Volume 30 | Supplement 1

Virtual Conference

August 28-31, 2021

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

Disclosure Information: In order to help readers, form their own judgments of potential bias in published abstracts, authors are asked to declare any competing financial interests. Contributions of up to EUR 10 000.- (Ten thousand Euros, or equivalent value in kind) per year per company are considered "Modest". Contributions above EUR 10 000.- per year are considered "Significant".

Presenting author names are bolded in the contributor lists.

e-Posters P01 Reproductive Genetics/Prenatal Genetics

P01.001.A Frequency of Y chromosome microdeletions in Turkish infertile men: Single Center Experience

Aysel KalayciYigin, Gizem Erdogan, Deniz Agirbasli, Mehmet Seven

Department of Medical Genetics, Cerrahpasa Medical Faculty, Istanbul University-Cerrahpasa, Fatih, Turkey.

Objective: Y chromosome microdeletions are the leading genetic cause of male infertility and their detection is clinically relevant for appropriate genetic counseling. Y chromosome includes genes for testicular development and spermatogenesis. The aim of this study was to establish the frequency of the Y chromosome microdeletions in Turkish infertile men who referred to our center with severe oligozoospermia and azoospermia.

Materials and Methods: In our study, 396 infertile men referred to İstanbul University- Cerrahpaşa, Cerrahpaşa Medical Faculty Department of Medical Genetics (GETAM) between 2016 to 2020 with azoospermia/severe oligospermia. We evaluated microdeletions of the Y-chromosome STS markers AZFa, AZFb and AZFc, ZFX/ZFY, terminal sY160 regions by using DNA Fragment analysis.

Results: Among the 396 infertile men, we determined 30 cases of Y chromosome micro- deletions (7.57%). Among 30 cases, AZFc microdeletions were found in 18 cases (60%), AZFa microdeletions in 4 cases (13.3%), AZFb microdeletions in 1 case (3.3%), AZFa,b,c in 4 cases (13.3%), AZFb,c in 3 cases (10%). Our findings are consistent with the literature.

Conclusion: Our results are similar to the previous studies which have mostly reported a frequency of less than 10% for Y chromosome microdeletions. The etiology of infertility remains unknown and novel genes other than y chromosome microdeletions should be identified with high throughput techniques.

A. KalayciYigin: None. G. Erdogan: None. D. Agirbasli: None. M. Seven: None.

P01.002.B Serotonin transporter 5-HTTLPR genotypes and trinucleotide repeats of androgen receptor exert a combinatorial effect on hormonal milieu in patients with lifelong premature ejaculation

Shahzad Bhatti, Haroon Latif Khan, Sana Abbas, Yousuf Latif Khan

Lahore Institute of Fertility and Endocrinology, Hameed Latif Hospital, Lahore, Pakistan.

Premature ejaculation is one of the most common sexual disorders in men due to the uncontrolled modulation of spinal reflexes. In this study, we investigate the combinatorial effects of trinucleotide repeats of androgen receptor and allelic variants of the 5-HTTLPR gene on sex steroids, hypophyseal hormones, sexual performance, and premature ejaculation assessment parameters among evidence-based lifelong premature ejaculation subjects. A total of 271 patients consulting for evidence-based lifelong premature ejaculatory dysfunction were selected in this study. The control group consists of 155 men with normal IELT (>4 min). The study revealed that the subjects who have the highest (≥26) CAG stretch depicted significantly higher serum oxytocin levels (102.1 pg/ml; n = 126, p < 0.001) compared with the control group (71.2 pg/ml; n = 75, p = <0.001). Almost 33 (26.1%) lifelong premature ejaculatory patients had AR variant of longer (≥26) CAG repeats was homozygous for S alleles (SS), 45 (35.7%) was homozygous for L allele (LL), and 48 (38%) had the L/S or S/L genotype of 5-HTTLPR gene. Homozygous (SS) alleles have a significant positive correlation (r = 0.44, p < 0.0001) with the high score of BDI-II (39.1, n = 126, p < 0.001). However, LL alleles have shown a significant positive correlation with PEDT (r = 0.46, p < 0.001) and a negative correlation with self-estimated IELT. The study design elaborates that androgen receptor trinucleotide repeats and 5-HTTLPR genotypes have a combinatorial impact on hormonal milieu and sexual function regarding evidence-based lifelong premature ejaculatory dysfunction patients.

S. Bhatti: None. H. Latif Khan: None. S. Abbas: None. Y. Latif Khan: None.

P01.003.C Challenge in prenatal diagnostics of severe skeletal dysplasias: a case of Achondrogenesis type 2

Natalija Krasovskaja 1,2, Aušra Matulevičienė1,2, Kamilė Šiaurytė1,2, Gabrielė Žukauskaitė2, Algirdas Utkus1

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

Introduction: Achondrogenesis type 2 (ACG2) belongs to a group of most severe type 2 collagen related skeletal dysplasias with autosomal dominant inheritance. Characteristic phenotype features include short stature, extreme micromelia, narrow chest, pulmonary hypoplasia, edema. The condition can be diagnosed prenatally.

Materials and methods: Primegravida, 23 years of age, was referred at 21st week of gestation due to asymmetrical fetal growth restriction. Fetal ultrasound revealed micromelia, narrow chest, prominent abdomen, brachydactyly, talipes, polyhydramnios. Pedigree was uninformative. Sanger sequencing of DNA from amniotic fluid identified no pathogenic changes in the FGFR3 gene. Next generation sequencing (NGS) of skeletal dysplasia gene panel was performed.

Results: NGS skeletal dysplasia gene panel identified variant NM_001844.5:c.[4387_4389del];[4387=] (rs527236145) in exon 54 of COL2A1 gene of uncertain clinical significance (in silico analysis: Provean: deleterious, Varsome: likely pathogenic). Segregation analysis in the family showed de novo origin. Several therapeutic amniocentesis were done to reduce severe polyhydramnios. Pregnancy was carried to 37 gestational weeks. Male newborn was delivered by Caesarean section, birth weight - 2050 g, height - 33 cm, Apgar score - 5/8. Condition quickly derteriorated due to severe pulmonary hypoplasia. Palliative care was administered. Baby died in 11 days.

Conclusions: Precise fetal ultrasound is essential for early suspicion of ACG2. Clinical diagnosis enables advanced diagnostic methods to be applied in timely manner and informed decisions on prenatal and postnatal care to be made.

N. Krasovskaja: None. A. Matulevičienė: None. K. Šiaurytė: None. G. Žukauskaitė: None. A. Utkus: None.

P01.004.D The impact of FMR1 allelic score in infertile females with preferential X-chromosome inactivation

Bárbara Rodrigues 1,2, Emídio Vale-Fernandes2,3, Daniela Sousa3, Raquel Brandão3, Nuno Maia1,2, Isabel Marques1,2, Rosário Santos1,2, Carla Leal3, Márcia Barreiro3, António José Arsénia Nogueira4, Paula Jorge1,2

1Molecular Genetics Unit, Centro de Genética Médica Dr. Jacinto Magalhães (CGMJM), Centro Hospitalar Universitário do Porto (CHUP), Porto, Portugal, Porto, Portugal, 2Unit for Multidisciplinary Research in Biomedicine (UMIB), Institute of Biomedical Sciences Abel Salazar (ICBAS), University of Porto, Porto, Portugal, Porto, Portugal, 3Centre for Medically Assisted Procreation/PublicGameteBank, Centro Materno-Infantil do Norte Dr. Albino Aroso (CMIN), Centro Hospitalar Universitário do Porto (CHUP), Porto, Portugal, Porto, Portugal, 4Center for Environmental and Marine Studies (CESAM), Department of Biology, University of Aveiro, Aveiro, Portugal, Aveiro, Portugal.

Introduction: X-chromosome inactivation (XCI) occurs randomly; however, skewing can occur in 3.2-3.5% of females. The association of XCI skewing with premature ovarian failure and implication of a low FMR1 gene CGG number (CGGs < 26) in ovarian dysfunction are still controversial. Aiming to test the effect of the AGG interspersions, our group developed a mathematical model that combines the AGG interspersion number and pattern as well as the FMR1 total repeat length. We then tested if the FMR1 allelic score obtained correlates with XCI pattern in females with idiopathic infertility.

Material and Methods: Blood samples from females at reproductive age, in an infertility clinic setting: 40 infertile and 27 potentially fertile females. Allelic score of each FMR1 allele was determined using our mathematical model, as was XCI pattern using HUMARA.

Results: No significant difference was observed between the proportion of infertile cases in each equivalent and dissimilar allelic combinations, respectively 25/41 and 15/26. In the equivalent group one sample carried a 56 CGG premutated allele with two AGG interspersions. The dissimilar group was enriched with low FMR1 CGG genotypes (16/26; 62%), 63% being infertile females as opposed to 39% (16/41) and 50% respectively in the equivalent group.

Conclusions: The incidence of highly-skewed XCI (>90:10) was statistically higher in the dissimilar group (80% versus 50%), suggesting an association between allelic score and preferential XCI. Although exploratory, this study suggests that such association may result from a protective FMR1 AGG interspersion pattern-related effect or unknown X-chromosome-linked anomaly that likely correlates with female infertility.

B. Rodrigues: None. E. Vale-Fernandes: None. D. Sousa: None. R. Brandão: None. N. Maia: None. I. Marques: None. R. Santos: None. C. Leal: None. M. Barreiro: None. A.J.A. Nogueira: None. P. Jorge: None.

P01.006.B How simple is a simple genetic counseling?

Moran Echar 1, Amir Peleg1, Amalia Harari-Shacham1, Shirley Modan1, Lena Sagi-Dain1,2

1Carmel Medical Center, Haifa, Israel, 2The Ruth and Bruce Rappaport Faculty of Medicine, Technion - Israel Institute of Technology, Haifa, Israel.

Introduction: Prenatal genetic counseling before amniocentesis in uneventful pregnancies is considered to be a "simple" counseling. In some medical centers the duration of such consultations is limited (to 15 minutes or less), to provide only the basic explanation. The purpose of this study was to evaluate the time required for such supposedly "simple" genetic consultations.

Material and Methods: Data was collected from January 2018 until August 2020 of all patients undergoing genetic counseling before amniocentesis in uneventful pregnancies, and were given by four genetic counselors and two medical geneticists. We have estimated the time required for each consultation.

Results: Of the 1085 consultations, 60.5% required additional explanation. The reasons for extended counseling included medical disorders of the woman or spouse (21.2%), carrier state for autosomal recessive diseases (18.6%), genetic conditions of a child or previous pregnancy (9.6%), or medical disorders in the extended family (79.1%). In 31.0% of patients, carrier screening tests were recommended or added. The additional explanations were estimated as short (up to 5 minutes) in 36.9% of the cases, intermediate (5 to 15 minutes) in 59.9%, and long (over 15 minutes) in 2.6% of cases. The consultation’s length was not affected from it being a first or a recurrent consultation.

Discussion: This study reflects the need for a proper genetic consultation for all seemingly simple indications, with an emphasis on detailed personal and family history. As over half of the consultees required extended counseling beyond the basic explanation, assigning sufficient time for the consultation is important.

M. Echar: None. A. Peleg: None. A. Harari-Shacham: None. S. Modan: None. L. Sagi-Dain: None.

P01.009.A Genetic analysis of azoospermic men by an integrated NGS panel

Monika Logara Klarić 1, Lovro Trgovec-Greif1, Lucija Žunić2, Filip Rokić1, Ana Vičić3, Tihana Marić4, Ana Merkler5, Ana Katušić Bojanac4, Robert Belužić1, Feodora Stipoljev3, Oliver Vugrek1, Maja Barbalić2,6

1Ruđer Boškovic Institut, Zagreb, Croatia, 2Genom Ltd., Zagreb, Croatia, 3Department of Obstetrics and Gynecology, Clinical Hospital "Sveti Duh", Zagreb, Croatia, 4Department of Medical Biology, University of Zagreb School of Medicine, Zagreb, Croatia, 5Department of Laboratory Diagnostics, University Hospital Centre Zagreb, Zagreb, Croatia, 6Department of Medical Biology, School of Medicine, University of Split, Split, Croatia.

Introduction: Y chromosome microdeletions, Klinefelter syndrome and CFTR mutations are the leading genetic causes of azoospermia and are all analyzed by different molecular methods. In addition, a number of candidate genes were related to infertility in the last two decades.

Materials/Methods: We designed an NGS amplicon-based panel that simultaneously analyzes all the known above-mentioned genetic variants as well as 11 additional genes recently being associated with azoospermia and ran the analysis on 44 azoospermic men. Twelve samples with known genetic aetiology were used to evaluate the performance of the NGS amplicon-based test. Remaining thirty-two samples consisted of azoospermic men with no defined cause of infertility. The panel consisted of 393 amplicons covering regions of interest. In house bioinformatic pipeline was developed to analyse the raw data.

Results: We correctly detected all genetic variants in men with known genetic aetiology. In 32 samples with no defined cause of infertility, we detected three Y chromosome microdeletions and 6 variants in selected genes that passed our filtering criteria for functional impact (in CFTR, SYCE1L, TEX15 and AR). Altogether, we detected a genetic cause of azoospermia in 4 individuals and likely causative variants in another 4 out of 32 individuals by running our NGS amplicon-based panel.

Conclusions: This work showed that genetic variants associated with male infertility could be detected by running only one assay. Moreover, customization of the panel with newly discovered genes increases the chance of finding the genetic cause of male patient infertility. Funding: The European Regional Development Fund (KK.

M. Logara Klarić: None. L. Trgovec-Greif: None. L. Žunić: A. Employment (full or part-time); Significant; Genom Ltd.. F. Rokić: None. A. Vičić: None. T. Marić: None. A. Merkler: None. A. Katušić Bojanac: None. R. Belužić: None. F. Stipoljev: None. O. Vugrek: None. M. Barbalić: A. Employment (full or part-time); Significant; Genom Ltd.

P01.010.B Molecular genetic carrier screening of in the Republic of Sakha (Yakutia) (Russia)

Aitalina Sukhomyasova 1,2, Anastasia Danilova1, Tatyana Grigorieva1,2, Lutcia Gotovtseva1,2, Polina Golikova1, Nadezhda Maksimova1

1North-Eastern Federal University named after M.K. Ammosov, Medical Institute, Yakutsk, Russian Federation, 2"Republican Hospital No. 1-National Center of Medicine", Yakutsk, Russian Federation.

Introduction: The Republic of Sakha (Yakutia) is a region of Russia with a high prevalence of some hereditary diseases among the indigenous population, due to genetic and population reasons and the founder effect.With the Medical Genetic Center of the National Center of Medicine for people of Yakut nationality, molecular genetic carrier screening of major mutations with 7 frequent autosomal recessive diseases is carried out: Three M-syndrome, SOPH-syndrome, tyrosinemia type 1, neuronal ceroid lipofuscinosis type 6, non-syndromic type 1 A deafness, type 1 methemoglobinemia, mucopolysaccharidosis-plus syndrome.

Materials and Methods: peripheral blood samples with free informed consent were taken from 404 pregnant women at early prenatal screening and 341 women consulted for pregnancy planning using in vitro fertilization. To detect mutations in 7 diseases all 745 samples were analyzed by real-time PCR.

Results: out of 404 pregnant women examined, 105 (26%) were heterozygous carriers of at least 1 hereditary disease, and 3 pregnant women were carriers of 3 diseases. 3 couples were carriers of the same disease as mucopolysaccharidosis-plus syndrome and SOPH syndrome. They were offered prenatal diagnosis.

Conclusions: a high frequency of mutation carriers is revealed in the Yakut population, which is important both for family planning, as well as for the provision of medical and genetic assistance and the expansion of molecular genetic screening among the population of the Republic of Sakha (Yakutia). The work was carried out within the framework of the state assignment of the Ministry of Science and Higher Education of the Russian Federation (project FSRG-2020-0№14).

A. Sukhomyasova: None. A. Danilova: None. T. Grigorieva: None. L. Gotovtseva: None. P. Golikova: None. N. Maksimova: None.

P01.012.D Personalised non-invasive prenatal diagnosis (NIPD) for maternally inherited variants in rare conditions using droplet digital PCR

Joe Shaw1, Ben Paternoster 1, Maureen Ramos1, Sarah Nesbitt1, Sophie Sheppard1, Lyn S. Chitty1,2, Natalie Chandler1

1NHS North Thames Genomic Laboratory Hub, London, United Kingdom, 2UCL Great Ormond Street Institute of Child Health, London, United Kingdom.

Introduction: NIPD for maternally-inherited variants is challenging due to the high background of the maternal variant in cell free DNA. Relative haplotype dosage is used clinically for common X-linked and recessive conditions, but is not suitable for consanguineous couples or those where there is no proband DNA available, and is too expensive to permit validation for rare disorders. Droplet digital PCR (ddPCR) offers the potential for development of NIPD assays personalised to maternal variants, regardless of inheritance type, through relative mutation dosage.

Methods: ddPCR assays were designed for 24 pregnancies at risk of X-linked recessive (14), X-linked dominant (1), autosomal dominant (7) and autosomal recessive (2) conditions. Assays were optimised using maternal genomic DNA, before testing cfDNA extracted from stored maternal plasma obtained from 10 weeks gestation. Fetal fraction was determined using ZFY or a paternally inherited SNP for pregnancies bearing male and female fetuses, respectively.

Results: ddPCR testing was concordant with fetal genotype determined following invasive testing in 20 cases. Four cases, all with fetal fractions <4%, were inconclusive. The analysis was modified to reflect the different inheritance patterns, including a case where both parents were affected with the common achondroplasia variant, FGFR3 c.1138G>A. In this scenario, ddPCR correctly predicted the fetus to be homozygous for the reference allele with a fetal fraction of 2.6%.

Conclusion: ddPCR offers personalised NIPD for maternally inherited variants, using only maternal samples regardless of inheritance pattern. Funding was from the GOSH NIHR Biomedical Research Centre. Samples were obtained from the RAPID sample bank.

J. Shaw: None. B. Paternoster: None. M. Ramos: None. S. Nesbitt: None. S. Sheppard: None. L.S. Chitty: None. N. Chandler: None.

P01.015.C Chromosomal abnormalities in prenataly identified cases with amniocentesis from south of Turkey

Ayfer Pazarbasi 1, Davut Alptekin1, Sabriye Kocaturk-Sel1, Inayet N. Uslu1, Nermin S. Ilgaz1, Gulsevinc Ay-Aksoy1, Osman Demirhan1, Umit Luleyap1, Mehmet B. Yilmaz1, Selim Buyukkurt2

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

Amniocentesis is a medical procedure used in prenatal diagnosis of chromosomal abnormalities and very crucial for preventing the birth of genetically defective fetuses in order to decrease the prevalence of genetic diseases in populations. A retrospective review of our amniocentesis database for the period from January 2000 to February 2021 was carried out. The karyotyping of 8635 fetuses was carried out in Department of Medical Biology from the samples of amniotic fluids which were sent from Department of Gynecology and Obstetrics of Balcali Hospital. A standart nomenclature has been developed to describe each of types of abnormality found in human chromosomes. A total of 8635 amniocentesis specimens were processed during the study period. 638 fetuses (7.38%) had various chromosomal abnormalities. 54.23% of abnormal karyotypes (346 cases) were numerical and 43.57% (278 cases) were structural. Both numerical and structural chromosomal aberrations were observed in 14 cases (2.19%). The ratios were as: trisomy 21 (49.42%), trisomy 18 (18.49%), monosomy X (9.24%), trisomy 13 (6.64%), Triploidy (4.62%), Klinefelter Syndrome (3.46%), Trisomy X (1. 15%), XYY Syndrome (0.86%), and the others in all numerical abnormalities. The frequent structural abnormalities were as: 46,XX/XY, inv(9) (p11;q12)/(p11;q13)(27.69 %), 46,XX/XY, 1qh(+)(12.58%), 46,XY, Yqh(-)(7.19%), 46,XX/XY, 16qh(+)(6.83%), 46,XX/XY, 9qh(+)(4.31%) and 46,XY, Yqh(+)(4.31%). Balanced and unbalanced translocations, deletions and duplications were also found in less ratio. According to the literature and our results, advanced maternal age is the main cause of fetal chromosomal abnormalities. Fetal chromosomal abnormality ratio that we found was 7.38%. This ratio emphasize the importance of prenatal diagnosis.

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

P01.016.D Is it time to report carrier state for recessive disorders in every microarray analysis? - A pilot model based on hearing loss genes deletions

Lena Sagi-Dain1, Idit Maya 2, Lina Basel2

1Carmel Medical Center, Haifa, Israel, 2Rabin Medical Center, Petach Tikva, Israel.

Purpose: To examine the implications of reporting heterozygous losses of recessive genes in Chromosomal Microarray Analysis (CMA), based on the incidence of microdeletions of three common hearing impairment genes in the local cohort and the prevalence of sequence variants in these genes in worldwide databases.

Methods: Prevalence of heterozygous microdeletions in OTOA and STRC genes, as well as deletions in the DFNB1 locus encompassing GJB6 gene, was determined using electronic database of Rabin Medical Center. ClinVar archive and Deafness Variation Database were used to generate a list of clinically significant sequence variants in these three genes, as well as GJB2 gene, and estimation of the frequency of sequence variants was performed.

Results: Of the 19,189 CMA tests were performed in our laboratory, 107 STRC microdeletions were found (0.56%), followed in frequency by OTOA deletions (39, 0.2%), and DFNB1 locus deletions (10, 0.05%). The estimated risk for a hearing loss in the examined individual carrying the microdeletion was estimated as 0.11-0.67% for STRC, 0.016-0.13% for OTOA, and 1.9-7.5% in the DFNB1 locus (including double heterozygocity with GJB2 clinically significant sequence variants). The risks were higher in specific populations.

Conclusions: We believe that that general decision whether to report or to disregard such incidental findings cannot be part of a uniform policy, but rather based on a detailed evaluation of origin-specific variants for each gene, with a careful consideration and discussion whether to include the microdeletion in the final report for each patient.

L. Sagi-Dain: None. I. Maya: None. L. Basel: None.

P01.018.B Prenatal craniofacial malformations should be analysed by whole exome sequencing in addition to chromosomal microarray analysis

Rachel Michaelson-Cohen 1, Amihood Singer2, Lena Sagi-Dain3, Reeval Segel1

1Shaare Zedek Medical Center, Hebrew University, Jerusalem Israel, Jerusalem, Israel, 2Department of Community Genetics, Public Health Services, Ministry of Health, Jerusalem, Israel, 3Genetics Institute, Carmel Medical Center, Rappaport Faculty of Medicine, Technion Institute of Technology, Haifa, Israel.

Introduction: Craniofacial malformations (CFMs) account for approximately 15% of congenital malformations. Genetic evaluation is important for decision-making processes in couples dealing with fetal CFMs, yet previous assessments are limited. Our objective was to examine the detection rate of clinically significant chromosomal microarray analysis (CMA) findings in pregnancies with CFMs.

Methods: Data from all CMA tests in pregnancies with sonographic diagnosis of CFMs (cleft lip and/or palate, malformations of eyes, nose or ears, micro-retrognathia etc) performed between January 2016 and April 2020 were retrospectively obtained from the Israeli Ministry of Health computerized database. Rates of clinically significant CMA results in fetuses with CFMs were compared to baseline risk, based on a local cohort of pregnancies with no major sonographic anomalies.

Results: A total of 111 CMA tests were performed due to fetal CFMs. In the 18 pregnancies with non-isolated CFMs, three (16.7%) clinically significant CMA results were detected, a significantly higher frequency compared to the control cohort, for whom the rate of pathogenic CMA is 1.4% (RR 14.1 (95% CI 3.7-54.2)). Of the 93 cases with isolated CFMs, four (4.3%) clinically significant pathogenic CMA results were detected, a rate slightly increased compared to the control population (RR 3.17 (95% CI 1.02-9.83)).

Discussion: Fetal CFMs diagnosed by sonogram, whether isolated or associated with additional sonographic defects, are associated with abnormal CMA findings. However, when isolated, abnormal CMA rate is only slightly higher than the background risk. Therefore, combining CMA and whole exome sequencing should be considered for optimizing genetic evaluation of CFMs.

R. Michaelson-Cohen: None. A. Singer: None. L. Sagi-Dain: None. R. Segel: None.

P01.019.C Web-based interactive educational tool to prepare couples for prenatal chromosomal-microarray-analysis (CMA)

Hagit Hochner1, Talya Millo2, Gil Siegal3, Shiri Shkedi-Rafid 4

1Braun School of Public Health, the Hebrew University of Jerusalem, Jerusalem, Israel, 2Hebrew University, Jerusalem, Israel, 3Ono Academic Center, Center for Health Law, Bioethics and Health Policy, Kiryat Ono, Israel, 4Hadassah Hebrew University Medical Center, Jerusalem, Israel.

Introduction: Results from prenatal Chromosomal-microarray-analysis (CMA) include variants with uncertain clinical significance (VUS), low-penetrance susceptibility-loci (SL) and risks for late-onset conditions. Some medical centers offer parents the choice if to be informed about these findings. We set-out to design and implement a web-based interactive educational tool for women/couples undergoing prenatal CMA aimed to assist in making informed decisions and improve their preparation for potential findings.

Methods: Development of the tool was based on interviews with women/their partners (N = 42) following prenatal CMA to explore their experience with parental choice. Knowledge and feedback questions were incorporated into the web-based tool, that was offered to women/couples prior to the CMA-test. Uptake, knowledge and satisfaction were evaluated on the first 180 users.

Results: About 80% of the women who logged into the system completed the process. Distribution of choices made using the educational tool was 88.9%, 63.6%, and 57.6% for disclosure of late-onset, SL and VUS findings, respectively, similar to distribution of choices published prior the tool’s implementation. The majority of respondents answered the knowledge questions correctly (range 88.8% to 98.7%). Reported satisfaction was highest for the use of animated videos (85.1%), 76.0% of the respondents felt they were better prepared for CMA testing and 78.1% indicated that they would recommend the tool to others. Decision-aid based on interviews data was reported helpful by 63.4% of respondents.

Conclusions: A pre-CMA test interactive web-based educational tool is well received and valued by women/couples and assists in making informed decisions regarding the disclosure of complex genomic-results.

H. Hochner: None. T. Millo: None. G. Siegal: A. Employment (full or part-time); Modest; This author is the founder and CEO of Consent MD. S. Shkedi-Rafid: None.

P01.020.D Prenatal findings of cataract and arthrogryposis: recurrence of cerebro-oculo-facio-skeletal syndrome and review of differential diagnosis

Fabio Sirchia 1, Ilaria Fantasia2, Agnese Feresin3, Elisa Giorgio1, Moira Barbieri3, Valentina Guida4, Alessandro De Luca4, Tamara Stampalija3,2

1University of Pavia, Pavia, Italy, 2Burlo Garofolo Hospital, Trieste, Italy, 3University of Trieste, Trieste, Italy, 4Division of Medical Genetics, Fondazione IRCCS-Casa Sollievo della Sofferenza, San Giovanni Rotondo, Italy.

Cerebro-oculo-facio-skeletal syndrome (COFS) is a severe and progressive neurologic condition characterized by prenatal onset of arthrogryposis, cataract, microcephaly and growth failure. The aim of this study was to present a case of recurrence of the COFS syndrome and to propose a differential diagnosis flow-chart in case of prenatal findings of arthrogryposis and cataract.

We report a case of recurrence of COFS3 syndrome within the same family, with similar diagnostic features. In the first case the COFS syndrome remainedundiagnosed, while in the second case, due to prenatal findings of arthrogryposis and cataract, genetic investigation focusing on responsible genes of COFS (ERCC5, ERCC6 and FKTN genes) was carried out. The fetus was found to be compound heterozygous for two different ERCC5 mutations, confirming the clinical suspect of COFS syndrome. A review of the literature on possible causative genes of prenatal cataract and arthrogryposis was performed and we present a flow-chart to guide differential diagnosis and possible genetic testing in case of these findings.

COFS syndrome is a rare autosomic recessive condition. However, it can besuspected and diagnosed prenatally. The flow-chart illustrates a pathway to guide differential diagnosis according to the prenatal findings. Main syndromes, key testing and specific genes are included. Targeted molecular testing should be offered to the couple in order to reach a diagnosis and assess the recurrence risk for future pregnancies.

F. Sirchia: None. I. Fantasia: None. A. Feresin: None. E. Giorgio: None. M. Barbieri: None. V. Guida: None. A. De Luca: None. T. Stampalija: None.

P01.021.A Hypomorphic variants in FLNA cause isolated congenital anomalies of kidney and urinary tract in a large family

Shalini S. Nayak, Shravya MS, Katta M. Girisha

Kasturba Medical College, Manipal, Manipal, India.

Introduction: FLNA encodes filamin A, an actin binding protein that regulates the reorganization of the actin cytoskeleton by interacting with integrins, transmembrane receptor complexes and secondary messengers. FLNA is known to cause several X-linked allelic diseases. Recently unrelated four individuals were noted to have isolated congenital anomalies of the kidney and urinary tract (CAKUT) and hypomorphic variants in FLNA. We hereby report a family with six affected offspring’s with CACUT.

Methods: We evaluated a consanguineous family with three-years-old male living child and five abortions. We performed chromosomal microarray followed by duo exome sequencing for the living proband and sixth abortuses.

Results: The family had bilateral renal pyelectasis in first pregnancy, gross ascites in second conceptus and cardiac anomalies in fifth pregnancy. However, a detailed postnatal examination was not possible. Autopsy from third pregnancy revealed bladder outlet obstruction whereas the sixth pregnancy had bilateral tortuous ureters. The living child has hydroureteronephrosis, mildly impaired functioning of left kidney and preserved functioning of right kidney with non-obstructive clearance. Exome sequencing identified a novel hemizygous variant, c.7282G>A; p. (Gly2428Arg) in exon 44 of FLNA, mother is a carrier and father has wild type allele.

Conclusion: The hypomorphic variant c.7282G>A in FLNA is the likely genetic cause responsible for CAKUT seen in this family. However, the segregation analysis in other affected fetuses was not possible due to unavailability of DNA. Also, there was limited phenotypic data in fifth pregnancy to explain the cardiac anomaly.

S.S. Nayak: None. S. Ms: None. K.M. Girisha: None.

P01.022.B Two prenatal cases with a variant of loss in homozygosity involving the CRPPA (ISPD) gene

Cíntia Ventura 1, Raquel Lemos1, Patríca Costa1, Marisa Teixeira1, Joaquim Sá1, Gabriela Soares1, Martin Mistrík2, Rita Cerqueira1

1CGC Genetics UNILABS Portugal, Porto, Portugal, 2Alpha Medical UNILABS Eslováquia, Zilinsky, Slovakia.

Introduction: Homozygous mutations of the CRPPA gene [MIM 614631] are associated with congenital muscular dystrophy-dystroglycanopathy with brain and eye anomalies (type A7) described by OMIM [MIM 614643] including features such as hydrocephalus and being the cause of the most severe phenotype of Walker-Warburg syndrome. We present, in the context of prenatal diagnosis, two cases with aCGH results involving a homozygous deletion that includes the CRPPA gene. The fetus in case 1 presented hydrocephalus and abnormal morphology - Dandy Walker malformation. The fetus in case 2 presented hydrocephalus.

Methodology: The aCGH was performed using Affymetrix Cytoscan 750K. PCR amplification of exons 1 and 9 of the CRPPA gene (chromosome 7) was performed.

Results: Case1 - A female genomic profile was detected with a loss in homozygosity (zero copies) in 7p21.2p21.1 of 360 Kbp involving the CRPPA, CRPPA-AS1 and SOSTDC1 genes. No amplification of exons 1 and 9 of the CRPPA gene was observed, confirming the findings of aCGH. Case 2 - A female genomic profile was detected with a loss in homozygosity (zero copies) in 7p21.2p21.1 of 360 Kbp involving the CRPPA, CRPPA-AS1 and SOSTDC1 genes. These variants are classified as pathogenic and may correspond to these fetuses’ phenotypes.

Conclusions: These findings reinforce the importance of aCGH as a first-line diagnostic test in fetuses with ultrasound anomalies. Genetic Counseling is imperative to guide the couple in the best decision as well as to warn of the need for further analysis in the family.

C. Ventura: None. R. Lemos: None. P. Costa: None. M. Teixeira: None. J. Sá: None. G. Soares: None. M. Mistrík: None. R. Cerqueira: None.

