cento Hospitalar Lisboa Central, Lisbon, Portugal, 7Unidade de Genética Médica, Hospital de Braga, Braga, Portugal, 8Serviço de Pediatria, Hospital Dr. Nélio Mendonça, Funchal, Portugal, 9Associação Portuguesa de Osteogénese Imperfeita, Sacavém, Portugal, 10Serviço de Pediatria and Centro de Referência para Doenças Metabólicas, Centro Hospitalar Universitário do Porto, Oporto, Portugal, 11Serviço de Ortopedia Infantil, Hospital Dona Estefânia, Cento Hospitalar Lisboa Central, Lisbon, Portugal, 12Serviço de Ortopedia Infantil, Hospital CUF, Lisbon, Portugal, 13Serviço de Reumatologia, Hospital Garcia de Orta, Almada, Portugal

Introduction: Osteogenesis imperfecta (OI) is a rare genetic bone fragility disorder. Although mutations in COL1A1/2 account for 90% of cases, up to 17 different genes were associated with OI. Our aim was to characterize the clinical and mutational spectrum of OI in Portugal and to correlate genotype and phenotype.

Materials and Methods: Clinical data of 201 OI individuals (135 adults, 58 children, and 8 fetuses) from 157 families were collected through clinical evaluation and/or medical records analysis. Sanger and/or different NGS based strategies and MLPA (COL1A1/2) were used for molecular analysis.

Results: One hundred thirty-seven individuals had mild, 31 moderate, 24 severe, and 9 extremely severe OI. A total of 123 different variants (65 novel) were identified. Variants in COL1A1 (n=91, including one multi-exonic deletion) and COL1A2 (n=29, including one homozygous case) account for 85.1% of all molecularly diagnosed families (66 with quantitative and 48 with qualitative variants), followed by SERPINF1 (n=4), LRP5 (n=4), IFITM5 (n=3), FKBP10 (n=3), WNT1 (n=2), CRTAP (n=1), TMEM38B (n=1), P3H1 (n=1), PPIB (n=1), and BMP1 (n=1). Interestingly, heterozygous variants in severe autosomal recessive OI genes were found in 3 patients with mild OI.

Conclusions: This is the first large-scale study on OI in Portugal. In accordance with other populations, quantitative variants predominate in mild COL1A1/2 cases, and IFITM5 and FKBP10-related cases had recognizable phenotypes. The identification of a homozygous variant in COL1A2 and heterozygous variants in autosomal recessive genes complicate genetic counselling. No variant was identified in 6.5% of probands.

A.M. Travessa: None. P. Dias: None. M. Aza-Carmona: None. J. Rosmaninho-Salgado: None. T. Saraiva: None. A. Grangeia: None. M. Amorim: None. M. Gonçalves-Rocha: None. G. Araújo: None. H. Santos: None. M. Rodrigues: None. A. Medeira: None. I. Cordeiro: None. J. Dupont: None. O. Moldovan: None. A. Beleza: None. J. Sá: None. J.M. Saraiva: None. L. Ramos: None. M. Venâncio: None. S. Maia: None. S. Fernandes: None. G. Soares: None. J.P. Freixo: None. F. Díaz: None. C. Barreiros: None. C. de la Torre: None. A. Bandeira: None. J. Campagnolo: None. F. Godinho: None. M. Cassiano-Neves: None. V. Tavares: None. F. Teixeira: None. T. Kay: None. R. Oliveira: None. A. Fortuna: None. S.B. Sousa: None. K.E. Heath: None. A.B. Sousa: None.

P04.51C Next generation sequencing performance in osteogenesis imperfecta

S. Monnot1,2, G. Baujat1,2, C. Michot1,2, J. Litzler1, A. Tourre1, J. Bonnefont1,2, J. Steffann1,2, V. Cormier-Daire1,2

1Department of genetics, Necker Hospital, Paris, France, 2INSERM UMR1163, Paris Descartes University, Imagine Institute, Paris, France

Osteogenesis imperfecta (OI) is a connective tissue disorder characterized by bone fragility. An important clinical heterogeneity exists ranging from severe antenatal letal form to mild postnatal affection. For now, 19 genes are known to be responsible for OI: 3 dominant autosomal forms (COL1A1, COL1A2 and IFITM5), 14 recessive autosomal forms and 2 X-linked forms (PLS3, MBTPS2). Importance of a proper molecular diagnosis for genetic counseling led us to design a panel of targeted resequencing of the 19 gene coding regions, substituting for the former Sanger analyses. Some genes implicated in differential diagnoses like hypophosphatasia or overlapping phenotypes were added, increasing to 39 the total number of genes. All requests were submitted to the Reference Center for Constitutional Bone Diseases with clinical and radiological details. Libraries were prepared using capture technology (SureSelectXT Custom - Agilent) and medium throughput sequencing on a NextSEq sequencer (Illumina). Since 2015, 368 mutations were identified in 517 patients (71%): 187 in the COL1A1 gene (50%), 98 in the COL1A2 gene (26%), 15 in IFITM5 (4%), 48 in recessive genes responsible for OI (13%), 3 in X-linked gene. All these mutations were confirmed by Sanger sequencing. The results are discussed during a feedback meeting with clinicians and biologists. This panel allows efficient screening for all OI genes, leading to time and cost-savings. Using a panel strategy avoid incidental findings. The main limit is the occasional difficulty to determine the pathogenicity of the identified variant. Whole exome sequencing analysis can be proposed for patient without mutation.

S. Monnot: None. G. Baujat: None. C. Michot: None. J. Litzler: None. A. Tourre: None. J. Bonnefont: None. J. Steffann: None. V. Cormier-Daire: None.

P04.52D Diagnostic yield of NGS analysis of a panel of Osteogenesis imperfecta-related genes in 550 patients with Osteogenesis imperfecta, (early-onset) nonsyndromic osteoporosis and related disorders

A. Maugeri1, E. Voorhoeve1, N. M. Appelman-Dijkstra2, A. T. H. van Dijk3, F. S. van Dijk4, E. M. W. Eekhoff5, M. W. Elting1, A. van Haeringen6, A. Harsevoort7, M. Isrie1, G. J. M. Janus7, R. T. de Jongh5, J. M. van de Kamp1, M. C. van Maarle8, C. L. M. Marcelis9, M. E. H. Simon10, S. Simsek11, C. T. R. M. Stumpel12, P. A. Terhal10, H. E. Veenstra-Knol13, M. C. Zillikens14, E. J. Meijers-Heijboer1,8, E. A. Sistermans1, M. M. Weiss1,8, G. Pals1, D. Micha1

1Dept. of Clinical Genetics, Amsterdam UMC, Vrije Universiteit Amsterdam, Amsterdam, Netherlands, 2Department of Endocrinology, Leiden Universitary Medical Center, Leiden, Netherlands, 3Dept. of Paediatrics, University Medical Center Utrecht, Utrecht, Netherlands, 4London North West Thames Regional Genetics Service, London North West Healthcare NHS Trust, Harrow, London, United Kingdom, 5Dept. of Internal Medicine, sect. Endocrinology, Amsterdam UMC, Vrije Universiteit Amsterdam, Amsterdam, Netherlands, 6Dept. of Clinical Genetics, Leiden Universitary Medical Center, Leiden, Netherlands, 7Dept. of Orthopaedics, Isala Clinics Zwolle, Zwolle, Netherlands, 8Dept. of Clinical Genetics, Amsterdam UMC, University of Amsterdam, Amsterdam, Netherlands, 9Dept. of Human Genetics, Radboud University Medical Center, Nijmegen, Netherlands, 10Dept. of Genetics, University Medical Center Utrecht, Utrecht, Netherlands, 11Dept. of Internal Medicine, Noordwest Ziekenhuisgroep, Alkmaar, Netherlands, 12Dept. of Clinical Genetics and GROW-School for Oncology and Developmental Biology, Maastricht University Medical Center, Maastricht, Netherlands, 13Dept. of Genetics, University Medical Center Groningen, Groningen, Netherlands, 14Dept. of Endocrinology, Erasmus Medical Center, Rotterdam, Netherlands

Introduction: Our Genomediagnostics laboratory in Amsterdam UMC offers targeted NGS analysis of genes involved in Osteogenesis imperfecta (OI) and other connective tissue disorders. OI is characterized by bone fragility and fractures, bone deformities, short stature, dentinogenesis imperfecta, hearing loss, and blue sclerae. The phenotype is highly variable, from mild to perinatal lethal. Osteoporosis is a common, multifactorial disorder, characterized by reduced bone-mass and fractures. A few genes have been associated with (early-onset) monogenetic non-syndromic osteoporosis (OP). Here we present the results of the analysis of a panel of 19 OI/OP-related genes in 550 patients referred with OI, OP or a related disorder.

Material and Methods: A solution-based enrichment kit was designed to capture exons and splice sites of the target genes. Data were analysed using an in-house pipeline and Cartagenia, and an NGS-based CNV analysis tool. 68% of the patients (mean age 37y) had osteoporosis and/or fractures; no additional OI-related features were reported (OP-cohort). 23% of the patients (mean age 22y) had at least one other OI-related symptom reported (OI-cohort). Results Diagnosis was molecularly confirmed in 67% of the patients in the OI-cohort, versus 6% in the OP-cohort. In the OP-cohort, a suspicious VUS was identified in 12% of the patients. Variant distribution among genes was different between OI- and OP-cohorts.

Conclusions: As expected, the yield of genetic testing was significantly higher in the OI-cohort versus the OP-cohort. However, the proportion of patients with a (potential) genetic cause in the OP-cohort is relevant and warrants further investigation of these patients.

A. Maugeri: None. E. Voorhoeve: None. N.M. Appelman-Dijkstra: None. A.T.H. van Dijk: None. F.S. van Dijk: None. E.M.W. Eekhoff: None. M.W. Elting: None. A. van Haeringen: None. A. Harsevoort: None. M. Isrie: None. G.J.M. Janus: None. R.T. de Jongh: None. J.M. van de Kamp: None. M.C. van Maarle: None. C.L.M. Marcelis: None. M.E.H. Simon: None. S. Simsek: None. C.T.R.M. Stumpel: None. P.A. Terhal: None. H.E. Veenstra-Knol: None. M.C. Zillikens: None. E.J. Meijers-Heijboer: None. E.A. Sistermans: None. M.M. Weiss: None. G. Pals: None. D. Micha: None.

P04.53A Between LSD and skeletal dysplasia - a rare case of Touraine-Solente-Golé syndrome caused by new mutation

H. M. Kathom1, D. Avdjieva-Tzavella1, T. Todorov2, A. Todorova2

1Department of Clinical Genetics, University Pediatrics Hospital, Medical University Sofia, Sofia, Bulgaria, 2Genetic Medico-Diagnostic Laboratory "Genica", Sofia, Bulgaria

Introduction: Primary hypertrophic osteoarthropathy (PHO), also known as pachydermoperiostosis (PDP) or Touraine-Solente-Golé syndrome, is a rare genetic osteo-arthro-dermopathic syndrome with familial and idiopathic forms differentiating it from secondary (pulmonary) hypertrophic osteoarthropathy. Though the diagnosis can be made on the basis of the classic clinical and radiological findings, it is often missed due to variable presentations PHO is characterized by digital clubbing, periostosis, acroosteolysis, painful joint enlargement, and skin manifestations that include thickened facial skin, a thickened scalp, and coarse facial features. The disorder has estimated prevalence of 0.16%, and usually manifests in adolescence, occurring almost exclusively in males, with a M: F ratio of 7:1.

Materials and Methods: We present a 15 years old boy, from Afghanistan, born to consanguine parents who presented for the first time in our department with painful swelling of hands, feet, and face, coarse facial features, hypertrichosis, short and broad palms, and soles. He reports on elder brother with similar swelling, and alopecia. Radiological examinations show wide and thick metacarpals, short distal phalanges, square-like vertebras.

Results: Many tests were performed to rule out lysosomal storage disorders, and skeletal dysplasia. After performing an NGS-analysis (clinical exome sequencing), the patient was found to carry, in homozygous state, a new mutation (c.290G>A) in SLCO2A1 gene causing PHO type 2.

Conclusions: PDP type 2 has diverse radiological and clinical features. It may remain undiagnosed for long time and progress until there are significant facial, joint, and digital deformities that finally make the patient seek medical attention.

H.M. Kathom: None. D. Avdjieva-Tzavella: None. T. Todorov: None. A. Todorova: None.

P04.54B Dental Management Experience for Patients Suffering from Papillon-Lefévre Syndrome

Y. M. Khalil1, M. R. Abouzeid1, M. A. Abd Elkader1,2, P. M. Abdelmassieh1

1national research center, giza, Egypt, 2Misr International University, Cairo, Egypt

Introduction: Papillon–Lefévre syndrome (PLS) is a rare autosomal recessive disorder caused by mutation of the Cathepsin C gene. Patients mainly suffer from palmoplantar keratosis, periodontitis, and early loss of teeth. The present study is concerned with the dental management of PLS patients through periodontal and prosthetic intervention.

Materials and Methods: Nine PLS patients were included in this study. The patients were referred to our Orodental Genetics Clinic complaining of early loss of the deciduous dentition and were in different stages of permanent teeth eruption. Our management protocol included; conventional scaling and root planning every 3 month, diode laser curettage and pocket sterilization monthly sessions and construction of a removable prosthesis to restore the lost teeth. Pocket depth, bleeding index and mobility was assessed every 6 month during the 2-year period of the study.

Results: All patients showed good response to the laser treatment. Gingival inflammation was controlled with less bleeding, decreased mobility of teeth and no teeth were lost over 2 years. However, panoramic radiographs showed bone loss in the pocket areas.

Conclusion: Laser pocket disinfection was effective in reducing gingival tissue inflammation, yet the progressive bone loss was a concerning outcome that needs further investigations.

Y.M. Khalil: None. M.R. Abouzeid: None. M.A. Abd Elkader: None. P.M. Abdelmassieh: None.

P04.55C Abnormal activation of mutated complement 1 subunits C1r/C1s in periodontal Ehlers-Danlos syndrome

R. Gröbner1, E. Brunner1, R. Redolfi1, A. Amberger1, H. Stoiber1, N. Thielens2, C. Gaboriaud2, I. Kapferer-Seebacher1, J. Zschocke1

1Medical University, Innsbruck, Austria, 2Institut de Biologie Structurale, Grenoble, France

Introduction: Periodontal Ehlers–Danlos syndrome (pEDS) is a connective-tissue disorder characterized by early severe periodontitis and various joint and skin manifestations. Most EDS types are caused by mutations in collagens or protein-modifying enzymes. In contrast, periodontal type is caused by heterozygous missense or in-frame insertion/deletion mutations in C1R or C1S, indicating a previously unknown connection between the inflammatory complement pathway and connective tissue homeostasis. pEDS is autosomal dominant and involves gain-of-function effects; loss-of-function variants in C1R/C1S are asymptomatic when heterozygous and can cause a lupus-like phenotype when homozygous.

Methods and Results: In-vitro overexpression in HEK293T cells demonstrated that all pathogenic C1R variants except a mutant in the C1q binding motif cause abnormal intracellular processing and secretion of enzymatically active serine protease. Unlike C1r wild type, mutations in the CUB1 and CCP1 domains of C1r show intracellular retention of the N-terminal fragment. Mutations in the CUB2 domain cause secretion of aggregated N-terminal fragments, whereas CCP2 domain mutations induce a new cleavage site. Importantly, the C-terminal catalytic fragment from all C1r mutants was secreted and enzymatically active in the supernatant. Western blot analysis of patient-derived skin and gingival fibroblasts confirmed aberrant activation and secretion of mutated C1r even in the presence of C1s and C1 inhibitor. C1s activation was confirmed by in-vitro complement activation assays.

Conclusion: pEDS is caused by gain-of-function mutations that cause abnormal activation of complement 1 independent of microbial triggers. We hypothesize that secreted catalytic fragments cleave extracellular matrix proteins with adverse consequences on connective tissue homeostasis.

Funded by FWF I-2909-B30.

R. Gröbner: None. E. Brunner: None. R. Redolfi: None. A. Amberger: None. H. Stoiber: None. N. Thielens: None. C. Gaboriaud: None. I. Kapferer-Seebacher: None. J. Zschocke: None.

P04.57A Genotype of Bruck syndrome with phenotype of Osteogenesis Imperfecta, separate syndromes or expansion of the spectrum

G. A. Otaify1, V. Ruiz-Perez2, P. Lapunzina2, S. Temtamy1, M. S. Aglan1

1Clinical Genetics Department, Human Genetics and Genome Research Division, National Research Centre, Cairo, Egypt, 2Instituto de Investigaciones Biomedicas, Consejo Superior de Investigaciones Cientificas, Universidad Autonoma de Madrid, Madrid, Spain

Introduction: Osteogenesis Imperfecta (OI) is heterogenous group of disorders with increased bone fragility. Bruck Syndrome (BS) is a rare autosomal recessive syndrome characterized by OI with congenital contractures due to mutations in FKPB10 and PLOD2 genes (BS1 and BS2 respectively). FKPB10 mutations were found to cause moderately severe OI type XI and PLOD2 was reported before to cause fractures without contractures. Herein we present two patients from two Egyptian families with recurrent fractures but without joint contractures having PLOD2 mutations.

Materials and Methods: two unrelated Egyptian consanguineous families with repeated fractures were studied. Mutation analysis was performed using NGS panel, homozygosity mapping, then candidate gene approach.

Results: the first family included a 15 years male who presented with repeated fractures around 10 times/year, deformities and severe kyphoscoliosis (clinical severity score; CSS 17). The second family had a 9 years old girl milder in severity with 1-2 fractures/year (CSS 14). Both patients had no joint contractures. They were short and had Wormian bones in their skull x-rays and osteoporosis confirmed by DEXA. Molecular testing for both patients revealed homozygous donor splice site mutation in PLOD2 in intron 12(c.1358+5G>A). The mutation was heterozygous in the parents.

Conclusion: This study and previous reports from other centers identified different PLOD2 mutations with repeated fractures and variable severity but without contractures and this expands the phenotypic spectrum of PLOD2 associated mutations. Accordingly, it’s important to reconsider the current classification to include BS as a variant of OI subtypes rather than being a separate syndrome.

G.A. Otaify: None. V. Ruiz-Perez: None. P. Lapunzina: None. S. Temtamy: None. M.S. Aglan: None.

P04.58B Clinical and molecular characteristics of GNAS inactivation disorders observed in 18 Korean patients

J. Ko, S. Han, Y. Lee, C. Shin, S. Yang, B. Lim, T. Cho

Seoul National University Hospital, Seoul, Korea, Republic of

The GNAS gene on chromosome 20q13.3 is a complex, imprinted locus regulated in a tissue-specific manner. GNAS inactivation disorders are a heterogeneous group of rare disorders caused by mutations and methylation defects. These are divided into pseudohypoparathyroidism (PHP) types 1a and 1b, pseudo-pseudohypoparathyroidism (PPHP), and progressive osseous heteroplasia (POH), depending on the presence or absence of hormone resistance, Albright’s hereditary osteodystrophy (AHO), and ectopic ossification. This study analyzed the clinical characteristics and molecular genetic backgrounds of 18 Korean patients from 16 families with a genetically confirmed GNAS defect. Auxological parameters, AHO phenotypes, types of hormonal resistance, family history, and molecular genetic disturbances were reviewed retrospectively. Nine (90%) patients with PHP1a showed resistance to parathyroid hormone (PTH) and all patients showed elevated thyroid-stimulating hormone (TSH) levels at diagnosis. Eight (80%) patients were managed with levothyroxine supplementation. Three of six patients with PHP1b had elevated TSH levels, but none of whom needed levothyroxine medication. AHO features were absent in PHP1b. Patients with PPHP and POH did not show any hormone resistance. Among the 11 families with PHP1a, PPHP, and POH, eight different (three novel) mutations in the GNAS gene were identified. Among the six patients with PHP1b, two were sporadic cases and four were familial cases. This, the largest single-center series study of GNAS inactivation disorders in Korea, summarizes the detailed clinical and molecular genetic characteristics of these diseases. Identification of molecular genetic backgrounds, along with clinical phenotypes, enables appropriate management and proper genetic counseling.

J. Ko: None. S. Han: None. Y. Lee: None. C. Shin: None. S. Yang: None. B. Lim: None. T. Cho: None.

P04.59C Compound screening for PXE using zebrafish abcc6a mutant models: a proof-of-concept study

M. Van Gils1,2, A. Willaert1,2, P. J. Coucke1,2, O. M. Vanakker1,2

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

Introduction: Pseudoxanthoma elasticum (PXE) is an ectopic mineralization disease due to biallelic ABCC6 mutations. As no curative therapy is available, we characterized a zebrafish abcc6a CRISPR/Cas9 knockout model (Cmg52) for drug screening purposes, identifying spinal hypermineralization as an early, quantifiable phenotype. As a proof-of-concept we tested our screening workflow on two compounds (80µM Vitamin K1 [VK1] and 30µM Sodium Thiosulfate [STS]), both implicated in PXE.

Materials and Methods: Following Cmg52+/- incross, Cmg52+/+, Cmg52+/-and Cmg52-/- embryos were collected. At 3 days post-fertilization (dpf), embryos are distributed per 20 in baskets. From 3-10dpf, 60 embryos are treated with 8ml compound in 1x E3-medium and refreshed daily. Following euthanasia at 10dpf, embryos are fixed, bleached and stained for mineralization. Photos are taken of embryos under identical conditions and via ImageJ mineralization is quantified. DNA is extracted from embryos and mineralization values are linked to respective genotypes for statistical analysis.

Results: VK1 significantly reduced spinal mineralization in Cmg52-/- ([Mean ± SD] Controls: 19.371 ± 12.794; VK1: 10.322 ± 5.519; P<0.05). STS-treated larvae had no spinal mineralization or higher mortality compared to controls. However, 33% of STS-treated animals had spotty abdominal mineralization regardless of genotype, indicating putative toxicity.

Conclusions: We confirmed the Cmg52-/- mineralization phenotype to be a good read-out for compound screening. Our data suggests a role for VK1 in PXE pathogenesis though further validation is required. The STS data underscores the importance of proper controls and that compound screening data need to be interpreted cautiously.

M. Van Gils: None. A. Willaert: None. P.J. Coucke: None. O.M. Vanakker: None.

P04.61A Retinoic acid catabolism defects affecting skeletogenesis and resembling craniosynostosis syndromes

I. Chacon Fonseca1, R. Babul-Hirji1, E. Campos2, P. Kannu1

1Division of Clinical and Metabolic Genetics, Toronto, ON, Canada, 2The Hospital for Sick Children Research Institute, Toronto, ON, Canada

Introduction: Homozygous missense mutations in the CYP26B1 gene, have been described in very few individuals with phenotypes ranging from a lethal generalised skeletal dysplasia characterised by severe skull defects and craniosynostosis, radiohumeral fusions, oligodactyly and narrow thorax to a milder presentation described in one adult affected by multiple craniosynostosis, characteristic facies, radiohumeral joint limitation, hearing loss and intellectual disability. The enzyme coded by CYP26B1 is involved in retinoic acid catabolism and regulates its precise temporo-spatial gradient during embryogenesis. Case presentation: We describe a healthy non-consanguineous couple with 2 children with distinctive craniofacial features including turribrachycephaly, shallow orbits, malar hypoplasia, low-set and protruding ears, small mouth, high palate with crowding teeth and pointy chin. Both siblings have bilateral conductive hearing loss, arachnodactyly, reduced movement of the radio-ulnar joints and mild to moderate learning disability. Their phenotype resembles that of the reported adult.