P01.023.C Three foetuses with Cornelia de Lange diagnosis: prenatal findings and genetic diagnosis

Fe Amalia García Santiago 1,2, Elena Mansilla1,2, Eugenia Antolin3, Fernando Santos Simarro1,2, Roberto Rodriguez3, Julian Nevado4,2, Maria Palomares-Bralo4,2, Karen Heath1,2, Rita Maria Regojo5, Carmen Rodriguez-Jimenez6, Isabel Vallcorba6, Angela del Pozo4, Pablo Lapunzina4,2

1INGEMM, Hospital Universitario La Paz, Idipaz, UMDE, Ciberer, Madrid, Spain, 2Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER, U753), Instituto Carlos III, Madrid, Spain., Madrid, Spain, 3Department of Obstetrics and Gynaecology, Hospital Universitario La Paz, Idipaz, UMDE, Madrid, Spain, 4INGEMM, Hospital Universitario La Paz, Idipaz, Ciberer, Madrid, Spain, 5Department of Pathology, Hospital Universitario La Paz, UMDE, Madrid, Spain, 6INGEMM, Hospital Universitario La Paz, Madrid, Spain.

Introduction: Cornelia de Lange syndrome (CdLS; MIM #12270, 300590, 610759, 614701, 300882) is a rare and clinically variable disorder that affects multiple organs. Ultrasound findings are highly variable and include distinctive facial features, prenatal growth retardation (FGR), malformations of the upper limbs, diaphragmatic hernia, heart defects and genitourinary malformations. To date, five genes (NIPBL, SMC1A, SMC3, RAD21 and HDAC8) have been associated with CdLS. We report 3 foetuses with ultrasound characteristics and pathology examination with abnormalities associated with CdLS.

Materials and Methods: an NGS custom panel containing 1663 genes involved in common genetic disorders (RD seq (R) v6.0), exome trio and MLPA (multiplex-ligation dependent probe amplification) P141 (MRC-Holland) were performed after a CMA normal result.

Results: foetus 1 (23 weeks): severe FGR, ulnar hypoplasia and oligodactyly of right hand, hypospadias, left diaphragmatic hernia, prefrontal edema, retrognathia. Genetic analysis: a loss of dosage in heterozygosity is detected in the probe hybridising to exon 43 of the NIPBL gene by MLPA thecnique. Foetus 2: (21 weeks): severe FGR, micromelia, congenital heart, prefrontal edema, microcephalia, short corpus callosum. Genetic analysis: NIPBL NM_133433.3:c.5639_5642del, p.Pro1880Hisfs*10 heterocygosis. Foetus 3: (17 weeks): severe FRG, radius hypoplasia and oligodactyly of left hand, left diaphragmatic hernia, micrognathia. Genetic analysis: NIPBL NM_133433.3:c.598C>T;p./Gln200*) heterocygosis.

Conclusions: CdLS is a heterogeneous clinical and genetic condition. It is essential to have a thorough ultrasound examination and to perform this genetic study in cases of FGR that cannot be explained by chromosomal or vascular alterations.

F.A. García Santiago: None. E. Mansilla: None. E. Antolin: None. F. Santos Simarro: None. R. Rodriguez: None. J. Nevado: None. M. Palomares-Bralo: None. K. Heath: None. R.M. Regojo: None. C. Rodriguez-Jimenez: None. I. Vallcorba: None. A. del Pozo: None. P. Lapunzina: None.

P01.024.D Transcriptome landscape of the human decidual cells

Anastasia A. Babovskaya, Ekaterina Trifonova, Maria Swarovskaya, Viktoria Serebrova, Alexei Zarubin, Vadim Stepanov

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

Human reproductive success depends on a properly decidualized uterine endometrium that allows implantation and the formation of the placenta. At the core of the decidualization process are endometrial fibroblasts that differentiate to decidual stromal cells (DSCs). Characterizing transcriptome of DSC, which is crucial for a pregnancy’s outcome, can serve as a basis for identifying the mechanisms underlying physiological and pathological pregnancy. In our study for the first time for native cells high-throughput sequencing (RNA-seq) was applied to analyze the global transcriptome of the human DSCs during uncomplicated pregnancies. DSCs were obtained by Laser capture microdissection. During analyses we obtained 17960 transcripts expressed with CPM >1 in each sample. Most of the analyzed transcripts corresponded to protein coding regions of the human genome (13683). Antisense RNAs, long noncoding RNAs, and processed pseudogenes predominated in the remaining cluster. To gain a better understanding of the biological implications, the assembled transcripts were annotated using DAVID Bioinformatics Resources. The top 5 represented GO terms for the biological process were cell-cell adhesion, transcription, protein transport, rRNA processing and proteasome-mediated ubiquitin-dependent protein catabolic process. The findings suggest that human DSCs play a key role in the induction of maternal-fetal communication. We applied an upstream analysis approach implemented in geneXplain platform and identified top 10 master regulators (DUSP10, MOS, ROCK2, MAPK6, HTT, SGK1, PRKCI, PASK, DUSP22, and CUX1). These key genes may be potential biomarkers of diagnosis or new therapeutic targets for pregnancy complications.The reported study was funded by RFBR №20-34-90128, №18-29-13045.

A.A. Babovskaya: None. E. Trifonova: None. M. Swarovskaya: None. V. Serebrova: None. A. Zarubin: None. V. Stepanov: None.

P01.025.A Novel pathogenic c.34G>C mutation in GATA4 gene detected in 46,XY DSD patient from Ukraine. The evidence for autosomal dominant DSD inheritance with incomplete penetrance in women

Ludmila Livshits 1, Dmytro Sirokha1, Olexandra Gorodna1, Kamila Kusz-Zamelczyk2, Patrick Sproll3, Anna Lauber Biason3, Serge Nef4, Nataliya Zelinska5

1Institute of Molecular Biology and Genetics, National Academy of Sciences of Ukraine, Kyiv, Ukraine, 2Institute of Human Genetics, Polish Academy of Sciences, Poznań, Poland, 3Section of Medicine, University of Fribourg, Fribourg, Switzerland, 4Faculty of Medicine, University of Geneva, Geneva, Switzerland, 5Ukrainian Scientific and Practical Center for Endocrine Surgery, Transplantation of Endocrine Organs and Tissues, Ministry of Health of Ukraine, Kyiv, Ukraine.

Disorders of sexual development (DSD) are an important group of rare human diseases. To date there are more than 150 known genes involved in DSD and up to 1000 candidates possibly implicated in gonadal development. The aim of the research was to identify novel DSD genetic variants using whole exome sequencing (WES). The WES was performed for a 46,XY SRY positive patient with gonadal dysgenesis. A c.34G>C (rs750597721) mutation in GATA4 gene was identified and confirmed as pathogenic using bioinformatic tools. This is a heterozygous missense substitution that leads to Gly12Ala mutation in a protein sequence that corresponds to transactivation domain 1. Sanger sequencing analysis conducted in family members revealed that healthy mother and healthy maternal grandmother are heterozygous carriers of c.34G>C mutation as well. Previously, another heterozygous mutations in GATA4 were detected in 46,XY DSD patients. Interestingly, the same mutation was previously shown in patient with Atrioventricular septal defect 4. Obtained data are the evidence of phenotypic variation and incomplete penetrance in women. It is in line with previously obtained explanation for possible molecular mechanisms of such inheritance in mice (Bouma et. al., 2007). To our knowledge, this is the first report on c.34G>C in patient with 46,XY DSD and the results of family member analysis support the hypothesis that this variant is causative for 46,XY DSD with autosomal dominant inheritance and incomplete penetrance in women. Study was performed as a part of SCOPES 2013-2017: Joint Research Projects - Genetics of Human Disorders of Sexual Development.

L. Livshits: None. D. Sirokha: None. O. Gorodna: None. K. Kusz-Zamelczyk: None. P. Sproll: None. A. Lauber Biason: None. S. Nef: None. N. Zelinska: None.

P01.026.B Prenatal detection of a familial 640 kb microdeletion in chromosomal region 6q27 in a fetus with isolated severe bilateral ventriculomegaly

Yvonne Stratis 1, Cornelie Müller-Hofstede1, Malvina Kuschmann1, Matthias Meyer-Wittkopf2, Axel Bohring1, Albrecht Röpke1

1Institute of Human Genetics, Münster, Germany, 2Department of Gynecology and Obstetrics at the Health Center Rheine, Mathias Spital, Rheine, Germany.

The phenotype associated with terminal deletions of the chromosome 6q27 region includes intellectual disability, seizures and multiple brain malformations. The most common brain malformations are corpus callosum abnormalities, periventricular nodular heterotopia, polymicrogyria, hydrocephalus, ventriculomegaly and cerebellar malformations. The smallest region of overlap for the phenotype of brain malformations and intellectual disability is refined to a segment of 325 kb in 6q27, comprising the protein coding genes DLL1, PSMB1, TBP and PDCD2. Haploinsufficiency of DLL1, which is a NOTCH ligand, causes a neurodevelopmental disorder with nonspecific brain abnormalities with or without seizures. TBP is a candidate gene for ID and is linked to PDCD2 and PSMB1 in a conserved manner, suggesting a potential interaction between these genes. Here we report on an inherited 640 kb terminal 6q27 deletion in a 29-week fetus with isolated severe bilateral ventriculomegaly detected by SNP-array on uncultured amniocytes. The deletion comprises six genes, including the aforementioned protein coding genes DLL1, PSMB1, TBP and PDCD2 which define the minimal critical region. FISH-analysis of cultured amniocytes confirmed the deletion on metaphase cells. SNP-array and FISH analysis showed that the mildly affected mother harbors the same 6q27 deletion. Prenatal diagnosis of 6q27 deletion is extremely rare and to the best of our knowledge only nine patients have been reported yet. Of these, only four cases had isolated ventriculomegaly, whereas five cases had additional malformations. Our case clearly demonstrates the importance of performing prenatal array analysis also in cases of isolated bilateral ventriculomegaly.

Y. Stratis: None. C. Müller-Hofstede: None. M. Kuschmann: None. M. Meyer-Wittkopf: None. A. Bohring: None. A. Röpke: None.

P01.027.C Telomere length in individuals with early pregnancy losses

Nataliya Huleyuk 1, Danuta Zastavna1, Iryna Tkach1, Ivanna Haiboniuk1, Miroslaw Tyrka2

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

Introduction: Over the past decade, telomere biology has become an important topic in the field of human reproduction. Early pregnancy loss (EPL) occurs in ~ 15% of clinically-recognized pregnancies and is the most common complication of pregnancy. Spontaneously lost pregnancies are characterized by shortened telomeres. We focused on the relationship between relative telomere length (RTL) and tendency to EPL in humans.

Material and Methods: Relative telomere length was measured in DNA isolated from the blood samples using a real-time polymerase chain reaction approach. RTL was examined in control group (C) (N = 209) - women (CW) (N = 107) and men (CM) (N = 102) who had healthy pregnancies with no history of infertility or miscarriage, and in group with EPL (N = 445) - women (EPLW) (N = 223) and men (EPLM) (N = 212) who had single or more EPL. RTL data were analysed by gender and reproductive history.

Results: Women (CW+EPLW) have significantly higher RTL that men (CM+EPLM) (1.74 ± 0.06 in women and 1.40 ± 0.05 in men, Р = 0.000053). Average RTL were significantly lower in CM compared to CW (CW:2.27 ± 0.12 versus CM: 1.15 ± 0.08, Р = 0.0000001), and were similar in EPLW and EPLM (1.50 ± 0.06 in EPLW and 1.53 ± 0.06 in EPLM, Р = 0.47). The EPLW group had significantly lower RTL than control (EPLW:1.50 ± 0.06 versus CW:2.27 ± 0.12, P = 0.0000001). Average RTL were significantly lower in CM compared to EPLM (1.15 ± 0.08 in CM and 1.53 ± 0.06 in EPLM, P = 0.00006).

Conclusions: Women with no history of EPL have longer telomere that men. Woman with EPL have shorter telomere that women without miscarriage. In EPL group women and men have similar telomere length.

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

P01.028.D Genetic basis of endometriosis in the susceptibility of developing gynecological cancers

Aintzane Rueda-Martínez1,2, Bárbara Paola González-García1,2, Aiara Garitazelaia1,2, Ariadna Cilleros-Portet1,2, Sergi Marí1,2, Rebeca Arauzo1,2, Jokin de Miguel1,2, Nora Fernandez-Jimenez1,2, Jose Ramon Bilbao1,2,3, Iraia García-Santisteban 1,2

1University of the Basque Country (UPV/EHU), Leioa, Spain, 2Biocruces-Bizkaia Health Research Institute, Barakaldo, Spain, 3Spanish Biomedical Research Center in Diabetes and associated Metabolic Disorders, Madrid, Spain.

Introduction: Endometriosis is a common gynecological disorder in which the endometrium grows outside of the uterus. Despite being considered a benign condition, epidemiological evidence shows that women with endometriosis develop more frequently certain types of gynecological cancers, including endometrial, breast and ovarian carcinomas. However, the mechanisms underlying this relationship remain uncertain. Since genetic factors play a key role in these complex gynecological diseases, we hypothesized that the biological mechanisms behind this comorbidity could be mediated, at least in part, by shared genetic predisposition factors.

Materials and Methods: To test this hypothesis, we undertook a Bioinformatics approach that consisted of a cross-disorder meta-analysis and a Two-Sample Mendelian Randomization (2SMR) analysis of results from public GWASs on endometriosis and the abovementioned gynecological cancers.

Results: Firstly, our meta-analysis revealed novel susceptibility loci shared between endometriosis and endometrial, breast, and ovarian cancers, although the input of endometriosis was minor when compared to the cancer studies. Secondly, our 2SMR analysis confirmed previously reported genetic pleiotropy between endometriosis and endometrial cancer but gave inconclusive results about breast cancer, also in line with previous reports. Our research provides, for the first time, solid evidence of a causal genetic association between endometriosis and ovarian cancer, particularly clear cell type, and endometroid subtypes. Furthermore, we also identified genetic variants that could mediate in those associations, allowing future functional experiments.

Conclusions: This study represents the first Bioinformatics approach to elucidate the causal genetic relationship between endometriosis and gynecological cancers. Funding: GVSAN2020/111043, GVSAN2018/111086, and GVSAN2019/111085 to I.G.-S., J.R.B., and N.F.-J., respectively.

A. Rueda-Martínez: None. B.P. González-García: None. A. Garitazelaia: None. A. Cilleros-Portet: None. S. Marí: None. R. Arauzo: None. J. de Miguel: None. N. Fernandez-Jimenez: None. J. Bilbao: None. I. García-Santisteban: None.

P01.029.A Assisted reproductive technology can be a risk for epimutation-mediated imprinting disorders for mothers over 30 years

Kaori Hara-Isono 1, Keiko Matsubara1, Masashi Mikami2, Takahiro Arima3, Tsutomu Ogata4, Maki Fukami1, Masayo Kagami1

1National Research Institute for Child Health and Development, Tokyo, Japan, 2National Center for Child Health and Development, Tokyo, Japan, 3Tohoku University Graduate School of Medicine, Sendai, Japan, 4Hamamatsu University School of Medicine, Hamamatsu, Japan.

Backgrounds: The proportion of assisted reproductive technology (ART)-conceived livebirths of patients with imprinting disorders (IDs) is higher than that of the general population. Whether this is due to ART or confounding effects of advanced parental age was unknown. The aims of this study are 1) to clarify whether ART or maternal ages facilitates development of epimutation-mediated IDs (epi-IDs), and 2) to identify the differentially methylated region (DMR) that is vulnerable to the effect of ART and parental ages.

Results: We enrolled 136 patients with epi-IDs and obtained general population ART data from the Japanese robust nationwide registry. We compared the proportion of ART-conceived livebirths and maternal childbearing ages between patients with epi-IDs and the general population. The proportion of ART-conceived livebirths in patients with epi-IDs was higher than that in mothers aged ≥ 30 years, the age group in which more than 90% of ART procedures performed. The maternal childbearing ages of patients with epi-IDs were widely distributed from 19 to 45 (median: 32). In addition, we compared the proportion of ART-conceived livebirths and parental ages at childbirth across patients with eight epi-IDs. We demonstrated that most ART-conceived patients with epi-IDs were found in Silver-Russell syndrome (SRS) and Beckwith-Wiedemann syndrome (BWS) patients, and parental ages were almost consistent in patients with eight epi-IDs.

Conclusions: ART can be a risk factor for the development of epi-IDs for mothers aged ≥ 30 years. In addition, methylation status of SRS- and BWS- related DMRs may be vulnerable to the effects of ART.

K. Hara-Isono: None. K. Matsubara: None. M. Mikami: None. T. Arima: None. T. Ogata: None. M. Fukami: None. M. Kagami: None.

P01.030.B Analyzing the effects of ETV5 and CXCL12 genes in patients with Sertoli cell-only syndrome

O. Sena AYDOS 1, Dunya AYDOS2, Yunus YUKSELTEN3, Asuman SUNGUROGLU1, Kaan AYDOS4

1Department of Medical Biology, Ankara University School of Medicine, Ankara, Turkey, 2Ankara University Stem Cell Institute, Ankara, Turkey, 3Research Laboratories for Health Science, Y Gen Biotechnology Company Ltd., Ankara, Turkey, 4Department of Urology, Ankara University School of Medicine, Ankara, Turkey.

Introduction: Ets variant gene 5 (ETV5), belongs to a family of transcription factors, regulates several genes essential for spermatogonial stem cells (SSCs) self-renewal. Silencing of ETV5 in mice led to total loss of stem/progenitor spermatogonia, resulting in Sertoli cell-only (SCO) phenotype. ETV5-deficient Sertoli cells were also found to have decreased CXCL12 levels. In embryonic mouse gonads, CXCL12 was shown to direct the migration of primordial germ cells (PGCs) to the gonadal ridges. We aimed to investigate the role of interaction between ETV5 and CXCL12 genes in humans with SCO syndrome.

Materials and methods: Fold changes in expression levels of CXCL12 and ETV5 were determined by quantitative PCR in non-obstructive azoospermia (NOA) (n = 10) patients with SCOS and obstructive azoospermia (OA) (n = 2) as control cases. Testicular tissue samples were taken during microTESE attempt and testicular biopsy was performed for histopathological evaluation.

Results: Fold decreases in CXCL12 and ETV5 expression levels were found as 0.44 ± 0.1 and 0.27 ± 0.11, respectively, and the differences were significant compared to controls (p < 0.001).

Conclusions: According to our results, the decrease in CXCL12 and ETV5 expression levels in patients with SCOS indicated that CXCL12 and ETV5 work together in harmony to regulate the presence and maintenance of SSCs in humans. Whether the germ cell loss is due to inhibition of PGCs migration or impaired differentiation of SSCs needs further investigation.

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

P01.031.C Exome sequencing in structurally normal fetuses - yield and dilemmas

Hagit Daum, Tamar Harel, Shiri Gershon-Naamat, Adily Basal, Orly Elpeleg, Vardiella Meiner, Hagar Mor-Shaked

Hadassah, Jerusalem, Israel.

Abstract Introduction: Although there is a growing body of literature regarding the yield of chromosomal microarray analysis for structurally normal fetuses, there is no data regarding the yield of exome sequencing in this population. Exome sequencing (ES) is a powerful tool for identifying disease-causing single nucleotide variants (SNVs) and small indels. In the prenatal setup, when the clinical phenotyping of the fetus is frequently vague and non-specific, the contribution of exome sequencing to the diagnostic procedure is invaluable.

Material and methods: From February 2017 to February 2021, a total of 635 fetal exomes were analyzed at our center. Among these were 119 structurally normal fetuses. Analysis included SNVs, copy number variants and uniparental disomy. Reports included pathogenic and likely pathogenic findings, including ACMG secondary findings that are relevant during childhood.

Results: Two fetuses (1.7%) had molecular diagnoses of moderate to severe disease severity. Pathogenic compound heterozygous variants were identified in ATP7B gene and NR2E3, responsible for Wilson disease and for Enhanced S-cone syndrome, respectively. Notably, Wilson disease is a potentially treatable disease and early diagnosis is critical to postnatal management.

Conclusions: With careful management and restrictive analysis, prenatal ES should be considered as an adjunct to chromosomal microarray in sonographically normal fetuses. Further studies are called for in order to determine the yield of prenatal ES in this setting.

H. Daum: None. T. Harel: None. S. Gershon-Naamat: None. A. Basal: None. O. Elpeleg: None. V. Meiner: None. H. Mor-Shaked: None.

P01.032.D Genetic variants of MTHFR and TNF-α in fetal growth restriction

dema alset 1, Elena Butenko1, Ekaterina Zabanova2, Natalia Kuznetsova2

1southern federal university, Rostov-on-Don, Russian Federation, 2Rostov State Medical University, Rostov-on-Don, Russian Federation.

Introduction: Fetal Growth Restriction (FGR) is a multifactorial condition in which fetus cannot reach its genetically determined potential size. This syndrome is considered as an important cause of fetal morbidity and mortality and affects 5-10% of all pregnancies worldwide and 5-17.6% in Russia. Clinician trials are concerning maternal, fetal and placental polymorphisms as possible prenatal FGR-markers. This study aimed to investigate associations of two polymorphisms: 5,10–Methylenetetrahydrofolate reductase/ MTHFR(677T) and Tumor necrosis factor alpha/TNF-α(G308A) with FGR risk.

Material and methods: Using PRISMA statement, 20 studies were included in meta-analysis of MTHFR (C677T) and TNF-α (G308A) associations with FGR. Then MTHFR (C677T) genotyping was conducted with Allele-specific PCR to confirm meta-analysis result in FGR-diagnosed (n = 26) and healthy (n = 37) pregnant women.

Results: Meta-analysis showed no association of TNF-α(G308A) but a strong association of MTHFR(C677T) with high FGR risk (OR = 1.22, 95 % CI: 1.07-1.39, P = 0.002). However, Genotyping showed that in the studied population, MTHFR(C677T) has no significant association with FGR (OR = 0.917, 95 % CI: 0.328-2.560, P = 0.956).

Conclusion: To our knowledge, this is the first meta-analysis of TNF-α(G308A) in FGR. About MTHFR(C677T), genotyping result was inconsistent with meta-analysis. This suggests that association differs according to ethnicity, since some of the studies included in meta-analysis figured no association in their samples. However, due to our small sample size, further clinical trials are required to confirm results in the studied population.This study was funded by the Ministry of Science and Higher Education of the Russian Federation #0852-2020-0028.

D. alset: None. E. Butenko: None. E. Zabanova: None. N. Kuznetsova: None.

P01.033.A The FTO gene and adolescent puberty

Elena Mashkina, Maria Amelina, Michail Shkurat

Southern Federal University, Rostov-on-Don, Russian Federation.

Introduction: The time of the onset of puberty and obesity are multifactorial features of human. The aim of this work was to analyze the association between the FTO 23525T>A gene polymorphism and the delayed sexual development in boys aged 11-15 years.

Material and methods: DNA samples isolated from blood cells of 322 adolescents were used. The boys were divided into 2 groups - with a normal rate of sexual development and with a delay in sexual development. The stages of sexual development were determined using the Tanner scale. Allele-specific PCR was used to analyze the rs99305069 FTO.

Results: We analyzed the frequencies of genotypes for the FTO gene 23525T>A polymorphism among 11-15 year old boys depending on the stage of puberty. Heterozygotes 23525TA of the FTO gene predominated (45%) among boys with normal sexual development. The frequency of homozygotes for the 23525A allele was 28% in this group. In the group with delay sexual development the frequency of 23525AA genotype was 40%. Homozygotes 23525AA have an increased risk of delayed sexual development during puberty (OR = 1.38 CI 1.1-2.78). Also the 23525A allele of the FTO gene is more frequently recorded among adolescents with delay in sexual development (χ2 = 6.26 p = 0.01; OR = 1.49 (1.09-2.04)). Thus, an association of the 23525T>A polymorphism of the FTO gene with a disturbance in the rate of puberty in adolescents was revealed. This study was funded by the Ministry of Science and Higher Education of the Russian Federation #0852-2020-0028.

E. Mashkina: None. M. Amelina: None. M. Shkurat: None.

P01.034.B Genetic counseling and carrier screening of candidates for gamete donation at a public bank

Celia Azevedo Soares 1, Ana R. Soares1, Emídio Vale Fernandes2, Maria Abreu1, Cláudia Falcão Reis1, Ana M. Fortuna1, Natália Tkachenko1

1Centro de Genética Médica Jacinto Magalhães, Porto, Portugal, 2Centro Materno Infantil do Norte, Porto, Portugal.

Introduction: Genetic counseling and carrier screening of healthy candidates is part of gamete donors’ selection. We aim to review the findings of the genetic counseling of a cohort of patients at our public gametes bank.

Methods: Thirty-four male and 64 female candidates had genetic counseling with a medical geneticist before donation. Of these, one female candidate voluntarily dropped-out. Thirty-four males and 63 females performed karyotype and screening for the more common pathogenic variants of CFTR-related cystic fibrosis and spinal muscular atrophy (SMN1) in the Portuguese population. In addition, all females also performed Fragile X expansion screening (FMR1). Thirty patients with ancestry from Southern or Central Portugal, or with known or assumed African ancestry performed hemoglobinopathies screening.

Results: Six patients were withheld from the donation process given their family or personal history that required further investigation. Of the initial 97 candidates, 15.5% presented anomalous laboratory results (15/97). Ten patients were carriers for an autosomal recessive disorder - cystic fibrosis (5/97), sickle cell anemia (3/30), and spinal muscular atrophy (2/97). One female was an FMR1 pre-mutation carrier (1/63). One female patient presented with triple X mosaicism: 47,XXX[2]/46,XX[50]. Two patients presented with chromosomal instability of unknown origin. In one patient, a mosaic for the Philadelphia chromosome was detected, revealing the unexpected diagnosis of chronic myeloid leukemia.

Conclusions: From a cohort of 97 candidates, 21.7% presented a family/personal history or an anomalous laboratory result that required additional genetic counseling, stressing the importance of performing pre-donation genetic counseling in this population.

C. Azevedo Soares: None. A.R. Soares: None. E. Vale Fernandes: None. M. Abreu: None. C. Falcão Reis: None. A.M. Fortuna: None. N. Tkachenko: None.

P01.035.C Ascertainment of genome-wide androgenetic mosaicism after discordant results from primary fetal samples and cultured cells

Gioia Mastromoro 1, Daniele Guadagnolo1, Enrica Marchionni1, Francesca Di Palma1, Barbara Torres2, Marina Goldoni2, Annamaria Onori2, Laura Bernardini2, Alessandro De Luca2, Isabella Torrente2, Antonio Pizzuti1

1Sapienza University of Rome, Rome, Italy, 2Fondazione IRCCS Casa Sollievo della Sofferenza, San Giovanni Rotondo, Italy.

Introduction: Genome-wide androgenetic mosaicism is a rare condition in which two euploid cell lines coexist in the same individual, one with biparental content and one with genome-wide paternal isodisomy. It can present prenatally resembling Beckwith-Wiedemann syndrome (BWS). We report a fetus with a laborious diagnosis due to discordant results from cultured and uncultured samples.

Materials and Methods: A pregnant was referred at 15 gestational weeks for placental mesenchymal dysplasia and omphalocele. Karyotype, chromosomal microarray analysis (CMA) and BWS molecular testing (methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) analysis of 11p15 BWS critical region) were performed after amniocentesis. These tests were requested again on umbilical cord sample and on previously cultured amniocytes.

Results: Karyotype, CMA performed by an oligonucleotide-array and BWS MS-MLPA after amniocentesis were normal. BWS MS-MLPA from cultured amniocytes and umbilical cord sample showed KvH19 locus hypermethylation and KvDMR hypomethylation in mosaicism. These results, along with microsatellite analysis of BWS region, were consistent with mosaic paternal isodisomy of chromosome 11p15. Analysis performed to assess maternal contamination showed on cultured amniocytes and umbilical blood sample that the paternal alleles were constantly higher on all the microsatellite analyzed mapping in different chromosomes. This result leaded to suspicion of genome-wide androgenetic mosaicism, confirmed via SNP-array analysis from cultured amniocytes, with mosaic rate of 60%.

Conclusions: Assessment of genome-wide androgenetic mosaicism requires multiple laboratory approaches and an extension of the current diagnostic process and caution to low rate of mosaicism. Clinical acumen and an integrated testing approach are the key to a successful diagnosis.

G. Mastromoro: None. D. Guadagnolo: None. E. Marchionni: None. F. Di Palma: None. B. Torres: None. M. Goldoni: None. A. Onori: None. L. Bernardini: None. A. De Luca: None. I. Torrente: None. A. Pizzuti: None.

P01.036.D Sensitive screening of cell free DNA to determine the origin of trophoblastic tumours

Geoffrey J. Maher, Rosemary A. Fisher, Baljeet Kaur, Xianne Aguiar, Preetha Aravind, Natashia Cedeno, James Clark, Debbie Damon, Ehsan Ghorani, Foteini Kalofonou, Ravindhi Murphy, Rajat Roy, Naveed Sarwar, Mark R. Openshaw, Michael J. Seckl

Imperial College London, London, United Kingdom.

Introduction: Trophoblastic tumours that secrete human chorionic gonadotropin (hCG) are commonly gestational in origin and are typically diagnosed months to years after the causative pregnancy. However, hCG secretion can also occur in somatic or germ cell tumours. Despite the clinical importance of distinguishing between gestational and non-gestational trophoblastic tumours, which vary in their prognoses and treatment regimens, tumour biopsies are not always available for genotyping due to the risk of haemorrhage. Here we aimed to develop a sensitive cell free DNA (cfDNA) assay to non-invasively determine the origin of hCG-secreting tumours.

Materials and Methods: Genomic DNA and cfDNA from 23 women with hCG-secreting tumours underwent library preparation, probe capture and deep (>5,000x) Illumina sequencing of 195 common autosomal single nucleotide polymorphisms (SNPs) and 13 sex chromosome loci. Gestational tumours were identifiable by the presence of ‘non-host’ (i.e. paternal) alleles in cfDNA at SNPs that were homozygous in the genomic DNA.

Results: In gestational cases, non-host alleles were detected at multiple SNPs; non-host cfDNA comprised 0.3% - 41.4% of total cfDNA and correlated with serum hCG levels (906 – 3,042,881 IU/ml), although detection was variable below ~1,500 IU/ml. Non-host alleles were not detected in non-gestational cases, but the presence of circulating tumour DNA was confirmed by the identification of copy number alterations.

Conclusions: Previous methods for detecting cfDNA from hCG-secreting tumours lacked sensitivity or were patient-specific, making them unsuitable for routine diagnostic testing. Our sensitive non-invasive assay, applicable to any patient, will facilitate diagnosis at an earlier timepoint and improve patient management.

G.J. Maher: None. R.A. Fisher: None. B. Kaur: None. X. Aguiar: None. P. Aravind: None. N. Cedeno: None. J. Clark: None. D. Damon: None. E. Ghorani: None. F. Kalofonou: None. R. Murphy: None. R. Roy: None. N. Sarwar: None. M.R. Openshaw: None. M.J. Seckl: None.

P01.038.B Delivering accurate, reproducible non-invasive prenatal diagnosis (NIPD) for sickle cell disease using droplet digital PCR

Joe Shaw 1, Ben Paternoster1, Maureen Ramos1, Sarah Nesbitt1, Natalie Chandler1, Lyn S. Chitty1,2

1NHS North Thames Genomic Laboratory Hub, London, United Kingdom, 2UCL Great Ormond Street Institute of Child Health, London, United Kingdom.

Introduction: Sickle cell disease (SCD) is the most common single-gene indication for prenatal diagnosis in England. Non-invasive prenatal diagnosis (NIPD) for SCD is desired by patients, but complicated by the high background of the maternal mutation and frequent unavailability of paternal samples. Droplet digital PCR (ddPCR) offers the potential for NIPD using only a maternal sample via relative mutation dosage; however previous reports describe unacceptable rates (up to 10%) of incorrect genotype classifications.

Methods: A ddPCR assay was designed for the common SCD variant, HBB c.20A>T, and validated on heterozygous genomic DNA and paternal cell free DNA. Following optimisation, cell free DNA was extracted from 65 frozen maternal plasma samples from pregnancies at risk of SCD with known fetal genotypes. ddPCR analysis was performed blinded using a modified sequential probability ratio test (SPRT).

Results: ddPCR testing correctly predicted the fetal genotype in 48 cases (32 male, 16 female), with nine of 10 HbSS fetuses correctly identified. A sample from a dichorionic twin pregnancy was included, and the modified SPRT analysis correctly classified one fetus as affected whilst the other was heterozygous. The remaining 17 (26%) cases were inconclusive, with no incorrect fetal genotype predictions.

Conclusion: We have successfully optimised a ddPCR assay that accurately predicts the fetal genotype in pregnancies at risk of SCD. Further optimisation of cfDNA extraction and sampling is required to reduce the rate of inconclusive results and confirm accuracy. Funding was from the GOSH NIHR Biomedical Research Centre. Samples were obtained from the RAPID sample bank.