Results: Whole exome sequencing revealed both sibs inherited two missense mutations in trans affecting CYP26B1. Variant c.353C>T (p.P118L) was paternally inherited, not previously described and not seen in large population cohorts, and variant c.701G>A (p.R234Q), with frequency of 0.06% in large databases, was maternally inherited. Both variants were predicted to have a deleterious impact on the protein structure.

Conclusion: Our patients add to the wide phenotypic constellation seen with CYP26B1 pathogenic variants. We infer that higher retained enzyme activity is non-lethal and mostly associated with skeletal and neurological features including characteristic craniofacial features resembling craniosynostosis, hearing loss and intellectual disability. Funding source: Rare disease Foundation microgrant. Year 2018 #2803.

I. Chacon Fonseca: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Modest; Rare disease Foundation. R. Babul-Hirji: None. E. Campos: None. P. Kannu: None.

P04.62B The splicing variant c.1815G>A in KIAA0586 is associated with a phenotype overlapping short rib polydactyly and oral facial digital syndrome

D. Cocciadiferro1, E. Agolini1, L. Sinibaldi2, M. C. Digilio2, A. Dotta3, M. Castori4, A. Novelli1

1Laboratory of Medical Genetics, Bambino Gesù Children's Hospital, Rome, Italy, 2Medical Genetics Unit, Bambino Gesù Children's Hospital, Rome, Italy, 3Department of Neonatal Surgery, Bambino Gesù Children's Hospital, Rome, Italy, 4Division of Medical Genetics, Fondazione IRCSS Casa Sollievo della Sofferenza, San Giovanni Rotondo (Foggia), Italy

Ciliopathies are a group of hereditary disorders caused by abnormal structure/function of primary cilia, which are ubiquitously expressed organelles characterized by a mother centriole-derived basal body, a microtubule-based axoneme and a specialized membrane harboring proteins required for signal detection and amplification. Causative variants in KIAA0586, the human ortholog of chicken talpid3 essential for primary ciliogenesis and hedgehog signaling, were first associated with Joubert syndrome, a multisystem disorder displaying pathognomonic hindbrain malformation as well as variable skeletal, renal and ocular defects. KIAA0586 is also associated with other ciliopathies such as the short rib-polydactyly, a lethal bone dysplasia with severely hypoplastic thorax. Here we describe two Roma Gypsy siblings affected by a neonatal lethal short rib-thoracic dysplasia, harboring the KIAA0586 homozygous variant c.1815G>A (p.Gln605Gln), identified by targeted resequencing analysis. Clinical features of this family are not limited to the typical short rib-polydactyly syndrome but also include tongue/oral hamartomas, multiple frenulae and cleft palate. The same combination of intraoral findings have been already identified in patients with the esonic splice site variant p.Gln605Gln; suggesting continuity between short-rib-polydactyly and oral-facial-digital syndromes in the context of KIAA0586 clinical spectrum.

D. Cocciadiferro: None. E. Agolini: None. L. Sinibaldi: None. M.C. Digilio: None. A. Dotta: None. M. Castori: None. A. Novelli: None.

P04.63C Chipping away the challenges of diagnostic genomics applications for short stature

D. N. Azmanov1,2, G. M. Arscott1, M. B. Abraham3,4, J. Miller2, C. Connell2, G. Pathak5, S. Townshend5, B. Kamien5, A. Siafarikas3,4, G. Baynam4,5,6, J. Beilby1, C. S. Choong2,3,4

1Department of Diagnostic Genomics, PathWest, Nedlands, Australia, 2Faculty of Health and Medical Sciences, University of Western Australia, Crawley, Australia, 3Department of Endocrinology and Diabetes, Perth Children’s Hospital, Nedlands, Australia, 4Division of Paediatrics, Medical School, University of Western Australia, Crawley, Australia, 5Genetic Services of Western Australia, King Edward Memorial Hospital, Subiaco, Australia, 6Western Australian Register of Developmental Anomalies, King Edward Memorial Hospital, Subiaco, Australia

Introduction: As for many common complex disorders, diagnostic genomics applications for short stature (SS) face many challenges. In the absence of consensus international guidelines on genetic testing for SS, various clinical criteria are used to inform appropriate testing. The extent of genetic heterogeneity and complexity of SS are still being elucidated, challenging diagnostic approaches.

Materials and Methods: We employed a retrospective analysis of diagnostic genomics yield for SS patients receiving growth hormone therapy on the Western Australian Paediatric Endocrine Database. In addition, a prospective systematic study using chromosomal microarray and a clinical exome panel on cases with unknown genetic aetiology was initiated in 2018.

Results: A major genetic aetiology was identified in ~31% of cases prior to the systematic application of contemporary genomic technologies (35 out of 114 participants born since 2005 and receiving growth hormone for >2 years). Presence of composite genomic findings in some patients challenged interpretation. Referral to a local inter-disciplinary undiagnosed disease program helped in resolving the diagnostic odyssey for complex patients.

Conclusions: Diagnostic genomics applications for SS are still evolving and some of the challenges can be overcome by pre-test clinical selection, detailed phenotyping and contribution of an inter-disciplinary team input. The current efforts focus on the detection of high penetrance genetic causes, and the remaining challenge is to interpret moderate to low penetrance genetic risk factors for SS. Acknowledgements: Merck Serono Australia, grant 201603.4701.POT; DNA is supported by a Raine clinical research fellowship (CRF018, project “Diagnostic genomics applications for short stature”).

D.N. Azmanov: None. G.M. Arscott: None. M.B. Abraham: None. J. Miller: None. C. Connell: None. G. Pathak: None. S. Townshend: None. B. Kamien: None. A. Siafarikas: None. G. Baynam: None. J. Beilby: None. C.S. Choong: None.

P04.64D Identifications of novel variants affecting SHOX expression in Short Stature patients

A. Fanelli1, D. Babu1, S. Mellone1, F. Prodam1, S. Bellone1, G. Genoni1, S. Vannelli2, M. Giordano1

1Dipartimento di Scienze della Salute, Novara, Italy, 2Ospedale Regina Margherita, Divisione di Pediatria, Torino, Italy

Haploinsufficiency of the pseudoautosomal SHOX gene causes 70-90% of Léri-Weill dyschondrosteosis (LWD) and 2-10% of idiopathic short stature (ISS). Deletions removing the gene or enhancers and nonsense/frameshift mutations represent a well-established cause of disease. Otherwise, rearrangements not encompassing any described enhancer and 5’UTR variations are also identified in patients but their pathogenic role remains unclear. During diagnostic screening performed on 934 patients by standard methods (MLPA and sequencing) we identified SHOX alterations in 82 patients (8.8%). Among these, 65 (78%) carried deletions while 17 (22%) point mutations. Besides these pathogenic mutations we also identified small deletions not encompassing any already described enhancer (n=11) and variations within 5’UTR (n=5) in 24 LWD/ISS patients whose pathogenic role was less clear that were classified as VOUS. The aim of this work was to investigate through functional studies the pathogenic role of these variations. To better characterize the deletions not encompassing the enhancers we used a fine-tiling custom aCGH and performed in vitro functional assays to assess the potential enhancer activity of conserved sequences within the deleted areas. The variants within the 5’UTR were tested for their ability to interfere with correct gene expression. Our results showed that some of the VOUS might be responsible of SHOX deficiency either by removing putative regulatory elements or affecting the proximal promoter activity. In conclusion, the present study allowed us to reclassify as likely pathogenic some of the novel variants of uncertain significance, that we detected in the diagnostic screening, thus reducing them of at least 10%.

A. Fanelli: None. D. Babu: None. S. Mellone: None. F. Prodam: None. S. Bellone: None. G. Genoni: None. S. Vannelli: None. M. Giordano: None.

P04.66B Report of a novel variant causing a schneckenbecken-like dysplasia

O. W. Quarrell1, C. Rautengarten2, K. Stals3, R. Caswell4, E. De Franco5, E. Baple6, N. Burgess7, R. Jokhi8, J. L. Hazelwood9, A. C. Offiah10, B. Ebert2, S. Ellard6

1Dept Clinical Genetics Sheffield Children's Hospital, Sheffield S10 2TH, United Kingdom, 2School of BioSciences, The University of Melbourne, Victoria 3010, Australia, 3Royal Devon & Exeter NHS Foundation Trust, Exteter EX2 5DW, United Kingdom, 4College of Medicine and Health, University of Exeter, Exeter EX2 5DW, United Kingdom, 5Royal Devon & Exeter NHS Foundation Trust, Exeter EX2 5DW, United Kingdom, 6Royal Devon & Exeter NHS Foundation Trust and College of Medicine and Health, University of Exeter, Exeter EX2 5DW, United Kingdom, 7Dept Histology, Sheffield Children’s Hospital NHS Foundation Trust., Sheffield S10 2TH, United Kingdom, 8Dept. of Obstetrics and Gynaecology, Sheffield teaching Hospitals, Sheffield S10 2SF, United Kingdom, 9School of BioSciences, The University of Melbourne,, Victoria 3010, Australia, 10University of Sheffield, Academic Unit of Child Health, Sheffield Children's Hospital, Sheffield S10 2SF, United Kingdom

Introduction: A homozygous pathogenic variant in SLC35D1, an endoplasmic reticulum nucleotide sugar transporter, was identified as a cause of schneckenbecken dysplasia in 2007. We report a case of a skeletal dysplasia with inconclusive pathological and radiological features and a missense change in SLC35D1.

Material and Methods: A consanguineous family had 4 pregnancies affected by a skeletal dysplasia. Insufficient DNA from the 4 pregnancies meant that exome couple analysis was undertaken on the parents followed by in silico protein modelling using Phyre2 web and I-TASSER servers. Functional effects of variants were assessed using a proteo-liposome assay (Rautengarten et al., 2014 PNAS 111: 11563-11568).

Results: Both parents were heterozygous for a missense change in SLC35D1 c.398C>T p.(Pro133Leu). Reappraisal of the radiographs was consistent with schneckenbecken-like dysplasia. Testing of one of the fetuses and 2 healthy unaffected siblings was consistent with autosomal recessive inheritance but not sufficient to confirm pathogenicity. In silico protein modelling showed that p.Pro133 lies directly opposite p.Thr65, the site of the only previously reported pathogenic missense variant, at the mouth of the solute channel, implying pathogenicity. The prote-liposome assay demonstrated 2-4% in transport activity compared to wild-type but also showed SCLD231 is a general UDP-sugar transporter with clear activity towards UDP-Xylose, suggesting a mechanism by which the Golgi synthesized substrate can be imported into the endoplasmic reticulum for essential xylosylation reactions.

Conclusion: An agnostic approach to molecular testing together with protein modelling and a functional assay demonstrated pathogenicity of an SLC35D1 variant and increased the protein's biochemical characterization.

O.W. Quarrell: None. C. Rautengarten: None. K. Stals: None. R. Caswell: None. E. De Franco: None. E. Baple: None. N. Burgess: None. R. Jokhi: None. J.L. Hazelwood: None. A.C. Offiah: None. B. Ebert: None. S. Ellard: None.

P04.67C A novel POC1A variant in exon 2 causes classical SOFT syndrome: Clinical presentations of seven patients

N. Grüning1, M. Al-Shehhi2, A. Westenberger1,3, P. Scott4, O. Brandau1, L. Abbasi-Moheb1, Z. Yüksel1, C. Beetz1, P. Bauer1, A. Rolfs1, A. Al-Kindy4

1Centogene AG, Rostock, Germany, 2Royal Hospital, Muscat, Oman, 3University of Lübeck, Lübneck, Germany, 4Sultan Qaboos University, Muscat, Oman

Introduction: Biallelic pathogenic variants in POC1A (centriolar protein A) cause either SOFT (Short-stature-Onychodysplasia-Facial-dysmorphism-and-hypoTrichosis) syndrome or a milder and only partially overlapping phenotype, i.e., variant POC1A-related syndrome – with this pleiotropic effect depending on the respective exon affected by mutation. Pathogenic changes in a total of only 22 patients had been described underlining the disease rareness. Two large consanguineous families of Omani origin presented to the clinic with 7 individuals affected by primordial dwarfism resembling SOFT syndrome, but of unknown genetic cause and diagnosis.

Materials and Methods: Detailed clinical work-up to define the symptoms and whole-exome sequencing (WES) were performed in one of the index patients and two relatives, as well as subsequent Sanger sequencing for further segregation studies.

Result: WES yielded the variant POC1A (NM_015426.4): c.64G>T p.(Val22Phe) which showed segregation within the tested individuals and further family members. This variant affects the alternative exon 2, that is present only in two of three POC1A transcripts. Nevertheless, all of the investigated patients had comparable disease histories with severe growth retardation of prenatal onset, characteristic facial features and respiratory difficulties, the latter being most prominent in the index patient; which confirmed the diagnosis of SOFT syndrome.

Conclusion: The finding i) considerably increases the number of reported SOFT syndrome patients allowing for further clinical delineation of this rare disorder, and ii) add a novel POC1A missense variant in exon 2, allowing to refine the relevance of the functional domain affected by this change and expand the genotype- phenotype correlation in POC1A-related disorders.

N. Grüning: A. Employment (full or part-time); Significant; Centogene AG. M. Al-Shehhi: None. A. Westenberger: F. Consultant/Advisory Board; Significant; Centogene AG. P. Scott: None. O. Brandau: A. Employment (full or part-time); Significant; Centogene AG. L. Abbasi-Moheb: A. Employment (full or part-time); Significant; Centogene AG. Z. Yüksel: A. Employment (full or part-time); Significant; Centogene AG. C. Beetz: 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. E. Ownership Interest (stock, stock options, patent or other intellectual property); Significant; Centogene AG. A. Al-Kindy: None.

P04.69A Phenotypic overlap between spondyloepimetaphyseal dysplasia with joint laxity type 2 and Morquio syndrome type A: case report

M. Mijovic1, A. Miletic1, H. Janeski1, B. Dimitrijevic1, J. Ruml Stojanovic1, M. Lukic1, G. Cuturilo1,2

1University Children's Hospital, Department of Medical Genetics, Belgrade, Serbia, 2Faculty of Medicine, University of Belgrade, Belgrade, Serbia

Introduction: We present fifteen-year-old boy patient, with complex skeletal phenotype highly susceptible to mucoplysaccharidosis type IVA (Morquio syndrome). He has severe skeletal dysplasia including disproportionate short stature with short trunk, kyphoscoliosis with gibbus, joint hypermobility, bilateral congenital hip dislocations, knock-knee and ulnar deviation of the wrists. The patient is immobile since his sixth year. He doesn’t have hepatosplenomegaly, respiratory complications, cardiac valve abnormalities and dental abnormalities. No pathological ophthalmology findings, neither hearing loss.

Materials and Methods: We performed enzymes blood testing for several types of mucopolysaccharidosis in first line testing, and exome sequencing which targeted genes related to observed clinical presentation in second line testing.

Results: Unexpectedly, enzyme level of N-acetylgalatosamine-6-sulfate-sulphatease showed normal result and excluded Morquio syndrome. Subsequently, exome sequencing showed presence of heterozygous pathogenic missense variant in KIF22 gene (c.443C>T, p.Pro148Leu). Pathogenic variants in KIF22 gene represent an established cause of spondyloepimetaphyseal dysplasia with joint laxity type 2 (OMIM:603546). The clinical presentation of KIF22-associated conditions includes skeletal abnormalities, joint laxity, joint dislocations, spinal deformities and short stature, which is compatible with the clinical presentation in our patient.

Conclusion: Between Morquio syndrome and spondyloepimetaphyseal dysplasia may exist completely overlapping skeletal phenotype, which is confirmed literature data (ref. Biswas SN et al., 2017). The distinction between these diseases is very important because of available enzyme replacement therapy for Morquio syndrome type A and completely different pattern of inheritance and recurrence risk. Exome sequencing should be first genetic test in patient with severe skeletal dysplasia with overlapping phenotype.

M. Mijovic: None. A. Miletic: None. H. Janeski: None. B. Dimitrijevic: None. J. Ruml Stojanovic: None. M. Lukic: None. G. Cuturilo: None.

P04.70B Further evidence for COL9A3 associated autosomal recessive Stickler syndrome

A. Bohring, J. Horvath, P. Wieacker

Institut für Humangenetik, Münster, Germany

Heterozygous COL9A3 [MIM 120270] mutations were reported to cause multiple epiphyseal dysplasia-3 [MIM 600969] which is mainly characterized by stiffness and/or pain in the knees, due to flattened, irregular epiphyses, and varus or valgus deformity, waddling gait, and mild short stature in some patients. Here we report on the second case with bi-allelic COL9A3 nonsense mutations. The boy was born with club feet and moderate hearing loss to healthy parents. Radiographs revealed flat lumbar vertebrae, coxa retrotorta, and broad femoral metaphyses. At examination at age 2,5 years we saw a triangular face, bilaterally mild epicanthus, a high arched palate, and short fingers. In addition, myopia was diagnosed recently. Height was 92 cm, weight 15 kg (50. and 75. centile, respectively). Because of the phenotype, the provisional diagnosis of Stickler syndrome was made and molecular analyses revealed compound-heterozygosity for COL9A3 mutations c.268C>T; p.(Arg90*) and c.1739dup; p.(Gly581Trpfs*20). Bi-allelic COL9A1 [MIM 120210] and COL9A2 [MIM 120260] mutations are already known to cause autosomal-recessive inherited Stickler syndrome [MIM 614134; 614284]. However, as far as we know, only one family with bi-allelic occurrence of a COL9A3 nonsense mutation and Stickler syndrome is published until now. As in our patient, in this family the affected individuals showed a comparatively mild phenotype with no vitreoretinal involvement or cataracts so far and no cleft palate/Pierre-Robin sequence. Thus, the findings in our case further support the assumption that bi-allelic COL9A3 mutations are very likely sufficient to cause Stickler syndrome as well.

A. Bohring: None. J. Horvath: None. P. Wieacker: None.

P04.71C Stüve-Wiedemann syndrome: a rare clinical entity

O. Akgün Dogan1, Y. Kendir Demirkol1, M. Say2, T. Kizilboga Akgün3,4, L. Doğanay4

1Department of Pediatric Genetics, Health Sciences University, Umraniye Education and Research Hospital, İstanbul, Turkey, 2Bioinformatic Team, Gen-Era Diagnostic, İstanbul, Turkey, 3Department of Molecular Biology and Genetics, Istanbul Technical University, İstanbul, Turkey, 4GLAB (Genomic Laboratory), Health Sciences University, Umraniye Education and Research Hospital, İstanbul, Turkey

Introduction: Stüve-Wiedemann syndrome(SWS)(MIM601559) is an autosomal recessive skeletal dysplasia characterized by bowing of extremities, severe osteoporosis, joint contractures, dysautonomia, frequent respiratory infections, and feeding difficulty. Mutations in LIFR are responsible for the syndrome. Although the majority of patients are lost due to complications in infancy, patients who reach adolescent period are reported rarely. Here, we present a 9 years-old male with SWS with severe skeletal findings due to a novel homozygous mutation in LIFR.

Materials and Methods: The patient was the third child of healthy, consanguineous parents, born at term with a birth weight of 3600g(50-75th centile). Bowing in lower extremities was remarkable at birth, and progressed gradually with age. He was hospitalized many times for respiratory distress, feeding difficulties, and recurrent fever. Motor developmental milestones were delayed however, mental development was normal. Physical examination revealed facial dysmorphic features, bending of the extremities, laxity of the metacarpophalangeal joints, scoliosis, prominent heels, and pes planus. X-ray of extremities showed bowing of the limbs, bilateral valgus deformity, and widening of the femoral and tibial metaphysis.

Results: Next-generation sequencing was performed on Illumina MiSeq(v1.9) platform using the virtual panel for skeletal dysplasia consisting of 130 genes. The novel homozygous variant in LIFR(c.274C>T) was detected. Segregation within the family showed that parents were heterozygous carriers.

Conclusions: Although SWS is a rare genetic disease, it should be kept in mind in patients with congenital bowing in extremities. Targeted exome analysis has great importance in the fast and accurate setting in the diagnosis of heterogeneous groups of syndromes such as bent bone dysplasias.

O. Akgün Dogan: None. Y. Kendir Demirkol: None. M. Say: None. T. Kizilboga Akgün: None. L. Doğanay: None.

P04.73A Novel VPS33B mutation in a patient with autosomal recessive keratoderma-ichthyosis-deafness (ARKID) syndrome

S. Alter1, A. Hotz1, A. Jahn2, N. Di Donato2, E. Schröck2, M. Smitka3, M. von der Hagen3, J. Schallner3, M. Menschikowski4, C. Gillitzer3, M. W. Laass3, J. Fischer1, A. Tzschach1,2

1Institute of Human Genetics, Freiburg, Germany, 2Institute of Clinical Genetics, Technische Universität Dresden, Dresden, Germany, 3Children´s hospital, Medical Faculty Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany, 4Institute of Clinical Chemistry and Laboratory Medicine, Medical Faculty Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany

Autosomal recessive keratoderma-ichthyosis-deafness (ARKID) syndrome is a rare multisystem disorder caused by biallelic mutations in VPS33B; only three patients have been reported to date. ARKID syndrome is allelic to arthrogryposis-renal dysfunction-cholestasis (ARC) syndrome (MIM #208085), a severe disorder with early lethality whose phenotypic characteristics also include ichthyosis, hearing loss, severe failure to thrive, platelet dysfunction and osteopenia. We report on an 11-year-old male patient with ARKID syndrome and compound heterozygous VPS33B mutations, one of which [c.1440delG; p.(Arg481Glyfs*11)] was novel. Clinical features of this patient included ichthyosis, palmoplantar keratosis, hearing loss, intellectual disability, unilateral hip dislocation, microcephaly and short stature. He also had copper hepatopathy and exocrine pancreatic insufficiency, features that have not been associated with neither ARKID nor ARC syndrome so far. The patient broadens the clinical and molecular spectrum of ARKID syndrome and contributes to genotype-phenotype associations of this rare disorder.

S. Alter: None. A. Hotz: None. A. Jahn: None. N. Di Donato: None. E. Schröck: None. M. Smitka: None. M. von der Hagen: None. J. Schallner: None. M. Menschikowski: None. C. Gillitzer: None. M.W. Laass: None. J. Fischer: None. A. Tzschach: None.

P04.74B Analysis of a French cohort of patients with Werner syndrome; clinical and molecular features

B. Dauriat1, N. Uhrhammer2,3, H. Adamski4, C. Colson5, P. D’Anella6, F. Demurger7, H. Dollfus8, V. Drouin-Garraud9, C. Francannet10, M. Gerard5, F. Giuliano11, B. Isidor12, H. Journel7, P. Lacroix13, H. Levesque14, L. Mary8, C. Moraine1, X. Piguel15, M. Pistorius16, G. Plessis5, I. Reingeard17, M. Rio18, M. Ruivard19, G. Vera9, C. Vigouroux20, M. Vincent12, J. Bignon2,3, C. Yardin1,21

1Department of Cytogenetics and Medical Genetics, Dupuytren University Hospital, Limoges, France, 2Department of Oncogenetics, Centre Jean Perrin, Clermont-Ferrand University Hospital, Clermont-Ferrand, France, 3INSERM U1240, Imagerie Moléculaire et Stratégies Théranostiques, Clermont-Ferrand, France, 4Department of Dermatology, Rennes University Hospital, Rennes, France, 5Department of Genetics, Caen University Hospital, Caen, France, 6Department of Endocrinology, Avignon Hospital, Avignon, France, 7Department of Clinical Genetics, Rennes University Hospital, Rennes, France, 8Department of Genetics, Strasbourg University Hospital, Strasbourg, France, 9Department of Genetics, Rouen University Hospital, Rouen, France, 10Department of Genetics, Estaing University Hospital, Clermont-Ferrand, France, 11Department of Genetics, Nice University Hospital, Nice, France, 12Department of Genetics, Nantes University Hospital, Nantes, France, 13Department of Thoracic and Vascular Surgery-Vascular Medicine, Dupuytren University Hospital, Limoges, France, 14Department of Internal Medicine, University of Rouen, Institute for Research and Innovation in Biomedicine, Rouen, France, 15Department of Endocrinology, Poitiers University Hospital, Poitiers, France, 16Department of Internal Medicine, Nantes University Hospital, Nantes, France, 17Department of Endocrinology, Montpellier University Hospital, Montpellier, France, 18Department of Pediatrics, Neurology and Genetics, Hôpital Necker-Enfants-Malades, Paris, France, 19Department of Internal Medicine, Estaing University Hospital, Clermont-Ferrand, France, 20AP-HP Saint-Antoine Hospital, Molecular Biology and Genetics Laboratory, Endocrinology Department, National Reference Center for Insulin Secretion and Insulin Sensitivity Rare Diseases, Paris, France, 21Limoges University, CNRS, XLIM, UMR 7252, Limoges, France

Introduction: Werner syndrome (WS) belongs to adult onset progeroid diseases, leading to a premature ageing of mesenchymal and epithelial tissues, affecting one person per a million in the world. Its cardinal signs are short stature, lipodystrophy and atrophic skin, premature cataract and greying of hair; age-associated complications may occur in WS such as osteoporosis, chronic skin ulcers or diabetes mellitus, and patients usually die around 53 years of age from cardiovascular or neoplastic causes. This recessive genetic affection is linked to loss-of-function mutations of WRN gene, coding for a helicase involved in multiple DNA pathways. No recent clinical nor molecular description were available for French WS patients.

Methods: Data from French laboratories found 36 genetically confirmed WS cases, whose 34 for which molecular and clinical data were collected.

Results: 31 mutations including 14 newly described ones were identified, with some associated with a specific French region. The mean age for diagnosis was 40.5 years, the cardinal signs were found in all the patients, and underestimated features (deafness, hepatic, thyroid, valvular, hypertensive or capillary impairments) were commonly diagnosed. Death occurred at 53.6 years of age, mostly from cancerous etiologies. Despite the homogeneity of the main signs, it seems that phenotypes with less severe evolution may exist, without associated predictive or causal factors.

Conclusion: This cohort gives an exhaustive description of French patients presenting genetically confirmed WS since the beginning of WRN testing. It defines accurate frequencies for all the numerous symptoms and gives the opportunity for prospective investigations.

B. Dauriat: None. N. Uhrhammer: None. H. Adamski: None. C. Colson: None. P. D’Anella: None. F. Demurger: None. H. Dollfus: None. V. Drouin-Garraud: None. C. Francannet: None. M. Gerard: None. F. Giuliano: None. B. Isidor: None. H. Journel: None. P. Lacroix: None. H. Levesque: None. L. Mary: None. C. Moraine: None. X. Piguel: None. M. Pistorius: None. G. Plessis: None. I. Reingeard: None. M. Rio: None. M. Ruivard: None. G. Vera: None. C. Vigouroux: None. M. Vincent: None. J. Bignon: None. C. Yardin: None.

P04.75C Genetic predisposition to systemic sclerosis

N. Limaye1, M. Vanthuyne2, F. Houssiau3, V. Smith4, B. Lauwerys3

1de Duve Institute, Brussels, Belgium, 2Cliniques Universitaires St. Luc, Brussels, Belgium, 3Cliniques Universitaires St Luc & Institute of Experimental and Clinical Research, UCLouvain, Brussels, Belgium, 4Ghent University Hospital, Ghent, Belgium

Systemic sclerosis (SSc) is a multi-system disease of unknown cause, characterized by vascular and immune dysregulation that results in progressive fibrosis of the skin and other organs. While rare, it can be lethal, and there are currently no treatments. SSc is typically sporadic; familial forms are extremely rare. By applying genome-wide Next Generation Sequencing (NGS) to such families, we aim to identify genetic “drivers”, i.e., disease-causative or strongly-predisposing alleles. We hypothesize that these genes may also contribute to sporadic disease, when they carry strong de novo mutations, weak inherited variants (that cause disease only in combination with other genetic and environmental factors), or somatic mutations in affected tissues. We performed whole exome sequencing (WES) on blood-DNA from two affected first-degree relatives each from five families, and filtered for genes with missense or nonsense variants that (i) co-segregate with disease, (ii) are rare in public sequence databases and an in-house WES database, and (iii) predicted to affect protein function in silico. Between 18 and 40 such genes were identified per family. Candidate gene prioritization was performed using literature and database-mining, as well as intersection with RNASeq data from sporadic SSc cutaneous tissue biopsies. Variants in the two highest-ranked candidates (one a pro-fibrotic growth factor, the other a cytokine receptor with pro-fibrotic, pro-inflammatory effects) are currently being tested for their effects on protein function. Proof of an effect on protein function will be followed by testing for a role in a bleomycin-induced mouse model of SSc.

N. Limaye: None. M. Vanthuyne: None. F. Houssiau: None. V. Smith: None. B. Lauwerys: None.

P04.76D WNT10A mutations: refining genotype, phenotype, penetrance, clinical variability and inheritance manner

O. Patat1, I. Bailleul-Forestier2, J. Plaisancie1, D. Bonneau3, E. Colin3, C. Colson4, M. Cordier5, C. Coubes6, F. Demurger7, A. Dieux-Coeslier8, M. Fradin7, M. Gerard4, A. Goldenberg9, B. Isidor10, H. Journel10, D. Lacombe11, M. Lebrun12, D. Martin-Coignard13, M. Nizon10, S. Odent7, F. Petit8, N. Philip14, J. Piard15, J. Piard15, G. Plessis4, A. Putoux5, C. Quelin7, J. Thevenon16, A. Toutain17, C. Vanleberghe8, A. Verloes18, C. Vincent-Delorme8, Y. Capri18, F. Vaysse2, P. Calvas1, N. Chassaing1

1CHU Toulouse, Toulouse, France, 2Faculté de Chirurgie Dentaire, Toulouse, France, 3CHU Angers, Angers, France, 4CHU Caen, Caen, France, 5CHU Lyon HCL, Lyon, France, 6CHRU Montpellier, Montpellier, France, 7CHU Rennes, Rennes, France, 8CHRU Lille, Lille, France, 9CHU Rouen, Rouen, France, 10CHU Nantes-Hôtel dieu, Nantes, France, 11CHU Bordeaux, Bordeaux, France, 12CHU Saint-Etienne, Saint-Etienne, France, 13CHU Le Mans, Le Mans, France, 14CHU Marseille, Marseille, France, 15CHRU Besançon, Besançon, France, 16CHU Grenoble, Grenoble, France, 17CHRU Tours, Tours, France, 18CHU Paris Hôpital Robert Debré, Paris, France

Introduction: The agenesis of several teeth is a prevalent malformation in human. Hypodontia is defined by the agenesis of six teeth or less, whereas oligodontia refers to the agenesis of more than six teeth, excluding the third molars. Hypodontia and oligodontia can be associated with defects in the development of other ectodermal structures such as hairs, sweat glands and nails, defining the spectrum of ectodermal dysplasias. Mutations in WNT10A are known as a major cause of oligodontia with or without associated ectodermal dysplasia signs.

Patients and Methods: Based on the analysis of a large series of 413 probands with hypo or oligodontia and 190 of their relatives, we report 395 subjects with mutations in the gene WNT10A.

Results: We found a mutation in WNT10A in 227 of the 413 probands, among which two recurrent mutations the c.682T>A (p.Phe228Ile) and c.321C>A (p.Cys107Ter) are respectively involved in 178 and 33 unrelated probands and families. We show a pattern of tooth agenesis in patients with WNT10A mutations and a broad phenotypic variability despite a correlation between the number of mutations, the number of tooth agenesis and the presence of ectodermal signs. We confirm the incomplete penetrance of WNT10A mutation with an estimate of the penetrance and report several asymptomatic cases with biallelic mutations.

Discussion: Despite some degree of genotype-phenotype correlation, we highlight the incomplete penetrance and broad clinical variability associated with WNT10A mutations.

O. Patat: None. I. Bailleul-forestier: None. J. Plaisancie: None. D. Bonneau: None. E. Colin: None. C. Colson: None. M. Cordier: None. C. Coubes: None. F. Demurger: None. A. Dieux-coeslier: None. M. Fradin: None. M. Gerard: None. A. Goldenberg: None. B. Isidor: None. H. Journel: None. D. Lacombe: None. M. Lebrun: None. D. Martin-coignard: None. M. Nizon: None. S. Odent: None. F. Petit: None. N. Philip: None. J. Piard: None. J. Piard: None. G. Plessis: None. A. Putoux: None. C. Quelin: None. J. Thevenon: None. A. Toutain: None. C. Vanleberghe: None. A. Verloes: None. C. Vincent-delorme: None. Y. Capri: None. F. Vaysse: None. P. Calvas: None. N. Chassaing: None.

P05 Cardiovascular disorders

P05.01A European Reference Network For RareVascular Diseases (VASCERN) consensus statement for the screening and management of patients with pathogenic ACTA2 variants

I. van de Laar1, E. Arbustini2, B. Loeys3,4, E. Björck5, M. Groenink6, M. Kempers4, J. Timmermans4, J. Roos-Hesselink1, K. Benke7, G. Pepe8, B. Mulder6, Z. Szabolcs7, G. Teixidó-Turà9, L. Robert10, Y. Emmanuel10, A. Evangelista11, A. Pini12, Y. von Kodolitsch13, G. Jondeau14, J. De Backer15

1Erasmus MC, University Medical Center Rotterdam, Rotterdam, Netherlands, 2IRCCS foundatoin Policlinico San Matteo, Pavia, Italy, 3University Hospital of Antwerp University of Antwerp, Antwerp, Belgium, 4Radboud University Medical Center, Nijmegen, Netherlands, 5Karolinska University Hospital, Stockholm, Sweden, 6Academic Medical Center, Amsterdam, Netherlands, 7Semmelweis University, Heart and Vascular Center, Budapest, Hungary, 8Careggi Hospital, University of Florence, Florence, Italy, 9hospital universitari Vall D'Hebron, CIBER-CV, Barcelona, Spain, 10South East Thames Regional Genetic Service, London, United Kingdom, 11hospital universitari Vall D'Hebron, Barcelona, Spain, 12Centro Malattie Rare Cardilogiche, Milan, Italy, 13University Heart Center Hamburg, University Medical Center Hamburg-Eppendorf, Hamburg, Germany, 14Hopital Bichat-Claude Bernard, Paris, France, 15Ghent University Hospital, Ghent, Belgium

The ACTA2 gene encodes for smooth muscle specific α-actin, a critical component of the contractile apparatus of the vascular smooth muscle cell. Pathogenic variants in the ACTA2 gene are the most frequently encountered genetic cause of familial thoracic aortic aneurysm/dissection (FTAAD). Although TAAD is the main clinical manifestation, a variety of occlusive vascular disease and extravascular manifestations occur in ACTA2 related vasculopathy. Current data suggest possible mutation-specific manifestations of vascular and extra-aortic traits. Despite its relatively high prevalence, comprehensive recommendations on the care of patients and families with pathogenic variants in ACTA2 have not yet been established. We aimed to develop a consensus document to provide medical guidance for all health care professionals involved in the recognition, diagnosis and treatment of patients and relatives with pathogenic variants in ACTA2. The hereditary thoracic aortic disease (HTAD) Working Group of the European Reference Network for Rare Vascular Diseases (VASCERN) convened to review current literature and discuss expert opinions on clinical management of ACTA2 related vasculopathy. This consensus statement summarizes our recommendations on diagnosis, monitoring, treatment, genetic counselling and testing in patients with ACTA2 related vasculopathy. However, there is a clear need for additional prospective multicenter studies to further define proper guidelines.

I. van de Laar: None. E. Arbustini: None. B. Loeys: None. E. Björck: None. M. Groenink: None. M. Kempers: None. J. Timmermans: None. J. Roos-Hesselink: None. K. Benke: None. G. Pepe: None. B. Mulder: None. Z. Szabolcs: None. G. Teixidó-Turà: None. L. Robert: None. Y. Emmanuel: None. A. Evangelista: None. A. Pini: None. Y. von Kodolitsch: None. G. Jondeau: None. J. De Backer: None.

P05.02B Novel approach reveals celiprolol but not losartan as medical therapy for vascular Ehlers-Danlos syndrome

N. Dubacher1, J. Münger1, M. C. Gorosabel1, J. Crabb2, A. A. Ksiazek3,4, S. M. Caspar1, E. N. T. P. Bakker5, E. van Bavel5, U. Ziegler6, T. Carrel7, B. Steinmann8, S. Zeisberger3, J. Meienberg1, G. Matyas1

1Center for Cardiovascular Genetics and Gene Diagnostics, Foundation for People with Rare Diseases, Schlieren-Zurich, Switzerland, 2Institute of Mechanical Systems, Swiss Federal Institute of Technology Zurich, Zurich, Switzerland, 3Institute for Regenerative Medicine, University of Zurich, Zurich, Switzerland, 4Clinic for Small Animal Internal Medicine, University of Zurich, Zurich, Switzerland, 5Department of Biomedical Engineering and Physics, Academic Medical Center, University of Amsterdam, Amsterdam, Netherlands, 6Center for Microscopy and Image Analysis, University of Zurich, Zurich, Switzerland, 7Department of Cardiovascular Surgery, University Hospital, Berne, Switzerland, 8Division of Metabolism, University Children's Hospital, Zurich, Switzerland

Aims: Antihypertensive drugs are included in the medical therapy of vascular Ehlers-Danlos syndrome (vEDS). The β-blocker celiprolol has been suggested to prevent arterial damage in vEDS, but the underlying mechanism remains unclear. It is also unknown whether the widely-used AGTR1-antagonist losartan has a therapeutic effect in vEDS. We evaluated the impact of celiprolol and losartan on the biomechanical integrity of the vEDS thoracic aorta.

Methods: We (re-)characterised a murine vEDS model at molecular level using WGS and developed an objective approach to measure the maximum tensile force at rupture of uniaxially-stretched murine thoracic aortic rings. To assess treatment effect, heterozygous mice at 4 weeks of age underwent a 4-week treatment with celiprolol, losartan, and, as a proof-of-concept drug, the MMP-inhibitor doxycycline.

Results: Heterozygous mice showed a significant reduction in the rupture force compared to wild-type mice. Compared to age- and sex-matched untreated heterozygous mice, treatment with doxycycline or celiprolol resulted in a significant increase of rupture force, whereas no significant change was detected upon losartan treatment.

Conclusions: Our novel read-out system is suitable for the assessment of the biomechanical effect of candidate drugs. In a vEDS model, celiprolol or doxycycline, but not losartan, can improve the biomechanical integrity of the aortic wall. As doxycycline is a broad-spectrum antibiotic with considerable side effects, celiprolol may be more suitable for a long-term therapy and thus rather indicated for the medication of patients with vEDS. This is the first study demonstrating that celiprolol improves the rupture force of the thoracic aorta.

N. Dubacher: None. J. Münger: None. M.C. Gorosabel: None. J. Crabb: None. A.A. Ksiazek: None. S.M. Caspar: None. E.N.T.P. Bakker: None. E. van Bavel: None. U. Ziegler: None. T. Carrel: None. B. Steinmann: None. S. Zeisberger: None. J. Meienberg: None. G. Matyas: None.

P05.03C Next Generation Sequencing identifies novel variants in TJP1, TP63 and PPP1R13L genes in Arrhythmogenic Cardiomyopathy patients

M. Calore1,2, G. Poloni2, A. V. Postma3, A. Lorenzon2, G. Minervini4, G. Vazza2, I. E. A. Li Mura5, A. Telatin5, I. Zara5, B. Simionati5, J. Ponti2, G. Occhi2, L. Vitiello2, B. Bauce6, S. C. E. Tosatto4, P. J. van Tintelen3, A. Rampazzo2, M. De Bortoli2,7

1Department of Cardiology, Faculty of Health, Medicine and Life Sciences, Maastricht, Netherlands, 2Department of Biology, Padova University, Padova, Italy, 3Department of Medical Biology and Department of Clinical Genetics, Academic Medical Center, Amsterdam, Netherlands, 4Department of Biomedical Sciences, Padova University, Padova, Italy, 5BMR Genomics, Padova, Italy, 6Department of Biology Cardiac, Thoracic, and Vascular Sciences, Padova University, Padova, Italy, 7Institute for Biomedicine, Eurac Research, Bolzano, Italy, Affiliated Institute of the University of Lübeck, Lübeck, Germany

Among the most common inherited cardiomyopathies, arrhythmogenic cardiomyopathy (ACM) is characterized by progressive myocardial fibro-fatty replacement, arrhythmias and risk of sudden death. Mutations in genes encoding proteins of cardiac intercalated discs account for about 60% of ACM cases, but the remaining 40% is still genetically elusive.

Aim: We aim at identifying novel ACM genes by next generation sequencing.

Methods and Results: The DNA of 40 ACM patients was analyzed using a targeted gene panel consisting of 15 known
ACM genes and 53 candidate genes. We found two novel variants in TP63 (c.796C>T, p.R266*) and PPP1R13L (c.1858G>C, p.A620P) candidate genes, encoding for the proteins p63 and iASPP, respectively. The TP63 variant is
scored as likely pathogenic and the PPP1R13L variant as of uncertain significance and involves a conserved functional domain.
Importantly, the mutant TP63 allele leads to nonsense-mediated-mRNA decay, causing 

Also, novel variants in TJP1 gene were detected by whole exome sequencing in additional 4 ACM Italian and Dutch/German probands. Out of the 4 variants, p.Y669C in the encoded protein ZO-1 promotes structural rearrangements of the GUK domain, whereas the p.R265W, p.S329L, and p.D360V are predicted to impair the function of the involved the disordered region between PDZ2 and PDZ3 domains. Furthermore, rare variants in TJP1 are significantly enriched in patients with ACM relative to controls.

Conclusions: We provide the first evidence linking TP63 and TJP1 variants to ACM, while the functional involvement of PPP1R13L remains to be determined.

Grants: TRANSAC CPDA133979/13; Target Projects 331/12, RP 2014-00000394; (PRAT) CPDA133979; CVON2012-10 PREDICT;CVON2015-12 eDETECT

M. Calore: None. G. Poloni: None. A.V. Postma: None. A. Lorenzon: None. G. Minervini: None. G. Vazza: None. I.E.A. Li Mura: None. A. Telatin: None. I. Zara: None. B. Simionati: None. J. Ponti: None. G. Occhi: None. L. Vitiello: None. B. Bauce: None. S.C.E. Tosatto: None. P.J. van Tintelen: None. A. Rampazzo: None. M. De Bortoli: None.

P05.04D Aortic dilatation in patients with Traboulsi syndrome

L. Zahavich1,2, A. Ali3, R. Mendoza-Londono4, N. Tehrani3, L. Grosse-Wortmann1, R. K. Jobling4,5

1Division of Cardiology Labatt Family Heart Centre and Department of Paediatrics, The Hospital for Sick Children, University of Toronto, Toronto, ON, Canada, 2Department of Genetic Counseling, The Hospital for Sick Children, University of Toronto, Toronto, ON, Canada, 3Department of Ophthalmology and Vision Sciences, The Hospital for Sick Children, University of Toronto, Toronto, ON, Canada, 4Division of Clinical and Metabolic Genetics, The Hospital for Sick Children, University of Toronto, Toronto, ON, Canada, 5Ted Rogers Centre for Heart Research, Cardiac Genome Clinic, The Hospital for Sick Children, Toronto, ON, Canada

Traboulsi syndrome (OMIM 601552) is a rare autosomal recessive disorder caused by mutations in ASPH (OMIM 600582). It is characterized by ectopia lentis, spontaneous filtering blebs, other anterior segment anomalies and craniofacial dysmorphism. Thirteen patients have been reported, and aortic dilatation has not been noted, though details regarding aortic measurements have not been consistently provided. We report three paediatric patients from two families affected with Traboulsi syndrome presenting with mild-moderate aortic dilatation in addition to the ocular phenotype. The two affected individuals in Family 1 have dilation of the aortic root (Patient 1-I aortic sinus 30.2 Boston z-score 2.2, Patient 1-II aortic sinus 28.2 mm, Boston z-score 4.1, and sinotubular junction 23.7 mm, Boston z-score 4.7). In addition Patient 1-I has a myxomatous mitral valve with regurgitation, and Patient 1-II has a bicuspid aortic valve. Both patients harbor a homozygous variant in ASPH (NM_004318.3 c.2204 G>A (ep.Arg735Gln)). The substitution of tryptophan for arginine at this residue has been reported previously in Traboulsi syndrome. The ocular findings in Family 1 included both microspherophakia and ectopia lentis. The affected patient in Family 2 (Patient 2-I) has a dilated aortic root (aortic sinus 27.4 mm, Boston z-score of 4.1 and sinotubular junction at 20.9 mm, Boston z-score 3.2). Patient 2-I has a homozygous nonsense variant c.2181_2183dupATG (p.W728X) in ASPH discovered on whole exome sequencing. Comprehensive testing of FBN1 was negative in both families. The phenotype of this extremely rare disorder is continues to evolve and affected patients require a thorough cardiac assessment.

L. Zahavich: None. A. Ali: None. R. Mendoza-Londono: None. N. Tehrani: None. L. Grosse-Wortmann: None. R.K. Jobling: None.

P05.06B Metagenomic search for the protozoa in atherosclerotic plaques

A. Zarubin1, A. Markov1, D. Sharysh2, V. Puzyrev1,2, M. Nazarenko1

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

Background: Several reports have shown the relevance of infection in development of atherosclerosis. However, the bacterial DNA of atherosclerotic plaques mainly was investigated by metagenomic approaches. Objective: To search for the protozoa by using genome-wide sequencing data of human atherosclerotic plaques (AP).

Material and Methods: We used the results of whole genome sequencing of DNA isolated from 12 atherosclerotic plaques (SRA149235) and 2 blood samples from 1000 Genomes Project (HG00096, HG00099). Sequence reads were aligned to the human reference genome (hg19) using Bowtie2. The taxonomic classification of the reads was performed with Kraken2 using a curated microbial genome database containing archaea, bacteria, fungi, protozoa, viruses reference genome sequence. Results and discussion: Pre-alignment on the human genome, even with “very-sensitive” option of Bowtie2, cannot completely clean up human gene sequences. Kraken2 determined 13% to 92% of the human gene sequences in unmatched reads of different AP samples. We found sequences of Toxoplasma gondii genome in 8 samples (about 1678 reads per sample). These sequences were shown to be mapped to contigs (NW_017384310.1, NW_017384809.1). These contigs were also found in samples from 1000 Genomes Project.