J. Shaw: None. B. Paternoster: None. M. Ramos: None. S. Nesbitt: None. N. Chandler: None. L.S. Chitty: None.

P01.039.C Telomeres in TB is longer than in ICM in humans at the blastocyst stage

Anna A. Pendina 1, Andrei V. Tikhonov1, Vladimir A. Bogomolov2, Mikhail I. Krapivin1, Olga A. Efimova1, Irina D. Mekina1, Evgeniia M. Komarova1, Natalia P. Smirnova3, Olga G. Chiryaeva1, Alexander M. Gzgzyan1, Igor Yu. Kogan1, Vladislav S. Baranov1

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

Telomeres are complexes of short tandem DNA repeats and proteins at the ends of chromosomes. Their main function is the protection of chromosomes from shortening caused by DNA loss during each replication cycle. Correct regulation of telomere length (TL) is crucial for normal embryogenesis. Here, we performed a pairwise comparison of TLs in trophectoderm (TE) and inner cell mass (ICM) of human blastocysts. A total of 22 blastocysts were included in the study. All the blastocysts were not suitable for transfer because of genetic abnormalities revealed by preimplantation genetic testing. Each blastocyst was dissected into TE and ICM using a microsurgical laser and fixed on a glass slide. Telomeres were detected by qFISH (Telomere PNA FISH Kit/Cy3, Agilent). To avoid possible bias caused by different chromatin condensation, TLs were assessed as relative values by dividing the telomeric fluorescence by the fluorescence of reference region (21q22.13-q22.2) measured in ImageJ 1.51i. The relative TL values ranged between 0.089-0.607 in TE and between 0.061-0.414 in ICM. In TE, the relative TL appeared to be higher than in ICM (p = 0.029, paired t-test). Longer telomeres in TE may be linked to its crucial role in implantation and subsequent placentation, both accompanied by a high mitotic activity. Supported by RSF № 18-75-10046.

A.A. Pendina: None. A.V. Tikhonov: None. V.A. Bogomolov: None. M.I. Krapivin: None. O.A. Efimova: None. I.D. Mekina: None. E.M. Komarova: None. N.P. Smirnova: None. O.G. Chiryaeva: None. A.M. Gzgzyan: None. I.Y. Kogan: None. V.S. Baranov: None.

P01.040.D Are children born after medical assisted reproduction at greater risk of having an increased de novo mutation rate?

Roos M. Smits1, Manon S. Oud2, Aukje M. Meijerink1, Petra de Vries2, Giles S. Holt3, Bilal K. S. Alobaidi3, Lois Batty3, Kathrin Fleischer1,4, Didi D. M. Braat1, Liliana Ramos1, Miguel J. Xavier 3, Joris A. Veltman3

1Department of Obstetrics and Gynaecology, Radboudumc, Nijmegen, Netherlands, 2Department of Human Genetics, Donders Institute for Brain, Cognition and Behaviour, Radboudumc, Nijmegen, Netherlands, 3Biosciences Institute, Faculty of Medical Sciences, Newcastle University, Newcastle upon Tyne, United Kingdom, 4TFP Center of Reproductive Medicine, Düsseldorf, Germany.

Introduction: De novo mutations (DNMs) play a prominent role in sporadic disorders with reduced fitness such as infertility and intellectual disability. Advanced paternal age is known to increase disease risk in offspring by increasing the number of DNMs in their genome. Less is known about the effect of assisted reproduction techniques (ART) on the number of DNMs in offspring. With the on-going trend of delayed parenthood more children are now born both from older fathers and through ART.

Materials and Methods: We investigated 49 trios (mother, father and child) and 2 quartets (mother, father and 2 siblings) divided into children born after spontaneous conception (n = 18); born after in vitro fertilisation (IVF) (n = 17) and born after intracytoplasmic sperm injection combined with testicular sperm extraction (ICSI-TESE) (n = 18). Groups further divided by paternal age, young (<35) or old (>45 years of age at conception). Whole-genome sequencing was performed twice to independently detect and validate all DNMs in children.

Results: A clear paternal age effect was observed, with 70 DNMs detected on average in children born to young fathers and 94 DNMs in those born to older fathers (p = 0.001). No significant differences were observed between different methods of conception (p = 1) with paternal age affecting all methods equally.

Conclusions: Paternal age, not method of conception, had a major effect on the observed number of DNMs in offspring. Given the role DNMs in disease risk, this negative result is good news for IVF and ICSI-TESE born children, if replicated in larger cohorts.

R.M. Smits: None. M.S. Oud: None. A.M. Meijerink: None. P. de Vries: None. G.S. Holt: None. B.K.S. Alobaidi: None. L. Batty: None. K. Fleischer: None. D.D.M. Braat: None. L. Ramos: None. M.J. Xavier: None. J.A. Veltman: None.

P01.041.A The relevance of loss-of-function variants in the X-chromosomal gene TEX13B in azoospermia is questionable

Margot J. Wyrwoll 1,2, Manon S. Oud3, Matthias Vockel4, Sabine Kliesch2, Corinna Friedrich1, Frank Tüttelmann1

1Institute of Reproductive Genetics, University of Münster, Münster, Germany, 2Centre of Reproductive Medicine and Andrology, Department of Clinical and Surgical Andrology, University Hospital Münster, Münster, Germany, 3Department of Human Genetics, Radboud University Medical Centre, Nijmegen, Netherlands, 4Institute of Human Genetics, University of Münster, Münster, Germany.

Introduction: Azoospermia is often assumed to be of genetic origin, with constantly emerging genes in the context of male infertility. Previously, a chromosomal translocation in two infertile brothers with breakpoints close to TEX13B was described and a recent publication reported a stop-gain variant in TEX13B as cause for azoospermia in one man. According to RNA-sequencing data, TEX13B is a germ cell-specific gene with highest expression in spermatogonia. These findings suggest an association of the X-linked gene TEX13B with male fertility.

Materials and Methods: Because these studies were limited in size, we screened the exome data of our Male Reproductive Genomics (MERGE) cohort of currently 1,003 men with various infertility phenotypes and some fertile controls for variants in TEX13B. Additionally, we utilised the exome data of 5,784 genetically proven fathers. The data were filtered for rare stop-gain, frameshift and splice-site variants (minor allele frequency [MAF] <1% in gnomAD database) in TEX13B.

Results: We detected nine azoospermic men carrying the hemizygous stop-gain variant c.382C>T;p.(Gln128Ter) in TEX13B. A fertile control, a man with asthenoteratozoospermia and a proven father were also hemizygous for this variant. Another proven father carried the hemizygous splice-donor variant c.459+1G>A in TEX13B.

Conclusions: Based on these findings, we question whether pathogenic variants in TEX13B are a monogenic cause for azoospermia. We are currently screening additional fertile men for the recurrent stop-gain variant c.382C>T;p.(Gln128Ter) to determine the frequency of this variant in the fertile male population. This work was supported by the DFG Clinical Research Unit 326 ‘Male Germ Cells’.

M.J. Wyrwoll: None. M.S. Oud: None. M. Vockel: None. S. Kliesch: None. C. Friedrich: None. F. Tüttelmann: None.

P01.043.C Impact of cigarette smoking on the expression of oxidative stress-related genes in cumulus cells retrieved from healthy women undergoing IVF

Fani Konstantinidou 1,2, Maria Cristina Budani3, Annalina Sarra4, Gian Mario Tiboni3, Liborio Stuppia1,2, Valentina Gatta1,2

1School of Medicine and Health Sciences, "G. d’Annunzio" University of Chieti-Pescara, Chieti, Italy, 2Unit of Molecular Genetics, Center for Advanced Studies and Technology (CAST), "G. d’Annunzio" University of Chieti-Pescara, Chieti, Italy, 3Department of Medical, Oral and Biotechnological Sciences, “G. d’Annunzio” University of Chieti-Pescara, Chieti, Italy, 4Department of Philosophical, Pedagogical and Quantitative Economic Sciences, "G. d’Annunzio" University of Chieti-Pescara, Chieti, Italy.

Increased production of reactive oxygen species is an important factor in the pathophysiology of fertility decline. Cigarette smoking, for example, can directly derange folliculogenesis by ROS production. Delicate communication between the germline and cumulus cells (CCs) is also the basis for all processes in ovarian physiology. Several studies have aimed to discover non-invasive tools to indirectly evaluate oocyte quality by focusing on CCs as a mirror of oocyte characteristics. However, the general lack of uniformity among them indicates that their utility in the clinical setting remains controversial due to their high sensitivity to intrinsic and extrinsic factors related to the patient.On these premises, we focused our attention on evaluating the impact of tobacco smoking on gene expression of CCs in two categories of individuals, 5 overall healthy smokers and 5 non-smokers (<35 years of age) undergoing IVF treatments. Total RNA was extracted from CCs of all subjects and subsequently reverse transcribed. Predesigned oxidative stress 96-well plates were used to perform qRT-PCR analysis. A gene was considered differentially expressed in smokers’ CCs versus control CCs when showing a fold change >1.4 or <0.7 and a P-value < 0.05 (ANOVA). Statistical analysis showed a significant down-regulation of genes protecting and repairing cells against oxidative damage in CCs of all or the majority of smoking females compared to their corresponding age-matched controls, indicating a cigarette smoke-derived impact on the oxidative stress pathway.In conclusion, an important interaction between cigarette smoking and oxidative stress-related genes in possible fertility biomarkers is clearly evidenced.

F. Konstantinidou: None. M. Budani: None. A. Sarra: None. G. Tiboni: None. L. Stuppia: None. V. Gatta: None.

P01.044.D To be or not to be pathogenic - distinguishing pathogenic mutations from benign missense variants in NR5A1

Jana Emich* 1, Corinna Friedrich*1, Christina Burhöi1, Thaís Fenz Araujo2, Ann-Christin Tewes3, Susanne Ledig3, Albrecht Röpke3, Sabine Kliesch4, Jörg Gromoll5, Frank Tüttelmann1

1Institute of Reproductive Genetics, University of Münster, Münster, Germany, 2Department of Genetics, Ribeirao Preto Medical School, University of Sao Paulo, Ribeirao Preto, Brazil, 3Institute of Human Genetics, University of Münster, Münster, Germany, 4Centre of Reproductive Medicine and Andrology, Department of Clinical and Surgical Andrology, University Hospital Münster, Münster, Germany, 5Centre of Reproductive Medicine and Andrology, Institute of Reproductive and Regenerative Biology, University of Münster, Münster, Germany.

*These authors contributed equally.

Introduction: Infertility affects 10-15% of all couples. The most severe form of male infertility is azoospermia, of which most cases are suspected to be of genetic origin. The transcription factor SF1, encoded by NR5A1, plays a central role in gonadal development. Severe variants are associated with gonadal dysgenesis and sex reversal, while mild missense variants have been reported in isolated forms of male infertility.

Materials and methods: The exome data of 1,003 otherwise healthy infertile men from the Male Reproductive Genomics (MERGE) cohort was filtered for heterozygous missense variants in NR5A1 with a minor allele frequency of ≤0.1% in the gnomAD database. The identified variants were forwarded to functional analysis by site-directed mutagenesis and a luciferase reporter assay.

Results: We detected eight potentially relevant missense variants in nine patients with severe oligozoospermia or azoospermia, resulting in a detection rate of 1.0% for NR5A1 in the MERGE cohort. In the luciferase assay, two of these variants showed reduced SF1 transcriptional activity.

Conclusions: The assessment of missense variants is challenging, warranting functional analyses to distinguish pathogenic mutations from benign variants. The luciferase reporter assay supports that two of the investigated variants are relevant in the pathogenesis of spermatogenic failure and male infertility.

This work was supported by the DFG Clinical Research Unit 326 ‘Male Germ Cells’.

J. Emich*: None. C. Friedrich*: None. C. Burhöi: None. T. Fenz Araujo: None. A. Tewes: None. S. Ledig: None. A. Röpke: None. S. Kliesch: None. J. Gromoll: None. F. Tüttelmann: None.

P01.045.A FKBP6 has an essential role in human spermatogenesis

Godfried W. van der Heijden 1,2, Manon Oud2, Rytis Stakaitis3, Ieva Golubickaite3, Channah Gaasbeek2, Nadja Rotte4,5, Sabine Kliesch4, Liliana Ramos1, Kristian Almstrup3, Miguel J. Xavier6, Joris A. Veltman6, Frank Tüttelmann5

1Division of Reproductive Medicine, Department of Obstetrics and Gynecology, Radboud University Medical Centre, The Netherlands, Nijmegen, Netherlands, 2Department of Human Genetics, Donders Institute for Brain, Cognition and Behavior, Radboud University Medical Centre, Nijmegen, Netherlands, 3Copenhagen University Hospital - Rigshospitalet, Department of Growth and Reproduction, Copenhagen, Denmark, 4Centre of Reproductive Medicine and Andrology, Department of Clinical and Surgical Andrology, University of Münster, Münster, Germany, 5Institute of Reproductive Genetics, University of Münster, Münster, Germany, 6Biosciences Institute, Faculty of Medical Sciences, Newcastle University, Newcastle, United Kingdom.

We performed exome sequencing in an infertile patient and his fertile parents to identify the genetic cause of his germ cell arrest in spermatogenesis. A homozygous pathogenic 22-bp insertion was identified in the FKBP6 gene. In an independent cohort, a second patient with a different homozygous variant predicted to be pathogenic was also identified in FKBP6. RNA isolated from testicular tissue was used to show that FKBP6 expression levels were severely reduced in both patients, confirmed by immunostaining. In male mice, Fkbp6 functions in the fetal piRNA-pathway and localizes to the synaptonemal complex (SC) during meiosis. In adult male KO mice, failure to complete chromosome synapsis causes meiotic arrest, presumably due to its absence from the SC. To ascertain if these features also underlie infertility in humans, we analyzed meiotic progression in the two cases. Germ cell loss did occur during meiosis but not due to incomplete synapsis of the chromosomes. In control samples, we were unable to detect FKBP6 at the SC, altogether suggesting a function for FKBP6 outside SC-biology. To determine if a role for FKBP6 in the piRNA-pathway was evident in humans, we assessed its co-localization with piRNA-pathway factors PIWIL1 and TDRKH. In controls, FKBP6 localized to cytoplasmic granules together with PIWIL1 and TDRKH. Loss of FKBP6, however, did not affect the localization of these factors. Our data indicates that FKBP6 is required for human spermatogenesis and that it might play a role in the piRNA-pathway rather than in the establishment of the SC.

G.W. van der Heijden: None. M. Oud: None. R. Stakaitis: None. I. Golubickaite: None. C. Gaasbeek: None. N. Rotte: None. S. Kliesch: None. L. Ramos: None. K. Almstrup: None. M.J. Xavier: None. J.A. Veltman: None. F. Tüttelmann: None.

P01.047.C Effects of polymorphisms of energy metabolism and angiogenesis genes on intrauterine growth restriction

Irina Novikova, Inna Pokudina, Elena Mashkina, Tatiana Shkurat

Southern Federal University, Rostov-on-Don, Russian Federation.

Introduction: Intrauterine growth restriction (IUGR) is a condition in which the growth rate of the fetus during pregnancy is less than expected. Energy metabolism genes play an important role in the regulation of both maternal and fetal body weight and thus in the development of IUGR. Another placental key factor of IUGR physiopathology is angiogenesis. The aim of research is to study the polymorphisms of genes of energy metabolism (LEPR; FTO) and angiogenesis (NOS3; VEGFA) in women with IUGR compared to the control group to identify it`s possible functional significance in the pathogenesis of IUGR.

Materials and Methods: The material for the study was blood samples from 36 pregnant women diagnosed with IUGR and 30 healthy women. Genotyping of targeted SNPs: A223G (LEPR), A23525T (FTO), C786T (NOS3), C634G (VEGFA) were detected by RT-PCR.

Results: No statistically significant associations of LEPR and FTO gene polymorphisms with IUGR were found (p = 0.765; p = 0.461, respectively). Genotypes CT (NOS3) and CC (VEGFA) were significantly associated with low risk of developing IUGR. OR were 0,309 (CI 95% 0,110-0,868 p < 0.001) and 0.14 (CI 95% 0.04-0.46 p = 0.003) respectively. Genotypes TT (NOS3) and GG (VEGFA) were associated with higher relative risk of developing IUGR. OR were 2,400 (CI 95% 1,462-3,339 p < 0,001) and 3.67 (CI 95% 1.26-10.7 p = 0.003) respectively.

Conclusions: The data obtained indicate the need for further investigation to search for genetic determinants of the development of IUGR. This study was funded by the Ministry of Science and Higher Education of the Russian Federation #0852-2020-0028

I. Novikova: None. I. Pokudina: None. E. Mashkina: None. T. Shkurat: None.

P01.048.D KIR-HLAC genotyping in married couples with unexplained early reproductive losses

Kateryna Sosnina, Danuta Zastavna, Oresta Terpyliak, Bogdan Tretiak

Institute of Hereditary Pathology, NAMS of Ukraine, Lviv, Ukraine.

The immune interaction between the uterus and the embryo is crucial to achieve a pregnancy. Maternal immunotolerance is provided by the interaction between the cells of the maternal immune system in the uterus (uterine NK cells) and the embryo. These cells recognize the embryo through KIR receptors, interacting with their HLA-C ligands on the embryo. The aim of this study was to establish and analyze the combinations of KIR and HLA-C genes in married couples with unexplained early reproductive losses.

Results: KIR-HLAC genotyping was performed in 43 married couples with unexplained reproductive losses. Depending on the number and type of genes, the KIR genotype of women (AA, AB and BB) was established. The results were compared with the parental HLAC genotype, and the risk of reproductive losses in such a pair was calculated. According to the results, 23.3% of women have the AA genotype (high risk of reproductive loss), 76.7% of women had the AB genotype (medium risk). According to the results of KIR-HLAC genotyping of couples with early reproductive losses, 58.14% of married couples have a high risk of reproductive losses. Such married couples had the KIR AA genotype in women and/or the C2/C2 HLAC genotype in women and/or men.

Conclusions: KIR-HLAC genotyping is a genetic test that allows to assess the risks of the embryo being rejected by the maternal immune system, and thus to direct medical interventions in order to achieve a successful pregnancy.

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

P01.050.B Identification of two novel point mutations in DPY19L2 leading to total globozoospermia in two unrelated patients

Fatma Abdelhedi 1,2, Emmanuel Dulioust3, Nourhène Gharbi2, Sandrine Barbaux4, Ahmed Ziyyat4, Leila KESKES2, Jean Michel Dupont1,4

1Laboratoire de Cytogénétique, Hôpital Cochin, APHP, Paris, France, 2Laboratoire de Génétique Moléculaire Humaine, Faculté de Médecine, Sfax, Tunisia, 3Laboratoire d’Histologie Embryologie Biologie de la Reproduction, Hôpital Cochin, APHP, Paris, France, 4Génomique, Epigénétique et Physiopathologie de la Reproduction, U1016 INSERM-UMR 8104 CNRS, Institut Cochin, Université Paris Descartes, Paris, France.

Background: Globozoospermia is a rare and severe teratozoospermia characterized by the presence of round-headed spermatozoa lacking an acrosome. So far, only three genes have been well documented as responsible for this phenotype in human, namely DPY19L2, SPATA16, and recently GGN. Genetic causes remain unexplained for 20% to 30% of patients with globozoospermia suggesting that this phenotype is likely genetically heterogeneous.

Methods: As alterations of DPY19L2 gene represent the main cause of globozoospermia in human, we first screened for DPY19L2 molecular defects in a cohort of 10 patients of African and European origin, diagnosed with complete or partial globozoospermia. Whole genome analysis with SNP array was carried out in patients lacking genetic defects of DPY19L2.

Results: Molecular analyses performed on 9 genetically independent individuals showed that four (44%) were homozygous for the DPY19L2 deletion, two were heterozygous composite for two novel non-synonymous mutations (p.R686G and p.D687E) in exon 21. No genetic causes were identified in four patients with partial globozoospermia.

Conclusion: Our results highlight the importance of screening for DPY19L2 mutations in the absence of DPY19L2 deletions and strongly suggest that partial globozoospermia is not due to genetic defects on DPY19L2. Alghough the genetic diagnosis of globozoospermia does not yet provide any clear therapeutic indications, a molecular diagnosis remains useful to provide adequate genetic counseling. Considering these points, we recommend the search for DPY19L2 defects as the first-line genetic analysis in complete globozoospermia but not in its partial form.

F. Abdelhedi: None. E. Dulioust: None. N. Gharbi: None. S. Barbaux: None. A. Ziyyat: None. L. Keskes: None. J.M. Dupont: None.

P01.051.C Causal inference analyses reveal population risks and benefits of extending female reproductive lifespan through manipulation of biological processes

Katherine S. Ruth 1, Felix R. Day2, Anna Murray1, John R. B. Perry2, ReproGen consortium (www.reprogen.org)

1Genetics of Complex Traits, University of Exeter Medical School, University of Exeter, Exeter, United Kingdom, 2MRC Epidemiology Unit, University of Cambridge School of Clinical Medicine, Institute of Metabolic Science, Cambridge, United Kingdom.

Introduction: Recently we demonstrated the potential for new therapeutics to extend female reproductive lifespan by manipulating DNA damage response (DDR) pathways (MedRxiv https://doi.org/10.1101/2021.01.11.20248322). We investigated the wider population health risks and benefits of prolonged sex hormone exposure from such interventions.

Materials and Methods: We performed Mendelian Randomization using a genetic instrument for age at menopause (ANM) comprised of 290 variants from GWAS in >200,000 women. We tested outcomes associated with hormone replacement therapy, as being analogous to longer hormone exposure from later ANM.

Results: One-year later genetically-instrumented ANM increased the odds of female-specific hormone-sensitive cancers by up to 5%. We identified individual variants with effects on DDR and cancer risk independently of ANM and sex hormone exposure - loss-of-function alleles in BRCA2 and CHEK2 resulted in ANM 1.69 years earlier (95% CI 0.12-3.26, p = 0.03) and 2.36 years later (1.31-3.41, p = 1 × 10-5). We used MR-Radial to identify and exclude such variants from analyses, strengthening the evidence for an association with ER+ breast cancer (OR = 1.04, from p = 4 × 10-9 to p = 3 × 10-16). Later ANM had protective effects on Type 2 diabetes (OR = 0.98, p = 1 × 10-3) and bone health (fracture, OR = 0.98, p = 3 × 10-4). There was little evidence for associations with cardiovascular disease, longevity or Alzheimer’s disease.

Conclusions: Later ANM is detrimental for hormone sensitive cancers yet benefits metabolic and bone health, consistent with effects of oestrogen therapy in trials. However, our results do not support associations from observational studies. Novel therapeutic approaches to extending reproductive lifespan are likely to have wider effects on population health over prolonged fertility.

K.S. Ruth: None. F.R. Day: None. A. Murray: None. J.R.B. Perry: None.

P01.052.D Locus-specific methylation of the ESR1 gene promoter region as a marker for the prognosis of placental insufficiency and perinatal loss

Zoia I. Rossokha 1, Ludmila I. Vorobey2, Valeriy V. Kaminskyi2, Tetjana V. Kolomiichenko2, LIlia Y. Fishchuk1, Natalia L. Medvedieva1, Ludmila I. Brisevac2, Olexandr I. Zhdanovych2, Natalia G. Gorovenko2

1State Institution "Reference-centre for molecular diagnostic of Public Health Ministry of Ukraine", Kyiv, Ukraine, 2Supic Medical Acadeny of Postgraduate Education, Kyiv, Ukraine.

Introduction: Chronic stress and polluted environment are known direct determinants in prenatal maternal promoter hypermethylation of ESR1 gene and blockade of estrogen-dependent processes in the placenta. ESR1 gene expression is important for embryo implantation and pregnancy outcome, neuroprotection throughout life from antenatal period, and also has an organizational impact on the formation of long-term behavioral and cognitive functions, as well as maintaining energy homeostasis. The aim of the study was to investigate the effect of hypermethylation of the ESR1 gene promoter region on the risk of early pregnancy and perinatal loss.

Materials and Methods: 31 women with pregnancy loss and 10 women without reproductive failure were involved in the study. 24 women had early pregnancy loss and 7 women had perinatal loss. Methylation of the promoter region of the ESR1 gene was determined by methyl-specific PCR using DNA after bisulfide conversion.

Results: Hypermethylated ESR1 promoter region was detected in 35,48% patients with pregnancy loss. 29,16% patients with early pregnancy loss had hypermethylated status in ESR1. More often hypermethylated status was found among patients with perinatal loss - 57,14%. And no hypermethylated cases were detected in women without reproductive failure. Determination of hypermethylated ESR1 promoter region was significantly higher in patients with perinatal loss and 75% of them had had placental insufficiency resulting in antenatal death at 26-28 weeks of gestation.

Conclusion: Hypermethylation of the ESR1 gene promoter region in women was associated with placental insufficiency and perinatal loss. Further research is needed to develop personalized methods of perinatal loss prevention.

Z.I. Rossokha: None. L.I. Vorobey: None. V.V. Kaminskyi: None. T.V. Kolomiichenko: None. L.Y. Fishchuk: None. N.L. Medvedieva: None. L.I. Brisevac: None. O.I. Zhdanovych: None. N.G. Gorovenko: None.

P01.053.A The role of miR 196b in the pathogenesis of endometriosis

Victoria Spasova 1, Vesela Karamisheva1,2, Olga Antonova1, Zora Hammoudeh1, Liliya Koleva3, Rada Staneva1, Mihail Ganev1, Desislava Nesheva1, Draga Toncheva1, Savina Hadjidekova1

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

Introduction: Endometriosis is a chronic disease affecting about 10% of women worldwide. There is still a discussion among scientist on the exact molecular pathogenesis of the disease appearance. MiRNAs as posttranscriptional expression regulators may contribute to the development of endometriosis.

Materials and methods: Total RNA was isolated from tissue samples of ectopic endometrium of 30 patients with endometriosis and healthy endometrium of 15 controls (miRNeasy-MiniKit,Qiagen). Three pool samples were constructed based on the disease stage - each containing 15 RNA samples: early stage endometriosis, late stage endometriosis, and controls. Reverse transcription was conducted on each of the pool samples via miScript-II-RT Kit,Qiagen. SYBR-Green-based Real-time PCR assay was used to determine the expression profile of 84 miRNAs (Human miFinder miRNA PCR Array,Qiagen). QIAGEN’s GeneGlobe Data Analysis Centre was used to perform the data analysis. The prediction of target genes was accomplished using miRBase.org, Targetscan.org and Diana tools.

Results: We detected miR-196b-5p downregulated in both stages of endometriosis compared to the healthy controls (fold change = -19,28 for the early stage, fold change = -139,54 for the late stage). HOX genes are a known target of miR-196b-5p. These genes encode proteins that act as transcription factors. Changes of HOX genes expression are associated with altered endometrium development.

Conclusion: We suggest that the altered expression of miR-196b may contribute to reveal the etiology of endometriosis occurrence and progression.Funding. Contract No.212/12.12.2018, grant No.4731/03.07.2018, Council of Medical Science at the Medical University of Sofia, Bulgaria; Bulgarian Ministry of Education and Science, National Program for Research “Young Scientists and Postdoctoral Students.”

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

P01.054.B Comparative analysis of cytogenetic abnormalities revealed by karyotyping and interphase FISH in chorion of first-trimester miscarriages

Andrei V. Tikhonov 1, Olga A. Efimova1, Mikhail I. Krapivin1, Yanina M. Sagurova1, Anna A. Pendina1, Alla S. Koltsova1, Anna A. Smirnova2, Olga E. Talantova1, Olga G. Chiryaeva1, Lubov’ I. Petrova1, Vera S. Dudkina1, Vladislav S. Baranov1

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

Abnormal karyotype is a frequent cause of first-trimester miscarriage. In ~20% of cases conventional karyotyping is not possible because of absence of mitotic activity in chorion. Here, we compared the results of karyotyping and interphase fluorescence in situ hybridization (FISH) with centromeric/locus-specific DNA-probes specific to all chromosomes excluding chromosomes 1 and 19 (Abbott Molecular) on chorion of first-trimester miscarriages. A total of 3511 cases were retrospectively analyzed. In 2816 cases, conventional karyotyping was performed on direct metaphase preparations. In 695 cases, interphase FISH was performed because of no metaphases on direct preparations. The frequency of abnormal karyotype reached 64.4%(1814/2816) in the karyotyped group and 57.7%(401/695) in the FISH-analyzed group. Expectably, structural chromosomal abnormalities were detected only in the karyotyped group at the frequency of 2.9% (53/1814). The spectrum of revealed numerical karyotype abnormalities did not differ between the groups. However, their frequencies differed between the karyotyped and FISH-analyzed groups. Aneuploidies were revealed with higher frequency in the karyotyped compared to the FISH-analyzed group: 73.4%(1332/1814) vs 63.8% (256/401) (p = 0.0001). In contrast, polyploidy and mosaicism were more frequent in FISH-analyzed compared to the karyotyped group: 26.2% (105/401) vs 16.9% (306/1814) (p < 0.0001) and 8.0% (32/401) vs 4.4% (80/1814) (p = 0.0047). Thus, FISH can be used for the detection of numerical chromosomal abnormalities in chorion of first-trimester miscarriages, when karyotyping is not possible. Difference in frequencies of the same cytogenetic categories between the samples with and without mitotic activity suggests that viability of chorionic cell depends on the type of chromosomal abnormality. Supported by RSF№19-75-00023.

A.V. Tikhonov: None. O.A. Efimova: None. M.I. Krapivin: None. Y.M. Sagurova: None. A.A. Pendina: None. A.S. Koltsova: None. A.A. Smirnova: None. O.E. Talantova: None. O.G. Chiryaeva: None. L.I. Petrova: None. V.S. Dudkina: None. V.S. Baranov: None.

P01.055.C Case report: Prenatal mosaic trisomy 9 as result of a meiotic non-disjunction event

Sebastian Rusch 1,2, Melanie Gass2, Sevgi Tercanli3, Isabel Filges2,4

1Medical Genetics, Institute of laboratory medicine, Kantonsspital Aarau, Aarau, Switzerland, 2Medical Genetics, Institute of Medical Genetics and Pathology, University Hospital Basel, University of Basel, Basel, Switzerland, 3Centre for Prenatal Ultrasound, Basel, Switzerland, Basel, Switzerland, 4Department of Clinical Research, University Hospital Basel, Basel, Switzerland.

Introduction: Prenatal mosaic aneuploidy remains a challenge for counseling clinicians. The impact on embryonic and fetal development is depending on the level of mosaicism and tissue distribution of the aneuploid cell line; hence, clinical consequences are difficult to predict. We present the rare instance of mosaic trisomy 9 in a fetus with multiple malformations. We discuss how CMA-SNP data can contribute to understand the mechanism of aneuploidy.

Methods: Prenatal sonography of a 44-year-old woman at 14 weeks of gestation (WG) showed tricuspid regurgitation, a small branchial cyst, single umbilical artery and retrognathia, at 19 WG additional heart, renal and brain anomalies. FTT found increased risk scores of 1:102 and 1:2 for trisomy 21 and 13/18 respectively. CMA was used on amniotic fluid cells to detect copy number variants.

Results: The CMA result on amniotic fluid cells was in respect to copy number state (2.5) and B-Allele Frequencies (BAF) of approx. 0, 40, 60 and 100% concordant with mosaic trisomy 9 of about 50%. Additionally, with BAF of 20 and 80% in several chromosomal regions of chromosome 9 SNP-data signals indicated the presence of an additional haplotype.

Conclusions: CMA-SNP data reveal regions containing an additional haplotype which most likely arose from meiotic crossing over events. The distribution of the additional haplotype in the centromeric region suggests a meiotic I non-disjunction event followed by a postzygotic trisomic rescue for the normal cell line. We increase our knowledge on the fetal phenotype caused by the aberrant cell line which is only rarely reported.

S. Rusch: None. M. Gass: None. S. Tercanli: None. I. Filges: None.

P01.056.D Mosaicism for copy number variations in the placenta is even more difficult to interpret than mosaicism for whole chromosome aneuploidy

Ida Charlotte Bay Lund 1,2, Naja Becher1,2,3, Jesper Graakjaer4, Dorte Lildballe4, Niels Uldbjerg5,6, Pauline Bogaard7, Astrid Petersen7, Else Marie Vestergaard8, Ida Vogel1,2,6

1Department of Clinical Genetics, Aarhus University Hospital, Aarhus N., Denmark, 2Center for Fetal Diagnostics, Aarhus University Hospital, Aarhus N., Denmark, 3Department of Biomedicine, Aarhus University, Aarhus N., Denmark, 4Clinical Genetics, University Hospital of Southern Denmark, Sygehus Lillebaelt, Vejle, Denmark, 5Department of Obstetrics and Gynaecology, Aarhus University Hospital, Aarhus N., Denmark, 6Department of Clinical Medicine, Aarhus University, Aarhus N., Denmark, 7Department of Pathology, Aalborg University Hospital, Aalborg, Denmark, 8Department of Clinical Biochemistry, Horsens Regional Hospital, Horsens, Denmark.