Conclusions: There were no protozoa in genome-wide sequencing data of human atherosclerotic plaques. The reads that mapped to the Toxoplasma genome are false positive. Some microbial reference genomes probably to contain human DNA sequences not presented in the human reference genome assembly.

A. Zarubin: None. A. Markov: None. D. Sharysh: None. V. Puzyrev: None. M. Nazarenko: None.

P05.07C Gain of function mutation in the cardiac Kv4.2 potassium channel underlies paroxysmal atrial fibrillation

M. Drabkin, N. Zilberberg, S. Menahem, W. Mulla, D. Halperin, Y. Yogev, O. Wormser, Y. Perez, R. Kadir, Y. Etzion, A. Katz, O. Birk

Ben-Gurion University, Beer-Sheva, Israel

Introduction: Three generations of a kindred presented with autosomal dominant early-onset paroxysmal atrial fibrillation (pAF), with recurrent nocturnal self-terminating palpitations.

Methods: Whole exome sequencing, linkage analysis, electrophysiological assays in Xenopus oocytes.

Results: Through genetic studies we identified a disease-causing p.S447R mutation in KCND2, encoding the pore-forming (α) subunit of the Kv4.2 cardiac potassium channel. Kv4.2, with Kv4.3, contributes to the cardiac fast transient outward K+ current, Ito. Ito underlies the early repolarization phase in the cardiac action potential, setting the initial potential of the plateau phase and governing its duration and amplitude. In Xenopus oocytes, the p.S447R mutation increased inactivation time constant of the channel and affected its regulation: the mutation resides in a protein-kinase C (PKC) phosphorylation site, which normally attenuates Kv4.2 membrane expression. Mutant Kv4.2 exhibited impaired response to PKC, resulting in augmented Kv4.2 membrane expression and enhanced potassium currents. Moreover, in a hybrid channel composed of Kv4.3 and Kv4.2, simulating the mature endogenous hetero-tetrameric channel underlying Ito, the Kv4.2 mutation exerted gain-of-function effect on Kv4.3. Thus, the mutation exerts gain-of-function effect on both Kv4.2 homo-tetramers and Kv4.2-Kv4.3 hetero-tetramers.

Conclusions: Gain-of-function mutation in Kv4.2 causes nocturnal pAF. Interestingly, Kv4.2 expression was previously shown to demonstrate circadian variation, with peak expression at daytime in murine hearts (human nighttime), with possible relevance to the nocturnal onset of paroxysmal AF symptoms in our patients. The atrial-specific phenotype suggests that targeting Kv4.2 might be effective in the treatment of nocturnal paroxysmal AF, avoiding adverse ventricular effects.

M. Drabkin: None. N. Zilberberg: None. S. Menahem: None. W. Mulla: None. D. Halperin: None. Y. Yogev: None. O. Wormser: None. Y. Perez: None. R. Kadir: None. Y. Etzion: None. A. Katz: None. O. Birk: None.

P05.08D Novel ABCC9 missense mutation in a Chinese infant with Cantu syndrome without skeletal manifestations

R. Guo1,2,3,4, C. Hao1,2,3,4, S. Qian1, W. Li1,2,3,4

1Beijing Children's Hospital, Capital Medical University, Beijing, China, 2Beijing Key Laboratory for Genetics of Birth Defects, Beijing Pediatric Research Institute, Beijing, China, 3Genetics and Birth Defects Control Center, National Center for Children's Health, Beijing, China, 4MOE Key Laboratory of Major Diseases in Children, Beijing, China

Background: Cantu syndrome (CS) is a congenital rare disorder characterized by congenital hypertrichosis, neonatal macrosomia, a distinct osteochondrodysplasia, and cardiomegaly. It is a dominant condition caused by heterozygous variants in ABCC9 or KCNJ8. Here, we report a baby boy patient who was referred to Cantu syndrome without skeletal manifestations.

Methods: Blood samples and clinical data were collected after informed consent. Trio whole exome sequencing was performed in this patient and his parents. Public population SNP databases (dbSNP147, gnomAD, ExAC and 1000 Genomes project) and in silico predictive algorithms were used to identify the pathogenic variants.

Results: We detected a novel de novo heterozygous missense, likely pathogenic variant (c.3203T>C) in ABCC9 gene. This mutation was located in transmembrane cytoplasmic domain 2 (TMD2) which may be gain-of-function and increase KATP channel activity.

Conclusion: This is the first report of a Chinese baby patient with pathogenic mutation in ABCC9. Our description of the patient’s phenotype would be a part of a spectrum of features associated with Cantu syndrome. The unreported mutation extends the genetic spectra of ABCC9 and emphasizes the usefulness of WES for genetic diagnosis in clinical context. Keywords: Cantu syndrome, ABCC9 gene, whole exome sequencing, cardiovascular abnormalities.

Grant references: This work was partially supported by the Ministry of Science and Technology of China (2016YFC1000306), and the Beijing Municipal Commission of Health and Family Planning Foundation (PXM2017_026274_000001).

R. Guo: None. C. Hao: None. S. Qian: None. W. Li: None.

P05.09A RASA1 mosaic mutations in patients with capillary malformation - arteriovenous malformation

N. Revencu1, E. Fastre1, M. Ravoet1, R. Helaers2, P. Brouillard2, A. Bisdorff-Breson3, C. W. Chung4, M. Gerard5, V. Dvoravoka6, A. D. Irvine6, L. Boon7, M. Vikkula2

1Center for Human Genetics, Cliniques universitaires Saint-Luc, Université catholique de Louvain, Brussels, Belgium, 2Human Molecular Genetics, de Duve Institute, Université catholique de Louvain, Brussels, Belgium, 3Service de Neuroradiologie, Hopital Lariboisière, Paris, France, 4Department of Clinical Genetics, Liverpool Hospital, Liverpool, Australia, 5Service de Génétique, Centre hospitalier universitaire de Caen – Hôpital Clémenceau,, Caen, France, 6Paediatric dermatology, Our Lady’s Children’s Hospital Crumlin, National Children’s Research Centre and Trinity College, Dublin, Ireland, 7Centre for Vascular Anomalies, Division of Plastic Surgery, Cliniques universitaires Saint-Luc, Université catholique de Louvain, Brussels, Belgium

Background: Capillary malformation-arteriovenous malformation is an autosomal dominant disorder, characterised by capillary malformations and increased risk of fast-flow vascular malformations, caused by loss-of-function mutations in the RASA1 or EPHB4 genes. Around 25% of the patients do not seem to carry germline mutation in either one of these two genes. While some of those 25% of patients may have mutations in as-yet-unidentified genes, mutations in RASA1 or EPHB4 that escape detection by less sensitive techniques, such as post-zygotic mosaic mutations, are also possible explanations.

Methods: DNA was extracted from peripheral blood lymphocytes, saliva or vascular malformation tissues from 4 patients. RASA1 and EPHB4 coding regions and exon/intron boundaries were analysed by targeted custom gene panel sequencing. A second panel and/or Sanger sequencing were used to confirm the mutations identified.

Results: Four distinct mosaic RASA1 mutations, with an allele frequency ranging from 3% to 25%, were identified in the 4 index patients with classical capillary malformation - arteriovenous malformation phenotype. Three mutations were known, one was novel. In one patient, a somatic second-hit was also identified. One index case had three affected children, illustrating germline mosaicism.

Conclusion: This study shows that RASA1 mosaic mutations can cause capillary malformation - arteriovenous malformation. Thus, highly sensitive sequencing techniques should be considered as diagnostic tools, especially for patients with no family history. Even low-level mosaicism can cause the classical phenotype and increased risk for offspring. In addition, our study further supports the second-hit pathophysiological mechanism to explain the multifocality of vascular lesions in this disorder.

N. Revencu: None. E. Fastre: None. M. Ravoet: None. R. Helaers: None. P. Brouillard: None. A. Bisdorff-breson: None. C.W. Chung: None. M. Gerard: None. V. Dvoravoka: None. A.D. Irvine: None. L. Boon: None. M. Vikkula: None.

P05.10B Unique cardiogenetic clinic- our experience from 300 patients from Israeli diverse ethnic population

N. Ruhrman Shahar1, A. Oz1, D. Monakier2, I. Maya1, L. Salzer Sheelo1, L. Basel - Salmon1,3, Y. Goldberg1, L. Bazak1

1Recanati Genetic Institute, Rabin Medical Center, Petah Tikva, Israel, 2Cardiology Dept. Beilinson Hospital, Rabin Medical Center, Petah Tikva, Israel, 3Sackler Faculty of Medicine, Tel Aviv Universit, Tel Aviv, Israel

Introduction: Genetic heart diseases are a major public health problem worldwide. Cardio-genetics is emerging as a main discipline for deciphering multiple cardiac pathologies, with immediate clinical effects on diagnosis, treatment, prevention and prognosis for patients and their family members.

Methods: We have established the first designated cardio-genetics clinic in Israel as part of a tertiary hospital two years ago. We retrospectively reviewed all our patient's files from the last 2 years.

Results: Three hundred patients from 200 families visited our clinic and they represent variant population from all over Israel both from Jewish and non Jewish origins. The patients were referred from both pediatric and adults cardiologists, cardiovascular surgeons and primary care physicians.

Most visits (90%) in our clinic were as outpatients and only 10% were acutely hospitalized.

The patients were referred due to: congenital heart diseases in 10%, hypertrophic cardiomyopathy in 52%, dilated cardiomyopathy in 14%, arrhythmogenic right ventricle dysplasia in 8%, left ventricular non- compaction in 4%, channelopathies in 10% and 2% due to aborted sudden death.

The patients underwent a genetic investigation in different methods according to the clinical presentation: Sanger sequencing for known familial/founder mutation, single gene sequencing, NGS based gene panel sequencing, Whole Exome Sequencing and chromosomal microarray.

We have identified the underlying genetic cause in approximately 40% of the families - the percentage varies between the different cardiac pathologies.

Summary: Cardio-genetics clinic is a useful tool both for the patient, families and the primary care physician in various Genetic heart disease.

N. Ruhrman Shahar: None. A. Oz: None. D. Monakier: None. I. Maya: None. L. Salzer Sheelo: None. L. Basel - Salmon: None. Y. Goldberg: None. L. Bazak: None.

P05.11C Molecular diagnostic testing of inherited cardiomyopathies by targeted Next-Generation Sequencing in a public hospital

M. J. Gamundi, B. Mañé, A. I. Jaber, D. López, M. D. Martínez, J. Martínez, C. Moure, A. Sánchez, A. Vancells, J. M. Viguer, I. Hernan, E. Borràs, M. Carballo

Hospital de Terrassa (CST), Terrassa, Spain

Introduction: Inherited cardiomyopathies (CMs) are a major cause of heart disease. They can be classified in hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM), arrhythmogenic right ventricular cardiomyopathy (ARVC), restrictive cardiomyopathy (RCM) and left ventricular non-compaction (LVNC). Due to their clinical and genetic heterogeneity, targeted-gene Next-Generation-Sequencing (NGS) can be a helpful tool in their diagnosis and to early detect them in pre-symptomatic carriers.

Materials and Methods: 39 DNA samples of patients affected with different CMs were analyzed by targeted-NGS using Cardio-GeneSGKit® (n=238 genes) or Cardio-GeneSGKit® MCP (n=90 genes). Results were filtered and analyzed with GeneSystems® platform and informed to patients. Co-segregation analysis and/or carrier testing were performed when possible.

Results: 7 pathogenic/probably pathogenic variants were detected in 5 HCM patients and 2 DCM patients. In HCM patients, mutations were found in MYBPC3 (p.Arg495Gln, p.Gly532Alafs*23 and p.Arg597Gln), CAV3 (p.Thr78Met), TNNC1 (p.Ala8Val) and TNNI3 (p.Arg145Gln). In DCM patients, mutations were detected in DSP (p.Leu1773Tyrfs*8) and TTN (p.Pro28946Leufs*6). Also, 17 variants of unknown significance (VOUS) were detected in 14 patients affected with HCM (n=10), DCM (n=3) and ARVC (n=1). These VOUS were mainly detected in MYBPC3 (n=7) but also in other genes as DSC2 (n=1), FHL1 (n=1), FLNC (n=1), LDB3 (n=1), LMNA (n=1), MYH7 (n=2), PKP4 (n=1), SCN5A (n=1) and TNNT2 (n=2).

Conclusions: 19 of the 39 analyzed patients (48.7%) presented a clinically significant variant in one or two genes associated with CMs. Detection of pathogenic/probably pathogenic variants in probands can help to improve the diagnostic, prognostic and treatment in them and in pre-symptomatic carriers.

M.J. Gamundi: None. B. Mañé: None. A.I. Jaber: None. D. López: None. M.D. Martínez: None. J. Martínez: None. C. Moure: None. A. Sánchez: None. A. Vancells: None. J.M. Viguer: None. I. Hernan: None. E. Borràs: None. M. Carballo: None.

P05.12D Something old, something new: two syndromes associated with pediatric-onset cardiomyopathy

J. C. Herkert, A. J. van Essen, I. M. van Langen

University of Groningen, University Medical Center Groningen, Groningen, Netherlands

Introduction: Hypertrophic and dilated cardiomyopathies (HCM/DCM) are inherited heart muscle disorders. They are isolated in the majority of patients, but are often accompanied by extracardiac features in children. Distinguishing children with one syndromic disorder from those with two inherited conditions is important because this may affect management options, cardiac surveillance in relatives and reproductive decision-making.

Methods and Results: We present two patients with intellectual disability (ID) and cardiomyopathy. Patient 1 presented with paroxysmal ventricular tachycardia at age 3 months. At age 5 she showed severe HCM, prolonged QTc, severe ID, epilepsy and cerebral vision impairment. Exome sequencing (ES) revealed a damaging c.247C>T, p.(Arg83Cys) variant in NAA10, which encodes a ribosomal protein involved in N-terminal acetylation. Several X-linked NAA10 variants have been associated with genetic disorders, but cardiomyopathy had not described until recently. Patient 2 was diagnosed with severe DCM at age 4 months, mild ID, microcephaly (-6.2SDS), failure to thrive and clinodactyly. ES revealed compound heterozygous variants in CEP135. Biallelic variants in CEP135 have also been reported in two families with microcephaly. Interestingly, most of Patient 2’s clinical features overlap with those of five previously reported unrelated patients with autosomal recessive inherited microcephaly-cardiomyopathy syndrome with unknown genetic etiology. Genematcher identified another patient with neonatal cardiomyopathy, failure to thrive, microcephaly and biallelic variants in CEP135. CEP135 may thus have a role in cardiomyopathy.

Conclusions: These two cases illustrate that ES is a valuable diagnostic tool for pediatric-onset cardiomyopathy that can identify novel features in well-known syndromes or novel genes associated with pediatric cardiomyopathy.

J.C. Herkert: None. A.J. van Essen: None. I.M. van Langen: None.

P05.13A Cardiomyopathy: genomic-diagnostic approach

L. Piherova1, K. Hodanova1, H. Hartmannova1, D. Musalkova1, M. Kubanek2, A. Krebsova2, S. Kmoch1

1RURD, Prague 2, Czech Republic, 2Institute for Clinical and Experimental Medicine (IKEM), Department of Cardiology, Prague, Czech Republic

Cardiomyopathy is a disease of the heart muscle associated with a disorder of its function. This is a heterogeneous group of diseases with various clinical signs that can ultimately lead to heart failure. A significant proportion of cardiomyopathies is genetically determined. In this group can be found congenital heart defects, muscular dystrophy and congenital myopathy, hereditary disorders of metabolism and genetic syndromes, (rasopathies). The use of the whole exome sequencing is currently one of the most effective tools for elucidating the genotype of individual patients. We have successfully used this method for studying of genetic architecture of dilated cardiomyopathy cohort where in about 80% of the 460 examined patients we have found probably causal variant. Most frequently affected genes were TTN (18%), FLNC (4%), MYBPC3 (4%), MYH7 (4%), DSP (4%), RBM20 (3%), TNNT2(3%), DES (2%), LMNA (1%). We have also found CNV variations in DMD, LAMP2, FLNC, LMNA and MYH7 genes, which were predicted to cause major structural and functional abnormalities of the affected genes. We are also focusing on paediatric forms of cardiomyopathy which are more complex and the clarification is below 50%. Successful molecular biology diagnostic helps to identify the risk of occurrence of the disease in the family and to provide prenatal diagnosis. In selected cases, the phenotype can also be studied in cell models, which contribute to the understanding of the molecular mechanism of the disease and allow a more accurate interpretation of extreme clinical or laboratory findings. LM201509 NCLG, AZV 15-27682A, AZV 15-28208A, SVV-260367

L. Piherova: None. K. Hodanova: None. H. Hartmannova: None. D. Musalkova: None. M. Kubanek: None. A. Krebsova: None. S. Kmoch: None.

P05.14B 11 years of the Queensland Cardiac Genetics Service

R. Jonathan1, J. McGaughran1, J. Atherton2,3

1Genetic Health Queensland, Brisbane, Australia, 2Royal Brisbane and Women's Hospital, Brisbane, Australia, 3University of Queensland, Brisbane, Australia

Introduction: A dedicated conjoint cardiac genetics clinic (CGC) was established through Genetic Health Queensland and the Cardiology Department at the Royal Brisbane and Women’s Hospital in 2007. The aim of this study was to characterize the patient cohort seen and assess the uptake of genetic testing and diagnostic rates achieved.

Methods: We explored the local databases and patient charts to characterise the age, sex, referral phenotype and incidence of genetic testing for all patients who had attended the CGC. The outcomes of genetic testing, and number of at risk relatives screened were also calculated. Unsolved cases who would benefit from further testing with Whole Genome Sequencing were identified.

Preliminary Results: Over 1400 individuals were seen for the first time during the 11-year period and over 900 have been characterized to date. 488 were probands, the majority (265) of whom were referred for a cardiomyopathy. 141 were referred for an arrhythmia syndrome, and 72 had had a cardiac arrest or were the relative of an individual with a sudden cardiac death. 385 probands were tested, with a genetic diagnosis being achieved in 164 (43%). The highest diagnostic rate was achieved in patients with cardiomyopathies (47%). Predictive testing was performed in a further 415 patients.

Conclusions: We have reviewed the characteristics of a large cohort of cardiac genetics patients and compared our diagnostic rates to those in the literature. We have identified unsolved families who will benefit from further testing with contemporary technologies.

R. Jonathan: None. J. McGaughran: None. J. Atherton: None.

P05.15C Structural variants in Alpha-actinin 2 are associated with cardiomyopathy and hypertrophy in human cardiac tissue and iPSC-derived cardiomyocytes

M. E. Lindholm, H. Zhu, Y. Huang, E. A. Ashley, M. Wheeler

Stanford Cardiovascular Institute, School of Medicine, Stanford University, Stanford, CA, United States

Background: In striated muscle, alpha-actinin 2 is a critical cytoskeletal protein that anchors actin filaments within the sarcomere. Rare mutations in ACTN2 have been associated with cardiac abnormalities, including arrhythmias and cardiomyopathy. However, the mechanisms behind how dysfunctional ACTN2 causes cardiac malfunction is not known. The aim of the present study was to investigate the effects of two novel ACTN2 variants on human cardiac tissue and patient-specific iPSC-derived cardiomyocytes.

Methods and Results: We identified patients in the Stanford Center for Inherited Cardiovascular Disease database with novel ACTN2 variants using a custom mutation pipeline optimized for rare variant discovery. We identified one patient homozygous for a stop-gain mutation (p.Q860X) in ACTN2 and a family with an exon 8-10 deletion. In heart transplant tissue of the homozygous patient, we observed mild hypertrophy and interstitial fibrosis. There was no evidence of variation in ACTN2 protein expression, indicating absence of nonsense mediated decay. We used siRNA to knock down ACTN2 in neonatal rat ventricular cardiomyocytes and a human myoblast cell line and observed dramatic changes in cell size and morphology. Patient-derived iPSC-cardiomyocytes were hypertrophic, displayed sarcomeric structural disarray and had slower contractile velocity compared to control iPSC-cardiomyocytes. Using Co-Immunoprecipitation for ACTN2, followed by mass-spectrometry, we identified missing protein-protein interactions in the patient with the truncated ACTN2.

Conclusions: Here, we provide evidence that two structural genetic variants in ACTN2 are associated to contractile dysfunction and lead to cardiac abnormalities, including hypertrophy and arrhythmia.

No specific grant funded this study.

M.E. Lindholm: None. H. Zhu: None. Y. Huang: None. E.A. Ashley: E. Ownership Interest (stock, stock options, patent or other intellectual property); Significant; Personalis, DeepCell. F. Consultant/Advisory Board; Modest; SequenceBio, Genome Medical. M. Wheeler: None.

P05.16D Genetic cardiomyopathies revisited: reevaluating current gene panel testing

D. Dooijes, M. A. Siemelink, J. J. van der Smagt, R. L. E. van Loon, J. G. Post, J. F. van der Heijden, F. W. Asselbergs, A. F. Baas, J. P. van Tintelen

UMC Utrecht, The Netherlands, Utrecht, Netherlands

Introduction: There is an ongoing debate on the genes that should be examined during genetic evaluation of idiopathic cardiomyopathy patients. Although several genes have sufficient evidence to be cardiomyopathy-related, for other genes this remains unclear. These genes may however cause a significant burden on variant evaluation. In order to make an evidence-based estimation of the value of genes examined, we assessed results of cardiomyopathy gene panels after reevaluation and reclassification of variants according to current criteria.

Materials and Methods: All results of a 64-gene NGS-cardiomyopathy panel performed between 2014 and 2018 in the UMC Utrecht, the Netherlands were retrieved. The pathogenicity of all gene variants was reevaluated according to current criteria by an experienced clinical laboratory specialist in cardiogenetics. Benign or likely benign variants were excluded from analysis. Summary results were determined for all individual genes.

Results: 1264 cardiomyopathy panels performed showed 848 variants (657 unique) in 638 patients (overall yield 50.5%). Of the 848 variants, 124 variants were reclassified (15%), of which 117 were downgraded (94%). Reclassification was mostly due to a high frequency in control populations or based on in-house clinical data (e.g. non-segregation). 28 genes were determined to be of limited value in regular pan-cardiomyopathy gene panel testing because of the absence of relevant variants, published literature (including ClinGen gene adjudication).

Conclusions: Cardiomyopathy NGS-gene panels performed on 1264 patients, identified 28 genes of limited value in regular genetic testing for cardiomyopathy. In addition, state-of-art reevaluation of variants resulted in a notable downgrading of previously identified variants.

D. Dooijes: None. M.A. Siemelink: None. J.J. van der Smagt: None. R.L.E. van Loon: None. J.G. Post: None. J.F. van der Heijden: None. F.W. Asselbergs: None. A.F. Baas: None. J.P. van Tintelen: None.

P05.17A Circadian clock genes and myocardial infarction in patients with type 2 diabetes mellitus

I. Škrlec1, J. Milić2, I. Cilenšek3, D. Petrovič3, B. Peterlin4

1Department of Biology and Chemistry, Faculty of Dental Medicine and Health, J. J. Strossmayer University of Osijek, Osijek, Croatia, 2Faculty of Medicine, J. J. Strossmayer University of Osijek, Osijek, Croatia, 3Institute of Histology and Embryology, Faculty of Medicine, University Ljubljana, Ljubljana, Slovenia, 4Clinical Institute of Medical Genetics, University Medical Center Ljubljana, Ljubljana, Slovenia

Disruption of circadian clock may trigger the onset of diabetes mellitus and myocardial infarction. Type 2 diabetes mellitus (T2DM) is well-known risk factors for cardiovascular diseases and myocardial infarction. Several physiological factors can stimulate the emergence of T2DM and myocardial infarction, and some of these factors are known to oscillate with circadian rhythms. This study aimed to explore a possible association of the genetic variability in the circadian clock genes ARNTL, CLOCK, and PER2 with myocardial infarction in patients with type 2 diabetes mellitus as an additional risk factor for myocardial infarction. The study group consisted of 231 patients with myocardial infarction and T2DM and a control group of 426 T2DM patients. Altogether, 8 SNPs were tested, three in ARNTL gene (rs3789327, rs4757144 and rs12363415), three in CLOCK (rs11932595, rs6811520 and rs13124436), and two in PER2 (rs35333999 and rs934945). The significance of association for individual SNP was calculated to compare the allelic frequency and genotype distribution in patients and control participants using the Chi-Square test. After using Bonferroni correction significant difference in the distribution of ARNTL rs12363415 polymorphism genotypes were found in patients with myocardial infarction and T2DM in comparison to controls, with a p-value of 1.42x10-4 and odds ratio equal to 7.37 (95% CI: 4.15 to 13.08). Other SNPs in ARNTL, CLOCK, and PER2 genes were not significant additional risk factors for myocardial infarction in T2DM patients. We provide evidence that genetic variation in ARNTL gene might be an additional risk factor for myocardial infarction in T2DM patients.

I. Škrlec: None. J. Milić: None. I. Cilenšek: None. D. Petrovič: None. B. Peterlin: None.

P05.18B Clinical Utility of Next Generation Sequencing (NGS) Panel Testing in Patients with a Clinical Suspicion of Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT)

E. H. Seppälä, I. Saarinen, J. Tallila, J. Hathaway, S. Tuupanen, H. Turpeinen, T. Kangas-Kontio, J. Schleit, J. Tommiska, E. Salminen, P. Salmenperä, J. Sistonen, M. Gentile, S. Myllykangas, J. Paananen, T. Alastalo, J. Koskenvuo

Blueprint Genetics, Helsinki, Finland

Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT) is a rare, but potentially fatal channelopathy. Genetic testing may be used to confirm a diagnosis in unclear cases and therefore, is increasingly being performed in a heterogeneous patient population. This is a retrospective review of 134 patients with clinical suspicion of CPVT referred for genetic testing at Blueprint Genetics over a 5-year period (2013-2018). Genetic test results were compiled. A sub-analysis of diagnostic RYR2 variants (location, segregation) was performed. A pathogenic (P) or likely pathogenic (LP) variant was identified in 27 patients (20.1%). Twenty patients (14.9%) had a diagnostic finding in a CPVT-associated gene: 62.9% in RYR2, 7.4 % in CALM1, and 3.7% in CASQ2 (biallelic). Four patients (14.8%) had a P or LP variant in KCNQ1, KCNJ2 or SCN5A and three (11.1%) had a P or LP variant in a cardiomyopathy-associated gene (DSG2, DSP or PLN). All P/LP RYR2 variants were missense, except a deletion encompassing exon 3. Parental testing was performed in 11/17 cases where P/LP RYR2 variants were found; 8 (72.7%) variants were de novo. Enrichment of P/LP RYR2 variants in the four described hotspots (OR 50, 95% CI 29-85, P<0.0001) was observed. In conclusion, 37% of patients with a diagnostic test result had a clinically significant variant in a gene other than RYR2. Interestingly, 26% of these patients had genetic diagnosis of a cardiomyopathy or a channelopathy other than CPVT. This study supports the utilization of broad NGS panels for patients with a clinical suspicion of CPVT.

E.H. Seppälä: A. Employment (full or part-time); Significant; Blueprint Genetics. I. Saarinen: A. Employment (full or part-time); Significant; Blueprint Genetics. J. Tallila: A. Employment (full or part-time); Significant; Blueprint Genetics. J. Hathaway: A. Employment (full or part-time); Significant; Blueprint Genetics. S. Tuupanen: A. Employment (full or part-time); Significant; Blueprint Genetics. H. Turpeinen: A. Employment (full or part-time); Significant; Blueprint Genetics. T. Kangas-Kontio: A. Employment (full or part-time); Significant; Blueprint Genetics. J. Schleit: A. Employment (full or part-time); Significant; Blueprint Genetics. J. Tommiska: A. Employment (full or part-time); Significant; Blueprint Genetics. E. Salminen: A. Employment (full or part-time); Significant; Blueprint Genetics. P. Salmenperä: A. Employment (full or part-time); Significant; Blueprint Genetics. E. Ownership Interest (stock, stock options, patent or other intellectual property); Significant; Blueprint Genetics. J. Sistonen: A. Employment (full or part-time); Significant; Blueprint Genetics. M. Gentile: 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. J. Paananen: A. Employment (full or part-time); 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. J. Koskenvuo: A. Employment (full or part-time); Significant; Blueprint Genetics. E. Ownership Interest (stock, stock options, patent or other intellectual property); Significant; Blueprint Genetics.

P05.19C Mutations in genes involved in MAPK pathway cause lymphatic anomalies

D. Li

Center for Applied Genomics, Philadelphia, PA, United States

Complex lymphatic anomalies include a variety of diagnoses: lymphangiectasia, central conducting lymphatic anomaly (CCLA), generalized lymphatic anomaly, kaposiform lymphangiomatosis, and Gorham Stout disease. The overlapping of diagnostic criteria for these disorders suggests that a common pathway rather than a common gene is responsible for the various clinical syndromes. Upon sequencing 45 such patients, we identified seven pathogenic variants in five genes encoding components of RAS/MAPK signaling (BRAF, KRAS, SOS1, RASA1, and PTPN11) in seven unrelated patients. Additionally, we identified a recurrent somatic missense mutation in a candidate gene, ARAF (c.640T>C:p.S214P), as the basis for CCLA in two unrelated patients. ARAF encodes for serine/threonine-protein kinase A-Raf. Little is known about ARAF apart from the enzyme being involved in MAPK pathway with no previous involvement in lymphatic disease report. Primary endothelial cell studies showed the mutation altered actin skeleton and VE-cadherin organization, which were fully reversed by inhibition of MEK signaling. Functional relevance of the mutation was also validated by recreating a lymphatic phenotype in a zebrafish model, with rescue of the anomalous phenotype using a MEK inhibitor. Subsequent therapy of the lead proband with a MEK inhibitor led to dramatic clinical improvement, with remodeling of the patient’s lymphatic system with resolution of the lymphatic edema, marked improvement in his pulmonary function tests, cessation of supplemental oxygen requirements, and near normalization of daily activities. Taken together, our work demonstrates the realization of precision medicine approach enabled through a novel genetic discovery in a patient with lymphatic anomaly of a previously unknown etiology.

D. Li: None.

P05.21A Copy number variants detection by microarray and multiplex ligation-dependent probe amplification in congenital heart diseases

O. Nagy1, K. Szakszon2, D. Nagy3, G. Mogyorósy2, B. Biro4, B. Nagy5, I. Balogh1, A. Ujfalusi1

1Division of Clinical Genetics, Department of Laboratory Medicine, Faculty of Medicine, University of Debrecen, Debrecen, Hungary, 2Department of Pediatrics, Faculty of Medicine, University of Debrecen, Debrecen, Hungary, 3Department of Medical Genetics, Faculty of Medicine, University of Szeged, Szeged, Hungary, 4First Department of Obstetrics and Gynecology, Semmelweis University, Budapest, Hungary, 5Department of Human Genetics, Faculty of Medicine, University of Debrecen, Debrecen, Hungary

Congenital heart diseases (CHDs) are the most common birth defects among life births, which could be presented as isolated or syndromic with other congenital malformations. The etiology of CHD largely unknown, genetic and environmental factors contribute to the disease. Recurrent copy number variants (CNVs) have been reported in the pathogenesis of CHD. The aim of this study was to evaluate the clinical utility of multiplex ligation-dependent probe amplification (MLPA) and microarray analyses on isolated and syndromic CHD cases and to explore the relationship between identified CNVs and CHD.

Materials and Methods: Cytoscan 750K array (Affymetrix) and MLPA SALSA P250 and P311 kits (MRC-Holland) were used in the study. The identified CNVs were confirmed by fluorescence in situ hybridization. Results. Eighteen prenatal samples, 16 isolated and 33 syndromic patients with mild to severe CHD phenotype were studied. Prenatal and isolated CHD cases did not show pathogenic CNVs. Clinically significant CNVs were detected in 7/33 (21%) syndromic CHD patients: del 22q11.2 (n=2), 8p23.1 duplication (n=2), deletion 5p (n=1), deletion 6q21-q22 (n=1), unbalanced translocation causing partial deletion of 4q34.3 and duplication of 6q25.1 (n=1).

Conclusion: The identified genomic imbalances contain genes that has been associated with human CHD before. The present study demonstrates that using microarray and MLPA analysis increases the detection rate of causal CNVs in individuals with syndromic CHD. This study was supported by the Ministry of National Economy, Hungary (GINOP-2.3.2-15-2016-00039).

O. Nagy: None. K. Szakszon: None. D. Nagy: None. G. Mogyorósy: None. B. Biro: None. B. Nagy: None. I. Balogh: None. A. Ujfalusi: None.

P05.24D Adipokine profile of adipocytes isolated from different fat depots of coronary artery disease patients

M. Sinitsky1,2, Y. Dyleva1, E. Uchasova1, E. Belik1, O. Gruzdeva1,3, A. Ponasenko1

1Research Institute for Complex Issues of Cardiovascular Diseases, Kemerovo, Russian Federation, 2Federal Research Center of Coal and Coal Chemistry of SB RAS, Kemerovo, Russian Federation, 3Kemerovo State Medical Academy, Kemerovo, Russian Federation

Introduction: Adipose tissue is interesting in the context of its role in pathogenesis of cardiovascular diseases. Human adipokinome consists of hundreds of molecules many of which have not yet been well characterized and have variable expression in different adipose depots.

Materials and Methods: We compared DPP4, LCN2, NAMPT, ITLN1, APLN and ADIPOQ mRNA levels in adipocytes isolated from the biopsies of subcutaneous (SCAT), epicardial (EAT) and perivascular (PVAT) adipose tissue obtained from 25 patients with coronary artery disease. Gene expression signature was determined by RT-qPCR with hydrolysis probes.

Results: We found that DPP4 and APLN mRNA expression was significantly (P<0.05) increased only in adipocytes isolated from epicardial adipose tissue compared to the subcutaneous fat. The ITLN1 gene was overexpressed in epicardial adipose tissue compared to both subcutaneous and perivascular tissues and expression of ADIPOQ gene was reduced both in EAT and PVAT compared to SCAT. Moreover, APLN mRNA expression was positively correlated with total and LDL cholesterol plasma level, and DPP4 mRNA expression - with VLDL cholesterol concentration. We found no significant changes in LCN2 and NAMPT mRNA level between studied adipose depots.

Conclusions: Adipocytes isolated from different adipose depots are characterized by differential gene expression of adipokines. EAT is of particular interest in the context of its function, molecular and genetic mechanisms of regulation of the cardiovascular system and as a therapeutic target for correction of adipose tissue-induced effects on health. This work was supported by RSF grant No. 17-75-20026.

M. Sinitsky: None. Y. Dyleva: None. E. Uchasova: None. E. Belik: None. O. Gruzdeva: None. A. Ponasenko: None.

P05.26B Cardiac arrhythmias at baseline predict a clinically relevant genetic yield in idiopathic non familial DCM affecting long term outcome

J. Verdonschot1, M. T. H. M. Henkens1, A. G. Raafs1, P. Wang1, J. J. Merken1, G. R. F. Claes1, I. P. C. Krapels1, E. K. Vanhoutte1, A. van den Wijngaard1, H. G. Brunner1,2, M. R. Hazebroek1, S. R. B. Heymans1

1Maastricht University Medical Center, Maastricht, Netherlands, 2Radboud University Medical Center, Nijmegen, Netherlands

Background: Current guidelines recommend genetic analysis only in familial dilated cardiomyopathy (DCM) patients without acquired disease. The genetic yield in so-called acquired and/or non-familial DCM remains largely unknown.

Methods: This study included 689 DCM patients who all underwent genetic evaluation using a 47 cardiomyopathy-associated gene panel and had complete cardiac diagnostic work-up including echocardiography, cardiac MRI, endomyocardial biopsies and holter monitoring. Environmental triggers were predefined as viral, inflammatory, toxic, electrical and systemic auto-immune disease.

Results: At least one etiology (genetic and/or environmental) was found in 530 patients (77%) after complete diagnostic workup, of which 159 (23%) had a genetic mutation. One in five of the DCM patients (n=90; 20%) had a combination of a genetic and acquired (environmental) trigger. All of the acquired triggers had a genetic yield around 20%. Although familial DCM had a significant higher yield of genetic mutations compared to non-familial DCM (43% versus 16%, p<0.001), pathogenic mutations were still present in 1 of 6. In non-familial DCM patients with arrhythmias at baseline without environmental trigger, the genetic yield was even 39%, comparable to the yield in familial DCM (43%; p=0,17). Also, genetic DCM has a worse prognosis compared to non-genetic DCM (Log-Rank=0.008), irrespective of familial history (Log-Rank=0.29).

Conclusions: One in five DCM patients with an acquired trigger and/or absence of familial history has an underlying genetic mutation. Non-familial DCM patients with arrhythmias and no clear etiology have a comparable genetic yield compared to familial DCM.

J. Verdonschot: None. M.T.H.M. Henkens: None. A.G. Raafs: None. P. Wang: None. J.J. Merken: None. G.R.F. Claes: None. I.P.C. Krapels: None. E.K. Vanhoutte: None. A. van den Wijngaard: None. H.G. Brunner: None. M.R. Hazebroek: None. S.R.B. Heymans: None.

P05.27C A novel truncating mutation in DSP (Desmoplakin) causes autosomal dominant dilated cardiomyopathy with variable penetrance

L. Camerota1, V. Ferradini2, R. Petroni3,4, R. Monetta4,1, S. Romano3, G. Ussia5, F. Sangiuolo6, F. Brancati1,4

1Medical Genetics Division, Department of Life, Health and Environmental Sciences, University of L'Aquila, L'Aquila, Italy, 2Department of Biomedicine and Prevention, University of Rome Tor Vergata, Rome, Italy, 3Department of Cardiology, University of L'Aquila, L'Aquila, Italy, 4Laboratory of Molecular and Cell Biology, Istituto Dermopatico dell'Immacolata IDI-IRCCS, Rome, Italy, 5Facoltà Dipartimentale di Medicina e Chirurgia, University Campus Bio-Medico, Rome, Italy, 6Department of Biomedicine and Prevention, University of Rome Tor Vergata, L'Aquila, Italy

Background: Arrhythmogenic Cardiomyopathy (AC) is mainly determined by mutations in desmosomal genes. Approximately 75% of AC patients show bi-ventricular involvement and left-dominant forms are not uncommon. Loss-of-function mutations in Desmoplakin (DSP) cause a form of AC often manifesting with severe arrhythmias and sudden cardiac death (SCD). Less is known about DSP mutations in dilated cardiomyopathy (DCM) regarding (endo)phenotype, penetrance, and management.

Materials and Methods: A family with DCM and SCD was referred to our cardiogenetics outpatient clinic. Accurate genetic and cardiologic assessments were performed. DNA sequencing was initiated in the proband using NGS (cardiopanel).

Results: A novel variant in DSP c.4639dupC was identified, absent in gnomAD, locus-specific databases and predicted as deleterious by dedicated software, affecting cardiac-specific isoform and leading to frameshift p.(Gln1547ProfsTer80). The 60-year-old proband and her three nieces (aged 32-45) were diagnosed with DCM and ICD implanted. Five siblings (aged 25-61) had SCD. Segregation analysis in asymptomatic relatives identified two mutated siblings (31 and 47 years): one presented an episode of acute myocarditis. Despite detailed cardiologic evaluation, they did not meet current diagnostic guidelines for AC/DCM.

Conclusions: Loss-of-function DSP mutations cause different phenotypes including AC and DCM. A diagnostic grey zone between left-dominant AC and arrhythmogenic DCM exists, indicating the need for updating current classification. Moreover, genotype-based definition is warranted to develop personalized management in patients/asymptomatic-mutated subjects. Intrafamilial phenotypic variability and lack of penetrance demand modifier variants in additional, yet unknown, genes or epigenetic factors.

Funding: Fondi Ricerca Premiale from University of L’Aquila, Italy

L. Camerota: None. V. Ferradini: None. R. Petroni: None. R. Monetta: None. S. Romano: None. G. Ussia: None. F. Sangiuolo: None. F. Brancati: None.

P05.28D Double heterozygosity in a group of patients with rare cardiovascular diseases

S. Josifovska1,2, R. Vazharova3, L. Balabanski2, M. Malinov2, A. Kaneva4, S. Panov1, M. Ganev5, D. Toncheva5

1Laboratory of Molecular Biology, Faculty of Natural Sciences and Mathematics, Ss. Cyril and Methodius University, Skopje, Macedonia, The Former Yugoslav Republic of, 2Genome Centre, GARH Malinov, Sofia, Bulgaria, 3Department of Biology, Medical Genetics and Microbiology, Faculty of Medicine, Sofia University St. Kliment Ohridski, Sofia, Bulgaria, 4Department of Pediatric Cardiology, National Heart Hospital, Sofia, Bulgaria, 5Department of Medical Genetics, Medical University of Sofia, Sofia, Bulgaria

Introduction: Rare cardiovascular diseases (CVD) are heterogeneous and this complicates their diagnosis. The more complex forms of rare CVD tend to result from the integrated effect of multiple genetic variants that create a background of susceptibility requiring sometimes additional factors for the disease to manifest. Whole exome, whole genome and target sequencing offer new possibilities to discover the complex etiology of such diseases. The aim of this study was to identify genetic variants associated with the rare CVD using next generation sequencing (NGS).

Materials and Methods: NGS with TruSight panels (Illumina) was used to analyze DNA samples from 24 patients with rare CVD: Long QT Syndrome, Tetralogy of Fallot, Familial Hypercholesterolemia, suspected Marfan Syndrome, Pathology of great vessels, Cardiomyopathies and other.

Results: In 14 (58%) of the 24 patients, at least one pathogenic or likely pathogenic variant was found. Of those, in 8 patients we detected only one variant, in 3 patients there was a combination of two or more only pathogenic/likely pathogenic variants, and in 3 patients the combination was of pathogenic/likely pathogenic and VUS. Of the remaining 10 patients, in 5 (21%) patients, only VUS were detected (3 with two VUS and 2 with one VUS) and in 5 (21%) patients, we did not detect significant mutations. Overall, in 9 (47%) of the patients with detected variants, more than one variant was found indicating multiple heterozygosity. In conclusion, NGS has a potential to detect multiple heterozygosity in complex rare cardiovascular diseases.

S. Josifovska: None. R. Vazharova: None. L. Balabanski: None. M. Malinov: None. A. Kaneva: None. S. Panov: None. M. Ganev: None. D. Toncheva: None.

P05.29A The causal relationship between dyslipidemia and cardiovascular disease using Mendelian randomization

E. Wan1, T. Wu2, W. Wu1, S. Chen3, A. Yen3, Y. Lee4, C. Tse5, T. Liu6, H. Chen7, M. Lin1

1Institute of Public Health, National Yang-Ming University, Taipei, Taiwan, 2Department of Public Health, Chung-Shan Medical University, Taichung, Taiwan, 3School of Oral Hygiene, College of Oral Medicine, Taipei Medical University, Taipei, Taiwan, 4Department of Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan, 5Bureau of Health and Welfare, Lienchiang County Government, Matsu, Taiwan, 6Lienchiang County Government, Matsu, Taiwan, 7Institute of Epidemiology and Preventative Medicine, College of Public Health, National Taiwan University, Taipei, Taiwan

Background: Cardiovascular disease (CVD) is ranked as the second leading causes of death in Taiwan. Dyslipidemia is one of the risk factors for CVD. The LPL and APOE genes were found to be involved in lipid metabolism. Many studies uncovered the association between dyslipidemia and CVD, however, conventional epidemiological studies may suffer from un-measured confounders and reverse causality. We aimed to elucidate the causal relationship between dyslipidemia and CVD using Mendelian randomization design.

Methods: A total of 1951 Chinese adults were recruited from the Matsu community-based integrated health screening project during 2015-2017. Two SNPs (LPL rs328 and APOE rs157580) were selected as the instrumental variables. Both logistic regression and two-stage least-square instrumental variable (IV) regression were applied to estimate the relationship between the lipid profiles and CVD.

Results: We found rs328 was significantly associated with decreased triglyceride levels (β=-9.105, p=0.012) and increased HDL-C levels (β=1.317, p=0.037), but not with LDL-C levels. The rs157580 was marginally associated with increased LDL-C levels. We further found a significantly protective effect of LDL-C levels on cardiovascular disease (OR=0.987, 95% CI=0.980-0.994), but not with triglycerides, HDL-C and CVD. The IV analysis revealed an increasing risk of LDL-C levels on cardiovascular disease by using rs157580 and allele score as the IVs (rs157580: OR=1.028, 95% CI=1.018-1.038; allele score: OR=1.028, 95% CI=1.019-1.038). Triglyceride and HDL-C were also associated with cardiovascular disease.

Conclusion: We identified the causal relationships between dyslipidemia and cardiovascular disease by using APOE rs157580 and allele score as the instrumental variables. Grant No: MOST 106-2314-B-010-020-MY3

E. Wan: None. T. Wu: None. W. Wu: None. S. Chen: None. A. Yen: None. Y. Lee: None. C. Tse: None. T. Liu: None. H. Chen: None. M. Lin: None.