Objective: To compare mosaicisms in prenatal chorionic villus samples with corresponding postpartum placental samples.

Method: We collected placentas from 15 consecutive cases of mosaicism detected in chorionic villus samples and obtained five standardized samples on each placenta after delivery. All pre- and postnatal placental samples were uncultured and analyzed by high-resolution chromosomal microarray.

Results: Ten cases of mosaicism for whole chromosome aneuploidy(mWC) and five cases with mosaicism for (sub)chromosomal copy number variations(mCNVs) were included. In 5/10 mWC cases and in 4/5 mCNV cases the prenatally detected aberration was confirmed in the postpartum placenta. Three postpartum placentas revealed various complex aberrations differing from the prenatal results: 1) mosaicisms for different deletions/duplications on 9p and 9q in all samples (prenatal: mosaic 5.3 Mb duplication on 9p24), 2) different regions with deletions/duplications/loss of heterozygosity on 1p in all samples (prenatal: mosaic 2.3 Mb 1p36 duplication), and 3) mosaicism for a duplication on 5q and a deletion on 6p in one out of five samples (prenatal: mosaic trisomy 7).

Conclusion: CNVs constitute a complex subgroup in placental mosaicism. Counseling of these couples after chorionic villus sampling shouldn´t focus on the specific CNV involved, but on the nature of mosaicism and the option of amniocentesis and ultrasound.

Funding: This work was funded by The Foundation of Aase and Ejnar Danielsen, The Foundation of 17-12-1981 and The Novo Nordisk Foundation.

I. Lund: None. N. Becher: None. J. Graakjaer: None. D. Lildballe: None. N. Uldbjerg: None. P. Bogaard: None. A. Petersen: None. E. Vestergaard: None. I. Vogel: None.

P01.057.A Prenatal diagnosis of Neu-Laxova syndrome through identification of a novel exonic-deletion and missense mutation in the PHGDH gene

Aruna Marchetto 1, Moneef Shoukier1, Katja Gahle1, Anne Janke1, Justin Seidel1, Ursula Hiener2, Vera Link3, Kai Wagner3, Sabine Minderer1, Karl Philipp Gloning1

1Prenatal-Medicine Munich, Munich, Germany, 2Klinikum Dritter Orden - Department of Radiology, Munich, Germany, 3Rotkreuzklinikum - Institute of Pathology, Munich, Germany.

Mutations in the PHGDH gene disrupt the serine biosynthesis and are known to be associated with the autosomal recessive disorder Phosphoglycerate dehydrogenase deficiency (PHGDH) and the more severe form Neu-Laxova syndrome 1 (NLS1). To date, there is no clear genotype-phenotype correlation between PHGDH and NLS1. PHGDH is characterized by congenital microcephaly, psychomotor retardation and seizures whereas it has been assumed that NLS1 could be caused by more severe PHGDH-mutations with main clinical features of intrauterine growth retardation (IUGR), microcephaly, cutaneous and craniofacial abnormalities. We present a case of a prenatal conspicuous microcephaly linked to two novel mutations in the PHGDH gene likely associated with NLS1.

We report on a pregnant 42-year-old patient, 23rd gestational week, whose fetus presented with severe microcephaly, IUGR, cleft lip, dilated intestinal loops and mild hydronephrosis. MRI confirmed sonographic findings while array-CGH was normal (arr(1-22)x2,(XY)x1). A multigene panel analysis (brain malformations panel, 400 genes) revealed two likely disease-causing variants; a maternal inherited hemizygous missense mutation c.766G>A, p.Gly256Arg in exon 7 and a paternal inherited heterozygous deletion of exon 6 and 7 of the PHGDH gene. The pregnancy was terminated in the 25th week. Immunohistological staining of the fetuses’ skin biopsy revealed ichthyosis and hyperkeratosis. Both detected mutations in the PHGDH gene were localized in the nucleotide-binding domain (NBD).

Considering the molecular and clinical findings, we assume these mutations to be causative for a Neu-Laxova syndrome. Thus, our results support previous findings indicating that mutations in NBD mostly cause the more severe phenotype of PHGDH-associated disorders (NLS1).

A. Marchetto: None. M. Shoukier: None. K. Gahle: None. A. Janke: None. J. Seidel: None. U. Hiener: None. V. Link: None. K. Wagner: None. S. Minderer: None. K. Gloning: None.

P01.058.B Neurofibromatosis type 1 and the next generation: is preimplantation genetic testing the solution?

Vivian Vernimmen, Aimee D. C. Paulussen, Constance T. R. M. Stumpel, Ron J. T. van Golde, Christine E. M. de Die

Maastricht University Medical Center, Maastricht, Netherlands.

Introduction: Future parents affected with Neurofibromatosis type 1 (NF1) can opt for preimplantation genetic testing (PGT) to avoid NF1 in their offspring. We aim to identify challenges and pitfalls in PGT for NF1.

Methods: We collected data on PGT cycles from the medical files of couples requesting PGT for NF1 between January 1997 and January 2020.

Results: PGT was the reproductive option of choice for 96 couples. PGT was not possible for 14 couples, mostly because the causative variant was not identified or of unknown significance. The origin of the NF1 mutation was (presumed) sporadic in 63% of the 82 couples proceeding with PGT. PCR with multiple polymorphic markers was most frequently applied, combined with direct mutation analysis if needed. PGT/PCR work-up showed several exceptional situations: in one family two different NF1 mutations turned out to be causal and in one family with a sporadic male index the mutation was not detected in the sperm cells during PGT work-up. The ‘OnePGT’ genome wide haplotyping method, based on next generation sequencing, was used for 2 recent families with a familial mutation. A successful PGT test could be developed for 78 couples with 71 different variants in the NF1 gene. Together, 65 couples underwent 141 PGT cycles and the transfer of 162 unaffected embryos resulted in 39 ongoing pregnancies (pregnancy rate 24,1%/embryotransfer).

Conclusions: PGT is a successful reproductive option for couples with NF1. Test development was possible in almost all cases reviewed despite the fact that most variants were unique and sporadic.

V. Vernimmen: None. A.D.C. Paulussen: None. C.T.R.M. Stumpel: None. R.J.T. van Golde: None. C.E.M. de Die: None.

P01.059.C Prenatal diagnostics of NF2 mutation in the fetus, associated with the development of endometrial cancer

Kalina Belemezova 1,2, Kunka Kamenarova3, Kalina Mihova3, Mariela Hristova-Savova1, Albena Todorova4, Radka Kaneva3, Ivanka Dimova1,3

1Genetic Laboratory, SAGBAL Dr. Shterev, Sofia, Bulgaria, 2Medical University of Sofia, Sofia, Bulgaria, 3Molecular Medicine Center, Medical University of Sofia, Sofia, Bulgaria, 4Laboratory Genika, Sofia, Bulgaria.

Endometrial carcinoma (EC) is rarely diagnosed during pregnancy. An electronic search of the literature revealed just 25 cases of EC diagnosed during or after pregnancy, for the period between 1995 and 2019. Ten percent of endometrial carcinomas harbor NF2 mutations. This observation is of interest since mutated NF2 can activate mTOR, the downstream effector of PIK3CA, and in a high percentage of this cancer type (>80%), there are PI3K pathway aberrations. NF2 is a complex gene with somatic mutations being associated with various cancers. Germline mutations cause the autosomal dominant disorder neurofibromatosis 2 (NF2). Individuals with hereditary NF2 do develop schwannomas, ependymomas, and meningiomas. Although the somatic mutations sometimes overlap with those in hereditary NF2, no published papers are documenting an increased risk of other cancer types in neurofibromatosis patients. Here we report a young pregnant woman, affected by classical NF2 (presence of bilateral schwannomas) and carrying mutation c.592C>T (p.Arg198Ter) in exon 6 of the NF2 gene. Because of the high risk for inheritance (50%), CVS was performed and DNA from the fetus was analyzed for the presence of mutation with Sanger sequencing - the fetus was found to be a carrier of the same pathogenic mutation. In the meantime, ultrasound examination suspected tumor formation in the uterus, which was confirmed to be endometrial cancer after biopsy and pregnancy was terminated. This case suggests an adverse effect of the simultaneously NF2-affected fetus and mother for endometrial malignancy and suggesting PGD when the mother is affected by NF2.

K. Belemezova: None. K. Kamenarova: None. K. Mihova: None. M. Hristova-Savova: None. A. Todorova: None. R. Kaneva: None. I. Dimova: None.

P01.060.D Patient-friendly integrated first trimester screening by NIPT and fetal anomaly scan

Karin E. M. Diderich, Malgorzata I. Srebniak, Maarten F. C. M. Knapen, Marieke Joosten, Sander Galjaard, Diane Van Opstal

ErasmusMC, Rotterdam, Netherlands.

Many major structural fetal anomalies can be diagnosed by first trimester fetal anomaly scan. NIPT can accurately detect aneuploidies and large chromosomal aberrations in cfDNA in maternal blood plasma. This study shows how a patient-friendly first trimester screening for both chromosomal and structural fetal anomalies in only two outpatient visits can be provided. Genotype-first approach assures not only the earliest diagnosis of trisomy 21 (the most prevalent chromosome aberration), but also completion of the screening at 12–14 weeks. To ensure proper management and avoid unnecessary anxiety abnormal NIPT different from trisomy 21, 18 and 13 should be referred for genetic counseling.

K.E.M. Diderich: None. M.I. Srebniak: None. M.F.C.M. Knapen: None. M. Joosten: None. S. Galjaard: None. D. Van Opstal: None.

P01.061.A NIPD for translocation carriers - yes please or no go?

Malgorzata I. Srebniak 1, Fernanda S. Jehee1, Marieke Joosten1, Marjan Boter1, Walter de Valk1, Robert van der Helm1, Erik Sistermans2, Els Voorhoeve2, Shama Bhola2, Mariette Hoffer3, Nicolette den Hollander3, Merryn Macville4, Diane Van Opstal1

1Erasmus MC, Clinical Genetics, Rotterdam, Netherlands, 2Amsterdam UMC, Vrije Universiteit Amsterdam, Clinical Genetics, Amsterdam, Netherlands, 3Leiden University Medical Center, Clinical Genetics, Leiden, Netherlands, 4Clinical Genetics, GROW School for Oncology and Developmental Biology, Maastricht University Medical Center, Maastricht, Netherlands.

The presence of an unbalanced familial translocation can reliably be assessed in the cytotrophoblast of chorionic villi. However, carriers of a balanced translocation often refuse invasive testing due to the miscarriage risk. Because the cytotrophoblast is also the source of cell free (cf) DNA in plasma, this study aimed to investigate whether an unbalanced translocation can also be diagnosed in cfDNA by whole-genome NIPT.Pregnant women carrying a fetus with an unbalanced familial translocation, from whom NIPT as well as microarray data were available, were included in this retrospective assessment. NIPT was performed in the course of the TRIDENT study, but at the time of screening, carrier status of a parental translocation was unknown (exclusion criterion for NIPT in the Netherlands). In 12 cases both NIPT and microarray data were available. In 10/12 cases the unbalanced translocation was correctly identified without prior knowledge on parental translocation. One was missed due to too low fetal fraction. One was missed due to technical restrictions in calling 16p gains. Both missed cases were later identified by invasive diagnostic testing after ultrasound anomalies were found.This study supports the hypothesis that routine NIPT may be used for prenatal diagnosis of unbalanced inheritance of familial translocations, especially with prior knowledge of the translocation allowing focused examination of the involved chromosomal regions. Our study showed that routine shallow sequencing designed for aneuploidy detection in cfDNA may be sufficient for higher resolution NIPT, if specialized copy number software is used and if sufficient fetal fraction is present.

M.I. Srebniak: None. F.S. Jehee: None. M. Joosten: None. M. Boter: None. W. de Valk: None. R. van der Helm: None. E. Sistermans: None. E. Voorhoeve: None. S. Bhola: None. M. Hoffer: None. N. den Hollander: None. M. Macville: None. D. Van Opstal: None.

P01.062.B Non-invasive prenatal diagnosis of paternally-inherited disorders: three years clinical experience

Mathilde Pacault1,2, Camille Verebi 1, Maureen Lopez1, Nicolas Vaucouleur1, Lucie Orhant1, Nathalie Deburgrave1, France Leturcq1, Dominique Vidaud1, Emmanuelle Girodon1, Thierry Bienvenu1, Juliette Nectoux1

1Service de Génétique et Biologie Moléculaires, Hôpital Cochin, APHP.Centre Université de Paris, Paris, France, 2Laboratoire de Génétique Moléculaire et d’Histocompatibilité, Centre Hospitalier Régional Universitaire, Brest, France.

Introduction: Cell-free fetal DNA analysis is now performed routinely for aneuploidy screening, fetal RhD genotyping or sex determination. However, applications to single gene disorders (SGD) remain sparse. We previously described a standardized protocol for non-invasive exclusion of paternal mutation in SGD using a targeted, droplet digital PCR-based approach. Here, we report our three-year clinical experience since offering this service.

Patients and Methods: Patients were referred by clinicians nationwide for several monogenic disorders, paternally-inherited or de novo, dominant or recessive, such as cystic fibrosis, neurofibromatosis type I or FGFR3-related disorders (thanatophoric dysplasia and achondroplasia). Non-invasive prenatal diagnosis was performed using custom assays for droplet digital PCR. Results, turn-around time and cost-effectiveness were evaluated.

Results: Our tests proved very robust and highly discriminant, as a result was obtained for every 205 referral except one, where fetal fraction was too low to conclude, but returned « unaffected fetus » on a subsequent sample. All referred pathogenic variants could be targeted except one located in a repetitive genomic region. Mean time between order and validation of an assay was 14 days. Mean result reporting time was 6 days.

Conclusion: This genetic testing service was implemented as a routine practice with a simple development and interpretation process that could be offered for any paternally-inherited or de novo SGD. Thanks to a short turn-around time, an affordable price and a great robustness, this method has been widely adopted by clinicians nationwide for dominant paternally-inherited disorders, or as a first test for recessive disorders.

M. Pacault: None. C. Verebi: None. M. Lopez: None. N. Vaucouleur: None. L. Orhant: None. N. Deburgrave: None. F. Leturcq: None. D. Vidaud: None. E. Girodon: None. T. Bienvenu: None. J. Nectoux: None.

P01.063.C Implementing non-invasive prenatal testing for aneuploidy in a national healthcare system in Russia. Moscow experience

Olesya Sagaidak 1, Alexandra Galaktionova1, Anton Olenev2, Ekaterina Kuznetsova1, Madina Kaplanova1, Maksim Belenikin1, Ekaterina Songolova3, Elena Baranova1,4

1LLC Evogen, Moscow, Russian Federation, 2Moscow City Health Department, City clinical hospital № 24, Moscow, Russian Federation, Moscow, Russian Federation, 3Moscow City Health Department, City clinical hospital № 67 named after L.A. Vorokhobova, Moscow, Russian Federation, Moscow, Russian Federation, 4Federal State Budgetary Educational Institution of Further Professional Education “Russian Medical Academy of Continuous Professional Education” of the Ministry of Healthcare of the Russian Federation, Moscow, Russian Federation.

Since March 2020, NIPT has become available for women at risk up to 1:2500 in Moscow. Objectives. To evaluate the clinical efficacy of non-invasive prenatal testing (NIPT) versus prenatal screening in the first trimester of pregnancy.

Materials. According to the results of a biochemical blood test, patients were dividedinto two groups: the high-risk group (threshold ≥ 1:100) (n = 338) and the average-risk group (threshold 1:101 - 1:2500) (n = 8567). NIPT was performed for all patients. In case of high risks evaluatedby NIPT, the results were confirmed by karyotyping in the high-risk group and groupof the average-risk patients.

Results: The results of karyotyping were available in 280 patients (82.8%) of the high-risk group. High risks of trisomies by NIPT were confirmed in 66/70 cases for trisomy 21,in 22/23 cases for trisomy 18 and in 4/4 cases for trisomy 13. A high risk of fetal aneuploidies by NIPT was identified in 29 cases in the average-risk group with karyotyping available19 (65.5%) cases: 12 cases of trisomy 21 and 3 cases of trisomy 13 were confirmed, other 4 cases were false-positives in the average-risk group (including 1 case of trisomy 21 and 3 casesof trisomy 13).

Conclusion: NIPT implementation in Moscow demonstrates high efficiency and accuracy. More data is needed to choose an implementation strategy in a national healthcare system. The work was supported by Moscow City Health Department grant.

O. Sagaidak: None. A. Galaktionova: None. A. Olenev: None. E. Kuznetsova: None. M. Kaplanova: None. M. Belenikin: None. E. Songolova: None. E. Baranova: None.

P01.064.D Simple and complex aneuploidy in premeiotic oocytes detected by aCGH & NGS: evidence for genetic influence and effect on fertility

Harita Ghevaria 1, Sioban SenGupta1, Roy Naja2, Rabi Odia3, Paul Serhal4, Xavier Vinals Gonzalez3, Xuhui Sun1, Joy Delhanty1

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

Aneuploidy is the major cause of embryonic & fetal death. Most errors arise in meiosis I/II in the adult female, strongly correlated with maternal age. Our application of array comparative genomic hybridization (aCGH) and next generation sequencing (NGS) has shown that 10-15% of oocyte aneuploidy is in fact premeiotic (PM) and present in the early embryo, leading to a risk of an aneuploid conception in adult life irrespective of age. Mosaic aneuploidy maybe present in the primordial germ cells or may arise during the extensive mitotic divisions of the oogonia. A substantial individual variation in the incidence of PM errors is likely to be related to the genetic background of the oocyte donor. NGS or aCGH was performed on 141 immature oocytes [germinal vesicles (GV) & metaphase I (MI) oocytes] and 61 mature oocytes [Metaphase II - 1st PB complexes]. Fifteen (19.2%) of 78 donors had oocytes with PM errors. Oocyte aneuploidy incidence was correlated with the reproductive histories of female partners. In categories 1 & 2 most couples have fertility issues & very similar incidence of PM errors. Groups 3, 5 & 6 from couples with no known fertility issues all have lower incidences. Two cases in Group 4 stand out, with a much higher incidence which may be related to the genetic factors responsible for the onset of breast cancer in their 30s.

Table: Classification of female partners donating oocytes
Classification of female partners based on reproductive histories Total No. of donors Total oocytes tested Oocytes with premeiotic aneuploidy No. of donors contributing oocytes with premeiotic aneuploidy No. of oocytes with Simple (SE)/ Complex (CE) Errors
1. Female fertility status unknown 27 76 11 (14.5%) 6 donors 7(SE); 4(CE)
2.Primary/secondary female factor infertility 25 48 6 (12.5%) 4 donors 3(SE); 3(CE)
3.Oocyte preservation due to social reasons 3 23 1 (4.3%) 1 donor 1(SE)
4.Oocyte preservation due to breast cancer 2 10 5 (50%) 2 donors 1(SE); 4(CE)
5.Female carriers of structural rearrangements or monogenic disorders (non-cancer related) 17 30 2 (6.66%) 2 donors 1(SE); 1(CE)
6.Females at increased risk of developing breast/ovarian cancer due to BRCA1/2 gene mutations 4 15 0 0 0
Total 78 202 25 (12.38%) 15 donors 13 (SE); 12 (CE)
Grant Support - CRGH

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

P01.065.A Evaluation of the Telomere Length and its Effect on the Ovarian Reserve in a Sample of Colombian Women

Johana Marin Suarez 1, Harold Moreno-Ortiz2, Lissette Otero3, Stephen Matlin3, Clara Inés Esteban-Pérez2, Maribel Forero-Castro1

1Universidad Pedagogica y Tecnologica de Colombia, Tunja, Colombia, 2Departamento Biogenética reproductiva. Invitro, Colombia, Bogota, Colombia, 3Life Length, Madrid, Spain.

Introduction: Studies have been carried out where they relate longevity in humans with higher fertility, and indicate that delays in the onset of menopause in longer-lived women could be due to slower cellular aging; which has led us to think about the importance of the effect of telomeric shortening in reproductive aging. In addition, it has been observed that alterations in telomere length (TL) is turn related to a decrease in ovarian reserve (OR). To evaluate telomere length and its effect on OR values in healthy women with a diagnosis of PCOS.

Methodology: 66 healthy women and 44 women with PCOS, LT was quantified in mononucleated cells using the HT-QFISH technique at the Life Length Laboratory. EB was calculated from the measurement of LT. Individual RO markers were integrated with LT and EB results. The chronological and biological age of the participants was compared as well as the reproductive status.

Results: LT in both groups was in the normal range, there were no significant differences between the LT (11.7 vs 11.87, p = 0.33). The mean CD was 24 years for controls and 27 years for women with PCOS. There were no significant differences between CD and BE in each group (control: 24 vs 26, p = 0.46, and PCOS: 27 vs 26, p = 0.9). OR markers were higher in women with PCOS.

Conclusion: The evaluation of TL is a tool that should be explored to evaluate the reproductive status in healthy women with PCOS.

J. Marin Suarez: None. H. Moreno-Ortiz: None. L. Otero: None. S. Matlin: None. C. Esteban-Pérez: None. M. Forero-Castro: None.

P01.066.B Systematic review and meta-analysis of genetic association studies of pelvic organ prolapse

John W. F. Chua 1, Kristina Allen-Brady2, Romana Cuffolo3, Marianne Koch4, Felice Sorrentino5, Rufus Cartwright6,7

1Zhongshan Hospital, Fudan University, Shanghai, China, 2Genetic Epidemiology, Department of Internal Medicine, University of Utah, Salt Lake City, UT, USA, 3Department of Obstetrics & Gynaecology, John Radcliffe Hospital, Oxford University Hospitals NHS Trust, Oxford, United Kingdom, 4Department of Obstetrics and Gynecology, Medical University of Vienna, Vienna, Austria, 5Department of Medical and Surgical Sciences, Institute of Obstetrics and Gynecology, University of Foggia, Foggia, Italy, 6Department of Epidemiology & Biostatistics, Imperial College London, London, United Kingdom, 7Department of Urogynaecology, LNWH NHS Trust, London, United Kingdom.

Introduction and Objective: Family and twin studies demonstrate that pelvic organ prolapse (POP) is heritable, but the genetic etiology is poorly understood. This review aimed to identify genetic loci and specific polymorphisms associated with POP, while assessing the strength, consistency, and risk of bias among reported associations.

Methods: Updating an earlier systematic review PubMed and HuGE Navigator as well as relevant conference abstracts were searched using genetic and phenotype keywords from 2015-2020. Screening and data extraction were performed in duplicate. Fixed and random effects meta-analyses were conducted using co-dominant models of inheritance. We assessed credibility of pooled associations using interim Venice criteria.

Results: We screened 504 new abstracts and included 46 published and 7 unpublished studies. In pooled analyses we found significant associations for four polymorphisms: rs2228480 at the ESR1 gene (OR 0.67 95%CI 0.46-0.98, I2=0.0%, Venice Rating BAB), rs12589592 at the FBLN5 gene (OR 1.46 95%CI 1.11-1.82, I2=36.3%, Venice Rating BBB), rs484389 in the PGR gene (OR 0.61 95%CI 0.39-0.96, I2=32.4%, Venice Rating CBB), and rs1800012 at the COL1A1 gene (OR 0.80 95%CI 0.66-0.96, I2=0.0%, Venice Rating BAB). Further credible novel variants have also been recently identified in genome wide association studies.

Conclusion: The genetic contributions to POP remain poorly understood. Several biologically plausible variants have been identified, but much work is required to establish the role of these genes in the pathogenesis of POP, or to establish a role for genetic testing in clinical practice.

J.W.F. Chua: None. K. Allen-Brady: None. R. Cuffolo: None. M. Koch: None. F. Sorrentino: None. R. Cartwright: None.

P01.067.C Preimplantation genetic testing (PGT-M) for Parkinson disease (PD) risk reduction: Expanding of applications for selecting embryos

Shachar Zuckerman 1, Ari Zimran2, Talya Eldar-Geva3, Elina Farhi1, Shira Shaviv1, Elinor Hakam-Spector1, Tama Dinur2, Gheona Altarescu1

1Medical Genetics Institute, Shaare Zedek Medical Center, Jerusalem, Israel, 2Gaucher Center, Shaare Zedek Medical Center, Jerusalem, Israel, 3IVF unit, Shaare Zedek Medical Center, Jerusalem, Israel.

PGT is practiced worldwide, allowing to prevent transmission of a growing number of genetic conditions. For recessive disorders, both wild-type and carrier embryos are transferable. The justified applications for PGT are subject to ongoing debate. Mutations in the glucocerebrosidase (GBA) gene, causing Gaucher disease (GD), have emerged as a primary risk factor for PD in both patients and carriers. There is a differential effect of mild versus severe mutations: Mild mutations increase the risk of developing PD by 2.2-fold while severe mutations increase this risk by 13.6-fold. PGT counseling was given to a thirty-two years old woman with GD, compound heterozygote of N370S (mild mutation) and 84GG (severe mutation), whose mother (an 84GG carrier) died of early-onset PD associated with severe cognitive decline. GBA sequencing of her spouse was negative. The couple needed IVF. PGT-M was performed to select for N370S carrier embryos because of the reduced risk for PD, compare to 84GG carrier embryos. Eight embryos were sampled: Four were carriers for 84GG mutation; 1 aneuploid; 1 had no result; 2 were N370S carriers and thus transferable. This case report demonstrates the expansion of PGT-M use for selecting embryos with reduced risks for late-onset conditions. These novel indications for PGT-M will increase the numbers of people who would be candidates for PGT-M. The medical and bioethical consideration of these cases should be acknowledged by the professional community and discussed with couples in genetic counseling setting. Furthermore, Guidelines for PGT usage for risk reduction of late-onset disorders should be implemented.

S. Zuckerman: None. A. Zimran: None. T. Eldar-Geva: None. E. Farhi: None. S. Shaviv: None. E. Hakam-Spector: None. T. Dinur: None. G. Altarescu: None.

P01.068.D Structural fetal defects after preimplantation genetic testing - high throughput genetic investigations

Radostina Raynova 1,2, Petya Chaveeva2, Tanya Milachich2, Momchil Rizov2, Tanya Timeva2, Atanas Shterev2, Ivanka Dimova2,3

1National Genetic Laboratory, Sofia, Bulgaria, 2SAGBAL"Shterev", Sofia, Bulgaria, 3Medical University, Sofia, Bulgaria.

Preimplantaion genetic diagnostics (PGD) is a useful approach for reducing miscarriage and increase successful pregnancy rate in couples, carriers of balanced structural rearrangements. Robertsonian translocation (RT) is one of the most common balanced structural aberrations. Previous studies of embryos’ chromosomes in RT carriers subjected to PGD have shown abnormal chromosome segregation, resulting in high levels of mosaic embryos.

Here we report a case of PGD in a couple with two previous miscarriages. Cytogenetic analysis of the partners revealed RT in the woman: karyotype 45, XX der(14;22)(q10;q10). Because of the high risk for chromosomal abnormalities in the fetus, assisted reproduction with PGT-A was recommended and performed by Next Generation Sequencing after Whole Genome Amplification using Veriseq (Illumina) protocol.

Three embryos were tested at day 5, resulting in an euploid embryo, one complex aneuploidy; and a mosaic embryo - 46/46, −15; +17, dup(5)(q31.1qter). Transfer was done with the euploid embryo, but pregnacy was not realized. Mosaic embryo was transferred afterwards and pregnancy was detected. In the first trimester severe cleft palate was detected in the fetus and CVS was performed followed by array CGH analysis. The result showed terminal duplication of 12q- 46, XN, dup(12q24.23q24.33), not detected during PGT-A. Amniocentesis was performed to exclude placental mosaicism, followed by Vista™ Chromosome Sequencing technology - 12qterminal duplication was confirmed and pregnancy was terminated.

This case demonstrates the need for careful evaluation of pregnancies in which mosaic embryos are transferred; with emphasize on the use of high- throughout whole genome techniques for copy number analysis.

R. Raynova: None. P. Chaveeva: None. T. Milachich: None. M. Rizov: None. T. Timeva: None. A. Shterev: None. I. Dimova: None.

P01.069.A Embryo transfer decision dilemma in PGT-M: Dominant or recessive inheritance

Suleyman Aktuna, Merve Polat, Evrim Unsal, Leyla Ozer, Volkan Baltaci

Yüksek Ihtisas University, Ankara, Turkey.

Introduction: Among the PGT-M cases with a history of affected children homozygous for a recessive mutation, transfer decision of heterozygous embryos becomes challenging in case where the mutation is associated with an additional disease with autosomal dominant inheritance. Necessity of pretreament genetic counselling for these couples and the risk of heterozygous embryo transfer due to possible penetrance differences together with the patient management will be discussed in detail.

Materials and Methods: PGT-M was performed with direct mutation and haplotype analyses.

Results: This study investigates PGT-M results of 10 couples carrying different rare genetic conditions and 10 causative genes presented in Table 1. Eventough these genes are associated with multiple OMIM phenotypes with both autosomal dominant and autosomal recessive inheritance all reported cases presented autosomal recessive inheritance pattern. The total number of embryos analysed was 60 from 2 to 14 for each patient. PGT-M analysis revealed that, 15 embryos were normal, 33 were heterozygous and 11 were mutant.

Conclusions: Homozygous normal embryos should be prioritized for transfer to exclude clinical phenotype that may ocur due to potenital penetrance and expressivity differences. Normale mbryo was not detected in 3 cases, thus embryo transfer was cancelled with patient decision. This situation underlie the need for better understanding of this phenomenon during PGT-M management to prevent reduction of reproductive chance of patients coping with such tedious treatment procedures.

Table 1: Phenotype, gene, variant and variant classification details of all 10 PGT-M cases.
Phenotype Gene Variant classification
Cholestasis, progressive familial intrahepatic 3 ABCB4 Class 3
Hypophosphatasia, childhood
Hypophosphatasia, infantile
ALPL Class 2
Epidermolysis bullosa dystrophica COL7A1 Class 2
Cardiomyopathy, dilated, with woolly hair and keratoderma
Epidermolysis bullosa, lethal acantholytic
Skin fragility-woolly hair syndrome
DSP Class 2
Ellis-van Creveld syndrome EVC2 Class 1
Fumarase deficiency FH Class 1
Spondylocarpotarsal synostosis syndrome FLNB Class 2
Glanzmann thrombasthenia ITGA2B Class 3
Mucopolysaccharidosis type IIIB NAGLU Class 3
Hypotonia, infantile, with psychomotor retardation and characteristic facies 1 NALCN Class 3

S. Aktuna: None. M. Polat: None. E. Unsal: None. L. Ozer: None. V. Baltaci: None.

P01.071.C The shift towards rare: Increased frequency of rare PGT-M cases

Leyla Ozer, Evrim Unsal, Merve Polat, Suleyman Aktuna, Volkan Baltaci

Yuksek Ihtisas University, Ankara, Turkey.

Introduction: Initial step of PGT-M is the detection of causative mutation. This has been the limiting factor for patients with rare disorders due to complex phenotype or genetic heterogeneity. Improvements in genomic technologies such as WES has become an effective tool for delineating the mutations to enable PGT-M for rare disorders. This study summarizes the variability and distribution of genes from our 1085 PGT-M cases during 2014-2021 highlights the shift towards rare disorders.

Materials and Methods: PGT-M was performed using STR based protocol for direct mutation and haplotype analysis.

Results: Retrospective data show that PGT-M was frequently performed for 6 genetic loci (Table 1). Table 2 compares the frequency 6 common disorders at different time intervals.

Conclusions: Results show that PGT-M was performed for 94 different genes during 2014-2017 while it reaches 323 in 2021. The highlight of the results was decrease in the freqeuncy for total number of common disorder cases (50,2% to 42,4%). Furthermore resuts reveals that for 90% of genes only 1-4 PGT-M cases were refererred which underlines the shift towards rare disorders with the extended utilization of WES and increase in novel gene variants.