P05.30B Common atrium, atrioventricular canal defect/postaxial polydactyly: a mild clinical subtype of Ellis-van Creveld syndrome caused by compound heterozygosity for loss of function and hypomorphic EVC mutations

F. Piceci Sparascio1, M. C. Digilio2, A. Palencia-Campos3,4, I. Torrente1, V. Guida1, J. Rosati5, A. D'Anzi5, S. Briuglia6, P. Versacci7, B. Dallapiccola8, V. Ruiz-Perez3,4, B. Marino7, A. De Luca1

1Molecular Genetics Unit, Ospedale Casa Sollievo della Sofferenza, IRCCS, San Giovanni Rotondo, Italy, 2Medical Genetics, Department of Pediatrics, Ospedale Pediatrico Bambino Gesù, IRCCS, Rome, Italy, 3CIBER de enfermedades Raras (CIBERER), Insitituto de Salud Carlos III, Madrid, Spain, 4Instituto de Investigaciones Biomédicas de Madrid, Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid, Madrid, Spain, 5Cellular Reprogramming Unit, Ospedale Casa Sollievo della Sofferenza, IRCCS, San Giovanni Rotondo, Italy, 6Department of Human Pathology of Adult and Childhood "Gaetano Barresi", Unit of Emergency Pediatrics, University of Messina, Messina, Italy, 7Department of Pediatrics, Università Sapienza, Rome, Italy, 8Genetics and Rare Diseases Research Division, Ospedale Pediatrico Bambino Gesù, IRCCS, Roma, Italy

Clinical expression of Ellis van Creveld syndrome (EvCS) is variable with a classic form associated with homozygous/compound heterozygous mutations in EVC and EVC2 genes, and the milder Weyers acrodental dysostosis linked to specific heterozygous mutations in the last coding exon of EVC2. Additional transitional phenotypes have been described, including patients with prevalently cardiac and limb involvement, presenting with atrioventricular canal defect with common atrium (AVCD-CA) and postaxial polydactyly. We report on the results of molecular analysis of a family with vertical transmission in father and daughter of AVCD-CA and postaxial polydactyly. The father was compound heterozygous for the known recessive p.Arg622* nonsense variant and the novel p.Arg663Pro missense change in the EVC gene. His affected daughter was compound heterozygous for the same nonsense variant and the novel splice-site c.1316-7A>G variant. Two additional unaffected sisters were also examined. One was heterozygous carrier of the p.Arg622* variant, while the other was compound heterozygous for both the p.Arg663Pro change and the c.1316-7A>G variant. mRNA sequencing in the affected daughter’s fibroblast showed that c.1316-7A>G resulted in the in-frame insertion of 6 nucleotides between EVC exon 9 and 10, and that the expression of the p.Arg622* corresponding allele was greatly decreased. Consistently, western blot analysis showed a residual amount of EVC protein in the father's and daughter's fibroblasts. Present results suggest that p.Arg663Pro and c.1316-7A>G are hypomorphic EVC alleles that act as genetic modifiers together with a loss of function mutation to cause a milder clinical subtype of EvCS characterized by AVCD-CA and postaxial polydactyly.

F. Piceci Sparascio: None. M.C. Digilio: None. A. Palencia-Campos: None. I. Torrente: None. V. Guida: None. J. Rosati: None. A. D'Anzi: None. S. Briuglia: None. P. Versacci: None. B. Dallapiccola: None. V. Ruiz-Perez: None. B. Marino: None. A. De Luca: None.

P05.31C Heritability and family-based GWAS analyses of the circulating ceramide, endocannabinoid, and N-acyl ethanolamide lipidome

K. McGurk, A. Nicolaou, B. Keavney

University of Manchester, Manchester, United Kingdom

Introduction: Lipids of the endocannabinoid (EC), N-acyl ethanolamine (NAEA), and ceramide (CER) classes are potential novel biomarkers of coronary artery disease and type-2 diabetes. Major-gene effects have been discovered for certain lipid species, notably lipoprotein(a). We sought to establish the heritability of EC, NAEA, and CER species, and identify DNA variants influencing their concentrations in plasma.

Materials and Methods: We undertook heritability (QTDT, GCTA) and GWAS analyses (FaST-LMM) of 11 ECs and NAEAs, and 37 CERs in 1,016 plasma samples from 196 British Caucasian families ascertained through a hypertensive proband, using targeted mass spectrometry and Illumina 660W-Quad genotyping.

Results: NAEAs are more heritable than well-studied EC anandamide (AEA) (h2AEA = 32-35%; P<5.80x10-11). 24-46% of the variation in potential biomarker CER is due to genetic factors (P<1.00x10-7). GWAS identified associations with eQTLs of proteins in their metabolism (e.g. FAAH; PNAEADHEA <6.33x10-12, SPTLC3; PCER N(24)S(18) <8.99x10-19) and novel loci implicated in cancer risk and non-alcoholic fatty liver disease (e.g. FBXO28; PCERN(24)S(19)ratio <1.95x10-8, SULT1C4; PCERN(24)S(19) <8.99x10-19). Two-sample Mendelian randomisation suggests that a variant in FAAH (rs324420) influencing the level of plasma NAEAs (betaNAEADHEA = 0.39; P<6.33x10-12) is causally associated with both obesity and drug addiction.

Conclusions: We demonstrate for the first time estimates of heritability for this extended array of bioactive lipids, identify GWAS-significant SNPs associating with their levels in circulation, and implicate the lipid species studied here in cardiovascular disease, cancer, and drug addition.

K.M. is supported by a MRC Doctoral Award (MR/K501311/1) and Travel Grant from The Company of Biologists.

K. McGurk: None. A. Nicolaou: None. B. Keavney: None.

P05.32D A new zebrafish model for in vivo optical mapping of cardiac action potentials

D. Schepers1, E. Sieliwonczyk1, A. Schlaeppi2, B. Vandendriessche1, E. Simons1, M. Alaerts1, D. Knapen3, J. Huisken2, B. Loeys1

1Center of Medical Genetics, University of Antwerp and Antwerp University Hospital, Antwerp, Belgium, 2Morgridge Institute for Research, Madison, WI, United States, 3Zebrafishlab, Veterinary Physiology and Biochemistry, University of Antwerp, Antwerp, Belgium

Genetically encoded voltage indicators (GEVI) allow optical mapping of voltage changes and offer an alternative method for measuring alterations in cell membrane potentials. Initially driven by the neuroscience field, the development of GEVIs has now found applications in the cardiovascular sciences as well. Thanks to these GEVIs, it has become possible to image cardiac voltage dynamics at the cellular level in vivo in translucent organisms such as zebrafish. Unfortunately, up to now, the fluorescent signal of the cardiac voltage sensors used in zebrafish has been very dim. For this reason, we developed a new stable transgenic zebrafish model, expressing Ace2N-mNeon, a next generation voltage sensor with brighter fluorescence, under the control of the myocardial specific promoter myl7. Using Selective Plane lllumination Microscopy (SPIM), the conduction of individual action potentials in the heart of 3 dpf zebrafish larvae can be visualized in vivo, allowing the measurements of action potential duration and conduction speed. Next, we will apply this new transgenic zebrafish model to evaluate the pathogenicity of genetic variants of unknown significance identified in arrhythmia genes of patients with inherited primary electrical diseases.

D. Schepers: None. E. Sieliwonczyk: None. A. Schlaeppi: None. B. Vandendriessche: None. E. Simons: None. M. Alaerts: None. D. Knapen: None. J. Huisken: None. B. Loeys: None.

P05.33A Diagnostic Yield of Genetic Testing in an Unselected Cohort of 1,376 HCM Patients

J. Koskenvuo, J. Hathaway, I. Saarinen, J. Tallila, E. H. Seppälä, S. Tuupanen, H. Turpeinen, T. Kangas-Kontio, J. Schleit, J. Tommiska, E. Salminen, P. Salmenperä, J. Sistonen, M. Muona, M. Gentile, V. Kytölä, S. Myllykangas, J. Paananen, T. Alastalo

Blueprint Genetics, Helsinki, Finland

Introduction: Genetic testing in Hypertrophic Cardiomyopathy (HCM) is recommended by published guidelines. Genetic testing by NGS panels offers practical differential diagnostic solution.

Objective: Diagnostic yield in a heterogeneous cohort of patients with a suspicion of HCM

Methods: A retrospective review of patients with a suspected clinical diagnosis of HCM referred for genetic testing at Blueprint Genetics between 2013 and 2018 was undertaken. Variants classified as pathogenic (P) or likely pathogenic (LP) at the time of reporting were considered diagnostic.

Results: Diagnostic yield was 26.2% (361/1,376). In total, 373 P/LP variants were identified including 363 variants diagnostic for HCM whereas only 10 were diagnostic for another type of cardiomyopathy based on clinical and genetic interpretation (DES, DSP, LMNA, TTN). Approximately 86% (n=320) of diagnostic variants (23.3% of all tests) involved genes encoding the sarcomere. Seventeen P or LP variants (4.6% of variants and 1.2% of all tests) were in RASopathy genes and thirteen P or LP variants (3.5% of 0.9% of all tests) were in metabolic/infiltrative disease genes. In addition, three patients with non-diagnostic findings for cardiomyopathy had P or LP variants in genes linked to a channelopathy (RYR2, SCN5A) or neurofibromatosis (NF1).

Conclusions: The diagnostic yield of genetic testing in a heterogeneous cohort of patients with a suspected diagnosis of HCM analyzed is lower than what has been reported in well characterized patient cohorts. Importantly, 8% of all diagnostic findings were in metabolic and RASopathy genes which have significant systemic medical management implications.

J. Koskenvuo: None. J. Hathaway: None. I. Saarinen: None. J. Tallila: None. E.H. Seppälä: None. S. Tuupanen: None. H. Turpeinen: None. T. Kangas-Kontio: None. J. Schleit: None. J. Tommiska: None. E. Salminen: None. P. Salmenperä: None. J. Sistonen: None. M. Muona: None. M. Gentile: None. V. Kytölä: None. S. Myllykangas: None. J. Paananen: None. T. Alastalo: None.

P05.34B Psycho-social impact of predictive genetic testing in hereditary heart diseases (PREDICT Study)

C. Bordet1, S. Brice2, C. Maupain1, E. Gandjbakhch1,3, B. Isidor4, A. Palmyre5, A. Moerman6, A. Toutain7, S. Odent8, A. Brehin9, L. Olivier Faivre10, C. Rooryck Thambo11, E. Schaefer12, K. Nguyen13, D. Dupin Deguine14, C. Rouzier15, P. Jouk16, M. Port Lys17, I. Denjoy18, S. Staraci1, R. Mansouri1, M. Hebert1, A. Bekhechi1, I. Raji1, V. Fressart19, F. Ader19, P. Richard19, S. Tezenas du Montcel20,21, M. Gargiulo22,23, P. Charron1,24

1Referral Center for hereditary heart disease, Department of Genetics, Pitié Salpêtrière University Hospital,, Paris, France, 2INSERM, Sorbonne Université, Institut Pierre Louis d’Epidémiologie et de Santé Publique, F75013, Paris, France, 3Sorbonne Universités, UPMC Université Paris 6, AssistancePublique-Hôpitaux de Paris, Hôpital Pitié-Salpêtrière, ICAN, Département de Cardiologie,, Parsi, France, 4Department of Genetics, Nantes University Hospital,, Nantes, France, 5Department of Genetics, Ambroise Paré University Hospital,, Paris, France, 6Department of Genetics, Lille University Hospital, Jeanne de Flandre Hospital,, Lille, France, 7Department of Medical Genetics, Tours University Hospital, Tours, France, 8Department of Medical Genetics, Rennes University Hospital, Rennes, France, 9Department of Genetics, Normandy Centre for Genomic and Personalized Medicine, Rouen University Hospital, Rouen, France, 10Medical Genetics Unit, Dijon University Hospital, Dijon, France, 11Department of Medical Genetics, Bordeaux University Hospital, Bordeaux, France, 12Department of Genetics, Strasbourg University Hospital, Hautepierre Hospital, Strasbourg, France, 13Department of Medical Genetics, APHM, GMGF, Timone Hospital, Aix Marseille University,, Marseille, France, 14Department of Medical Genetics, Toulouse University Hospital, Toulouse, France, 15Department of Medical Genetics, Université Côte d'Azur, CHU, Inserm, CNRS, IRCAN, Nice, France, 16Department of Medical Genetics, Centre Hospitalo-Universitaire Grenoble Alpes, Grenoble, France, 17Clinical Genetics Unit, University Hospital, Pointe-à-Pitre University Hospital, Guadalupe Island,, Pointe à Pitre Guadeloupe, France, 18Department of cardiology, Referral Center for hereditary heart disease, Bichat Hospital, Paris, France, 19UF cardiogenetics, Pitié Salpêtrière University Hospital, Paris, France, 20Sorbonne Université, INSERM, Institut Pierre Louis d’Epidémiologie et de Santé Publique, AP-HP, Hôpitaux Universitaires Pitié Salpêtrière - Charles Foix, Département de Santé Publique, F75013,, Paris, France, 21INSERM UMR-1136, Paris, France, 22Institut of Myologie, Pitié Salpêtrière University Hospital,, Paris, France, 23Sorbonne Université, INSERM, Institut Pierre Louis de Santé Publique, Medical Information Unit, Pitié Salpêtrière University Hospital,, Paris, France, 24Sorbonne Université, INSERM, UMR_S 1166 and ICAN Institute for Cardiometabolism and Nutrition,, Paris, France

Introduction: Hereditary heart diseases are most often characterized by autosomal dominant inheritance and delayed cardiac expression. Predictive genetic testing is offered to asymptomatic relatives to allow targeted medical care with early therapeutics in order to reduce the risk of complications. The aim of this study was to evaluate the psychological and socio-professional impact of predictive genetic testing in hereditary heart diseases.

Patients and Methods: This multicentric French study involved 20 expert centers in hereditary heart diseases. We included 517 adult relatives (42.3±16.7 years, 60.6% females) who performed predictive genetic testing (prospective study: N=264, retrospective study: N=253). The opinion and experience were collected via auto-questionnaires, at various moments in the prospective study, with different items and validated scales (STAI and IES).

Results: In the prospective study, the main motivations for performing the test were: “to remove doubt” (65.3%), “for children” (64.0%), “to benefit from medical supervision” (34.9%). A mutation was present in 39.4% of relatives. No regret was expressed after testing (only 2.3% regrets). The result did not lead to a socio-professional change or family relationship change in 60.7%. Among those who had a change, it was perceived as unfavorable for only 3%. The level of anxiety (STAI scale) increases before the test result and decreased to return to baseline. Subjects with depression history were more likely to develop anxiety at long term (p=0.004).

Conclusions: Our results show no or marginal adverse psychological and socio-professional impact of genetic testing when performed by a team expert in predictive testing.

C. Bordet: None. S. Brice: None. C. Maupain: None. E. Gandjbakhch: None. B. Isidor: None. A. Palmyre: None. A. Moerman: None. A. Toutain: None. S. Odent: None. A. Brehin: None. L. Olivier Faivre: None. C. Rooryck Thambo: None. E. Schaefer: None. K. Nguyen: None. D. Dupin Deguine: None. C. Rouzier: None. P. Jouk: None. M. Port Lys: None. I. Denjoy: None. S. Staraci: None. R. Mansouri: None. M. Hebert: None. A. Bekhechi: None. I. Raji: None. V. Fressart: None. F. Ader: None. P. Richard: None. S. Tezenas du Montcel: None. M. Gargiulo: None. P. Charron: None.

P05.35C Development of a fast and cost effective genetic diagnostic method for familial hypercholesterolemia in Sweden

K. Duvefelt1, E. Hagström2, E. Bachus3, V. Hamrefors3, M. Rehnberg4, S. Romeo5, M. Linde1, P. Kiviluoma1, B. Angelin1, P. Benedek1, J. Kere1, M. Eriksson1

1Karolinska University hospital, Stockholm, Sweden, 2Uppsala University, Uppsala, Sweden, 3Skånes University hospital, Malmö, Sweden, 4Linköping University, Linköping, Sweden, 5Gothenburg University, Gothenburg, Sweden

Familial hypercholesterolemia (FH) is an autosomal dominant disease causing elevated levels of low-density lipoprotein cholesterol, carrying a high risk of premature coronary heart disease such as myocardial infarction. The prevalence of FH is approximately 1/250. There is an obvious need for improving early detection and treatment of FH, which is currently an underdiagnosed condition.

We aimed to characterize the disease-causing mutation spectrum in Sweden, and to develop a cost-effective diagnostics. This was done through iterative improvement of the analytical strategy, consisting of (i) targeted mutation analysis using a panel based on Agena mass spectrometry-based genotyping; (ii) sequencing of samples failing to show mutations; and (iii) redesign of the panel to include new, recurrently found mutations.

To date, we have included 1,143 patients from 14 hospitals across Sweden in a nationwide collaboration using a panel of 113 pathogenic/likely pathogenic mutations in the LDLR, PCSK9 and APOB genes.

Dutch Lipid Clinical Network (DLCN) score was available for 482 patients in whom mutations were detected in 27%. If restricted to patients with probable or definite FH (score ≥6; n=309) pathogenic mutations were detected in 35%. The two most prevalent mutations were NM_000384.2(APOB):c.10580G>A(p.Arg3527Gln) and NM_000527.4(LDLR):c.259T>G(p.Trp87Gly). In total, 38 different mutations were detected by the panel. 142 panel negative samples were sequenced with the SEQPPRO-LIPO method, yielding 15 additional mutations in 16 patients.

In conclusion, our FH-panel detected mutations in approximately one fourth of Swedish patients with suspected FH. The number was increased if analyses were restricted to patients with high DLCN score.

Vinnova 2016-02972

K. Duvefelt: None. E. Hagström: None. E. Bachus: None. V. Hamrefors: None. M. Rehnberg: None. S. Romeo: None. M. Linde: None. P. Kiviluoma: None. B. Angelin: None. P. Benedek: None. J. Kere: None. M. Eriksson: None.

P05.36D A novel candidate gene of familial hypertrophic cardiomyopathy with therapeutic potential

P. Phowthongkum, S. Tongkobpetch, K. Suphapeetiporn, V. Shotelersuk

Faculty of Medicine, Bangkok, Thailand

TNNI3K encodes cardiac troponin I-interacting kinase (TNNI3K), forming homo-dimers or homo-oligomers and involving in the cardiac contractility regulation. Loss-of-function mutations in TNNI3K can lead to dilated cardiomyopathy (MIM616117). No TNNI3K mutations have been found to cause hypertrophic cardiomyopathy in (HCM: MIM192600). We performed a 178 gene-panel enriched and next-generation sequencing in 8 Thai families with HCM. 8 pathogenic (P)/likely pathogenic (LP) variants were found in 7 families (MHY7(4), TPM1(2),TNNI2(1), TTR(1)). One family harbored two variants in MYH7 and TPM1. The remaining family without identified P/LP variants was sent for whole-exome sequencing. We identified a rare spliced TNNI3K (c.1178-2A>G) variant in the 41-year-old male with severe eccentric HCM who presented with syncope from ventricular arrhythmia. His asymptomatic 60-year-old father with apical HCM also harbored this variant. The variant was observed once in the ExAC database and once in our in-house Thai-exome database (>2,000 cases). RNA analysis revealed an altered splicing mRNA predicted to create a protein with only N-terminal Ankyrin repeats without the kinase and the C-terminal serine-rich domain. Since the C-terminal serine rich domain is an inhibitory domain, the identified mutation may represent a gain-of-function variant by diminishing inhibitory signal of the kinase domain. A previous study expressing wild-type human TNNI3K in transgenic-mice found that the mice had cardiac hypertrophy. TNNI3K inhibitor which has undergone preclinical testing can serve as a potential therapy for patients with HCM. In summary, we report a family with a probably gain-of-function mutation in TNNI3K, suggesting that TNNI3K is a new disease gene for HCM.

P. Phowthongkum: None. S. Tongkobpetch: None. K. Suphapeetiporn: None. V. Shotelersuk: None.

P05.37A Circulating miRNAs profiling in diabetic patients with ischemic heart disease

A. Bielska1, M. Niemira1, A. Szalkowska1, J. Raczkowska1, D. Ostrowski1, W. Bauer1, P. Prokopczuk2, S. Dobrzycki2, A. Kretowski1,3

1Clinical Research Centre, Medical University of Bialystok, Białystok, Poland, 2Department of Invasive Cardiology, Medical University of Bialystok, Białystok, Poland, 3Department of Endocrinology, Diabetology and Internal Medicine, Medical University of Bialystok, Bialystok, Poland

Introduction: Due to a global increase of morbidity and mortality with ischemic heart disease (IHD) in type 2 diabetic patients, there is an urgent need to identify of early biomarkers, which would help to predict an individual risk of development of IHD. MiRNAs are small noncoding RNAs which regulate gene expression and the last few years appear as a key tool for understanding the pathophysiology of IHD. Here, we postulate that circulating serum-derived miRNA may serve as potential biomarkers for early IHD diagnosis and help to identify diabetic individuals with a predisposition to develop ischemic heart disease.

Materials and Methods: We obtained serum samples from 39 T2DM patients (22 with IHD and 17 free of complications). The levels of 798 miRNAs were analysed using NanoString nCounter Technology Platform. The miRNA regulatory network analysis was performed using the Ingenuity Pathway Analysis software.

Results: Our data showed that 28 miRNAs (including miR-615-3p, miR-1303, miRR-122-5p, miR-217) were significantly upregulated and 1 miRNA (miR-451a) was downregulated in T2DM IHD patients compared to T2DM patients without IHD (adj p < 0.05). Based on the above all miRNAs were classified into four interactive signalling networks. Targeted genes by the identified miRNAs were enriched in pathways associated with metabolic and cardiovascular diseases, cardiac dysfunction and cardiovascular system development.

Conclusions: Taken together, our findings suggest that circulating miRNAs might have a crucial role in the development of IHD in diabetic patients and may be used as a potential for early diagnosis.

A. Bielska: None. M. Niemira: None. A. Szalkowska: None. J. Raczkowska: None. D. Ostrowski: None. W. Bauer: None. P. Prokopczuk: None. S. Dobrzycki: None. A. Kretowski: None.

P05.38B Association of polymorphisms in PDE4D, ACE and GP1BA genes with clinical characteristics in patients with ischemic stroke and chronic cerebral ischemia

A. Ikonnikova1, A. Gunchenko2, S. Galkin2, A. Anisimova2, T. Nasedkina1

1Engelhardt Institute of Molecular Biology RAS, Moscow, Russian Federation, 2N.I. Pirogov Russian National Research Medical University, Ministry of Health of the Russian Federation, Moscow, Russian Federation

Introduction: A large data has been accumulated about the role of genetic factors in the development of cerebrovascular diseases, but the identified associations are often not reproducible in replication studies and meta-analyses. This may be due to the multifactorial nature of this group of diseases, the heterogeneity of pathogenic mechanisms, and the ethnicity of patients. The work was aimed to investigate the association of genetic polymorphisms with patient’s characteristics in well-defined clinical groups.

Patients and Methods: The study included 81 patients with chronic cerebral ischemia (without a history of stroke) and 69 patients with ischemic stroke (IS). Genotyping was performed using microarray to determine 21 SNPs in the АСЕ, SERPINE1, FGB, F5, F7, F12, GP1BA, GPIIIa, MTHFR, CYP11B2, PON1, PON2, NOS2, NOS3, PDE4D, HIFla, LTA, ALOX5AP genes. Stroke subtypes were defined according to TOAST criteria.

Results: In patients with chronic cerebral ischemia, the TT genotype of the PDE4D (rs966221, SNP83C>T) was associated with rapidly progressive arterial hypertension (OR=6.22; 95%CI=1.9-20.8; p=0.0036). In patients with IS the D allele of the ACE gene (rs1799752, I>D) and the DD genotype were associated with cardioembolic subtype of stroke (OR=2.67, 95%CI=1.23-5.8, p=0.02 and OR=7.14, 95%CI=1.7-29.7, p=0.0057). In patients with IS the C allele of the GP1BA gene (rs2243093, -5T>C) and TC genotype were associated with large-artery atherosclerosis subtype of stroke (brachiocephalic artery stenosis >75% and large-artery occlusion) (OR=3.39, 95%CI=1.1-10.2, p=0.03 and OR=4.44, 95% CI=1.3-15.5, p=0.023).

Conclusions: Detailed genetic analysis in the context of clinical features of cerebrovascular disease will allow the identification of significant associations.

A. Ikonnikova: None. A. Gunchenko: None. S. Galkin: None. A. Anisimova: None. T. Nasedkina: None.

P05.39C Apolipoprotein E e4 associates to age of ischemic stroke onset but not to stroke outcome

C. Lagging1,2, E. Lorentzen3, A. Pedersen1,2, T. M. Stanne1, M. Söderholm4,5, J. M. Maguire6,7, A. Lindgren4,5, C. Jern1,2

1Department of Laboratory Medicine, Institute of Biomedicine, the Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden, 2Department of Clinical Genetics and Genomics, Sahlgrenska University Hospital, Gothenburg, Sweden, 3Bioinformatics Core Facility, University of Gothenburg, Gothenburg, Sweden, 4Department of Clinical Sciences Lund, Neurology, Lund University, Lund, Sweden, 5Department of Neurology and Rehabilitation Medicine, Skåne University Hospital, Lund, Sweden, 6Faculty of Health, University of Technology Sydney, Sydney, Australia, 7Hunter Medical Research Centre, Newcastle, Australia

Introduction: APOE genotype is a candidate predictor for stroke outcomes. We evaluated whether common APOE alleles associate with ischemic stroke outcome, severity and age at onset, within the large multicenter Genetics of Ischaemic Stroke Functional Outcome (GISCOME) study.

Methods: This study comprises 6,165 ischemic stroke cases with genotype data from SNP arrays. Baseline stroke severity was scored according to the NIH Stroke Scale (NIHSS). Functional outcome at 3 months was scored according to the modified Rankin Scale (mRS). We derived common APOE allele status (ε2,ε3,ε4) by combining information from SNPs rs7412 and rs429358. Effects of minor allele count (ε4,ε2) on age at stroke, stroke severity and outcome were estimated separately in comparison to the most common ε3/ε3 genotype.

Results: We found no evidence of a direct effect of ε4 on outcome. There was, however, an inverse association between ε4 allele count and age at stroke (β-1.8, P=0.00017). This association was significant in both sexes. The ε2 allele was independently associated with poor functional outcome (mRS>2) in men (odds ratio[OR]1.47, P=0.008), but not in women (OR 0.87, P=0.44).

Conclusion: This is the largest meta-analysis on APOE genotype and ischemic stroke outcome to our knowledge. We found a lower age at stroke onset in ε4 carriers and a worse functional outcome in male ε2 carriers. Even larger studies are warranted to further investigate the effects of APOE alleles on ischemic stroke outcome in different age and sex strata.

Study funding: The Swedish Research Council, Swedish Heart and Lung Foundation, Swedish State; ALF-agreement.

C. Lagging: None. E. Lorentzen: None. A. Pedersen: None. T.M. Stanne: None. M. Söderholm: None. J.M. Maguire: None. A. Lindgren: D. Speakers Bureau/Honoraria (speakers bureau, symposia, and expert witness); Modest; Bayer, BMS Pfizer. F. Consultant/Advisory Board; Modest; Bayer, Astra Zeneca, Boehringer Ingelheim, BMS Pfizer, Reneuron. C. Jern: None.

P05.40D Functional characterization of new missense and intronic variants in LDLR

C. Rodríguez-Jiménez1, N. Agra2, L. Reinares-garcía3, C. Guijarro4, A. Carazo-Álvarez1, B. Gil Fournier5, F. García-Iglesias6, C. Alonso-Cerezo7, J. Mostaza8, S. Rodríguez-Nóvoa1

1Department of Genetics of metabolics diseases. Institute of Medical & Molecular Genetics (INGEMM), Hospital Universitario La Paz, Universidad Autónoma de Madrid, IdiPAZ, Madrid, Spain; Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERE, Madrid, Spain, 2Vascular Malformations Section. Institute of Medical & Molecular Genetics (INGEMM), Hospital Universitario La Paz, Universidad Autónoma de Madrid, IdiPAZ, Madrid, Spain; Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERE, Madrid, Spain, 3Department of Internal Medicine, Hospital Clínico San Carlos, Madrid, Spain., Madrid, Spain, 4Hospital Universitario Fundacion Alcorcon, Madrid, Spain, Madrid, Spain, 5Unidad de Genética del Hospital Universitario de Getafe, (Madrid), Spain, Madrid, Spain, 6Department of Internal Medicine, Hospital Carlos III, Madrid, Spain, Madrid, Spain, 7Department of Genetics, Hospital de La Princesa, Madrid, Spain, Madrid, Spain, 8Lipid and Vascular Unit, Hospital Carlos III, Madrid, Spain, Madrid, Spain

Introduction: About 70% of human plasma cholesterol circulates as a component of low-density-lipoproteins (LDL). Most LDL is cleared from the circulation through the hepatic LDL-receptor(1). Reduction in activity or number of LDLR gives rise to Familial hypercholesterolemia (FH; MIM#143890). Early detection of patients with FH allows initiation of treatment, thus reducing the risk of coronary heart disease. In this study we performed in vitro characterization of new LDLR variants found in FH patients.

Materials and Methods: DNA samples from FH patients were analyzed by Next Generation Sequencing (NGS) using a customized panel of 198 genes. The LDLR missense variants were generate into the expression vector LDLR_NM_000527-Human-cDNA-GFPSpark®-tag by site-directed-mutagenesis and transfected in LDLR-deficient cell line CHO-ldlA7(2,3). Activity and expression of cell surface LDLR were measured by flow cytometry. LDLR expression was detected by Western Blot and immunofluorescence. In order to characterize the LDLR intronic variant, the RNA extracted from patient´s peripheral-blood-mononuclear-cells was analyzed by RT-PCR followed by Long-Range-PCR. All transcripts were validated by Sanger sequencing.

Results:Seven new variants at LDLR were characterized. The missense variants c.776A>G;p.(Tyr259Cys), c.851G>A;p.(Cys284Tyr), c.1072T>G;p.(Cys358Gly) showed significant differences in activity and expression of LDLR regarding to the wildtype(WT). The variants c.2279C>T;p.(Thr760Ile) and c.2579C>A;p.(Ala860Glu) did not showed any differences. The intronic variant c.2389+4A>G revealed impact on splicing of LDLR resulting as pathogenic.

Conclusions: The functional in vitro characterization of rare variants at the LDLR allow us to confirm the genetic diagnosis of FH, avoiding the classification as “uncertain significant variants”, and therefore, to allow for cascade family screening.

C. Rodríguez-Jiménez: None. N. Agra: None. L. Reinares-garcía: None. C. Guijarro: None. A. Carazo-Álvarez: None. B. Gil Fournier: None. F. García-Iglesias: None. C. Alonso-Cerezo: None. J. Mostaza: None. S. Rodríguez-Nóvoa: None.

P05.41A Divergent degradation pathways for corresponding LDLR and VLDLR disease-causing mutants

P. Kizhakkedath, A. John, L. Al-Gazali, B. R. Ali

United Arab Emirates University, Al-Ain, United Arab Emirates

Introduction: Misfolded proteins in the early secretory pathway are mainly degraded by the ubiquitin-proteasome systems known as ER associated degradation (ERAD) which has been implicated in the pathogenesis of numerous congenital disorders. Therefore, ERAD has been a promising target for therapy of such diseases. In this study we compare and contrast the degradation behavior of equivalent disease-causing mutations in LDLR and VLDLR, two LDLR family receptors.

Materials and Methods: HRD1-SEL1L knockout (KO) HEK293 cell lines have been generated using CRISPR/Cas9. The missense mutants were generated by QuikChange site-directed mutagenesis. The expressed proteins were analyzed biochemically and by microscopy.

Results: We found that the disequilibrium syndrome-causing VLDLR missense mutants (p.D487Y, p.D521H and p.C706F) are retained in the ER whereas only p.D482H and p.C667F of the LDLR corresponding mutants are retained. Two of the ER-retained VLDLR mutants were found to be aggregation-prone leading to ER stress as measured by spliced-XBP mRNA levels. However, there was no differences between LDLR wild type and mutants with regard to their aggregation status or ER stress. Treatment with inhibitors of autophagy seemed to stabilize the degradation of wild type receptors. In addition, p.C706F VLDLR mutant stabilized during early phase of both proteasomal and autophagy inhibition. Cycloheximide chase analysis in SEL1L-KO cells revealed that the HRD1-SEL1L complex could be involved in the clearance of ER-retained VLDLR mutants.

Conclusions: The downstream degradation and ER stress induction behavior of ER-retained corresponding LDLR and VLDLR mutants are divergent suggesting different clearance mechanisms despite their structural similarities.

P. Kizhakkedath: None. A. John: None. L. Al-Gazali: None. B.R. Ali: None.

P05.42B Phenotype-driven strategy of DNA diagnostics in patients with LVNC and different types of myocardium remodeling

M. Polyak1, A. A. Bukaeva1, A. G. Shestak1, O. V. Blagova2, L. B. Mitrofanova3, E. A. Mershina4, Y. V. Frolova1, S. L. Dzemeshkevich1, E. V. Zaklyazminskaya1,5

1Petrovsky Russian Research Center of Surgery, Moscow, Russian Federation, 2Sechenov First Moscow State Medical University, Moscow, Russian Federation, 3Federal Almazov North-West Medical Research Centre, Saint Petersburg, Russian Federation, 4Medical Scientific and Educational Center of Lomonosov Moscow State University, Moscow, Russian Federation, 5Pirogov Russian National Research Medical University, Moscow, Russian Federation

Introduction: Left ventricular noncompaction (LVNC) is a cardiomyopathy with causative genetic variants identified in more than 20 genes. It's often accompanied by other types of myocardium remodeling. Several guidelines recommend DNA-diagnostics for LVNC patients but strategy remains unclear. The aim of our study is to evaluate efficiency of DNA-diagnostic in LVNC patients using gene panel (13 candidate genes).

Materials and Methods: We observed 67 probands diagnosed with LVNC. Deep phenotyping was performed including evaluation of cardiac remodeling (dilatation, hypertrophy or isolated noncompaction). Genetic screening by NGS (Ion Torrent) sequencing of 13 genes was performed for all patients. Sanger sequencing of additional genes and whole exome sequencing (WES) were performed if needed. Pathogenecity of genetic variants was analyzed according to ACMG Recommendations (2015). Results. Overall 33 genetic variants in 30 probands (45% of cases) were ascribed to III-V classes of pathogenicity: 12 variants were classified as likely pathogenic/pathogenic, and 21 - as variants of unknown significance. Efficiency of DNA diagnostic was 22.4% in whole cohort but it varied significantly depending on cardiac remodeling. The highest rate of V-IV class variants was found in “LVNC+cardiac hypertrophy” subgroup (53%). In subgroup “LVNC+cardiac dilation” variants of V-IV class were detected in 14% of probands. We detected no mutations in isolated LVNC cases.

Conclusion: Phenotype-driven strategy might be useful for decision-making in LVNC patients. Screening of 13 genes might be cost-effective in “LVNC+cardiac hypertrophy” subgroup. In patients with isolated LVNC/LVNC and dilatation WES might be preferred. This work was supported by RSF grant № 16-15-10421

M. Polyak: None. A.A. Bukaeva: None. A.G. Shestak: None. O.V. Blagova: None. L.B. Mitrofanova: None. E.A. Mershina: None. Y.V. Frolova: None. S.L. Dzemeshkevich: None. E.V. Zaklyazminskaya: None.

P05.43C Molecular analysis confirmed common ancestor of 10 Czech families with long QT syndrome carrying C926T-KCNQ1 variant

I. Synková1,2, I. Valášková1,2, R. Gaillyová1,2, T. Novotný3,2, M. Bébarová4, I. Andršová3,2, A. Floriánová3, P. Vít5,2, R. Navrátil6

1Department of Medical Genetics, University Hospital, Brno, Czech Republic, 2Faculty of Medicine, Masaryk University, Brno, Czech Republic, 3Department of Internal Medicine and Cardiology, University Hospital, Brno, Czech Republic, 4Department of Physiology, Faculty of Medicine, Masaryk University, Brno, Czech Republic, 5Department of Pediatrics, University Hospital, Brno, Czech Republic, 6Repromeda, Biology Park, Brno, Czech Republic

Introduction: Long QT syndrome (LQTS) is a hereditary arrhythmic syndrome characterized by abnormal prolongation of QT interval, increased risk of ventricular arrhythmias, and sudden death. It is the most often diagnosed hereditary arrhythmic disorder with prevalence 1:2000. The KCNQ1 gene is one of the 3 major genes (KCNQ1, KCNH2 and SCN5A) which account for 75 % of the genetically identified LQTS cases. The same KCNQ1 mutation c.926C>T (p.T309I) was identified in 10 putatively unrelated families.

Materials and Methods: 11 highly polymorphic short tandem repeats (STR) markers were chosen for haplotype analysis in 10 families. Multiplex PCR and fragment analysis were performed to identify variant linked to the mutation across families. Single nucleotide polymorphism (SNP) analysis was performed with HumanKaryomap-12 DNA Analysis Kit (Illumina) in one member of each family. 6219 SNPs on p arm of chromosome 11 were analysed.

Results: The same haplotype was identified in the nearest region of the mutation spot in every family by STR analysis and then confirmed by SNP analysis, which also identified possible crossing-overs. The maximum size of the area shared by all families is 658407 bp and contains the whole sequence of KCNQ1 gene. The maximum size of the area shared by two families is 12633501 bp.

Conclusion: Allelic frequencies of identified alleles in STR markers in control population suggest that there is only one common ancestor with mutation c.926C>T in the group of families investigated in this study.

Supported by grant of the Ministry of health of the Czech Republic: NV16-30571A.

I. Synková: None. I. Valášková: None. R. Gaillyová: None. T. Novotný: None. M. Bébarová: None. I. Andršová: None. A. Floriánová: None. P. Vít: None. R. Navrátil: None.

P05.44D Novel LOX mutations in five probands with thoracic aortic/arterial aneurysm and dissection with variable connective tissue findings

M. Perik1, I. Van Gucht1, A. Krebsova2, B. Diness3, R. Zhurayev4, D. Adlam5, M. Kempers6, I. Luyckx7, N. Peeters1, L. Van Laer1, A. Verstraeten1, B. Loeys1,6

1Center of Medical Genetics, Faculty of Medicine and Health Sciences, University of Antwerp, Antwerp University Hospital, Antwerp, Belgium, 2Department of Cardiology, IKEM, Prague, Czech Republic, 3Department of Clinical Genetics, Copenhagen University Hospital, Copenhagen, Denmark, 4Department of Clinical Pathology, Lviv National Medical University after Danylo Halytsky, Lviv, Ukraine, 5Acute and interventional Cardiology, University of Leicester, Leicester, United Kingdom, 6Department of Human Genetics, Radboud University Medical Center, Nijmegen, Netherlands, 7Center of Medical Genetics, Faculty of Medicine and Health Sciences, University of Antwerp, Anwerp University Hospital, Antwerp, Belgium

Loss-of-function variants in LOX, encoding lysyl oxidase, were reported to cause familial thoracic aortic aneurysm and dissection (TAAD). Using gene panel and exome sequencing, we identified five additional probands carrying likely pathogenic LOX variants, including three missense variants, affecting highly conserved amino acids and absent from gnomAD. The two stopcodons, (c.(351delC)) and p.(Gly149*), were identified in males with type A dissection at 19 and 51 years without family history. The first proband also suffered from splenic rupture, pneumothorax and varicose veins whereas the second presented with flat feet and inguinal hernia surgery. A p.(Thr99Ala) missense variant was found in a Marfan syndrome-like male with TAA. The second missense variant, p.(Met298Arg), was discovered in 46 year old female presenting with left carotid and prior coronary artery dissection. Interestingly, the identical LOX variant previously segregated in five affected members of a TAAD family. The woman presented skin hyperelasticity with recurrent shoulder dislocations. Her mother is known with berry aneurysm, but not available for genetic testing. The last missense variant, p.(Leu306Pro), was found in a tall male with dilatation of the aorta sinus and ascendens with elective surgery at age 15. Family history is significant for aortic dissection at paternal side of the family. Segregation studies revealed the absence of p.(Leu306Pro) in unaffected mother. The latter two missense variants are both located within the LOX catalytic domain.

Our data demonstrate that loss-of-function LOX variants cause a wide spectrum of aortic and arterial aneurysmal disease (including coronary artery dissection), combined with connective tissue findings.

M. Perik: None. I. Van Gucht: None. A. Krebsova: None. B. Diness: None. R. Zhurayev: None. D. Adlam: None. M. Kempers: None. I. Luyckx: None. N. Peeters: None. L. Van Laer: None. A. Verstraeten: None. B. Loeys: None.

P05.46B Left ventricular non-compaction and Ebstein anomaly in a patient with a variant in the sarcomere gene MYH7

I. Loddo, F. Barbera, G. Di Gesaro, D. Bellavia, E. La Franca, G. Mamone, G. Gentile, F. Clemenza, D. Di Carlo

IRCCS Mediterranean Institute for Transplantation and Advanced Specialized Therapies (ISMETT), Palermo, Italy

Introduction: Left ventricular non-compaction (LVNC) is a rare hereditary cardiomyopathy, resulting from abnormal embryonic myocardial development.

The most prevalent Congenital Heart Disease in LVNC is Ebstein anomaly (EA), characterized by apical displacement and partial fusion of the septal and posterior leaflet of the tricuspid valve with the ventricular septum.

Materials and Methods: We present the case of 46 years old male affected by EA and LVNC.

MR imaging shows an Ebstein anomaly, in which the origin of the septal leaflet is displaced downward into the right ventricle, dividing it into a proximal atrialized and distal ventricularized (true or functional ventricle) chambers. These features, in addition to annular dilatation, result in valve regurgitation and right chambers dilatation. MR imaging shows the coexistence of left ventricle hypertrabeculation with spongy appearance of the myocardium consistent with left ventricular non-compaction.

Genetic counselling was offered. He reported that his nephew was affected by EA and she died at three months of age. Molecular analysis by NGS target panel was performed to our patient.

Results: We identified a heterozygous missense variant (c.728G>A) p.Arg243His in MYH7 gene, classified as pathogenic according to ACMG Guidelines.

This variant was originally reported in an individual with hypertrophic cardiomyopathy, has subsequently been reported in multiple individuals with LVNC and in an individual with isolated EA.

Conclusions: The association between EA, LVNC and mutations in MYH7, seems to represent a subtype of Ebstein anomaly with autosomal dominant inheritance and variable penetrance.

NGS target resequencing represents a valuable tool in cardiomyopathy genetic testing.

I. Loddo: None. F. Barbera: None. G. Di Gesaro: None. D. Bellavia: None. E. La Franca: None. G. Mamone: None. G. Gentile: None. F. Clemenza: None. D. Di Carlo: None.

P05.47C Genetic screening of cardiovascular genes in a heart transplantation cohort

E. Cuesta-Llavona1, J. Gómez1, B. Díaz-Molina1, J. Lambert1, B. Alonso1, R. Lorca2, J. Reguero1, E. Coto1

1Hospital Universitario Central de Asturias, Oviedo, Spain, 2Hospital General Universitario Gregorio Marañón, Madrid, Spain

Heart failure is considered one of the main causes of death in developed countries. Moreover, it is a disease that is influenced by multiple genetic risk factors and the only effective treatment when there are no medical or surgical alternatives would be heart transplant. The aim of this study is to genetically characterized patients whose undergo heart transplantation. We sequenced 65 transplanted patients for a comprehensive cardiovascular gene panel of 209 genes, using Ion semiconductor chips technology in a Ion GeneStudio S5 Sequencer. We identified 29 patients with rare variants (either not described or gnomAD frequency ≤ 0.0001). Twenty-two of them were single carriers (75.9%), six harbor two variants (20.6%), and in one case we identified three variants (3.5%). Regarding to its pathogenicity, eight patients were carriers of at least a variant classified as pathogenic by ClinVar database, six as variants of unknown significance, and the others have no classification. The reason for transplantation of these carriers was due to ischemic heart disease in 48% of cases, 29% to dilated cardiomyopathy, 4% valvular, 4% hypertrophic and 15% to another type of heart disease. In conclusion, we have identified a significant percentage of new or rare genetic variants in genes that would be associated with heart diseases in a heart transplanted cohort. Therefore, detection of these variants might be helpful to achieve an early diagnosis of these diseases. Further co-segregation and functional studies have to be performed in order to accomplish an accurate variant classification.

E. Cuesta-Llavona: None. J. Gómez: None. B. Díaz-Molina: None. J. Lambert: None. B. Alonso: None. R. Lorca: None. J. Reguero: None. E. Coto: None.

P05.48D Analysis of endothelin-1 (EDN-1) UTR regions

C. Solarat1, M. Lago-Docampo1, A. Baloira2, D. Valverde1

1University of Vigo, Vigo, Spain, 2Servicio de Neumología, Complexo Hospitalario Universitario de Pontevedra, Vigo, Spain

Pulmonary Arterial Hypertension (PAH) is a disease characterized by an increase of secretion and deregulation of Endothelin-1 (ET-1). This peptide is secreted by the endothelium of blood vessels and promotes vasoconstriction. We carried out the characterization of the UTR regions of endothelin-1gene (EDN-1), in order to determine common variations that may modulate disease outcome. The analysis was carried out in 60 patients with different classes of PAH, testing a fragment of 2 kb for both UTR region. An in silico analysis was performed to evaluate binding transcription factors. Luciferase assay was done to evaluate in vitro the SNP influence in gene expression. Data revealed the presence of a deletion in the promoter region (rs397751713), while a transversion in the 3’ UTR region was found (rs2859338). The distribution of the genotype frequencies in our PAH patients were: for rs397751713: A/A: 0.08; A/-: 0.27; -/-: 0.66; for rs2859338: A/A: 0.15; A/G: 0.60; G/G: 0.25. Variations are located in a KLF4 binding sequence and a vitamin D receptor binding sequence respectively. Both transcription factors are related to PAH development. In conclusion, these SNPs in the UTR regions of EDN1 are related with gene expression levels, as we measured higher expression rates for patients with A/A and G/G genotype. Moreover, we hypothesized that this overexpression is due to the inability of KLF4 and vitamin D receptor to attach the target sequence and to regulate the expression of EDN1, as KLF4 is probe to avoid PAH when present and vitamin D is an anti-hypertrophic factor.

C. Solarat: None. M. Lago-Docampo: None. A. Baloira: None. D. Valverde: None.

P05.49A Rare ABCC8 variants identified in Spanish pulmonary arterial hypertension patients

M. Lago Docampo1,2,3, J. Tenorio4,5, C. Pérez Olivares6, I. González Hernández7,8, P. Escribano Subías6,9, G. Pousada2, A. Baloira10, M. Arenas1,2,3, P. Lapunzina4,5, D. Valverde1,2,3

1University of Vigo, Vigo, Spain, 2IIS Galicia Sur, Vigo, Spain, 3Centro de Investigaciones Biomédicas (CINBIO), Vigo, Spain, 4Instituto de Genética Médica y Molecular (INGEMM), Madrid, Spain, 5Centro de Investigación Biomédica en Red de enfermedades Raras (CIBERER), Madrid, Spain, 6Hospital 12 de Octubre, Madrid, Spain, 7Fundación Jiménez Díaz, Madrid, Spain, 8Centro de Investigación Biomédica en Red de Enfermedades Cardiovasculares (CIBERCV), Madrid, Spain, 9Centro de Investigación Biomédica en Red de Enfermedades Cardiovasculares (CIBERCV), Madrid, Spain, Madrid, Spain, 10Complejo Hospitalario Universitario de Pontevedra, Pontevedra, Spain

Introduction: Pulmonary Arterial Hypertension (PAH) is a rare and fatal disease consisting in the obliteration of the pulmonary precapillary arteries, leading to right heart failure and death.