Table 1
Distribution of cases and gene  
Number of cases Total gene number
1 case 192 (59,5%)
2-4 cases 99 (30,6%)
5-15 cases 26 (8,1%)
over 15 case (common disorders) (HBB, SMN1, DMD, CFTR, HLA, FMR1) 6 (1,8%)
Total 323
Table 2
  PGT-M cases perfomed during 2014-2017 PGT-M cases perfomed during 2017-2021
PGT-M number for common disorders 123 (50.2%) 356 (42.4%)
Total number of PGT-M 245 840

L. Ozer: None. E. Unsal: None. M. Polat: None. S. Aktuna: None. V. Baltaci: None.

P01.072.D Placental tissue co-expression networks across Russians and Yakuts identify key genes and pathways for preeclampsia

Ekaterina Trifonova, Anastasia Babovskaya, Aleksei Zarubin, Anton Markov, Vadim Stepanov

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

Population transcriptomics is a promising approach for finding loci of susceptibility to diseases which frequency depends from the ethnicity. Here we investigate the population transcriptomics in a preeclampsia (PE) model using WGCNA. The study included whole-genome expression samples from 21 Russian (Indo-Europeans population) and 23 Yakut (Mongoloids population) women with PE and physiological pregnancy. We found 10 clusters for Russian containing 7968 genes associated with PE. The main categories and pathways in these clusters are processes of the cytokine signaling (FDR = 0,005). For Yakut we found 9 clusters yielded 7966 PE-genes. The main GO-categories of these genes are amino-acid metabolism (FDR = 0,003), regulation of receptor interactions (FD = 0,047) and PI3K-Akt signaling pathway (FDR = 0,023). According to the MCC analyze of CytoHubba there were 10 hub genes with rank = 1 for Russians (CUL1, ANAPC11, LNX1, CDC20, UBE2L6, FBXO9, KLHL13, UBA3, KCTD7, RNF111) and Yakut (KLHL3, FB11PSXL, ASB2, LRRC41, LMO7, RNF7, SKP2, FBXO2). These genes are responsible for the regulation of the ubiquitin-ligase complex. In the second step we pick-out 1701 common genes for both populations. Most functionally active (rank = 1-2) remained the genes of the ubiquitin-ligase family and key processes belong to the pathways of interferon signaling (FDR = 0,004). Thus, we found population-specific pathways of PE: these are the processes of immune response for Indo-Europeans and the processes of ligand-receptor interaction for Mongoloids. In addition, we have identified common PE-genes for populations which are associated with the processes of interferon activity and operation the ubiquitin-ligase complex. Supported by RFBR №18-44-700007, №18-29-1304.

E. Trifonova: None. A. Babovskaya: None. A. Zarubin: None. A. Markov: None. V. Stepanov: None.

P01.073.A Prenatal phenotype of PNKP-related primary microcephaly associated with variants in the FHA domain

Ilona Krey 1, Sonja Neuser1, Annemarie Schwan2, Tobias Bartolomaeus1, Jan Henje Döring3, Steffen Syrbe3, Margit Plassmann4, Stefan Rohde5, Christian Roth6, Helga Rehder7,8, Rami Abou Jamra1, Diana Le Duc1, Susanna Schubert1, Katharina Schoner8, Bernt Popp1

1Institute of Human Genetics, University of Leipzig Medical Center, Leipzig, Germany, 2MVZ Dr. Eberhard & Partner Dortmund, Dortmund, Germany, 3Department of Pediatrics, Hospital for Children and Adolescents, Heidelberg University Hospital, Heidelberg, Germany, 4Praxis für Pränatalmedizin, Dortmund, Germany, 5Department of Radiology and Neuroradiology, Klinikum Dortmund, Dortmund, Germany, 6Department for Pediatric Radiology, University of Leipzig Medical Center, Leipzig, Germany, 7Institute of Medical Genetics, Medical University Vienna, Wien, Austria, 8Institute of Pathology, Philipps-University Marburg, Marburg, Germany.

Biallelic polynucleotide kinase 3’-phosphatase (PNKP) variants cause different disorders ranging from neurodevelopmental disorder with microcephaly/seizures to adult-onset Charcot-Marie-Tooth disease. To date, only postnatal descriptions have been described.The index was a male fetus with prenatally diagnosed micro- and brachycephaly, brain malformations and microretrognathia at 13th gestational week. A recessive disorder was suspected because of previous termination of pregnancy (TOP) for similar abnormalities in a sister fetus. Prenatal exome sequencing using DNA derived from amniocentesis and from both unrelated parents identified compound-heterozygosity for the variants c.498G>A, p.[(=),0?] and c.302C>T, p.(Pro101Leu). Segregation analysis confirmed both variants in the affected previous fetus. Syndromic oriented fetopathology after TOP of the male index fetus revealed micrencephaly with pronounced hypoplastic frontal lobes, shortened occipital lobes, missing temporo-parietal lobulation and hypoplastic cerebellum. To characterize aberrant splicing of c.498G>A, we performed RT-PCR analysis on RNA from fetal muscle and a paternal PAXgene sample. This confirmed skipping of exon 4 affecting the FHA- and phosphatase-domains of PNKP (p.Leu67_Lys166del). To compare prenatal/postnatal phenotypes, we retrospectively investigated two unrelated male individuals diagnosed with biallelic PNKP-variants. Both carry the same splice-donor variant c.1029+2T>C and a second missense variant in the FHA-domain (c.311T>C, p.(Leu104Pro) and c.151G>C, p.(Val51Leu), respectively). RNAseq showed complex splicing events for c.1029+2T>C and c.151G>C. Computational modelling revealed significant clustering of the missense variants in the FHA-domain.Here, we present the first prenatally diagnosed PNKP-related primary microcephaly-/encephaly associated with variants affecting the N-terminal FHA-domain. Our detailed clinical and mutational characterisation extend the continuum of PNKP-manifestation to the prenatal stage.

I. Krey: None. S. Neuser: None. A. Schwan: None. T. Bartolomaeus: None. J.H. Döring: None. S. Syrbe: None. M. Plassmann: None. S. Rohde: None. C. Roth: None. H. Rehder: None. R. Abou Jamra: None. D. Le Duc: None. S. Schubert: None. K. Schoner: None. B. Popp: None.

P01.074.B Pregnancy loss and Exome sequencing analysis (WES)

Aicha Boughalem 1, Detlef TROST1, Constance Wells2, Audrey Lamouroux2, Viorica Ciorna-Monferrato3, Amandine Diakiese4, Pascale Kleinfinger4, Laurence Lohmann4, Armelle Luscan4, Mylene Valduga4, Jean-Marc Costa1, Patricia Blanchet2

1Laboratoire CERBA, Saint Ouen l’Aumône, France, 2Département de Génétique Médicale, Centre de Référence « Anomalies du Développement et Syndromes Malformatifs », CHU Hôpital Arnaud de Villeneuve, Montpellier, France, 3Génétique Médicale et Oncogénétique, Hôpital Femme Mère Enfant, Metz-Thionville, France, 4CERBA, Saint Ouen l’Aumône, France.

Background: Genetic abnormalities are known to cause pregnancy loss. In our cohort we evaluated the usefulness of exome sequencing (WES) in identifying the genetic etiology for pregnancy loss.

Methods: A cohort of 68 samples from products of conception or from fetal tissue, with normal karyotype and absence of pathogenic copy-number variants were selected for WES. WES data were analyzed based on the prenatally observed ultrasound findings and results of fetal autopsy.

Results: WES detected 12 pathogenic variants, 3 likely pathogenic variants, and 3 variants of uncertain significance (VUS) from this cohort. The Diagnostic yield for pathogenic and likely pathogenic variants was 22% and reached 26% with the inclusion of VUS. In 9 fetuses (50% of cases) the diagnoses followed an autosomal recessive inheritance pattern in nonconsanguineous couples (2 homozygous variants, 6 compound heterozygous variants and one variant with X-linked recessive inheritance) and in 9 cases the identified pathogenic variant occurred “de novo”. Affected genes included those associated with ciliopathic human genetic disorder, multisystem abnormalities, neurodevelopmental disorders, cardiac anomalies, renal diseases, skeletal dysplasia and metabolic disorders.

Conclusion: These results supported the clinical utility of WES for detecting the monogenic etiology of pregnancy loss. The identification of disease-associated variants provided information for follow-up genetic counseling of recurrence risk and management of subsequent pregnancies. Our cohort study illustrates how high-resolution obstetric scan, detailed observation of fetal features and application of exome sequencing; contribute to elucidate the etiology of pregnancy loss. Keywords: Exome sequencing (WES); foetopathology; pregnancy loss, medical termination of pregnancy.

A. Boughalem: None. D. Trost: None. C. Wells: None. A. Lamouroux: None. V. Ciorna-Monferrato: None. A. Diakiese: None. P. Kleinfinger: None. L. Lohmann: None. A. Luscan: None. M. Valduga: None. J. Costa: None. P. Blanchet: None.

P01.076.D Prenatal exome sequencing unravels unique co‐occurrence of Congenital Idiopathic arterial calcification and congenital Gaucher disease: A potential pitfall for counseling

Sara Hisham El-Dessouky 1, Mohamed Elhodiby2, Ghada M. H. Abdel-Salam3

1Prenatal Diagnosis & Fetal Medicine Department, Human Genetics and Genome Research Division, National Research Centre, Cairo, Egypt, 2Department of Obstetrics and Gynecology, Faculty of Medicine, M.U.S.T. University, Cairo, Egypt, 3Clinical Genetics Department, Human Genetics and Genome Research Division, National Research Centre, Cairo, Egypt.

Introduction: The presence of two or more genetic disorders represents a challenge for clinical and molecular diagnosis. If one condition remains undiagnosed, the risk of recurrence may not be appropriately assessed. In this study, we present a complex fetal phenotype that occurred in two successive pregnancies.

Materials and Methods: Prenatal ultrasound at 25 weeks revealed the constellation of polyhydramnios, hydrops fetalis, hepatosplenomegaly, arthrogryposis, microcephaly, and developmental brain anomalies in the form of intracranial calcifications, ventriculomegaly and cerebellar hypoplasia. Additionally, extensive vascular and cardiac calcification in the form of diffuse, echogenic cardiac outflow tracts, generalized hyperechogenicity in tricuspid, pulmonary valves, diffuse intrahepatic, renal and placental calcifications were observed. The fetus suffered intrauterine demise at 30 weeks gestation. Autopsy confirmed the prenatal findings. Prenatal exome sequencing (PWES), karyotyping and microarray were arranged from the fetal blood.

Results: PWES identified two novel homozygous missense variants in both the GBA gene (c.170G>A, p.Cys57Tyr) and the ABCC6 (c.3381G>A, p.Met1127 Ile) gene pointing to the co-occurrence of congenital idiopathic arterial calcification and congenital Gaucher disease. All identified variants were confirmed by Sanger sequencing and validated by parental testing

Conclusion: This is the first report of congenital idiopathic arterial calcification caused by homozygous missense pathogenic variant in ABCC6 gene and in combination with Gaucher syndrome. We believe that comprehensive phenotyping is essential for improving the diagnostic performance of PWES. Additionally our study emphasizes that unexplained phenotypes may result from the occurrence of pathogenic variants of two or more genetic disorders in the same patient.

S.H. El-Dessouky: None. M. Elhodiby: None. G.M.H. Abdel-Salam: None.

P01.077.A Exome sequencing in prenatal diagnosis: results from 88 cases

Emmanuelle Ranza 1, Xavier Blanc1, Federico Santoni1, Frédéric Guerry1, Bernard Conrad2, Isabelle Eperon3, Cécile Tissot4, Cécile Deluen2, Tanguy Araud2, Loïc Baerlocher2, Yasmine Sayegh Martin5, Anne-Claude Müller-Brochut6, Christian Bisch3, Joel Fluss7, Jean-Marie Pellegrinelli7, Marta Carrasco8, Thibaud Quibel9, Sylvie Lacroix9, Philippe Extermann3, Graziano Pescia10, Romaine Robyr Susini9, Stylianos E. Antonarakis1

1Medigenome, Swiss Institute of Genomic Medicine, Geneva, Switzerland, 2Genesupport, Geneva, Switzerland, 3Dianecho, Geneva, Switzerland, 4Clinique des Grangettes, Geneva, Switzerland, 5Centre d’Imagerie Jean-Violette, Geneva, Switzerland, 6Centre GynEcho, Fribourg, Switzerland, 7HUG, Geneva University Hospitals, Geneva, Switzerland, 8GHOL, Groupement hospitalier de l’Ouest Lémanique, Nyon, Switzerland, 9EchoFemme, Geneva, Switzerland, 10Genesupport, Lausanne, Switzerland.

Exome sequencing (ES) has become a standard test for undiagnosed developmental disorders, now increasingly used in prenatal diagnosis. We report here our experience with 88 consecutive prenatal cases through ES. The most common fetal signs were: increased nuchal translucency (25 %), congenital heart defect (22.7 %), skeletal malformations (22.7 %) and brain abnormalities (11.4 %). After performing a-CGH, 28 cases had solo and 60 trio ES. In 25 % of cases, we have identified a pathogenic or likely pathogenic variant (as per the ACMG guidelines) likely causative of the fetal phenotype. The diagnostic yield was 40 % for skeletal malformations, and roughly 20 % for nuchal translucency, congenital heart defect, and brain anomaly cases. In 18 of the solved cases, the pathogenic variants were SNVs, while 2 were pathogenic structural variants (SV). Furthermore, the trio ES has identified in two cases complete uniparental disomies UPD6 and UPD17, both including isodisomic segments with likely causative recessive genes. ES is a powerful and accurate method for the identification of causative variants in prenatal; trio sequencing reduces the turnaround time and likely increases the diagnostic yield. The VUS remain a diagnostic challenge and international guidelines are needed to assist in the interpretation and disclosure of the results. The discovery of novel mendelian genes and the introduction of additional laboratory and computational methods such as long-read sequencing and improvement of SV detection may further increase the diagnostic yield.

E. Ranza: A. Employment (full or part-time); Significant; Medigenome, Swiss Institute of Genomic Medicine. E. Ownership Interest (stock, stock options, patent or other intellectual property); Significant; Medigenome, Swiss Institute of Genomic Medicine. X. Blanc: A. Employment (full or part-time); Significant; Medigenome, Swiss Institute of Genomic Medicine. F. Santoni: A. Employment (full or part-time); Significant; Medigenome, Swiss Institute of Genomic Medicine. F. Guerry: A. Employment (full or part-time); Significant; Medigenome, Swiss Institute of Genomic Medicine. B. Conrad: A. Employment (full or part-time); Significant; Genesupport. Other; Significant; Medigenome, Swiss Institute of Genomic Medicine. I. Eperon: A. Employment (full or part-time); Significant; Dianecho. C. Tissot: A. Employment (full or part-time); Significant; Clinique des Grangettes. C. Deluen: A. Employment (full or part-time); Significant; Genesupport. T. Araud: A. Employment (full or part-time); Significant; Genesupport. L. Baerlocher: A. Employment (full or part-time); Significant; Genesupport. Y. Sayegh Martin: A. Employment (full or part-time); Significant; Centre d’Imagerie Jean-Violette. A. Müller-Brochut: A. Employment (full or part-time); Significant; Centre GynEcho. C. Bisch: A. Employment (full or part-time); Significant; Dianecho. J. Fluss: A. Employment (full or part-time); Significant; HUG, Geneva University Hospitals. J. Pellegrinelli: A. Employment (full or part-time); Significant; HUG, Geneva University Hospitals. M. Carrasco: A. Employment (full or part-time); Significant; GHOL, Groupement Hospitalier de l’Ouest Lémanique. T. Quibel: A. Employment (full or part-time); Significant; EchoFemme. S. Lacroix: A. Employment (full or part-time); Significant; EchoFemme. P. Extermann: A. Employment (full or part-time); Significant; Dianecho. G. Pescia: A. Employment (full or part-time); Significant; Genesupport. R. Robyr Susini: A. Employment (full or part-time); Significant; EchoFemme. E. Ownership Interest (stock, stock options, patent or other intellectual property); Significant; EchoFemme. S.E. Antonarakis: A. Employment (full or part-time); Significant; Medigenome, Swiss Institute of Genomic Medicine. E. Ownership Interest (stock, stock options, patent or other intellectual property); Significant; Medigenome, Swiss Institute of Genomic Medicine.

P01.078.B Mosaic Trisomy 7: A case report of discrepancies between noninvasive screening for fetal trisomy, array CGH, cytogenetics, and fetal ultrasound

Dorela Kroni1, Merita Xhetani 2, Marina Baldi3

1StemGen Partners Ltd, University of Medicine, Immunohematology Department, Tirana, Albania, 2University of Tirana, Faculty of Natural Sciences, Department of Molecular Biology, Tirana, Albania, 3Genoma Laboratories, Genetic Department, Roma, Italy.

Introduction: Here we report a case of mosaic trisomy 7 diagnosed incidentally, by the prenatal screening of cfDNA (CentoNIPT®) in a31 years old pregnant women resulted inconclusive for trisomy 21, 18, and 13 at 15 weeks of gestation. Array CGH was performed at 18 weeks of gestation. Male karyotype with mosaic chromosome 7 trisomy 7p22. 3q36. 3 was reported. Ultrasound reports on fetal growth and development were normal. Follow-up with cytogenetics karyotyping at 21 weeks of gestation was performed, which revealed a result of apparently normal male karyotype 46, XY in cultured amniocytes. Simultaneously DNA testing of both parents and fetal cultured amniocytes was performed by PCR and UPD was excluded. Two contradictions have led us to publish this case: first, confirmation of reported limitation of NIPT in the detection of mosaicism, a trisomy 7 would have been reported, taking into consideration that the analysis is performed on cfDNA with placental and fetal origin, the result of mosaic trisomy 7 might have been related to a placental mosaicism situation, which was confirmed from Array CGH analyses. Second, cytogenetics on cultured amniocytes confirmed that fetal cells don’t have trisomy 7 but either array CGH nor cytogenetics were diagnostic and confirmed the origin of the mosaic trisomy 7.

Conclusion: We suggest the necessity of a full panel NIPT instead of a narrow panel NIPT for better time management of pregnancy and in mosaic trisomy 7 where UPD is excluded.

D. Kroni: None. M. Xhetani: None. M. Baldi: None.

P01.079.C The contribution of chromosomal abnormalities in the formation of sporadic and recurrent early reproductive losses

Iryna Tkach 1, Nataliya Huleyuk1, Danuta Zastavna1, Thomas Liehr2, Ewa Ciszkowicz3

1Institute of Hereditary Pathology, NAMS of Ukraine, Lviv, Ukraine, 2Jena University Hospital, Friedrich Schiller University, Institute of Human Genetics, Jena, Germany, 3Department of Biotechnology and Bioinformatics, Faculty of Chemistry, Rzeszow University of Technology, Rzeszow, Poland.

Background: Spontaneous abortion occurs in 15-20% of clinically recognized pregnancy. The karyotype of a spontaneously aborted of products of conception (POC) provides valuable clinical information for the couple as well for basic research becouse provide important information for the recurrence risk of cytogenetic abnormalities in subsequent pregnancies Aim. The aim of the present study was to investigate the contribution of numerical chromosomal imbalances in products of conception from sporadic pregnancy loss (SPL) and recurrent pregnancy loss (RPL).

Methods: Banding cytogenetic and interphase mFISH with the probe panel for chromosomes 13, 14, 15, 16, 17, 18, 21, 22, X and Y.

Results: Cytogenetic studies of 1720 spontaneously aborted fetuses were performed. The results were stratified in 2 groups according to anamnesis: Group I (POC of first pregnancy and POC from lost pregnancy, with children in anamnesis) - 1199 samples and Group II (POC from RPL) - 521 samples. The contribution of chromosomal abnormalities was higher in the genesis of SPL (40.1%) compared to RPL (39.2%). Among most often diagnosed chromosomal change was triploidy - 24.53 % in SPL vs 24.51% in RPL, monosomy X - 21.62% vs 17.16% and trisomy 16 - 20.99% vs 26.96%.

Conclusions. Consequently, detected of chromosome aneuploidies in samples from products of conception is a key part of the investigations of reproductive failure in humans.

I. Tkach: None. N. Huleyuk: None. D. Zastavna: None. T. Liehr: None. E. Ciszkowicz: None.

P01.080.D Genetic testing of miscarriages using a QF-PCR /aCGH/ MLPA strategy: five years experiences from North-Eastern Slovenia

alenka erjavec škerget, boris zagradišnik, andreja zagorac, špela stangler herodež, nadja kokalj vokač

University Clinical Centre Maribor, maribor, Slovenia.

Introduction: Traditional testing of miscarriage samples involved culture of tissue followed by G-banded chromosome analysis; this approach has a high failure rate, is labour intensive and has a resolution of around 8-10 Mb. To improve diagnostic yield and efficiency we have updated our testing strategy in 2016. Since then we use more comprehensive strategy: QF-PCR assay for all samples, followed by array CGH or MLPA, depends on a gestational age of the embrional material. Here we describe the results from the last 5 years of our strategy.

Methods: Fetal tissue samples and maternal samples were tested using QF-PCR for the detection of aneuploidy for chromosomes 13, 15, 16, 18, 21, 22, X and Y. Confirmed fetal samples without aneuplody of tested chromosomes and less than 15 weeks of gestational age were then tested by MLPA while fetal samples older than 15 weaks were analyzed by aCGH.

Results: From 335 analysed samples, 266 samples were confirmed as fetal material (78,57%). In those QF-PCR analysis identified aneuploidy/triploidy in 21.42%. MLPA detected subtelomeric imbalances in a further 12.79% (11 out of 86) while aCGH analysis detected imbalance in 4.87% (6 out of 123) of samples. All detected imbalances by aCGH were submicroscopic (in range 0.19-2.6Mb) and 2 out of 6 (33.33%) were classified as causative for the spontaneous misscariage.

Conclusions: This efficient QF-PCR/aCGH/MLPA strategy has a lower failure rate and higher diagnostic yield than karyotype. It is therefore an efficient and cost-effective diagnostic testing strategy for misscarriage products.

A. erjavec škerget: None. B. zagradišnik: None. A. zagorac: None. Š. stangler herodež: None. N. kokalj vokač: None.

P01.081.A Deciphering the genetic cause of recurrent and sporadic pregnancy loss

Rick Essers 1,2, Ganesh Acharya3, Salwan Al-Nasiry2, H G. Brunner2,4, S. P. Deligiannis5, E. A. Fonova6, Alexander Hoischen4, Ants Kurg7, Igor N. Lebedev6, Merryn V. E. Macville1,2, T. V. Nikitina6, Andres Salumets3,5, E. A. Sazhenova6, Servi J. C. Stevens1,2, E. N. Tolmacheva6, Masoud Zamani Esteki1,2

1GROW School for Oncology and Developmental Biology, Department of Genetics and Cell Biology, Maastricht, Netherlands, 2Maastricht University Medical Center MUMC+, Department of Clinical Genetics, Maastricht, Netherlands, 3Karolinska Institutet and Department of Women’s Health- Karolinska- University Hospital, Division of Obstetrics and Gynecology- Department of Clinical Science- Intervention & Technology CLINTEC, Stockholm, Sweden, 4Radboud University Medical Center, Department of Human Genetics, Nijmegen, Netherlands, 5Institute of Clinical Medicine- University of Tartu, Department of Obstetrics and Gynecology, Tartu, Estonia, 6Tomsk National Research Medical Center, Research Institute of Medical Genetics, Tomsk, Russian Federation, 7Institute of Molecular and Cell Biology- University of Tartu, Department of Biotechnology, Tartu, Estonia.

Introduction: Spontaneous abortion (SA) occurs in 10-15% of clinically recognized pregnancies and recurrent pregnancy loss (RPL) in 1-3%. One potential cause for pregnancy loss is uniparental disomy (UPD). UPD can lead to imprinting disorders characterized by features affecting growth and development in liveborn offspring. This study aims to investigate the prevalence and effect of (mosaic) de novo genomic aberrations in RPL and SA.

Materials and Methods: We recruited 32 families with pregnancy loss (n = 16 RPL cohort, n = 16 SA cohort) with normal karyotyping results in both parents and the fetus. DNA was isolated from blood of both parents and placental tissues from the miscarried products of conception. The tissues originate from the extraembryonic mesoderm (EM) and the chorionic villi (CV). We performed SNP-genotyping (Illumina’s Global-Screening Array-24 v2.0 BeadChips) and applied haplarithmisis to delineate allelic architecture of fetal tissues.

Results: We analyzed 132 DNA samples (n = 32 families). Within the RPL cohort, we found aberrations in 6/16 families including: mosaic genome-wide hexaploidy and tetraploidy, non-mosaic genome-wide hetero UPD, mosaic UPD of chromosomes 14, 16, 6, and 5 in different families. Within the SA group, we found aberrations in 2/16 families including: mosaic UPD of chromosome 7, segmental UPD of chromosome 5. All UPDs were maternally inherited.

Conclusions: The prevalence of maternal UPD was 20.6% in fetal tissues. These findings could lead to a better understanding of causative factors for SA and RPL and the need for SNP-based non-invasive prenatal testing. Grants: EVA (KP111513), MUMC+; Horizon 2020 innovation (ERIN) (EU952516), European Commission; RFBR (20-315-90111)

R. Essers: None. G. Acharya: None. S. Al-Nasiry: None. H.G. Brunner: None. S.P. Deligiannis: None. E.A. Fonova: None. A. Hoischen: None. A. Kurg: None. I.N. Lebedev: None. M.V.E. Macville: None. T.V. Nikitina: None. A. Salumets: None. E.A. Sazhenova: None. S.J.C. Stevens: None. E.N. Tolmacheva: None. M. Zamani Esteki: None.

P01.082.B Regions of homozygosity in prenatal investigation: to sequence or not to sequence?

Andreea C. Tutulan-Cunita 1, Luiza Dimos1, Anca G. Pavel1, Florina M. Nedelea1,2,3, Azdasher Naszan4, Danae Stambouli1

1Cytogenomic Medical Laboratory, Bucharest, Romania, 2Filantropia Clinical Hospital, Bucharest, Romania, 3Carol Davila University of Medicine, Bucharest, Romania, 4CF2 Clinical Hospital, Bucharest, Romania.

Introduction: Many severe genetic disorders cannot be identified prenatally by cytogenomic analyses and, often, the parents are unaware of their carrier status. ACMG recommendations for prenatal diagnosis state that WES may be considered when standard CMA and karyotype analysis did not provide a diagnosis. We present the case of a fetus of consanguineous parents with ultrasound anomalies and normal CMA result, where WES uncovered two distinct pathogenic variants.

Materials and methods: The patient had two consecutive pregnancies with fetuses with short, bent femurs. DNA was isolated from amniotic fluid. CMA and WES were done on Affymetrix 750K and on Ion Torrent S5 platform (Thermo Fisher Scientific), respectively, while Sanger sequencing used BigDye Terminator kit.

Results: Following a normal CMA result, WES was recommended. Two novel homozygous variants were found in CRTAP gene (c.793+1G>T) and GNS (c.811A>G, p.Arg271Gly) and classified as pathogenic and likely pathogenic, respectively, according to ACMG criteria. CRTAP gene is associated with osteogenesis imperfecta type VII, while GNS – with mucopolysaccharidosis type III D. Sanger analysis confirmed that parents are carriers. Revising CMA, long runs of homozygosity (ROH), including these two genes, were found. Thus, an ultrasound anomaly led to the serendipitous discovery of the second pathogenic mutation, which otherwise would have been missed according to prenatal checks.

Conclusion: While this finding will allow the selection of the appropriate management of the future pregnancies, it invites to further discussion about when to proceed to WES in cases of normal CMA with long ROHs, regardless ultrasound results.

A.C. Tutulan-Cunita: None. L. Dimos: None. A.G. Pavel: None. F.M. Nedelea: None. A. Naszan: None. D. Stambouli: None.

P01.083.C Retrospective study of cartilage-hair hypoplasia carrier screening cases reveal frequent insertions in the promoter region of the RMRP gene

Ezen Choo, Trevor Smart, Nina Sanapareddy, Irina Rakova, J. Dianne Keen-Kim

Natera, Inc, San Carlos, CA, USA.

Introduction: Mutations in RMRP, a nuclear encoded, intronless gene, can lead to the autosomal recessive, multi-systemic disease cartilage-hair hypoplasia (CHH). Patient studies have revealed mutations in both the promoter and the transcribed region.

Materials and Methods: We retrospectively studied 51,599 healthy individuals tested with a 137 or 274 gene carrier screening panel. Samples were tested by next-generation sequencing with orthogonal confirmation by Sanger sequencing when Pathogenic or Likely Pathogenic variants were identified. Polymorphisms and Benign/ Likely Benign variants were not included in the analysis. We assessed the occurrence of alteration types and distribution across the gene.

Results: We identified 397 unique RMRP alterations, of which 20% (n = 78) were insertions. The distribution of insertions across the gene included 43 within the promoter, 8 involving the transcription initiation site, and 27 within the transcribed region. Unique insertions affecting the promoter and start (n = 51) were more prevalent and involved larger insertions (base pairs, mean = 14.4, range:1-64) than those in the transcribed region (n = 27, mean = 2.5, range:1-17).

Conclusions: Given that RNA polymerase III promoters are usually very short, it was surprising to find larger and more diverse alterations in the promoter than in the rest of the gene in our carrier population. Up to 30bp promoter insertions have been reported in CHH patients. We identified 3 alterations >30bp in the promoter region. The predicted functional significance is promoter inefficiency and reduced transcription; however, the clinical significance and risk to patient offspring is to be determined.

E. Choo: A. Employment (full or part-time); Significant; Natera, Inc.. E. Ownership Interest (stock, stock options, patent or other intellectual property); Significant; Natera, Inc.. T. Smart: None. N. Sanapareddy: A. Employment (full or part-time); Significant; Natera, Inc.. E. Ownership Interest (stock, stock options, patent or other intellectual property); Significant; Natera, Inc. I. Rakova: A. Employment (full or part-time); Significant; Natera, Inc.. E. Ownership Interest (stock, stock options, patent or other intellectual property); Significant; Natera, Inc. J. Keen-Kim: A. Employment (full or part-time); Significant; Natera, Inc.. E. Ownership Interest (stock, stock options, patent or other intellectual property); Significant; Natera, Inc..

P01.088.D Russian women’s preferences about invasive prenatal testing

Elena Baranova 1, Elizaveta Zaiaeva1, Gaukhar Zobkova2, Lyudmila Zhuchenko1, Svetlana Shelykalina3, Vera Izhevskaya4

1Federal State Budgetary Educational Institution of Further Professional Education "Russian Medical Academy of Continuous Professional Education" of the Ministry of Healthcare of the Russian Federation, Moscow, Russian Federation, 2LLC Evogen, Moscow, Russian Federation, 3National Research Medical University named after N.I. Pirogov of the Ministry of Health of Russia, Moscow, Russian Federation, 4Research Centre for Medical Genetics, Moscow, Russian Federation.

Introduction: In early prenatal screening, regardless of the technology used, confirmation of the fetal chromosomal abnormality (CA) is required by invasive prenatal diagnosis. Russian women’s preferences about invasive prenatal testing (IPT) are poorly understood. The aim of study was to find out the readiness of pregnant women to undergo IPT to diagnose fetal CA and terminate pregnancy if pathology is detected. The material of the research: the results of a survey of 800 pregnant women from 16 regions in Russia.

Results: It was shown that 451 (56.3%) women are ready to undergo IPT with a high risk of fetal CA, 39 (4.8%) - will not do the procedure, 281 (35.1%) were undecided, 29 (3.6%) did not answer the question. Women who agreed to carry out an IPT, compared with those who decided not to carry out an IPT, more often noted the importance of obtaining the most complete and early information. Among the respondents, only a quarter of women, 209 (26.1%) are ready to terminate a pregnancy in the case of identified fetal CA, 84 (10.5%) will not terminate a pregnancy, 474 (59.2%) have not decided, 33 (4.1%) did not answer the question. Women who made the decision to terminate pregnancy in the case of identified fetal CA were older, had more children, among them there were more women with higher education.

Conclusions: The main preferences of pregnant women with regard to undergoing invasive prenatal testing and termination of pregnancy in case of detection of a chromosomal abnormality were revealed.

E. Baranova: None. E. Zaiaeva: None. G. Zobkova: None. L. Zhuchenko: None. S. Shelykalina: None. V. Izhevskaya: None.

P01.089.A A rare case of fetal skeletal dysplasia: TAR syndrome

Sofia Pérez 1, Bruno Carrilho2, Ana Bernardo2, Ana Teresa Martins2, Álvaro Cohen2, Inês Carvalho1

1Genetic Service, Pediatric Department, Hospital de Dona Estefânia, Centro Hospitalar Universitário de Lisboa Central, EPE, Lisboa, Portugal, 2Prenatal Diagnostic Center, Maternidade Alfredo da Costa, Centro Hospitalar Universitário de Lisboa Central, EPE, Lisboa, Portugal.

Introduction: Thrombocytopenia Absent Radius (TAR) syndrome is characterized by bilateral absent radii and thrombocytopenia, sometimes associated with other skeletal, cardiac and genitourinary anomalies. It´s an autosomal recessive disorder and results from compound heterozygosity of RBM8A pathogenic variants (one null and one hypomorphic allele). Case Report:We report the case of a 32-year-old female, with an ongoing pregnancy of 16 weeks, no relevant personal or family history, referred to our clinic due to an altered fetal genetic study. In the 1st trimester ultrasound, an increased nuchal translucency was identified, without other changes. For this reason, a CVS was performed and the fetal microarray revealed an interstitial deletion in the 1q21.1 region. Bearing in mind that the CNV in question encompassed the morbid RBM8A gene, in the medical genetics consultation we requested the sequencing of the RBM8A gene, since the simultaneous presence of the 1q21.1 deletion and a mutation in the other allele of the RBM8A gene is associated with TAR syndrome. In the 18-week ultrasound, bilateral agenesis of the radius, ulna and humerus was identified, with hands visible bilaterally. The couple opted for medical termination of pregnancy. Posteriorly, the sequencing of the RBM8A gene revealed the pathogenic variant c.-21G> A, in hemizygosis, confirming the diagnosis of TAR syndrome. The parents’ family study revealed a paternal origin of the 1q21.1 deletion and a maternal origin of the mutation in the RBM8A gene.

Conclusion: Fetal skeletal dysplasia comprises a complex group of disorders, where a multidisciplinary approach is essential for the diagnosis of rare clinical entities.

S. Pérez: None. B. Carrilho: None. A. Bernardo: None. A. Martins: None. Á. Cohen: None. I. Carvalho: None.

P01.090.B Telomere length dynamics during human preimplantation development

Mikhail I. Krapivin, Anna A. Pendina, Olga A. Efimova, Irina D. Mekina, Evgeniia M. Komarova, Andrei V. Tikhonov, Olga G. Chiryaeva, Alexander M. Gzgzyan, Igor Y. Kogan, Vladislav S. Baranov

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

Telomeres are complex structures that cap the ends of chromosomes and help to maintain genomic integrity and stability. Since telomere shorten after each cell division, their restoration during the transition of DNA to a new generation is of crucial importance. We analyzed telomere length (TL) in metaphases from 67 human embryos, unsuitable for transfer into the uterine cavity due to abnormal ploidy or morphology. For metaphase preparations, zygotes and embryos were treated with colchicines, hypotonic solution and fixed on the glass slides. We analyzed TLs in 84 metaphases from zygotes, 14 metaphases from the embryos at the 3-5-cell stage, 12 metaphases from the embryos at the 6-12-cell stage, and 49 metaphases from the blastocysts. To avoid the influence of chromatin compaction, TLs were measured as relative values by dividing telomeric by subtelomeric fluorescence intensity. The comparison of relative TL medians between the stages of preimplantation development showed significant differences: TL increased in the period from the zygote up to the 3-5-cell stage (Mann-Whitney test, p = 0.02) and from the zygote to the 6-12-cell stage (Mann-Whitney test, p < 0.0001). Then, TL decreased from the 6-12-cell stage to the blastocyst stage (Mann-Whitney test, p < 0.0001). Thus, we observed the waves of TL changes during preimplantation human development, with a peak at 6-12-cell stage followed by a decrease up to the blastocyst stage. These alterations of TL may be a result of telomeric region recombination and, thus, does not require telomerase activity which is absent until the blastocyst stage. Supported by RSF №18-75-10046.

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

P01.091.C Two Rare Cases of Miscarriage: Aneuploid Triploidy

Mustafa Oguz Acar 1, Sule Altiner1, Nuket Yurur Kutlay1, Ajlan Tukun2

1Ankara University, Ankara, Turkey, 2Duzen Laboratories Group, Ankara, Turkey.

Introduction: More than 50% of early pregnancy losses have a chromosomal abnormality. Triploidy is accounting for 10% of all spontaneous abortions.Here, two fetuses with atypical triploid karyotype referred to medical genetic department due to miscarriage at the 12th gestational week will be presented.

Materials and Methods: Conventional cytogenetic and FISH analyses of both abort tissues were carried out according to standard procedure, following tissue culture. STR analysis was used to determine the parental origins of the tetraploid chromosomes.

Results: Karyotyping revealed, 70,XXY,+18 and 71,XXXX,+6 formations, respectively. Presence of extra chromosomes was confirmed with FISH analysis. As expected, karyotypes of both parents were normal.

Conclusions: Although triploidy is common in abortion materials, the case accompanied by tetrasomy of some chromosomes has been rarely reported in the literature. Possible mechanisms to explain these chromosomal abnormalities will be discussed in detail.

M.O. Acar: None. S. Altiner: None. N. Yurur Kutlay: None. A. Tukun: None.

P01.092.D Triploidy/Vanished Twin Detection: Updated clinical experience following Single Nucleotide Polymorphism-based NIPT twins validation

Val Kantor, Kathryn Young, Wendy DiNonno

Natera, Inc, San Carlos, CA, USA.

Introduction: Occurring in ~1-2% of clinically recognized pregnancies, triploidy carries risk for miscarriage, fetal anomalies, and maternal complications. Detection is critical for medical management. Single nucleotide polymorphism (SNP)-based NIPT was previously validated to detect additional fetal haplotypes consistent with triploidy, vanished twin, or viable twin gestation. In October of 2017 viable twin gestations were validated for SNP-based NIPT. This study determines an updated PPV for triploidy using SNP-based NIPT, following the removal of known viable twin gestations.

Methods: Retrospective outcome was obtained on 1005 cases with vanished twin/triploidy results, following a SNP-based NIPT’s twin validation. Pregnancy outcomes were defined as: confirmed triploidy, suspected triploidy, confirmed vanished twin, suspected vanished twin, and normal singleton.

Results: Of 1005 cases, outcome was obtained for 786 (78.2%) cases. Including only cases of confirmed triploidy, data analysis indicates a triploidy PPV of 59/786 (7.5%). If cases of suspected triploidy are included the PPV improves to 85/786 (10.8%). The conservative PPV for vanished twin gestations was 460/786 (58.5%). When cases of suspected vanished twins were included, the PPV improved to 499/786 (63.5%).

Conclusions: With five times the number of cases as Curnow et al., this study shows an improved triploidy PPV (7.5% versus previously reported 5.3% PPV.) When triploidy is combined with vanished twin gestation, we observed an improved overall PPV (66.0% versus 47.4%). Our cohort confirms the ability of a SNP-based NIPT to detect triploidy and shows an improved PPV that can better inform post-test counseling for both fetal and maternal complications.

V. Kantor: A. Employment (full or part-time); Significant; Natera, Inc.. E. Ownership Interest (stock, stock options, patent or other intellectual property); Significant; Natera, Inc. K. Young: A. Employment (full or part-time); Significant; Natera, Inc.. E. Ownership Interest (stock, stock options, patent or other intellectual property); Significant; Natera, Inc. W. DiNonno: A. Employment (full or part-time); Significant; Natera, Inc.. E. Ownership Interest (stock, stock options, patent or other intellectual property); Significant; Natera, Inc..

P01.093.A Trisomy 8 mosaicism in the placenta: a Danish cohort study of 37 cases and a literature review

Simon H. Thomsen

Aarhus University, Aarhus, Denmark.

Objective: To evaluate the risk of fetal involvement when trisomy 8 mosaicism (T8M) is detected in chorionic villus samples (CVS).

Methods: A retrospective descriptive study of registered pregnancies in Denmark with T8M in CVS identified through a database search and a review of published cases of T8M found through a systematic literature search and inclusion of cross references. Pregnancies with T8M in CVS and no additional numerical chromosomal aberrations were included.

Results: A total of 37 Danish cases and 60 published cases were included. T8M detected in a CVS was associated with fetal involvement in 18 out of 97 pregnancies (18.6% [95%CI: 11.4-27.7]). Eight out of 70 (11.4% [95%CI: 5.1-21.3]) interpreted prenatally to be confined placental mosaicism (CPM) were subsequently found to be true fetal mosaicisms (TFM).

Conclusion: T8M detected in CVS poses a significant risk of fetal involvement, and examination of amniotic fluid (AF) and/or fetal tissue should be offered. However, a normal result of AF still has a considerable residual risk of fetal involvement. Genetic counselling at an early gestational age is essential, and follow-up ultrasonography should be performed to predict fetal involvement if possible.Funding: Ida Vogel is funded by a research grant from the Novo Nordic Foundation: NNF16OC0018772

S.H. Thomsen: None.

P01.094.B Mosaicism for genome-wide paternal uniparental disomy - two prenatal cases

Raquel Lemos1, Cíntia Ventura1, Fátima Torres1, Gabriela Fernandes1, Isabel Durães1, Áurea Pereira1, Patricia Costa1, Jorge Castro2, Conceição Brito2, Rita Cerqueira 1

1CGCgenetics UNILABS, Porto, Portugal, 2CHVNG, Obstetrícia, Vila Nova de Gaia, Portugal.

Introduction: Uniparental disomy (UPD) is the abnormal situation in which both members of a chromosome pair are inherited from one parent, and the other parent’s chromosome for that pair is missing. UPD of the whole genome is not consistent with life but mosaic genome-wide UPD might be compatible with life. We present 2 cases of prenatal diagnosis with paternal uniparental isodisomy (GWpUPD) after contradictory QF-PCR and fetal karyotype results. The fetuses died and the placentas had hydatidiform moles criteria.

Methods: Cytogenetic analysis was performed on 20 metaphases, stained with GTG bands, from three independent cultures. The most frequent aneuploidies by QF-PCR test was performed after DNA extraction from the fetus sample with STR markers specific for chromosomes 13, 18, 21, X and Y. Chromosomal microarray study (aCGH) was performed, after DNA extraction from the fetus sample, using the Affymetrix Cytoscan 750K platform. Results In both cases, the result of QF-PCR test revealed a profile compatible with triploidy discrepant from the karyotype that revealed a normal female chromosomal constitution. The aCGH on the Affymetrix Cytoscan platform allowed the confirmation of the mosaicism for uniparental disomy for all chromosomes. These results together are compatible with GWpUPD.

Conclusions: The articulation between the different techniques was essential for the distinction between triploidy, mosaic for GWpUPD and a normal result. A prenatal case with a normal fibroblasts karyotype and a suspected hydatidiform mole without further studies could be misdiagnosed.

R. Lemos: None. C. Ventura: None. F. Torres: None. G. Fernandes: None. I. Durães: None. Á. Pereira: None. P. Costa: None. J. Castro: None. C. Brito: None. R. Cerqueira: None.

P01.095.C Reassessing the clinical pathogenicity of genomic variant based on family history in two prenatal cases

Núria Capdevila 1, Neus Baena1, Silvia Pina1, Montserrat Pauta2, Maria Segura3, Antoni Borrell2

1Parc Taulí Sabadell, University Hospital, Sabadell, Spain, 2Clinic Hospital, Barcelona, Spain, 3Quantitative Genomic Medicine Laboratories, qGenomics, Esplugas del Llobregat, Spain.

Introduction: Whole exome sequencing (WES) is a diagnostic tool in postnatal settings for individuals with a suspected genetic condition. Recently, it is increasingly used as a diagnostic tool in prenatal settings with a diagnostic yield ranging from 15% to 35%. The aim is to evaluate the pathogenicity of two genetic variants found by WES and inherited from one parent and determinate the risk of recurrence for the couple.

Methods: WES was performed in fetal samples with ultrasound anomalies, previous prenatal CGH-array with normal result. MedExome analysis using NextSeq (Illumina). Variant and segregation studies were confirmed by Sanger sequencing.

ResultsCase 1: A 34-years-old pregnant woman was referred due to prenatal ultrasound of frontal antlers fusion, suspicion of holoprosencephaly, clubhand and facial proboscis. In the collection of family history, it is noticed that the father presents bilateral culbfeet and hypertelorism. WES detected a paternally inherited heterozygous variant c.3323G>T GLI2 (NM_005270). Case 2: Prenatal ultrasound at 18 weeks detected shortening upper limbs compatible with phocomelia in a 29-years-old woman. Family history shows the father has bilateral thumb hypoplasia and abnormal electrocardiogram. WES detected paternally inherited heterozygous variant c.663+1G>A TBX5 (NM_181486.4). After genetic counselling the pregnancies were terminated in both cases. Initially, the variants detected were not reported as a responsible of the foetal phenotype until the family history was collected.

ConclusionThe use of WES in foetuses with ultrasound defects previous an accurate compilation of family history is required to determine the pathogenicity of the variant and specific risk of recurrence.

N. Capdevila: None. N. Baena: None. S. Pina: None. M. Pauta: None. M. Segura: None. A. Borrell: None.

P01.096.D Prenatal TRIO WES sequencing for fetal structural ultrasound anomalies

Detlef TROST 1, Aïcha Boughalem1, Amandine Diakiese1, Patricia Blanchet2, Viorica Ciorna-Monferrato3, Pascale Kleinfinger1, Laurence Lohmann1, Mylène Valduga1, Armel Luscan1, Jean Marc Costa1

1Laboratoires CERBA, 95310 ST OUEN L’AUMONE, France, 2CHU Hôpital Arnaud de Villeneuve, 34295 Montpellier cedex 5, France, 3CHR Metz-Thionville, 57085 Metz Cedex 03, France.

The usefulness of whole exome sequencing (WES) for genetic testing in cases presenting with fetal structural ultrasound anomalies is currently evaluated. In our cohort WES was performed on 128 fetus -parents TRIOs with fetal structural anomalies in ongoing pregnancies and normal karyotype and CNV analysis. Genomic DNA was extracted from CVS or amniotic fluid samples. WES data were analyzed based on the prenatally observed ultrasound findings. Pathogenic, or likely pathogenic, single nucleotide variants were identified in 28 of 128 (35.8%) cases, all were compatible with respective fetal structural anomalies. In 15 cases the identified pathogenic variant occurred “de novo”, two times an autosomal variant was found to be of maternal origin with probably incomplete penetrance. In 11 of the studied fetuses recessive disorders were detected for couples unaware of their carrier status, these were homozygous in 7 cases (with 4 consanguineous couples) and composite heterozygous in 4 cases. We correlated the diagnostic yield to the major ultrasound findings, which may help to establish high risk ultrasound findings, needing investigation beyond fetal karyotyping or CNV analyses. Prenatal WES analyses enabled us to provide pertinent genetic counseling and to offer targeted prenatal diagnosis in case of a new pregnancy in 35.8 % of our couples.

D. Trost: None. A. Boughalem: None. A. Diakiese: None. P. Blanchet: None. V. Ciorna-Monferrato: None. P. Kleinfinger: None. L. Lohmann: None. M. Valduga: None. A. Luscan: None. J. Costa: None.

P01.097.A Prenatal diagnosis of Wieckaer-Wolff syndrome, a distinctive phenotype of arthrogryposis multiplex congenita caused by a ‘de novo’ ZC4H2 partial deletion

Armelle Duquenne 1, Charlotte Deneufbourg1, Jean Marc M. Biard2, Yves Sznajer1

1Center for Human Genetics, Cliniques universitaires Saint-Luc, UCLouvain, Brussels, Belgium, 2Obstetrics, Cliniques universitaires Saint-Luc, UCLouvain, Brussels, Belgium.

Introduction: Arthrogryposis multiplex congenita (AMC) defines phenotype when contractures are present in ≥ 2 joints. Monogenic conditions/cytogenetic CNV have been identified in a wide range of diagnosis with AMC (Hall 2017). A distinctive syndromic form was originally described postnatally in males with ‘club feet’, intellectual disability, ophthalmic dyspraxia and muscle atrophy (Wieckaer 1985). Intragenic deletion/point mutations in ZC4H2 gene have been identified responsible for this X-linked condition (OMIM 314580/ZARD: ‘ZC4H2 associated rare diseases’). Over time, case reports and recent largest review showed that females may be affected; haploinsufficiency was noted in 3; prenatal presentation in 6 unrelated fetuses (Frints 2019). The Belgian consortium in prenatal diagnosis (BEMAPRE) defined SNP-array as the first-tier diagnostic approach for congenital malformation (Vanakker 2013).

Method-Case Report: A pregnant woman was referred at 22 weeks to our tertiary care center for sporadic arthrogryposis, ‘unusual thin left leg’, ‘clinodactyly’, normal growth parameters in a female fetus. Ultrasound study was otherly normal. Invasive procedure (amniotic fluid puncture) was performed and SNP-array (Affymetrix CytoScan 750K) identified a heterozygous 262kbs deletion in Xq11.2. Breakpoint encompasses exon 1 of ZC4H2; no contiguous gene deletion was noted. SNP-array to parents confirmed a ‘de novo’ occurrence. Parents asked for termination of pregnancy. A short neck, anomaly on 4 limbs (distal fingers camptodactyly, unusual thin and amyotrophic appearance of left leg with homolateral club foot) were noted.

Conclusion: We report on the seventh fetus with Wieacker-Wolff syndrome. Cytogenetic work-up allows for precise genetic counselling. The recurrence risk is related to X-linked gonadal mosaïcism (±10%).

A. Duquenne: None. C. Deneufbourg: None. J.M. Biard: None. Y. Sznajer: None.

P01.098.B Impaired WNT/beta-catenin signaling pathway in the etiology of azoospermia

Dunya Aydos 1, Sena Aydos2, Yunus Yukselten3, Asuman Sunguroglu2, Kaan Aydos4

1Ankara University Stem Cell Institute, Ankara, Turkey, 2Department of Medical Biology, Ankara University Faculty of Medicine, Ankara, Turkey, 3Research Laboratories for Health Science, Y Gen Biotechnology Company Ltd., Ankara, Turkey, 4Department of Urology, Ankara University Faculty of Medicine, Ankara, Turkey.

Introduction: Dysregulated WNT signaling in Sertoli cells or in germ cells is associated with male infertility, in particular the Sertoli cell-only (SCO) pattern in testis. Secreted WNT ligands are important in development and maintenance of adult tissue homeostasis and are antagonized by multiple secreted inhibitors that prevent ligand-receptor interactions, such as Wnt-inhibitory factor 1 (WIF1). Understanding the association of WNT signaling components with spermatogonia proliferation/differentiation; induction of apoptosis in postmitotic germ cells could help elucidating the etiology of azoospermia. To interrelate perturbed WNT signaling control and germ cell differentiation, we profiled expression levels of canonical WNT ligand, WNT6 and WNT inhibitors, WNT5B and WIF1.

Materials and methods: Fold changes in expression levels of WNT5B, WNT6 and WIF1 were determined by quantitative PCR in testicular samples taken with microTESE intervention in NOA cases with SCO syndrome (n = 10) and control cases with OA (n = 2).

Results: WNT5B, WNT6 transcripts were found significantly upregulated (3.51 ± 1.11 and 30.23 ± 14.04 fold-change, respectively) as compared with control cases with OA (P < 0.05). WIF1 gene expression levels showed a significant (P < 0.001) decrease (0.41 ± 0.11 fold-change) compared with OA group.

Conclusions: In NOA cases increased WNT5B mRNA levels suggests an association with increased nuclear β-catenin in postmitotic germ cells and defective spermatogenesis. WNT6 secreted by Sertoli cells activates WNT/β-catenin signaling in spermatogonia. Also, a decrease in WIF1 levels causes β-catenin activation. In Sertoli cells, aberrant activation of canonical WNT signaling keeps them immature, which may interrupt male fertility via progressive degeneration of seminiferous tubules.

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

P01.099.C Hemizygous loss of Xq26.2-q26.3 including GPC3, GPC4 and PHF6 in a fetus with lethal complex malformations

Christa Überbacher, Irene Mutz-Dehbalaie, Renate Lunzer, Ingrid Weber, Johannes Zschocke, Christine Fauth

Medical University Innsbruck, Innsbruck, Austria.

Introduction: Hemizygous deletions of parts of the X-chromosome are rare and due to the nullisomy for essential genes often incompatible with life. Typically, affected males have a contiguous gene syndrome, which includes phenotypic features of different disorders. Here, we describe the prenatal and autopsy findings of a male fetus with lethal complex malformations due to a hemizygous deletion Xq26.2-q26.3.

Methods and Results: Routine first trimester screening detected several abnormalities (nuchal edema, ascites, dextroversio cordis, exomphalos, growth retardation) which prompted molecular karyotyping. A male karyotype with a 2 Mb hemizygous interstitial deletion Xq26.2-q26.3 harbouring several disease genes was found (GPC3, Simpson-Golabi-Behmel syndrome 1, SGBS1, MIM #312870; GPC4, Keipert syndrome, KPTS, MIM #301026; PHF6, Borjeson-Forssman-Lehmann syndrome BFLS, MIM #301900; HPRT1, Lesch-Nyhan syndrome, LNS #300322). The parents decided for termination of the pregnancy. Autopsy confirmed ultrasound findings and revealed additional features/malformations like hydrocephalus, hypertelorism, cleft lip and palate, bilobar lung, agenesis of the diaphragm, polysplenia, anal atresia, syndactyly of fingers and toes, and broad distal phalanges. The majority of these findings belong to the phenotypic spectrum of SGBS1 with the exception of intrauterine growth retardation. Another notable finding is the severity of the diaphragm defect: diaphragmatic hernia is frequent in SGBS1 but agenesis of the diaphragm has not been previously reported.

Conclusion: The hemizygous interstitial deletion Xq26.2-q26.3 leads to a contiguous gene syndrome with congenital malformations mostly typical of SGBS1. Nullisomy of neighbouring genes like GPC4 and PHF6 may modulate/aggravate the severity of the phenotype.

C. Überbacher: None. I. Mutz-Dehbalaie: None. R. Lunzer: None. I. Weber: None. J. Zschocke: None. C. Fauth: None.

P01.100.D Analysis of preimplantation human and bovine embryos with regard to XIST repression on the future active X

Melis Atalar Aksit 1, Bo Yu2, Bernard A. J. Roelen3, Barbara Migeon1

1McKusick Nathans Department of Genetic Medicine, Johns Hopkins University, Baltimore, MD, USA, 2Farm Animal Health, Department of Population Health Sciences, Utrecht University, Utrecht, Netherlands, 3Embryology, Anatomy and Physiology, Department of Clinical Sciences, Faculty of Veterinary Medicine, Utrecht University, Utrecht, Netherlands.

X inactivation is the means of equalizing the dosage of X chromosomal genes in male and female mammals, so that there is only one active X in each cell. The XIST locus (in cis) on each additional X chromosome initiates its silence, making it an inactive X. Yet, how the active X in both males and females is protected from being silenced by its own XIST locus is not well understood in any mammal. Previous studies of autosomal duplications suggest that gene(s) on the short arm of human chromosome 19 genetically interact with the X chromosome to repress XIST function on the future active human X. Here, we examine the time of transcription of the candidate genes from human chromosome 19 and its ortholog, bovine chromosome 7, using single cell RNA sequence data from preimplantation human and qRT-PCR data from bovine embryos. Our results suggest that XIST on the future active X is repressed in both sexes just before, or at the time that, the pluripotent factors are upregulated during the 4-8 cell stage in the human and bovine embryo, well before the late blastocyst when XIST is upregulated on the inactive X in females. They also narrow the list of these putative candidate human and bovine genes.

M. Atalar Aksit: None. B. Yu: None. B.A.J. Roelen: None. B. Migeon: None.

P01.101.A Y-chromosome abnormalities in men with reproductive failure

Mariela Hristova-Savova, Kalina Belemezova, Yuri Batchvarov, Petya Andreeva, Daniela Savova, Maria Yunakova, Tanya Timeva, Atanas Shterev, Ivanka Dimova

SAGBAL"D-r Shterev", Sofia, Bulgaria.

Background: The human Y chromosome harbors genes that are responsible for testis development and also for initiation and maintenance of spermatogenesis in adulthood. Male infertility can be attributed to several factors such as cryptorchidism, varicocele, endocrinological disorders, obstruction/absence of seminal pathways, infections, alcohol consumption or chemotherapy. However, genetic alterations have also emerged as one of the leading cause of male infertility. The objective of our study was to investigate the type and frequency of Y-chromosome abnormalities in male infertility.

Materials and methods: We have analyzed by cytogenetic analysis 1063 men, attending reproductive clinic. Among them, we have applied also PCR analysis for Yq AZF microdeletions in 139 men with azoospermia and 195 men with oligoasthenozoospermia.

Results: Overall, 36 out of 1063 men were detected with some Y-chromosome cytogenetic aberrations - their types and frequency are shown in the Table.

Karyotype Number/%
47,XYY 4/0.4%
46,XY/47,XYY; 46,XY/48,XYYY; 46,XY/48,XXYY 13/1.3%
46,XYqh+ 9/0.8%
46,XYqh- 9/0.8%
46,Xinv(Y) 1/0.1%

Both numerical and structural Y-chromosome aberrations were detected in 1.7% of infertile men. Correlations with clinical and laboratory parameters were performed. PCR analysis revealed AZF a,b or c Y-deletions in 9 out of 139 azoospermic (6.5%) and in 2 out of 195 oligoasthenozoospermic men (1%).

Conclusion: Y-chromosome cytogenetic and molecular-genetic aberrations were found in 4.4% of infertile men. The accurate genetic diagnosis has a great impact on decision making for clinical management of these patients, offering in the affected patients PGT-A or sperm donor.

M. Hristova-Savova: None. K. Belemezova: None. Y. Batchvarov: None. P. Andreeva: None. D. Savova: None. M. Yunakova: None. T. Timeva: None. A. Shterev: None. I. Dimova: None.

P02 Sensory Disorders (Eye, Ear, Pain)

P02.001.B Identification of two deletions of the cis-regulatory region of the POU3F4 gene in patients with nonsyndromic sensorineural deafness from North Ossetia, Russia

Rena A. Zinchenko 1,2, Tatyana A. Vasilyeva1, Natalya V. Balinova1, Vitaly V. Kadyshev1, Elena F. Mikhailidi3, Nika V. Petrova1, Sergey I. Kutsev1

1Research Center for Medical Genetics, Moscow, Russian Federation, 2N.A. Semashko National Research Institute of Public Health, Moscow, Russian Federation, 3Republican Children’s Clinical Hospital, Vladikavkaz, Russian Federation.

Х-linked deafness 2 (DFNX2) represents the most common form of Х-linked deafness (OMIM PS304500). It is characterized by cochlear incomplete partition with fistulous communication with internal auditory canal. Mixed conductive and sensorineural deafness is developing. DFNX2 is associated with small intragenic POU3F4 mutations or chromosome rearrangements involving Xq21 chromosome region. Rearrangements comprise 50% of DFNX2 genetic causes.

Material and methods: 65 North Ossetian patients (Ironian Ossetian ethnic subgroup from the North Caucasus region of Russia) are included into the study. MLPA analysis of the Xq21 is implied.

Results: In two unrelated male patients representing sporadic cases of Х-linked deafness a deletion in Xq21.1 has been identified: hg18:chrX:g.(081676507_081728494)_(081728798_081866106)del. That removes distal cis-regulatory region in ~920 kb upstream from the POU3F4 gene and does not affect its coding sequence. Chromosome break points have not been determined. Size could vary from 300 bp to 200,000 bp. Earlier, two other deletions in the same area (~8 kb and ~200 kb of size) were identified in Europe.

Conclusions: Observations of similar chromosome region deletions in patients from different populations from West Europe and the North Caucasus indicate that Xq21 region with its multiple conserved noncoding sequences is a hot spot for chromosome breaks. That requires to include Xq21 deletions screening, as well as target Sanger sequencing of the POU3F4 gene in the routine analysis of the molecular causes of inherited deafness. The research was partially supported by RSF grant №17-15-01051 and within the state task of the Ministry of education and science of Russia

R.A. Zinchenko: None. T.A. Vasilyeva: None. N.V. Balinova: None. V.V. Kadyshev: None. E.F. Mikhailidi: None. N.V. Petrova: None. S.I. Kutsev: None.

P02.002.C Genetic and environmental factors influencing sensory decays during aging in a large Italian cohort

Giorgia Girotto 1, Massimiliano Cocca2, Anna Morgan2, Eulalia Catamo2, Paola Tesolin1, Agnese Feresin1, Paolo Gasparini1, Maria Pina Concas2

1University of Trieste-IRCCS-Burlo Garofolo, Trieste, Italy, 2IRCCS-Burlo Garofolo, Trieste, Italy.

Sensory perception changes over a lifetime and its impairment play a critical role in health and quality of life. Studies published until now have focused on single sensory impairments in elderly people while data on the significant “multisensory phenotype” (MS) are still lacking. Genetic and phenotypic data (hearing, taste and smell evaluated through sensory functions assessment) of 1152 individuals have been investigated. MS was calculated as the total number of impaired senses. The following steps have been applied: 1) regression models to assess the association between MS and personal/lifestyle characteristics, 2) GWAS meta-analysis and 3) gene-based analysis to find genetic influences on MS. Regarding (1), male gender, ageing, and low educational level were associated with MS higher values (p-value < 0.05) while no role was recognized for smoking habit and high alcohol consumption. For GWAS analysis (2), a total of seven genes resulted in being associated with MS (p-value < 1x10-6). In particular, BCL7C and MACROD2, both expressed in the brain and in the inner ear, have been recently associated with Lewy body dementia and neurological disorders, respectively. Finally, gene-based analysis (3) highlighted several genes implicated in sensory signalling pathways such as LSAMP, PRKG1, GNG7, RYR3, PTPRN2. Present data show that several factors- both environmental and genetic - influence concomitant sensory declines. Further investigation (GWAS replication combined with in vivo studies in animal models) are needed to confirm our results that will ultimately help to understand better the complex biological mechanisms underlying MIS and ageing.

G. Girotto: None. M. Cocca: None. A. Morgan: None. E. Catamo: None. P. Tesolin: None. A. Feresin: None. P. Gasparini: None. M. Concas: None.

P02.003.D Molecular Diagnosis of TYR Negative Albinism Patients by Clinical Exome Sequencing

Sezer Akyoney1, Ilayda Sahin 2,3, Büşra Ünal4, Nihat Buğra Ağaoğlu4, Abdulbaki Mudun5, Zeynep Parlakgüneş6, Engin Yılmaz7, Yasemin Alanay2,8, Uğur Özbek2,8, Özden Hatırnaz Ng1,8

1Acıbadem Mehmet Ali Aydınlar University, School of Medicine, Department of Medical Biology, Istanbul, Turkey, 2Acibadem Mehmet Ali Aydinlar University, School of Medicine, Department of Medical Genetics, Istanbul, Turkey, 3Acıbadem Mehmet Ali Aydinlar University, Department of Medical Biotechnology, Istanbul, Turkey, 4University of Health Sciences, Umraniye Training and Research Hospital (UEA), Department of Medical Genetics, Istanbul, Turkey, 5Acibadem Maslak Hospital, Department of Ophthalmology, Istanbul, Turkey, 6Yeditepe University Eye Center, Department of Ophthalmology, Istanbul, Turkey, 7Hacettepe University, Faculty of Medicine, Department of Medical Biology, Istanbul, Turkey, 8Acibadem University Rare Diseases and Orphan Drugs Application and Research Center (ACURARE), Istanbul, Turkey.

Introduction: Albinism is a group of rare genetic conditions inherited autosomal recessively and associated with reduced or no melanin production. Due to high clinical/genetic heterogeneity, genotype-phenotype correlation can only be carried out by genetic diagnosis that is vital especially for syndromic forms. More than 33 genes are related to albinism and these can explain the genetic background of 70-75% of all albinism cases. Among these TYR is the most detected gene. Here we aimed to determine the genetic background of TYR negative cases by clinical exome analysis.

Materials and Methods: Nine TYR negative cases were studied by clinical exome sequencing. Analyzes of the raw data were carried out both with ACURARE in-house pipeline and SOPHiA GENETICS software. The sequences were mapped and aligned with the GRCh38 reference genome. The detected variants were evaluated according to the ACMG guideline. Candidate variants were validated by Sanger sequencing in patients and segregation analyzes were performed in the family members that can provide samples.

Results: A homozygous variant was identified in all nine cases and eight of them were novel. The gene variants were summarized in Table1. The family members were heterozygous carriers of the same variant detected in the index case.

Conclusion: The results obtained reveal the importance of genetic diagnosis in albinism especially in syndromic forms who might need special follow-up

Table 6

S. Akyoney: None. I. Sahin: None. B. Ünal: None. N.B. Ağaoğlu: None. A. Mudun: None. Z. Parlakgüneş: None. E. Yılmaz: None. Y. Alanay: None. U. Özbek: None. Ö. Hatırnaz Ng: None.