Methods: We used targeted panel sequencing with a custom panel (HAP v1.2) including 21 genes in 318 PAH patients from the Spanish registry (REHAP). After detecting several changes in ABCC8 we carried out a functional analysis by minigene assay to evaluate possible splicing variants (8/10). Lastly, we used protein modeling by homology (Phyre2) to evaluate the pathogenicity of the changes at protein level assessing its stability with MODELLER.

Results: After validation, we identified ten variants in the ABCC8 gene that had never been related to PAH: c.298G>A:p.(Glu100Lys), c.2176G>A p.(Ala726Thr) and c.3238G>A:p.(Val1080Ile) were classified as neutral. c.2422C>A:p.(Gln808Lys) and c.3976G>A:p.(Glu1326Lys) were classified as likely pathogenic. c.1429G>A:p.(Val477Met), c.1643C>T:p.(Thr548Met), c.2694+1G>A, c.3288_3289del:p.(His1097ProfsTer16) and c.3394G>A:p.(Asp1132Asn) were classified as pathogenic. Minigenes confirmed the pathogenicity of c.3394G>A:p.(Asp1132Asn) inducing an exon skipping, and the correct processing of c.298G>A:p.(Glu100Lys) and c.1643C>T:p.(Thr548Met). But they were inconclusive for 5 variants, none of the encoded exons transcribed correctly even in the wild type. Protein modeling of the possible outcomes revealed that amino acid changes would not alter protein stability. The skipping of exons 20, 27 and 32 would yield unstable proteins, while skipping of exon 22 would barely affect stability.

Conclusions: We identified ten variants in ABCC8, confirmed experimentally the pathogenicity of c.3394G>A:p.(Asp1132Asn) and bioinformatically c.3288_3289del:p.(His1097ProfsTer16). Protein stability analysis allowed us to predict the possible outcomes of the unconfirmed splicing variants.

M. Lago Docampo: None. J. Tenorio: None. C. Pérez Olivares: None. I. González Hernández: None. P. Escribano Subías: None. G. Pousada: None. A. Baloira: None. M. Arenas: None. P. Lapunzina: None. D. Valverde: None.

P05.51C Familial hypercholesterolemia: functional characterization of new variants in PCSK9

S. Rodríguez-Nóvoa1, C. Rodríguez-Jiménez1, E. Sánchez-Nieves1, A. Carazo-Álvarez1, F. Arrieta2, L. Reinares-García3, P. Martínez-Hernández4, R. Cañas5, J. Mostaza6

1Department of Genetics of metabolics diseases. Institute of Medical & Molecular Genetics (INGEMM), Hospital Universitario La Paz, Universidad Autónoma de Madrid, IdiPAZ, Madrid, Spain; Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERE, Madrid, Spain, 2Department of Endocrinology and Nutrition. Unit Hospital Ramón y Cajal. Hospital Universitario Ramón y Cajal, Instituto Ramón y Cajal de Investigación Sanitaria (IRyCIS), E-28034. CIBER de Fisiopatología de la Obesidad y Nutrición (CIBEROBN), Madrid, Spain, 3Department of Internal Medicine, Hospital Clínico San Carlos, Madrid, Spain., Madrid, Spain, 4Department of Internal Medicine, Hospital Universitario la Paz, Madrid, Spain., Madrid, Spain, 5Department of Internal Medicine, Fundación Jiménez Díaz, Madrid, Spain, Madrid, Spain, 6Lipid and Vascular Unit, Hospital Carlos III, Madrid, Spain, Madrid, Spain

Introduction: Proprotein convertase subtilisin/kexin type 9(PCSK9) is a post-transcriptional regulator of the Low-density-lipoprotein receptor(LDLR). “Lost_of_function_variants” at PCSK9 has been related with lower LDL-cholesterol while the “gain_of_function_variants” result in autosomal dominant hypercholesterolemia-3 (FH3,OMIN#603776) whose prevalence is 0,1-2%(1,2).

Materials and Methods: The DNA samples from patients clinically classified as having probable or definitive familial hypercholesterolemia(FH), were analysed by NGS using a customized panel of 198 genes. The new variants found in PCSK9 were generated into the expression vector PCSK9_NM_174936.3-Human-cDNA-GFPSpark®-tag by site directed mutagenesis. The constructs were transfected in HepG2 cells. We measure both activity and expression of LDLR by flow cytometry in order to determine the impact of PCSK9 variants.

Results: In silico study showed six variants in PCSK9: c.289C>T;p.(Arg97Cys), c.1130C>G;p.(Thr377Ser), c.1495C>T;p.(Arg499Cys), c.1633A>G;p.(Ser545Gly), c.1978G>A;p.(Asp660Asn), c.1987A>G;p.(Thr663Ala) with potential effect in PCSK9. The variant c.1978G>A;p.(Asp660Asn) had a 15% decreased level of internalization of LDL and 20% decreased level of LDLR expression regarding WT-PCSK9. The rest of variants had the same or incremented level of LDL internalization and expression of LDLR regarding WT-PCSK9.

Conclusions: The functional characterization of variants in PCSK9 has allowed us to classify the variants avoiding the “uncertain significance” variants and thus to confirm the genetic diagnosis of hypercholesterolemia. The results suggest that the variant c.1978G>A;p.(Asp660Asn) could explain the raised level of LDL-c in the patient being a new case of FH3 due to gain-of-function variant in PCSK9.

S. Rodríguez-Nóvoa: None. C. Rodríguez-Jiménez: None. E. Sánchez-Nieves: None. A. Carazo-Álvarez: None. F. Arrieta: None. L. Reinares-García: None. P. Martínez-Hernández: None. R. Cañas: None. J. Mostaza: None.

P05.52D Polygenic risk information for coronary artery disease - P5.fi FinHealth

M. M. Marttila1, T. Paajanen1, H. Marjonen1, N. Kallio1, A. Haukkala2, H. Kääriäinen1, K. Kristiansson1, M. Perola1,3

11 National Institute for Health and Welfare, Helsinki, Finland, 22 Faculty of Social Sciences, University of Helsinki, Finland, Helsinki, Finland, 33 Research Program for Clinical and Molecular Metabolism, Faculty of Medicine, University of Helsinki, Finland, Helsinki, Finland

Introduction: We have tested how polygenic risk scores (PRS) for coronary artery disease (CAD), type 2 diabetes and venous thromboembolism affect the risk of disease within the following ten years in participants from a population-based study FINRISK. We will also utilize NMR metabolomic risk information in disease prevention.

Materials and Methods: Based on follow-up data from national health care registries we modelled PRS in whole genome genotyped population based FINRISK cohorts with multiple registry follow-up for incident cases (N=21726) using Cox regression models. We modelled the impact of genetic and traditional risk factors on a risk of disease within the next 10 years.

Results: We compared the CAD classification of Cox regression model with traditional risk factors and polygenic additive model with 6,6M variants. From basic model risk class 10-20% PRS reclassified 205 participants to highest >20% risk class. In FINRISK participants risk for CAD was higher for men and smoking adds to risk of disease in all PRS classes. Risk for disease increases with BMI being highest in BMI class 30-35.

Conclusions: The validation showed that adding PRS to the traditional risk factors significantly changed the risk enabling reclassification of as many as 17% of the participants. PRS and metabolic risk information are returned to volunteering participants through an internet portal. Changes in life style are followed up using questionnaires through the portal and the morbidity will be collected through health registers. P5 study is a pilot for P6 which will recruit 200 000 participants and study 10-20 diseases.

M.M. Marttila: None. T. Paajanen: None. H. Marjonen: None. N. Kallio: None. A. Haukkala: None. H. Kääriäinen: None. K. Kristiansson: E. Ownership Interest (stock, stock options, patent or other intellectual property); Significant; Negen oy. M. Perola: None.

P05.53A A heterogeneous molecular background of polymorphic ventricular tachycardia in pediatric patients with channelopathy and normal heart

M. Pelc1, P. Kowalski1, A. Madej-Pilarczyk1, D. Jurkiewicz1, J. Kosińska2, M. Rydzanicz2, M. Posadowska3, K. Pręgowska3, P. Stawiński2,4, M. Brzezińska3, E. Ciara1, D. Piekutowska-Abramczuk1, P. Halat-Wolska1, D. Siestrzykowska1, R. Płoski2, K. Chrzanowska1, K. Bieganowska3

1Department of Medical Genetics, The Children’s Memorial Health Institute, Warsaw, Poland, 2Department of Medical Genetics, Warsaw Medical University, Warsaw, Poland, 3Department of Cardiology, The Children’s Memorial Health Institute, Warsaw, Poland, 4Department of Genetics, Institute of Physiology and Pathology of Hearing, Warsaw, Poland

Introduction: Polymorphic ventricular tachycardia (PVT) is a life-threatening arrhythmia concomitant with the genetic disorders related to myocyte transmembrane ion channel dysfunction called “channelopathies”. They comprise long and short QT syndromes (LQTS/SQTS), Brugada syndrome, and catecholaminergic polymorphic ventricular tachycardia (CPVT). Although nearly 30 genes have been associated with arrhythmogenic channelopathies so far, the mutation detection rate in syndromes other than LQTS is relatively low (~15-65%), therefore their molecular profile remains largely unknown.

Patients and Results: A 62-gene panel comprising known and candidate genes was used for NGS screening of 12 patients with PVT-associated channelopathy. In 8 patients we identified 4 known and 6 novel, likely pathogenic variants in KCNH2, RYR2 and SCN5A. Additionally, rare variants of unknown significance in AKAP9, KCNA5, KCNH2, KCNT1, MYH6, SCNN1A, TRDN, TTN were found in 6 cases. Interestingly, in one CPVT patient two novel RYR2 variants inherited from healthy parents co-occured, suggesting recessive pattern of inheritance. In two other patients diagnosed with PVT and LQTS possibility of digenic inheritance or genetic synergism, associated with changes in RYR2 and KCNH2, has been postulated.

Conclusions: PVT-related channelopathies remain a diagnostic and therapeutic challenge. Phenotypic expression and/or variable penetration observed in the disease may result from the confluence of defects in different genes encoding or modulating ion channels’ function. NGS technology provides foundation to better understanding of the genetic profile, phenotype-genotype correlations and etiopathological mechanisms of PVT. Improved detection of risk factors will influence genetic counseling and therapeutic decision-making, preventing severe consequences, like sudden cardiac death.

Partially supported: CMHI-M31/18.

M. Pelc: None. P. Kowalski: None. A. Madej-Pilarczyk: None. D. Jurkiewicz: None. J. Kosińska: None. M. Rydzanicz: None. M. Posadowska: None. K. Pręgowska: None. P. Stawiński: None. M. Brzezińska: None. E. Ciara: None. D. Piekutowska-Abramczuk: None. P. Halat-Wolska: None. D. Siestrzykowska: None. R. Płoski: None. K. Chrzanowska: None. K. Bieganowska: None.

P05.54B RNF213 is a causative gene for pulmonary arterial hypertension and is associated with poor clinical outcomes

H. Suzuki1, M. Kataoka1, T. Hiraide1, M. Yamada1, T. Uehara1, T. Takenouchi1, N. Hirose1, S. Gamou2, K. Fukuda1, K. Kosaki1

1Keio University, Tokyo, Japan, 2Kyorin University, Tokyo, Japan

Introduction: Pulmonary arterial hypertension (PAH) is characterized by a strong genetic component. About 30% of patients with idiopathic/heritable PAH have variants in BMPR2. The causative genes, if any, in the remaining 70% of patients have yet to be clarified. Since we reported 2 unrelated patients with homozygosity for RNF213 p.Arg4810Lys who had Moyamoya disease and severe pulmonary hypertension in 2016, we suspected RNF213 would be causative gene for PAH.

Method: We performed whole-exome sequencing for 76 patients (19 males, 57 females) with idiopathic PAH who had been negative for BMPR2 and other known pathogenic gene mutations.

Result: We identified RNF213 p.Arg4810Lys in a heterozygous state in 7 (9.2%) of the 76 PAH patients. Since the minor allele frequency for the p.Arg4810Lys is 0.77% in the normal Japanese population, individuals who are heterozygous for the p.Arg4810Lys might be predisposed to PAH. From a therapeutic standpoint, patients with the RNF213 p.Arg4810Lys were low responders to PAH-specific vasodilators. The event-free rate of death or lung transplantation was significantly poorer in RNF213 p.Arg4810Lys variant carriers than in BMPR2 variants carriers (5-year event-free rate since the introduction of prostaglandin I2 infusion, 0% vs. 93%, P<0.001).

Conclusion: We demonstrated that nearly 10% of patients with PAH were heterozygous for the RNF213 p.Arg4810Lys. Documentation of an RNF213 p.Arg4810Lys might provide clinically relevant information when selecting pharmacologic interventions.

Grant references: This work was supported by KAKENHI from the MEXT, a grant from the Medical Department Collaborative Project of Kyorin University, and a grant from AMED (17kk0205002h0002) in Japan.

H. Suzuki: None. M. Kataoka: None. T. Hiraide: None. M. Yamada: None. T. Uehara: None. T. Takenouchi: None. N. Hirose: None. S. Gamou: None. K. Fukuda: None. K. Kosaki: None.

P05.55C Customized massive paralleled sequencing panel for diagnosis of Pulmonary Arterial Hypertension

J. A. Tenorio1, P. Arias1, I. Hernández2, N. Ochoa3, E. Granda1, P. Navas4, G. Gómez-Acebo1, N. Gallego1, PAH Spanish consortium, P. Lapunzina1, P. Escribano3

1INGEMM, Madrid, Spain, 2Hospital Universitario Fundación Jiménez Díaz, Madrid, Spain, 3Hospital Universitario 12 de Octubre, Madrid, Spain, 4Hospital Universitario Gregorio Marañón, Madrid, Spain

Introduction: Diagnosis of Pulmonary Arterial Hypertension (PAH) is challenging due to phenotypic overlapping and variable expressivity. Current classification based on clinical features, does not reflect the underlying molecular profiling of these groups, and patients from different groups can share clinical features that could be associated with their response. The advance in the massive paralleled sequencing technologies has allowed describing several new genes related to PAH, improving diagnosis ratio and a better clinical characterization Thus, our aimed was to address the molecular diagnosis of patients with any form PAH

Material and Methods: 318 patients were included in the analysis. 21 gene NGS custom panel was designed the sequencing was performed with a MiSeq. Custom script was developed to annotate and filter the variants.

Results: Pathogenic and likely pathogenic variants were found in 13% of the patients with a 14% of variants of unknown significance. Interesting, we have found variants in patients with connective tissue disease (CTD) and congenital heart disease (CHD). In CTD, we have found one pathogenic mutation and four VUS and, and for CHD two pathogenic variants and 6 VUS. In addition, in a small proportion of patients (0,93%) digenic mode of inheritance was detected.

Conclusions: These results highlight the importance of the genetic screening of PAH and allow to detect variants in PAH-associated forms not described previously. Molecular confirmation of the clinical suspicious is mandatory in cases with clinical overlapping and to perform a properly management and follow up of the patients. Grants FIS-PI15/02012 Actelion unrestricted grant FCHP-grant

J.A. Tenorio: Other; Significant; Actelion. P. Arias: None. I. Hernández: None. N. Ochoa: None. E. Granda: None. P. Navas: None. G. Gómez-Acebo: None. N. Gallego: None. P. Lapunzina: None. P. Escribano: C. Other Research Support (supplies, equipment, receipt of drugs or other in-kind support); Significant; Actelion.

P05.57A A case report of recessive restrictive cardiomyopathy caused by a novel mutation in cardiac troponin I (TNNI3)

M. P. Pantou1, P. Gourzi1, A. Gkouziouta2, I. Armenis2, C. Zygouri3, P. Constantoulakis3, S. Adamopoulos2, D. Degiannis1

1Molecular Immunopathology and Histocompatibility Unit, Molecular Genetics Facility, Kallithea-Athens, Greece, 2Heart Failure, MCS and Transplant Unit, Onassis Cardiac Surgery Center, Kallithea-Athens, Greece, 3Department of Molecular Genetics, BioAnalytica-Genotypes S.A., Athens, Greece

Introduction: Restrictive cardiomyopathy is a rare cardiac disease, for which several genes including TNNT2, MYPN, FLNC and TNNI3 have been associated with its familial form.

Materials and Methods: The index case and her relatives underwent full cardiological assessment. Genetic analysis of the index case was performed using Illumina's Trusight Cardio sequencing panel and Sanger sequencing was used to screen members of the family for the presence of the reported mutation.

Results: Here we describe a female proband with a severely manifested restrictive phenotype leading to heart transplantation at the age of 41 who was found homozygous for the novel TNNI3 mutation: NM_000363.4:c.586G>C, p.(Asp196His). Her parents were third-degree cousins originating from a small village and although they were found heterozygous for the same variant they displayed no symptoms of the disease. Her older sister who was also found heterozygous was asymptomatic. Her twin sister and her brother who were homozygous for the same variant displayed a restrictive and a hypertrophic phenotype, respectively.

Conclusion: These observations point to a recessive mode of inheritance reported for the first time for this combination of gene/disease.

M.P. Pantou: None. P. Gourzi: None. A. Gkouziouta: None. I. Armenis: None. C. Zygouri: None. P. Constantoulakis: None. S. Adamopoulos: None. D. Degiannis: None.

P05.58B The first Belgian SCN5A founder mutation: establishment of an iPSC-cardiomyocyte model to identify genetic modifiers

M. Alaerts1, A. Nijak1, E. Simons1, E. Sieliwonczyk1, D. Schepers1, B. Vandendriessche1, E. Van Craenenbroeck2, J. Saenen2, A. Labro3, D. Snyders3, P. Ponsaerts4, B. Loeys1,5

1Center of Medical Genetics, University of Antwerp and Antwerp University Hospital, Antwerp (Edegem), Belgium, 2Department of Cardiology, Antwerp University Hospital, Antwerp, Belgium, 3Laboratory for Molecular Biophysics, Physiology and Pharmacology, Department of Biomedical Sciences, University of Antwerp, Antwerp, Belgium, 4Laboratory of Experimental Hematology, Vaccine and Infectious Disease Institute, University of Antwerp, Antwerp, Belgium, 5Department of Human Genetics, Radboud University Medical Centre, Nijmegen, Netherlands

Introduction: The SCN5A gene encodes the α-subunit of the voltage-gated cardiac sodium channel Nav1.5. Mutations are detected in 20% of the patients with Brugada syndrome (BrS), an inherited cardiac arrhythmia that predisposes to sudden cardiac death (SCD). We have identified a loss-of-function SCN5A founder mutation (c.4813+3_4813+6dupGGGT) in 23 different Belgian families. We recruited 89 mutation carriers and their clinical spectrum ranges from completely asymptomatic to palpitations, syncopes and SCD. This provides us with a unique patient cohort to study modifiers that play a role in this variable expressivity and we decided to create patient-specific induced pluripotent stem cell (iPSC)-derived cardiomyocytes (CM) as a model for further investigations.

Materials and Methods: Dermal fibroblasts from a skin biopsy of two patients with different phenotypic severity and two unrelated control individuals were reprogrammed using Sendai viral vectors. The resulting iPSCs were validated using immunostaining, RT-qPCR and embryoid body formation, and for each individual three iPSC clones were established. For differentiation to iPSC-CMs we followed two published protocols and evaluated the cells using immunostaining and patch-clamp experiments.

Results: Optimization of the methods resulted in robust patient and control iPSC-CM models. The patient cells displayed a reduced sodium current compared to the control iPSC-CMs.

Conclusion: We established iPSC-CM models for a unique Belgian SCN5A founder mutation displaying remarkable variable expressivity. Further experiments including transcriptomics, whole-genome sequencing, electrophysiological and functional investigations will enable us to identify genetic modifiers and unravel their mechanism of action. This will hopefully stimulate the development of novel drugs for cardiac arrhythmias.

M. Alaerts: None. A. Nijak: None. E. Simons: None. E. Sieliwonczyk: None. D. Schepers: None. B. Vandendriessche: None. E. Van Craenenbroeck: None. J. Saenen: None. A. Labro: None. D. Snyders: None. P. Ponsaerts: None. B. Loeys: None.

P05.59C Clinical and genetic data of 151 individuals from 58 unrelated families with SMAD3 mutations

B. Chesneau1,2, T. Edouard2, Y. Dulac2, H. Colineau3,4, N. Hanna5, N. Chassaing1, S. Julia1, G. Jondeau5, J. Albuisson6, P. Khau Van Kien7, J. Plaisancié1,2

1Service de génétique médicale, Hôpital Purpan, CHU de Toulouse, Toulouse, France, 2Centre de Référence du syndrome de Marfan et des syndromes apparentés, Hôpital des Enfants, CHU de Toulouse, Toulouse, France, 3Department of Epidemiology, Health Economics and Public Health, Toulouse University Hospital, Toulouse, France, 4LEASP UMR1027, INSERM, Université Toulouse III, Toulouse, France, 5Centre de référence pour le syndrome de Marfan et apparentés, Assistance Publique-Hôpitaux de Paris, Hôpital Bichat, Faculté Paris Diderot, LVTS INSERM U1148, Paris, France, Paris, France, 6Service de génétique médicale, Hôpital Purpan, CHU de ToulouseAP-HP, Hôpital Européen Georges Pompidou, Département de Génétique, Service de Médecine Vasculaire et Centre de Référence des Maladies Vasculaires Rares, Paris, France, 7Service de génétique médicale, Centre Hospitalier Régional Universitaire de Nîmes, Nîmes, France

Pathogenic variants in SMAD3 are mainly responsible for a thoracic aortic disease, characterized by aneurysms and dissections, which has a major impact in terms of morbidity and causing early mortality. These vascular damages are associated with multisystemic signs including premature osteoarthritis. SMAD3 encodes one of the canonical TGF-β pathway members whose defect is involved in connective tissue disorders such as Loeys-Dietz syndrome and represents a rare cause (2%) of familial thoracic aneurysms and dissections. Clinical variability and incomplete penetrance are commonly associated with pathogenic SMAD3 variants. This prompted us to review all the individuals for which a SMAD3 mutation was identified in our Reference Centre and to compare these with published cases, to determine any genotype-phenotype correlation associated with mutations of this gene. Here, we report clinical and genetic data from 20 new cases from 8 families from our Reference Centre. After a complete review of the literature, we collected a total of 49 unique variants of different nature, from 151 individuals of 58 unrelated families, including missense, truncating and splicing variants. This report confirms the absence of correlation between the mutation type and the phenotype severity and highlights the important inter and intra familial clinical variability and incomplete penetrance described with S