P02.004.A Clinical and genetic analysis of new cases provides further characterisation of ALDH1A3-related anophthalmia/microphthalmia

Yesim Kesim 1, Fabiola Ceroni1,2, Alejandra Damián3,4, Fiona Blanco-Kelly3,4, Carmen Ayuso3,4, Kathy Williamson5, Véronique Paquis6, Dorine Bax1, Claudine Rieubland7, Chamlal Mostafa8, Marta Cortón3,4, Nicolas Chassaing9,10, Patrick Calvas9,10, Nicola Ragge1,11

1Department of Biological and Medical Sciences, Faculty of Health and Life Sciences, Oxford Brookes University, Oxford, United Kingdom, 2Department of Pharmacy and Biotechnology, University of Bologna, Bologna, Italy, 3Department of Genetics & Genomics, Instituto de Investigación Sanitaria-Fundación Jiménez Díaz University Hospital, Universidad Autónoma de Madrid (IIS-FJD, UAM), Madrid, Spain, 4Centre for Biomedical Network Research on Rare Diseases (CIBERER), Madrid, Spain, 5MRC Human Genetics Unit, MRC Institute of Genetics and Molecular Medicine, University of Edinburgh, Western General Hospital, Edinburgh, United Kingdom, 6Department of Medical Genetics, Nice Teaching Hospital, Nice, France, 7Department of Human Genetics, Inselspital, Bern University Hospital, University of Bern, Bern, Switzerland, 8Department of Pediatrics, Tangier Hospital, Tangier, Morocco, 9UDEAR, Université de Toulouse, UMR 1056 Institut National de la Santé et de la Recherche Médicale-Université Paul Sabatier, Toulouse, France, 10Department of Medical Genetics, Purpan University Hospital, Toulouse, France, 11Department of Clinical Genetics, West Midlands Regional Clinical Genetics Service and Birmingham Health Partners, Birmingham Women’s and Children’s Foundation Trust, Birmingham, United Kingdom.

Introduction: Anophthalmia and microphthalmia (AM) are a genetically heterogeneous group of disorders that can be isolated or syndromic. Biallelic ALDH1A3 variants are responsible for 11% of recessive AM cases, mostly described in consanguineous families, and present severe bilateral AM with variable neurodevelopmental anomalies. We present six families with biallelic ALDH1A3 variants, further characterising the associated phenotype.

Material and Methods: AM individuals from UK, France and Spain were analysed by WES, targeted gene screening and arrays.

Results: We identified 6 families: Family 1) with two brothers with bilateral anophthalmia, one present additional developmental delay, absent speech and autism with compound heterozygous ALDH1A3 variants (c.874G>T:p.(D292Y);c.1393A>T:p.(I465F)); 2) a boy with bilateral anophthalmia, developmental and intellectual delay, seizures and autistic features with compound heterozygous variants (c.845G>C:p.(G282A);c.1459A>G:p.(R487G)); 3) a girl with bilateral microphthalmia and coloboma with compound heterozygous variants (c.847_849del;p.(G283del);c.953C>A,:p.(S318Y)); 4) a girl with bilateral anophthalmia with a homozygous missense variant (c.1144G>A:p.(G382R)); 5) a boy with bilateral microphthalmia, unilateral coloboma and cataract, with a homozygous splice variant (c.1233+2T>C) and 6) a boy with bilateral microphthalmia, iris and chorioretinal coloboma, facial dysmorphism with a homozygous missense variant (c.434C>T:p.A145V).

Conclusions: Three of the six families presented with compound heterozygous variants, highlighting this mode of inheritance in ALDH1A3-related disorders. Five of 6 families had a variant in the catalytic domain, supporting the importance of this domain, which is critical for substrate selectivity. Severe neurodevelopmental phenotypes were present in two individuals, and variably penetrant in family 1, supporting that this can be an important feature of the ALDH1A3 syndrome.

Y. Kesim: None. F. Ceroni: None. A. Damián: None. F. Blanco-Kelly: None. C. Ayuso: None. K. Williamson: None. V. Paquis: None. D. Bax: None. C. Rieubland: None. C. Mostafa: None. M. Cortón: None. N. Chassaing: None. P. Calvas: None. N. Ragge: None.

P02.006.C Further evidence of involvement of SERPINB6 in autosomal recessive non-syndromic hearing loss

Barbara Vona 1, Thore Schade-Mann1, Petra Stöbe2, Philipp Gamerdinger1, Aboulfazl Rad1, Marc Sturm2, Hubert Löwenheim1, Tobias B. Haack2, Anke Tropitzsch1

1Dept of Otolaryngology—Head & Neck Surgery, Tübingen Hearing Research Centre, University of Tübingen, Tuebingen, Germany, 2Institute of Medical Genetics and Applied Genomics, University of Tübingen, Tübingen, Germany, Tuebingen, Germany.

Introduction: Non-syndromic hearing loss is a genetically heterogeneous sensory disorder. Autosomal recessive hearing loss is the most prevalent form of non-syndromic hearing loss with approximately 80 associated genes to date. SERPINB6 (serpin family B member 6, also called protease inhibitor 6) was mapped to the DFNB91 locus in 2010 and causally associated with moderate-to-severe high-frequency hearing loss.

Materials and Methods: As part of our hereditary deafness study, we ascertained a child with sensorineural hearing loss and a history of parental consanguinity. DNA from the proband was subjected to genome sequencing and bioinformatics analysis. Hearing evaluation and family history were recorded.

Results: A novel homozygous nonsense SERPINB6 variant (ENST00000335686:c.217C>T, p.(Gln76Ter)) was identified in a 24 Mb run of homozygosity. Moderate-to-severe high-frequency sensorineural hearing loss was diagnosed at approximately six years of age.

Conclusions: The limited literature about SERPINB6 in patients with biallelic nonsense or splicing variants hints to a postlingual onset and rapidly progressive hearing loss. Progression is consistent with loss of a functional intracellular protease inhibitor. We add to the limited clinical and molecular genetics knowledge, better characterizing SERPINB6-associated non-syndromic hearing loss.

B. Vona: None. T. Schade-Mann: None. P. Stöbe: None. P. Gamerdinger: None. A. Rad: None. M. Sturm: None. H. Löwenheim: None. T.B. Haack: None. A. Tropitzsch: None.

P02.007.D A new cases of Axenfelt-Rieger syndrome, caused by a novel FOXC1 mutation and 6p25 deletion

Esra Kilic

Department of Pediatric Genetics, University of Health Sciences, Ankara City Hospital, Ankara, Turkey.

Introduction: Axenfelt-Rieger syndrome (ARS) is an autosomal dominant genetic disorder characterised by ocular anterior segment disorders with systemic involvement. Neural crest origin dysgenesis of cornea, iris, anterior angle, glaucoma, oligodontia, conical incisor teeth, hypoplastic enamel, midface hypoplasia, hearing deficit, growth and development disorder, cardiac defects and intestinal malformations are clinical findings of this disorder. Mutations or deletions in forkhead box C1 (FOXC1, chromosome 6p25) are responsible 25% of ARS cases, pituitary homeobox 2 (PITX2, chromosome 4q25) 55% of ARS cases.

Patients, and Results: Case 1, 3-years-old boy with developmental delay, hypothroidism, iridocorneal dysgenesis, glaucoma, midface hypoplasia, thin upper lip and enamel hypoplasia on incisor teeth. Metabolic workup, hearing, abdominal ultrasonography, echocardiography and chromosomal analysis were normal. His microarray analysis revealed that 3,3Mb sized heterozygous deletion on 6p25 including FOXC1 gene. Case 2, 6-years-old boy with short stature, bilateral megalocornea, iris coloboma, crowded teeth with hypoplastic enamel, flat nasal bridge and everted lower lip. His developmental milestones and intellectual capacity was normal. Metabolic workup, hearing, abdominal ultrasonography, echocardiography, cranial MRI, chromosomal analysis, microarray analysis and PITX2 sequence analysis were normal. Sanger sequencing of FOXC1 gene revealed, novel heterozygous de novo c.76dupT (p.Y26Lfs *57) mutation.

Conclusion: Here we report two new patients of ARS, one had a novel FOXC1 mutation, and the other had 6p25 chromosomal deletion. We also observed more severe clinical phenotype in the deletion type ARS. Ocular anterior segment disorders are clinically and genetically heterogenous conditions, by demonstrating the underlying genetic cause we gave appropriate genetic counseling and follow-up.

E. Kilic: None.

P02.008.A Brittle cornea syndrome with a novel pathogenic variant of PRDM5 gene

Ezgi Susam 1, Nilgün Yıldırım2, Sinem Kocagil1, Oguz Çilingir1

1Eskisehir Osmangazi University Medical Genetics Department, Eskisehir, Turkey, 2Eskisehir Osmangazi University Ophthalmology Department, Eskisehir, Turkey.

Brittle cornea syndrome is a rare syndrome, characterized by extreme thinning of cornea with estimated prevalence less than 1 in 1,000,000. Biallelic pathogenic variants of PRDM5 and ZNF469 genes have been identified for etiology of the syndrome.Blue sclerae, corneal thinning with or without corneal rupture, myopia, early-onset keratoconus and keratoglobus are the main ophthalmological features of the disease. Although eyes are the most severely affected organs, it is a systemic disorder including hearing loss and some connective tissue manifestations like scoliosis, hyperelasticity, developmental dysplasia of the hip and rarely increased bone fractures. Here we report a 1-year-old male patient, referred to our clinic from ophthalmology department due to keratoglobus. He was born as third child of healthy first-degree cousin parents at term without any complication. At physical examination, he presented with corneal opacity on left eye, bilateral blue sclera, micrognathia and hyperelasticity. Echocardiography and hip ultrasonography were normal. Despite he had passed neonatal auditory screening, patient was consulted to ENT department again for hearing evaluation and diagnosed with moderate sensorineural type hearing loss. With these findings, brittle cornea syndrome was thought as the most accurate diagnosis and PRDM5 gene sequencing revealed a novel c.177+1G>A variant in homozygous state. This variant was classified as pathogenic by ACMG guidelines and segregation analysis was compatible with parents of the proband as carrier. According to his physical examination and the results of the molecular analyses we have concluded that this novel variant causes Brittle cornea syndrome and genetic counseling was given to family.

E. Susam: None. N. Yıldırım: None. S. Kocagil: None. O. Çilingir: None.

P02.009.B Two new BRPF1 variants associated with IDDDFP and previously unreported ocular findings

Erika Fiorentini 1, Samuela Landini2, Lucia Tiberi2, Angelica Pagliazzi2, Giulia Gori2, Elia Dirupo2, Elisa Marziali3, Pinuccia Fortunato3, Giacomo Bacci3, Roberto Caputo3, Sara Bargiacchi2, Viviana Palazzo2, Rosangela Artuso2

1Medical Genetics Unit, Department of Clinical and Experimental Biomedical Sciences ‘Mario Serio’, University of Florence, Firenze, Italy, 2Medical Genetics Unit, Meyer Children’s University Hospital, Firenze, Italy, 3Pediatric Ophthalmology Unit, A. Meyer Children’s Hospital, Firenze, Italy.

BRPF1 gene on 3p26-p25 encodes a protein involved in epigenetic regulation, through interaction with histone H3 lysine acetyltransferase KAT6A and KAT6B of the MYST family. Recently heterozygous variants in BRPF1 have been identified in subjects with IDDDFP (OMIM 617333), a disorder characterized by global developmental delay, intellectual disability, language delay and peculiar facial features (round face, flat profile, broad nasal root, hypertelorism, blepharophimosis, ptosis, abnormally shaped ears). Joint hypermobility, cervical spinal fusion, EEG abnormalities and epilepsy also occur. Reported ocular problems are strabismus, amblyopia and refraction errors. We found the de novo heterozygous variant c.330delC (p.Ile110fs) in BRPF1 by whole exome sequencing (WES), conducted in a patient (P1) with mild intellectual disability, ptosis and typical facies. Interestingly, the patient also had a Chiari Malformation type I and a subclinical optic neuropathy, which could not be explained by variations in other genes. WES performed in a second patient (P2) showing, as well as P1, intellectual disability, round face, ptosis and strabismus revealed the heterozygous variant c.1447_1450delGTCA (p.Val4834argfsTer11) in BRPF1; cerebral MRI at 1 and 3 years were normal. Having detected a peculiar ocular phenotype in P1, we suggested optical coherence tomography (OCT) for P2; such exam detected bilateral subclinical optic neuropathy also in this case. To date, only a few patients with BRPF1 mutations have been described and none was reported to have an optic neuropathy. Since subclinical optic nerve alterations can go easily undetected, our experience highlights the importance of a more detailed ophthalmologic evaluation in patients with BRPF1 variant.

E. Fiorentini: None. S. Landini: None. L. Tiberi: None. A. Pagliazzi: None. G. Gori: None. E. Dirupo: None. E. Marziali: None. P. Fortunato: None. G. Bacci: None. R. Caputo: None. S. Bargiacchi: None. V. Palazzo: None. R. Artuso: None.

P02.010.C Maternally inherited hemizygous 8.5 Mb Xq21.1 deletion in a male patient with choroideremia, deafness and intellectual disability found using NGS based CNV-calling approach

Ewelina Bukowska-Olech1, Alexander Pepler2, Aleksander Jamsheer 1,3

1Department of Medical Genetics, Poznan University of Medical Sciences, Poznan, Poland, 2CeGaT GmbH, Tubingen, Germany, 3Centers for Medical Genetics GENESIS, Poznan, Poland.

Background: Xq21 deletions associate with choroideremia-deafness-obesity syndrome (MIM: 303110), which main clinical features comprise choroideremia, obesity, moderate intellectual disability and hearing impairment. This contiguous gene deletion involves at least CHM and POU3F4 genes known to cause choroideremia and deafness, respectively.

Materials and methods: The male patient with choroideremia, deafness and intellectual disability underwent the whole-exome sequencing (WES). Next, he was subjected to confirmation studies using quantitative PCR (qPCR) and whole genome array comparative genomic hybridisation (array CGH) (SurePrint G3 Human CGH Microarray 1 × 1M; Agilent Technologies). Besides, we performed segregation analysis applying qPCR.

Results: We identified the maternally inherited hemizygous Xq21.1-q21.32 deletion (hg38 chrX:85003913-92618940) using WES based CNV-calling approach. The variant was calculated based upon observed versus expected coverage using exome sequencing data. Next, we confirmed the presence of aberration applying qPCR and narrowed down its size through array CGH method (hg38 chrX:84662472-93174172).

Conclusion: Our patient harbours a hemizygous Xq21 deletion of a smaller size than similar CNVs reported in the medical literature thus far. Our finding supports the contribution of CNVs to choroideremia, which is an orphan disease being tested for ocular gene therapy. Besides, we have shown the clinical utility of NGS based CNV-calling approach, which effectively allowed to detect of the CNV in the exome sequencing data.

This work was supported by the grant from the Polish National Science Centre, Poland UMO-2016/22/E/NZ5/00270 to Aleksander Jamsheer.

E. Bukowska-Olech: None. A. Pepler: A. Employment (full or part-time); Modest; CeGaT. A. Jamsheer: None.

P02.011.D Genome-wide association study identifies RNF123 locus as associated with chronic widespread musculoskeletal pain

Md Shafiqur Rahman 1, Bendik S Winsvold2, S.O. Chavez Chavez3, Sigrid Børte2, Yakov A. Tsepilov4, Sodbo Zh. Shapov4, HUNT All-In Pain, Yurii Aulchenko5, Knut Hagen6, Egil A. Fors6, Kristian Hveem6, John-Anker Zwart6, J.B.J. van Meurs3, Maxim B. Freidin1, Frances M.K. Williams1

1King’s College London, London, United Kingdom, 2Oslo University Hospital, Oslo, Norway, 3Erasmus Medical Center, Rotterdam, Netherlands, 4Novosibirsk State University, Novosibirsk, Russian Federation, 5PolyOmica, Maastricht, Netherlands, 6Norwegian University of Science and Technology, Trondheim, Norway.

Background and Objectives: Chronic widespread musculoskeletal pain (CWP) is a symptom of fibromyalgia and a complex trait with poorly understood pathogenesis. CWP is heritable (48-54%), but its genetic architecture is unknown and candidate gene studies have produced inconsistent results. We conducted a genome-wide association study to get insight into the genetic background of CWP.

Methods: Northern Europeans from UK Biobank comprising 6,914 cases reporting pain all over the body lasting more than 3 months and 242,929 controls were studied. Replication of three lead genome-wide significant single nucleotide polymorphisms (SNPs) was attempted in 6 independent European cohorts (N = 43,080; cases = 14,177). Genetic correlations with risk factors, tissue specificity, and colocalization were examined.

Results: Three genome-wide significant loci were identified (rs1491985, rs10490825, rs165599) residing within the genes RNF123, ATP2C1, and COMT. The RNF123 locus was replicated (meta-analysis p = 0.0002), the ATP2C1 locus showed suggestive association (p = 0.0227), and the COMT locus was not replicated. Partial genetic correlation between CWP and depressive symptoms, body mass index, age of first birth, and years of schooling were identified. Tissue specificity and colocalization analysis highlight the relevance of skeletal muscle in CWP.

Conclusions: We report a novel association of RNF123 locus with CWP and suggest a role of ATP2C1, consistent with a role of calcium regulation in CWP. The association to COMT, one of the most studied genes in chronic pain field, was not confirmed in the replication analysis.

M. Rahman: None. B. S Winsvold: None. S. Chavez Chavez: None. S. Børte: None. Y. A. Tsepilov: A. Employment (full or part-time); Modest; PolyOmica. B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Modest; Russian Foundation for Basic Research. S. Zh. Shapov: None. Y. Aulchenko: A. Employment (full or part-time); Significant; PolyOmica. K. Hagen: None. E. A. Fors: None. K. Hveem: None. J. Zwart: None. J. Meurs: None. M. B. Freidin: None. F. M.K. Williams: None.

P02.012.A Unexpected NGS findings in a case of Coats’ disease, combination of rare variants in the HMCN1 and NPHP4genes associated with other forms of retail dystrophy

Tatiana A. Vasilyeva, Vitaly V. Kadyshev, Nika V. Petrova, Andrew V. Marakhonov, Rena A. Zinchenko

Research Centre for Medical Genetics, Moscow, Russian Federation.

Coats’ disease (OMIM300216) is a form of retinal dystrophy which occurs due to congenital abnormality of retinal vessels. Patients, mainly young men, show unilateral retinal telangiectasia, retinal exudation and detachment. Coats’ disease occurs preferentially as sporadic cases, its genetic cause is still unknown. A 17-year-old Caucasian male patient with sporadic exudative vitreoretinopathy/Coats’ disease underwent complete ophthalmological examination, whole exome sequencing by NGS method was implied for searching causative genetic variants of the phenotype. Two heterozygous variants in different genomic loci associated with other forms of retinal dystrophy have been detected, a rare variant in the HMCN1 gene c.9571C>T p.(Arg3191Cys) and a known pathogenic variant in the NPHP4 gene c.2930C>T p.(Thr977Met). Pathogenic variants in the HMCN1 gene are responsible for dominant age-related macular dystrophy (#603075), variants in the NPHP4 gene cause recessive Senior-Loken syndrome 4 (#266900). Encoding proteins are involved in the regulation of integrity of blood/retina barrier at the levels of vascular endothelium and retinal pigment epithelium. The NPHP4 gene is expressed in connecting cilium, which normally performs important function of trafficking between the external and internal segments of photoreceptors. A mechanism of functional consequences of the detected variants combination is proposed to be accumulation of several defects, violations of blood/retina barrier, as well as diminishing of the ciliary transporting potential. The consequences can aggravate each other and lead to the observed phenotype. Carried out within the state assignment of Ministry of Science and Higher Education of the Russian Federation, supported in part by RFBR grant (No.20-015-00061).

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

P02.013.B Mutation analysis in frequent genes in a cohort of Russian patients with congenital glaucoma

Andrey V. Marakhonov 1, Alexander A. Sorokin2, Tatyana A. Vasilyeva1, Sofya A. Garifullina1, Darya M. Guseva1, Natalya A. Semenova1, Vitaly V. Kadyshev1, Rena A. Zinchenko1

1Research Centre for Medical Genetics, Moscow, Russian Federation, 2Moscow Helmholtz Research Institute of Eye Disease, Moscow, Russian Federation.

Primary juvenile glaucoma develops due to ocular hypertension with the onset either at birth or within the first few years of life. It arises due to abnormalities in the anterior chamber angle development, that obstructs aqueous outflow in the absence of systemic anomalies or other ocular malformations. According to Orphanet data, its birth prevalence in Europe is 2.2 cases per 100,000 newborns. Congenital glaucoma could be inherited in either autosomal recessive or autosomal dominant modes. The rationale of this study was to analyze mutations in frequent genes in a cohort of Russian patients with congenital glaucoma.Twenty-one Russian patients with a primary diagnosis of congenital glaucoma were included in the study. Targeted sequencing and MLPA analysis of FOXC1, PITX2 (with autosomal dominant inheritance), and CYP1B1 (with autosomal recessive inheritance) genes were performed.In eight patients, mutations in analyzed genes were detected. One patient has previously known homozygous variant in the CYP1B1 gene, NM_000104.3:c.685G>A. Four patients have heterozygous variants in the FOXC1 gene: two novel (NM_001453.2:c.246C>A, c.235C>T) and two previously described (c.379C>T, c.379C>T). Three more patients have heterozygous variants in the PITX2 gene: two novel (NM_001204397.1:c.114G>T and NM_153427.2:c.408_410delTCG) and one described earlier (NM_153427.2:c.191C>T). Thirteen patients lack variants in analyzed genes.According to our study, congenital glaucoma could be associated frequently with genes that are usually linked to anterior segment dysgenesis including Axenfeld-Rieger syndrome.Carried out within the state assignment of Ministry of Science and Higher Education of the Russian Federation, supported in part by RFBR grant (No. 19-015-00122).

A.V. Marakhonov: None. A.A. Sorokin: None. T.A. Vasilyeva: None. S.A. Garifullina: None. D.M. Guseva: None. N.A. Semenova: None. V.V. Kadyshev: None. R.A. Zinchenko: None.

P02.014.C Congenital insensitivity to pain: molecular characterization of a novel disrupting mutation in SCN9A

Margherita M. Marchi 1, Ilaria D’Amato1, Mirna Andelic1, Erika Salvi1, Daniele Cartelli1, Raffaella Lombardi1, Gumus Evren2, Giuseppe Lauria1,3

1Fondazione IRCCS Istituto Neurologico Carlo Besta, Milano, Italy, 2Department of Medical Genetics, Faculty of Medicine, University of Harran, Sanliurfa, Turkey, 3Department of Biomedical and Clinical Sciences "Luigi Sacco", University of Milan, Milan, Italy.

Aim of this study is to investigate the splicing consequence of a novel intronic mutation in SCN9A (NM_002977.3) in a case of congenital insensitivity to pain (CIP, #243000). CIP is an extremely rare condition caused by bi-allelic inactivating mutations in SCN9A, encoding the sodium channel Nav1.7, responsible for firing and transmission of noxious stimuli in the peripheral nociceptors. CIP presents clinically as the insensitivity to nociceptive pain and heat, often with anosmia.We report an 8-years-old girl, from an inbred family, diagnosed with CIP, showing absence of pain sensation, diminished temperature sensation, foot burns, normal olfaction, hearing and MRI. Next-generation-sequencing of SCN9A revealed a substitution c.377+7T>G in the donor splice-site of intron 3, homozygous in the girl and heterozygous in her healthy mother. Total RNA from the proband, her mother and one healthy unrelated control was extracted from blood and retrotranscribed. The cDNA region spanning exon 2 to 4 was amplified by PCR, followed by the fragments dimensional analysis on agarose gel and Sanger-sequencing. Sequencing revealed in the affected girl two mis-spliced transcripts: both lacking exon 3 and presenting a non-canonical isoform of exon 4, and one showing a partial retention of intron 2. The mis-spliced transcripts are predicted to induce a reading frame shift, which prematurely stops after two (p.Lys86fs2Stop) or twelve out-of-frame aminoacids (p.Lys86fs12Stop), resulting in the loss of NAv1.7.This study describes a novel mutation in SCN9A causing the Nav1.7 deficiency underlying the nociceptors dysfunction and highlights the importance of investigating intronic mutations outside the splicing consensus.

M.M. Marchi: None. I. D’Amato: None. M. Andelic: None. E. Salvi: None. D. Cartelli: None. R. Lombardi: None. G. Evren: None. G. Lauria: None.

P02.015.D Complex inheritance of retinal degeneration: At least three mutations segregate in a family with autosomal-dominant congenital stationary night blindness

Kalina Mihova 1, Krasimir Koev2, Kunka Kamenarova1, Silvia Cherninkova3, Radka Kaneva1

1Molecular Medicine Center, Department of Medical Chemistry and Biochemistry, Medical University - Sofia, Sofia, Bulgaria, 2University Hospital “Tsaritsa Yoanna", Medical University - Sofia, Sofia, Bulgaria, 3Department of Neurology, University Hospital “Alexandrovska”, Medical University – Sofia, Sofia, Sofia, Bulgaria.

Introduction: Congenital stationary night blindness (CSNB) comprises a group of genetically and clinically heterogeneous non-progressive retinal disorders (RD), mainly due to rod dysfunction. This study was performed to identify the genetic defect in a large family affected with autosomal-dominant (ad) CSNB.

Materials and Methods: Sixteen affected relatives of a large Gypsy pedigree segregating adRD were examined clinically by standard ophthalmological methods. Based on this, 15 patients were diagnosed as having CSNB and one presenting Retinitis pigmentosa (RP). Whole exome sequencing was performed in CSNB patients. A systematic filtering approach coupled with copy number variation (CNV) analysis was used to identify pathogenic variants, subsequently verified by Sanger sequencing and segregation analysis. Additionally, MLPA analysis was used to confirm presence of CNVs.

Results: A novel RHO-mutation (c.803A>G, p.Tyr268Cys) was identified in 5 patients with adCSNB. They had full vision under photopic conditions, showed no fundus abnormalities but presented night blindness with an altered scotopic ERG. A known RHO mutation, c.541G>A (p.Glu181Lys), was found in the patient presenting typical signs of RP. CNV and further MLPA analysis detected third, novel, IMPDH1-exon-17 heterozygous deletion in 11 patients, two of whom also carrying one of the RHO-mutations. No mutation was detected in one CSNB-patient suggesting the presence of another mutation segregating in the pedigree.

Conclusions: Here, we present a large family presenting two distinct RD-phenotypes and at least three mutations segregating across three generations. Although further functional studies are needed, this study adds a fifth rhodopsin mutation associated with CSNB. Grant references: KP-06-N33/12/18.12.2019 and D-131/24.06.2020.

K. Mihova: None. K. Koev: None. K. Kamenarova: None. S. Cherninkova: None. R. Kaneva: None.

P02.016.A There’s more than meets the eye: dual molecular diagnosis in complex hearing loss patients

Beatrice Spedicati 1, Anna Morgan2, Maria Teresa Bonati2, Giulia Severi3, Agnese Feresin1, Giulia Pelliccione2, Paola Tesolin1, Claudio Graziano3, Paolo Gasparini1,2, Flavio Faletra2, Giorgia Girotto1,2

1Department of Medicine, Surgery and Health Sciences, University of Trieste, Trieste, Italy, 2Medical Genetics, Institute for Maternal and Child Health - I.R.C.C.S. "Burlo Garofolo", Trieste, Italy, 3Medical Genetics Unit, S. Orsola-Malpighi University Hospital, Bologna, Italy.

Medical geneticists usually try to identify patients’ clinical conditions by recognizing the specific pattern of a syndrome. Whenever clinical features do not fit into a known model, the presence of two distinct conditions may be hypothesized and high-throughput sequencing technologies can allow a complete molecular characterization even in the most complex cases. Here we describe patients presenting with hearing loss (HL) and other signs that suggested the presence of a dual molecular diagnosis. Patients were divided in two categories: a) those with distinct phenotypes, i.e. syndromic or non-syndromic HL plus other clinical features related to a second condition; b) patients with overlapping phenotypes, with HL due to either of the two conditions. In group a) Patient-1 displayed HL and periventricular nodular heterotopia, due to a homozygous deletion of the STRC gene and a nonsense variant in the FLNA gene, respectively; Patient-2 was affected by Kabuki syndrome (KMT2D gene) and Bosma arhinia microphthalmia syndrome (SMCHD1 gene); Patient-3 was affected by Distal renal tubular acidosis with progressive sensorineural HL (ATP6V1B1 gene) and Marfan syndrome (FBN1 gene). In group b) Patient-4 presented sensorineural HL and retinitis pigmentosa: Whole Exome Sequencing revealed two compound heterozygous variants in the USH2A gene, responsible for Usher syndrome type 2A, and a nonsense variant in the EYA4 gene, associated with autosomal dominant non-syndromic HL. Overall, our findings highlighted that “genetic-first diagnostics” should be the gold standard for patients with syndromic conditions of unclear genetic origin, thus avoiding costly and distressing diagnostic procedures before reaching a final diagnosis.

B. Spedicati: None. A. Morgan: None. M. Bonati: None. G. Severi: None. A. Feresin: None. G. Pelliccione: None. P. Tesolin: None. C. Graziano: None. P. Gasparini: None. F. Faletra: None. G. Girotto: None.

P02.017.B NGS strategy for the analysis of genes and regions responsible for Early Onset High Myopia

Susana Noval1, Maria Nieves-Moreno1, Ana Lopez-Vazquez1, Fernando Santos-Simarro2, Irene Rosa-Perez1, Inmaculada Rueda-Arenas2, Marta Naranjo-Castresana2, Agela del Pozo2, Oriana D’Anna1, Victoria EF Montaño2, Elena Vallespin 2

1Department of Ophthalmology, Hospital Universitario La Paz, IdiPaz, Madrid, Spain, 2Medical and Molecular Genetics Institute (INGEMM), Hospital Universitario La Paz, IdiPaz, Rare Diseases Networking Biomedical Research Centre (CIBERER), ISCIII, Madrid, Spain.

Early Onset High myopia (eoHM) (−6.00 diopters or less) is one of the leading cause of vision loss or even irreversible blindness with pathologic complications such as myopic retinopathy, maculopathy, retinal detachment, cataract or primary open-angle glaucoma that is present before the age of ten. In UMOG (Ophtalmogenetics Multidiciplinary Unit) we have designed and developed a novel and comprehensive screening strategy for all genes and loci responsible for eoHM on next-generation sequencing (NGS) developing a systematic application and automation in the clinical routine. UMOG is a multidisciplinary diagnosis and research team with extensive experience in diagnosis, research and teaching in ophthalmogenetic diseases in Hospital La Paz (Madrid). Samples were screened using a customized NGS gene panel, OFT-v3-1, containing 419 genes associated with eye pathology of suspected genetic origin, including eoHM genes and loci. The patients study was carried out on all the genes in the panel as it has been observed by other researches that a significant proportion of eoHM is caused by mutations in RetNet genes. This panel was validated with at least 25 samples with excellent results. At this point, we already ran 30 eoHM families and the diagnostic yield is around 30%. The development of this project will introduce the use of these new technologies to Health Services for diagnosis and research and thereby will help to improve the diagnosis, treatment and care of patients with this and other genomic disorders. We are grateful to the patients and their families. Grants: PI18-1234-ISCIII and 2020/0197782-ONCE.

S. Noval: None. M. Nieves-Moreno: None. A. Lopez-Vazquez: None. F. Santos-Simarro: None. I. Rosa-Perez: None. I. Rueda-Arenas: None. M. Naranjo-Castresana: None. A. del Pozo: None. O. D’Anna: None. V. Montaño: None. E. Vallespin: E. Ownership Interest (stock, stock options, patent or other intellectual property); Modest; Genycell Biotech. F. Consultant/Advisory Board; Modest; Diaceutics, The Science Advisory Board.

P02.018.C LOXL1 and CACNA1A SNPs associated with exfoliation syndrome susceptibility in a sample of Northern Spanish population

Leire Escudero-Arrarás 1, Araceli Lara-López1, María Rodríguez-Hidalgo1, Txomin Alberdi2, Iñaki Rodríguez-Agirretxe2,3,4, Javier Mendicute2, Javier Ruiz Ederra5,4

1Biodonostia Health Research Institute, San Sebastián, Spain, 2Department of Ophthalmology, Donostia University Hospital, San Sebastián, Spain, 3Instituto Clínico-Quirúrgico de Oftalmología, Bilbao, Spain, 4RETICS OFTARED, National Institute of Health Carlos III, Ministry of Economy and Competitiveness, Madrid, Spain, 5Miramoon Pharma-Biodonostia HRI, San Sebastián, Spain, San Sebastián, Spain.

Introduction: Exfoliation syndrome (XFS) is a systemic disease characterized by whitish fibrillar substance deposition in the anterior segment of the eye. LOXL1 and CACNA1A are the main genes associated with an increased risk.

Materials and Methods: A case-control study with 235 Northern Spanish patients: 55 XFS patients and 180 controls. Genotypes of LOXL1 (rs1048661, rs3825942, rs2165241, rs16958477, rs12914489, rs11638944, rs7173049) and CACNA1A (rs4926244) SNPs were analyzed by direct sequencing.

Results: The G allele and the GG genotype of SNP rs3825942 were detected at a higher frequency in pseudoexfoliation patients (p = 0.00057, OR = 6.46; p = 0.00003, OR = 8.96, respectively). The T allele and the TT genotype of rs2165241 presented at significantly higher frequencies in XFS patients (p = 0.00473, OR = 2.10; p = 0.00001, OR = 4.49, respectively). The G allele and the GG genotype of rs1048661 were detected at a statistically higher frequency in XFS patients (p = 0.04587, OR = 1.78; p = 0.00179, OR = 2.66, respectively). The AA genotype of SNP rs12914489 was detected at a statistically lower frequency in XFS patients (p = 0.01898, OR = 5.503). The C allele and the CC genotype of rs11638944 presented at significantly lower frequencies in pseudoexfoliation patients (p = 0.00754, OR = 0.50; p = 0.00876, OR = 0.35, respectively). No significant association between XFS and LOXL1 rs16958477 and rs7173049 SNPs and CACNA1A rs4926244 was observed.

Conclusion: We found a significant association for several LOXL1 SNPs, whereas no differences were attributable to CACNA1A rs4926244 SNP. LEA is supported by fellowships from Fundación Jesús de Gangoiti Barrera and from the Basque Government (2018111062, MTVD19/BD/006 and MTVD20/BD/002). Supported by grants from the ISCIII and FEDER (PI18/00507) and BEGISARE.

L. Escudero-Arrarás: None. A. Lara-López: None. M. Rodríguez-Hidalgo: None. T. Alberdi: None. I. Rodríguez-Agirretxe: None. J. Mendicute: None. J. Ruiz Ederra: None.

P02.019.D The Fraser-complex pathologic spectrum: Familial cryptophthalmos in two families from GAFSA, TUNISIA

Nouha Bouayed ABDELMOULA, Sonda Kammoun, Fatma Abid Mzid, Saloua Ben Amor, Jamel Feki, Takwa Sammouda, Mouna Rekik

Genomics of signalopathies at the service of medicine UR17ES36, Medical University of Sfax, Sfax, Tunisia.

Recessive mutations in genes encoding members of the Fraser complex (FC) or associated proteins constitute an established genetic cause of Fraser syndrome in its three forms related to mutations in three different genes FRAS1, FREM2, and GRIP1 resulting in failure of the apoptosis program and disruption of the epithelial-mesenchymal interactions during embryonic development. We report in this study two Tunisian pedigrees from the town of Gafsa in which a recessive cyptophthalmos was detected.

Material and Methods: Two male newborns were referred to our genetic counselling for cryptophthalmos. One of them was also suspected to have Crigler-Najjar disease. Genetic exploration of FRAS1 and UGT1A1 genes was conducted.

Results: The two unrelated males were born to consanguineous parents from Gafsa. They had cryptophthalmos which is a condition of eyelid malformation associated to an underlying malformed eye. The first case had unilateral right complete cryptophthalmos associated with maldevelopment of the cornea and the crystalline as well as microphthalmia. At the left, he had posterior embryotoxon. Besides facial dysmorphism, he had bilateral syndactyly. Molecular analysis showed a homozygosity for an intronic sequence change in intron 22 of FRAS1 gene. The second patient as well as his brother had bilateral complete cryptophthalmos associated to anophthalmia, microphthalmia and iris coloboma. The patient had also a neonatal jaundice which was related to the homozygous mutation of UGT1A1 exon 3 (c.1070A>G).

Conclusion: More recently, bilateral anophthalmia and liver malformations with intrahepatic biliary atresia were described. FC screening is thus debated for the second family.

N.B. Abdelmoula: None. S. Kammoun: None. F. Abid Mzid: None. S. Ben Amor: None. J. Feki: None. T. Sammouda: None. M. Rekik: None.

P02.021.B Variants in FZD5 are primarily associated with non-syndromic phenotypes in individuals with ocular coloboma

Richard J. Holt 1, David Goudie2, Ajoy Sarkar3, Alejandra Damián4,5, Alejandra Tamayo4,5, Carmen Ayuso4,5, Marta Cortón4,5, Alice Gardham6, Virginia Clowes6, Julie Plaisancié7,8, Nicolas Chassaing7,8, Patrick Calvas7,8, Nicola K. Ragge1,9

1Oxford Brookes University, Oxford, United Kingdom, 2NHS Tayside, Dundee, United Kingdom, 3Nottingham University Hospitals NHS Trust, Nottingham, United Kingdom, 4Instituto de Investigación Sanitaria-Fundación Jiménez Díaz University Hospital - Universidad Autónoma de Madrid, Madrid, Spain, 5Centre for Biomedical Network Research on Rare Diseases, Madrid, Spain, 6Northwick Park and St Mark’s Hospital, Harrow, United Kingdom, 7Purpan University Hospital, Toulouse, France, 8Centre Hospitalier Universitaire (CHU) de Toulouse, Toulouse, France, 9Birmingham Women’s and Children’s Foundation Trust, Birmingham, United Kingdom.

Introduction: Ocular coloboma results from failure of optic fissure closure during development. It is genetically heterogenous, with variants in 30 genes implicated, many also associated with anophthalmia and microphthalmia. Most recently, heterozygous FZD5 variants have been reported in individuals with coloboma and microphthalmia, but are limited to frameshifts and inframe insertion/deletions. We describe two missense, two nonsense, and two frameshift variants in individuals with coloboma and/or microphthalmia.

Materials and methods: Variants were identified using whole exome sequencing (WES) or customised NGS panels of ocular development genes, in individuals from the UK (including the DDD Study [www.ddduk.org/access.html]), France and Spain.

Results: We identified six individuals with coloboma and heterozygous likely pathogenic variants: 1) male with bilateral microphthalmia, iris and chorioretinal coloboma, anal atresia, atrial and ventricular septal defect, cortical dysplasia, microcephaly, seizures, deafness, and tracheoesophageal fistula (NM_003468.4:c.539_540insG:p.(E181Rfs*88)), also diagnosed with a pathogenic SLC12A2 variant (NM_001046:exon4:c.C980T:p.A327V), 2) female with bilateral iris coloboma, patent ductus arteriosus, atrial septal defect, and volvulus (NM_003468:c.C577T:p.(R193C)), 3) female with bilateral iris and optic nerve colobomas, and downslanted palpebral fissures, (NM_003468:c.G1566A:p.(W522*)), 4) female with unilateral iris and chorioretinal coloboma (NM_003468.4:c.541G>T:p.(E181*)), 5) female with bilateral coloboma (NM_003468.4:c.1150G>C:p.(D384H)), and 6) female with unilateral iris and chorioretinal coloboma, and bilateral optic nerve colobomas (NM_003468.4:c.147delG:p.(H50Tfs*70)).

Conclusions: We present six individuals with FZD5 variants and ocular colobomas, providing the first evidence for FZD5 missense and stopgain variants in these disorders. As iris coloboma is the only consistent phenotype, these data highlight the importance of additional pathogenic variants underlying associated complex non-ocular phenotypes.

R.J. Holt: None. D. Goudie: None. A. Sarkar: None. A. Damián: None. A. Tamayo: None. C. Ayuso: None. M. Cortón: None. A. Gardham: None. V. Clowes: None. J. Plaisancié: None. N. Chassaing: None. P. Calvas: None. N.K. Ragge: None.

P02.022.C Clinical exome sequencing reveals different GUCY2D-related retinopathies in Bulgarian patients

Kunka Kamenarova 1, Nevyana Veleva2, Kalina Mihova1, Neda Sergeeva2, Alexander Oscar2, Radka Kaneva1

1Molecular Medicine Center, Medical University – Sofia, Sofia, Bulgaria, 2Department of Ophthalmology, University Hospital “Alexandrovska”, Medical University – Sofia, Sofia, Bulgaria.

Introduction: GUCY2D gene encodes the photoreceptor guanylate cyclase (GC-E) and different mutations can lead to cone-rod dystrophy (CRD), congenital stationary night blindness (CSNB), and Leber congenital amaurosis. In this study, we describe three unrelated families who carried different GUCY2D-variants and presented two types of retinopathy.

Materials and Methods: Two unrelated patients with autosomal-dominant (ad) and autosomal-recessive (ar) CRD, and one with arCSNB were examined clinically by standard ophthalmological methods. Targeted sequencing of clinical exome on Illumina® platform, followed by Sanger sequencing and segregation analysis, was used to identify pathogenic variants.

Results: All patients manifested decreased vision, photophobia and elevated thresholds of dark adaptation. Genetic analysis revealed three mutations in the GUCY2D gene segregating with the phenotype in the pedigrees. The common c.2512C>T (p.Arg838Cys) mutation presenting a relatively severe clinical phenotype of adCRD was found in one of the analyzed families. Mutations c.2900A>G (p.His967Arg) and c.3224+1G>C (p.?) were identified in two different combinations (c.2900A>G/c.3224+1G>C and c.3224+1G>C/c.3224+1G>C) in two unrelated probands affected by arCSNB and arCRD, respectively. GUCY2D mutations were accompanied by similar pattern of generalized cone (macular and peripheral) dysfunction with a tendency to less involvement of the rod photoreceptors in the two CRD-patients and a less severe phenotype in the proband with CSNB.

Conclusions: GUCY2D is a major gene responsible for progressive CRD which is estimated to affect 1 in 40,000 individuals. Here, we present phenotypes of adCRD, adCRD and arCSNB in three Bulgarian families carrying different pathogenic variants of GUCY2D. Grant references: KP-06-N33/12/18.12.2019 and D-131/24.06.2020.

K. Kamenarova: None. N. Veleva: None. K. Mihova: None. N. Sergeeva: None. A. Oscar: None. R. Kaneva: None.

P02.024.A Benefits of exome sequencing in patients diagnosed with isolated or syndromic hearing loss

Roxane van Heurck, Maria Teresa Carminho-Rodrigues, Emmanuelle Ranza, Caterina Stafuzza, Lina Quteineh, Corinne Gehrig, Eva Hammar, Michel Guipponi, Marc Abramowicz, Pascal Senn, Nils Guinand, Helene Cao-Van, Ariane Paoloni-Giacobino

hug, genève, Switzerland.

Purpose: Hearing loss is characterized by an extensive genetic heterogeneity and is a common disorder in children (1/500). Molecular diagnosis is of particular benefit and allows to identify clinically-unrecognized hearing loss syndromes, as well as appropriate management and follow-up, including genetic counselling.

Methods: We performed clinical whole exome sequencing, with analysis of a 189 gene panel associated with hearing loss, in a prospective cohort of 70 patients including 61 children and 9 adults presenting with hearing loss from 2017 to 2020.

Results: The overal diagnostic rate using exome sequencing reached 47,2 % - 50,8% in children and 22% in adults. In children with confirmed molecular results, seventeen out of 31 (54,8%) patients showed autosomal recessive inheritance patterns, thirteen out of 31 (41,94%) showed autosomal dominant and one case had X-linked hearing loss. While in adults the two patients showed autosomal dominant inheritance pattern. Out of the 31 children, 17 (54,84%) had non-syndromic hearing loss and 14 (45,16%) had syndromic hearing loss. Both adult cases were diagnosed with syndromic hearing loss. The most common causative genes were STRC (5 cases), GJB2 (3 cases), COL11A1 (3 cases), ACTG1 (2 cases), GATA3 (2 cases) and TMPRSS3 (2 cases).

Conclusion: Exome sequencing perfomed in hearing loss situations has a high diagnostic yield in children. This can reveal several hearing loss syndromes before involvement of other organs/systems, thus allowing the surveillance of present and/or future complications associated with these syndromes.

R. van Heurck: None. M. Carminho-Rodrigues: None. E. Ranza: None. C. Stafuzza: None. L. Quteineh: None. C. Gehrig: None. E. Hammar: None. M. Guipponi: None. M. Abramowicz: None. P. Senn: None. N. Guinand: None. H. Cao-Van: None. A. Paoloni-Giacobino: None.

P02.025.B Auditory development of patients with genetically determined hearing loss

Dominika Ozieblo 1,2, Anita Obrycka3, Henryk Skarżyński4, Monika Ołdak1

1Department of Genetics, Institute of Physiology and Pathology of Hearing, Warsaw, Poland, 2Postgraduate School of Molecular Medicine, Medical University of Warsaw, Warsaw, Poland, 3Department of 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: Every year, approximately 1-6/1000 children are born with severe to profound hearing loss (HL) and for this group of patients cochlear implantation (CI) is the treatment of choice. The aim of our study was to analyse the auditory development of DFNB1-negative CI patients.

Materials: The study group (n = 52) was recruited from patients with profound prelingual HL that were negative for DFNB1 locus pathogenic variants and had no environmental HL risk factors.

Methods: In all probands exome sequencing (WES) was performed and followed by bioinformatics analysis. Validation of selected variants and family segregation analysis were performed using standard Sanger sequencing or qPCR. Evaluation of patients auditory development was performed with the LittlEARS questionnaire (LEAQ) in three subsequent intervals - at the time of cochlear implant activation as well as in 5th and 9th month after CI.

Results: Causative variants were identified in 69% of patients (36/52). The majority of them are localized in the MYO15A (n = 6) and PAX3 (n = 5) genes. All patients presented a significant improvement of their auditory skills in subsequent intervals at 5th (p < 0.001) and 9th (p < 0.05) months after CI. There were no statistically significant differences between auditory development of patients with identified and unidentified genetic cause of HL.

Conclusions: Obtained results show a high heterogeneity of genetic HL causes in the population of Polish DFNB1-negative cochlea-implanted patients. All tested children were good candidates for CI as their HL causative genetic variants are localized in genes preferentially expressed in the cochlea. Supported by NCN grant: 2017/27/N/NZ5/02369

D. Ozieblo: None. A. Obrycka: None. H. Skarżyński: None. M. Ołdak: None.

P02.026.C Genetic Variant Curation in GJB2 and GJB6 genes from an Argentinean cohort of hearing loss patients

Paula I. Buonfiglio 1, Carlos D. Bruque2, Sebastián Menazzi3, Liliana Francipane3, Vanesa Lotersztein4, Ana B. Elgoyhen1, Viviana K. Dalamón1

1INGEBI, Ciudad Autónoma de Buenos Aires, Argentina, 2Hospital de Alta Complejidad SAMIC - El Calafate, El Calafate, Argentina, 3Servicio de Genética del Hospital de Clínicas “José de San Martín”., Ciudad Autónoma de Buenos Aires, Argentina, 4Servicio de Genética del Hospital Militar Central Cirujano Mayor “Dr. Cosme Argerich”, Ciudad Autónoma de Buenos Aires, Argentina.

Hereditary hearing impairment affects 1-500 newborn children. It is characterized by the large amount of genes involved (more than 100) and its phenotype heterogeneity. Despite the wide genetic variety of hearing impairment, the most commonly mutated genes in severe to profound autosomal recessive non-syndromic hearing loss are GJB2 and GJB6, accounting for nearly 50% of the cases in most populations around the Mediterranean Sea. Molecular diagnosis enables proper genetic counseling and medical prognosis to patients. Therefore, correct interpretation of the phenotypic consequences of genetic variants is crucial in genetic diagnosis, since discrepancies in sequence variant interpretation and classification has been reported to lead to serious impact in patient health maintenance.In this study we aimed to identify the genetic causes of hearing loss and performed a manual genetic variant curation following the American College of Medical Genetics and Genomics/Association for Molecular Pathology ACMG/AMP standards and hearing-loss-gene-specific criteria of the ClinGen Hearing Loss Expert Panel.A total of 600 patients were studied for genetic variants in GJB2 and GJB6 genes by Sanger Sequencing technique and Multiplex Gap-PCR, respectively.Overall, 48 different sequence variants were detected in our cohort of patients, being the c.35delG the most common causative variant identified. Besides, more than 50% of sequence variants were reclassified from their previous categorization in ClinVar after careful manual analysis. These results provide an accurately analysed and interpreted set of variants to be taken into account by clinicians and the scientific community, and hence, aid the precise genetic counseling to patients.

P.I. Buonfiglio: None. C.D. Bruque: None. S. Menazzi: None. L. Francipane: None. V. Lotersztein: None. A.B. Elgoyhen: None. V.K. Dalamón: None.

P02.027.D MYO6 intragenic deletion in a family with autosomal dominant deafness

Chiara Pescucci 1, Francesca Gerundino1, Costanza Giuliani1, Lucia Galli2, Giuseppina Marseglia1, Barbara Minuti1, Francesca Marin1, Francesca Buchi1, Monica Trafeli1, Paola Pelacani1, Chiara Castagnoli1, Elisabetta Pelo1, Alfredo Orrico2,3

1SOD Diagnostica genetica, AOU Careggi, Firenze, Italy, 2Molecular diagnosis and characterization of pathogenic mechanisms of rare genetic diseases, AOU Senese, Siena, Italy, 3Clinical Genetics ASL Toscana SudEst Ospedale della Misericordia, Grosseto, Italy.

Introduction: MYO6 loss-of-function variants can cause autosomal dominant progressive hearing loss with variable age of onset. Here we present a family with hereditary hearing loss transmitted in an autosomal dominant pattern. The proband, a 40-year-old female, began to show a progressive deafness since she was 17 years old. She was diagnosed with sensorineural hearing loss more pronounced in medium and high frequencies (500-8000Hz). Her mother and maternal grandmother also appeared with the same condition with an onset during the second decade.

Materials and Methods: libraries preparation by Illumina TSOne sequencing kit and massive parallel sequencing, followed by bioinformatic analysis of an in-silico panel of hearing loss genes were performed. Reads were aligned to the GRCh37/hg19 using BWA. Single-nucleotide variants and indels were called by SAMtools and GATK. Copy number variants analysis was performed by CONTRA. Variants were functionally annotated by ANNOVAR and interpreted by InterVar. ArrayCGH was perfomed using a custom array specifically designed to investigate intragenic CNVs in hearing loss related genes.

Results: a novel MYO6 intragenic deletion was identified in the proband. Segregation analysis showed that the deletion co-segregates with deafness within the family. ArrayCGH analysis allowed to confirm the presence of a 5,3 kb deletion encompassing exons 2 and 3 of the gene: arr[GRCh37]6q14.1(76515168x2,76527247_76532572x1,76537323x2).

Discussion: the identification of a novel deletion supports with additional evidence the known matter that a number of pathogenic variants in hereditary deafness are private. As a consequence, molecular diagnosis takes advantage from combined approaches for SNVs/CNVs analyses from sequencing data.

C. Pescucci: None. F. Gerundino: None. C. Giuliani: None. L. Galli: None. G. Marseglia: None. B. Minuti: None. F. Marin: None. F. Buchi: None. M. Trafeli: None. P. Pelacani: None. C. Castagnoli: None. E. Pelo: None. A. Orrico: None.

P02.028.A GJB2 sequencing, Multiplex Ligation Probe Amplification (MLPA) and Whole Exome Sequencing (WES) for the molecular diagnosis of Non-Syndromic Hearing Loss (NSHL): the experience of a cohort of 277 Italian families

Anna Morgan 1, Flavio Faletra1, Stefania Lenarduzzi1, Martina La Bianca1, Giulia Pelliccione1, Beatrice Spedicati2, Agnese Feresin2, Daniela Mazzà1, Alberto Sensi3, Claudio Graziano4, Marco Seri4, Umberto Ambrosetti5, Paolo Gasparini1,2, Giorgia Girotto1,2

1Institute for Maternal and Child Health – IRCCS “Burlo Garofolo", Trieste, Italy, 2University of Trieste, Department of Medicine, Surgery and Health Sciences, Trieste, Italy, 3Medical Genetics Unit, Department of Clinical Pathology, Pievestina, Cesena, Italy, 4Unit of Medical Genetics, S. Orsola-Malpighi Hospital, Bologna, Italy, 5University of Milano U.O.C. Audiologia/Fondazione IRCCS Cà Granda Ospedale Maggiore Policlinico, Milano, Italy.

Introduction: NSHL is the most common sensory disorder, with ~80% of congenital cases due to genetic causes. In addition to screening the most frequently mutated genes (GJB2/GJB6/MT-RNR1), the use of WES together with techniques able to detect copy number variants (CNVs) has proved to be efficient in the molecular diagnosis of NSHL.

Materials and Methods: We applied a multi-step approach for testing 277 NSHL families, which included: 1) an accurate clinical evaluation, 2) the analysis of GJB2, GJB6, and MT-RNR1, 3) the evaluation of STRC-CATSPER2 and OTOA CNVs via MLPA, 4) WES in patients negative to steps 2-3.

Results: About 20% of patients carried mutations in the GJB2 gene. MLPA and WES led to the characterization of an additional ~37% of cases. In particular, data analysis allowed to 1) confirm the relevant role of CNVs in NSHL, with ~8% of the positive cases carrying a pathogenic CNV, 2) unveil a series of unexpected scenarios, e.g. the detection of syndromes in patients displaying subtle phenotypic features, early diagnosis of late-onset diseases, identify mutations in different genes involved in the same phenotype, detect multiple genetic conditions in the same patient, 3) discover new disease genes (e.g. PSIP1, TBL1Y, SPATC1L, PLS1, SLC12A2), further exploring the complexity of NSHL.

Conclusions: Our approach proved to be efficient in identifying the molecular causes of NSHL, leading to an overall detection rate of ~50% in the Italian population. Furthermore, WES demonstrated its utility in identifying new disease-genes, deepening the knowledge of the biological mechanisms of NSHL.

A. Morgan: None. F. Faletra: None. S. Lenarduzzi: None. M. La Bianca: None. G. Pelliccione: None. B. Spedicati: None. A. Feresin: None. D. Mazzà: None. A. Sensi: None. C. Graziano: None. M. Seri: None. U. Ambrosetti: None. P. Gasparini: None. G. Girotto: None.

P02.029.B Use of OTO-NGS-v2 panel for the genetic diagnosis of hereditary hearing loss

María Lachgar 1,2, Matías Morín1, Manuela Villamar1, Miguel Ángel Moreno-Pelayo1

1Hospital Universitario Ramón y Cajal and IRYCIS and CIBERER, Madrid, Spain, 2Wolfson Centre for Age-Related Diseases, King’s College London, London, United Kingdom.

Hereditary hearing loss is the most common sensory deficit in humans. It has a highly heterogeneous genetic aetiology and, therefore, the use of Next Generation Sequencing (NGS) approaches is essential to undertake a genetic diagnosis.In this work, we use OTO-NGS-v2, a custom-designed NGS targeted panel containing 117 genes associated with hearing loss. Library preparation uses IDT probes to capture the regions of interest, followed by sequencing in the Illumina MiSeq and data analysis and variant interpretation in SOPHiA Genetics DDM, with all mutations confirmed by Sanger sequencing.OTO-NGS-v2 was validated using 16 previously genetically characterized samples for the identification of single-nucleotide variants (SNVs), small insertions and deletions (indels), and copy number variations (CNVs). We have studied 108 Spanish families with autosomal dominant sensorineural hearing loss (ADSNHL), 45 of which have been genetically diagnosed, thus constituting a diagnostic rate of 41.67%. The 58.33% of the cases remain undiagnosed and further studies are needed to identify the genetic cause of the hearing impairment in these patients. Our results indicate that WFS1 and MYO7A have the highest prevalence in the Spanish population (6.48%) followed by MYO6A (5.56%).We conclude that OTO-NGS-v2 is a robust diagnostic tool for the genetic diagnosis of hereditary hearing loss. In this study, we have laid the foundations for its implementation in clinical practice and contributed to the understanding of the genetic landscape of hearing impairment in the Spanish population.This research was funded by ISCIII (PI17/01659, PI20/0429, CIBERER, 06/07/0036) and by the Regional Government of Madrid (CAM,B2017/BMD3721).

M. Lachgar: None. M. Morín: None. M. Villamar: None. M. Moreno-Pelayo: None.

P02.030.C A RIPOR2 in frame deletion is a frequent and highly penetrant cause of adult onset hearing loss

Jeroen J. Smits 1, Suzanne E. de Bruijn1, Chang Liu2, Cornelis P. Lanting1, Andy J. Beynon1, Joëlle Blankevoort1, Jaap Oostrik1, Wouter Koole1, Erik de Vrieze1, DOOFNL Consortium, Cor W. R. J. Cremers1, Frans P. M. Cremers1, Susanne Roosing1, Helger G. Yntema1, Henricus P. M. Kunst1, Bo Zhao2, Ronald J. E. Pennings1, Hannie Kremer1

1Radboudumc, Nijmegen, Netherlands, 2Indiana University School of Medicine, Indianapolis, IN, USA.

Introduction: Hearing loss is one of the most prevalent disabilities worldwide. The adult-onset types of the condition is highly heritable, but the genetic causes are often unknown.

Methods: Family and cohort studies were performed and included exome sequencing and characterization of hearing phenotype. Ex vivo protein expression addressed the functional effect of a DNA-variant.

Results: We identified an in-frame deletion in RIPOR2, that co-segregated with hearing loss in twelve families of Dutch origin in an autosomal dominant pattern. Haplotype analysis indicated the in-frame deletion to be a founder variant, present in 18 of 22,952 individuals of an unselected cohort. This suggests that the deletion is a frequent cause of monogenic hearing impairment in the Netherlands, with potentially 8,000 affected individuals, and a significant cause of hearing impairment in neighboring countries. Hearing loss associated with the deletion in 63 subjects displayed an average age of onset of 30.6 years (SD 14.9 years) and variable audiometric characteristics. A functional effect of the variant was demonstrated by aberrant localization of the mutant RIPOR2 in the stereocilia of cochlear hair cells. Moreover, mutant RIPOR2 failed to rescue the morphological defects observed in RIPOR2-deficient hair cells, in contrast to the wildtype protein.

Conclusion: we identified a relatively common type of inherited hearing loss, with potentially thousands of individuals at risk in the Netherlands and beyond, which makes it an interesting target for developing a (genetic) therapy. This study was financially supported by grants from the DCMN Radboudumc, the Heinsius-Houbolt foundation and NIH/NIDCD (R01 DC017147).

J.J. Smits: None. S.E. de Bruijn: None. C. Liu: None. C.P. Lanting: None. A.J. Beynon: None. J. Blankevoort: None. J. Oostrik: None. W. Koole: None. E. de Vrieze: None. C.W.R.J. Cremers: None. F.P.M. Cremers: None. S. Roosing: None. H.G. Yntema: None. H.P.M. Kunst: None. B. Zhao: None. R.J.E. Pennings: None. H. Kremer: None.

P02.031.D GJB2-associated hearing loss in Northern Ossetians

Nika V. Petrova 1, Natalia V. Balinova1, Tatyana A. Vasilyeva1, E F. Mikhailidi2, Vitaliy V. Kadyshev1, Rena A. Zinchenko1,3

1Research Centre for Medical Genetics, Moscow, Russian Federation, 2Republican Children’s Clinical Hospital, North Ossetia-Alania, Vladikavkaz, Russian Federation, 3N.A. Semashko National Research Institute of Public Health, Moscow, Russian Federation.

Hearing loss (HL) is the most common sensorineural disorder worldwide. Pathogenic variants in the GJB2 gene are the main cause of congenital deafness in different populations. The aim was to determine the contribution of the GJB2 gene to the hereditary sensorineural hearing loss (HSNHL) incidence in Ossetians, including Ironians and Digorians, the main subethnic groups, from North Ossetia-Alania. Molecular genetic testing (sequencing and MLPA) was performed in 65 HL patients. In 27.7% HSNHL was associated with GJB2 variants. The c. 358_360delGAG variant frequency was 42.5% (19/42) in Ossetians with GJB2-associated HL, and 15.4% (20/130) in the general sample. The c.35delG variant accounts for 38.1% (16/42) in Ossetian patients with GJB2-associated HL and 12.3% (16/130) in the total sample. 368 healthy Northern Ossetian individuals, including 248 Ironians and 65 Digorians, were analyzed for variants c. 358_360delGAG and c.35delG. The frequencies of c.35delG and c.358_360delGAG GJB2 variants were 0.0061 and 0.0121 in Ironians, 0.0077 and 0.0154 in Digorians. Less than 30% of cases of hereditary sensorineural hearing loss in Ossetians were GJB2-associated. Also, as in other North Caucasian populations (for example, in Karachai), in Ossetians the most frequent GJB2 variant was c. 358_360delGAG. Two variants, c.358_360delGAG and c.35delG, made up 90% of alleles in GJB2-associated hearing loss in Ossetians. The summarized frequency of GJB2 pathogenic variants in Ossetian population exceeds 2% (1.8% in Iranians and 2.3% in Digorians). The research was partially supported by RSF grant №17-15-01051 and within the state task of the Ministry of education and science of Russia.

N.V. Petrova: None. N.V. Balinova: None. T.A. Vasilyeva: None. E.F. Mikhailidi: None. V.V. Kadyshev: None. R.A. Zinchenko: None.

P02.032.A A novel compound heterozygous mutation in RBP3 causes High Myopia

Maya Gombosh 1, Yuval Yogev1, Noam Hadar1, Libe Gradstein2, Ohad S. Birk3,1

1The Morris Kahn Laboratory of Human Genetics, National Institute of Biotechnology in the Negev, Beer Sheva, Israel, 2Department of Ophthalmology, Soroka Medical Center, Beer Sheva, Israel, 3Genetics Institute, Soroka Medical Center, Beer Sheva, Israel.

We studied a girl presenting with isolated high myopia. Whole exome sequencing was done and data were analyzed using our in-house tool, filtering through our in-house 582 ethnicity-matched controls and available databases, based on allele frequency, linkage locus, indel mutation analysis, etc. SNP arrays (750K) of family members yielded possible disease-associated loci on chromosomes 6 and 10. A single heterozygous missense mutation (c.3341G>A ; p. p.Arg1114Gln) in RBP3 was found within the chromosome 10 locus, analyzed using our in-house databases along with open access databases and verified through Sanger sequencing. In addition, CMA identified a ~5 million bp heterozygous deletion, encompassing RBP3, within that locus. Integrative Genomics Viewer (IGV) analysis of NGS data was used to confirm the deletion mutation, showing ~50% less reads in the deleted region compared to controls. Segregation analysis demonstrated that the missense mutation was inherited from the heterozygous father and that the deletion mutation was de-novo. Thus, the infantile high myopia phenotype was caused by novel compound heterozygous RBP3 mutations: an inherited heterozygous missense mutation and a large de-novo deletion mutation encompassing RBP3, that was identified through Indel analysis and low levels of NGS reads of the patient compared to controls. This is a first report of a large deletion mutation in RBP3, which we show to be an unusual "second hit" de-novo germline mutation. Genetic diagnosis is important in children presenting with infantile high myopia, which can be the presenting sign of a degenerative ocular disorder.

M. Gombosh: None. Y. Yogev: None. N. Hadar: None. L. Gradstein: None. O. Birk: None.

P02.033.B Differential methylation of microRNAs encoding genes may contribute to high myopia

Joanna Swierkowska 1, Sangeetha Vishweswaraiah2, Justyna A. Karolak3,1, Malgorzata Mrugacz4, Uppala Radhakrishna2, Marzena Gajecka1,3

1Institute of Human Genetics, Polish Academy of Sciences, Poznan, Poland, 2Department of Obstetrics and Gynecology, Oakland University William Beaumont School of Medicine, Royal Oak, MI, USA, 3Chair and Department of Genetics and Pharmaceutical Microbiology, Poznan University of Medical Sciences, Poznan, Poland, 4Department of Ophthalmology and Eye Rehabilitation, Medical University of Bialystok, Bialystok, Poland.

Introduction: High myopia (HM), an eye disorder with a refractive error of -6.0 D or higher, has multifactorial etiology with environmental and genetic factors involved. Research evidence supports the contribution of alterations in DNA methylation and genes encoding microRNAs (miRNAs) to myopia pathogenesis. Here, we combined both aspects to study the role of the miRNA gene methylation in HM.

Materials and Methods: From genome-wide methylation results of blood DNA of 18 Polish children with HM and 18 matched controls, we retrieved differentially methylated CG dinucleotides located in miRNA genes. Those miRNA genes and their targets were included in over-representation analyses in ConsensusPathDB-human. Expression of selected miRNAs’ target genes were also assessed using the RNA-seq data of human retinal ARPE-19 cell line.

Results: Significant differential methylation of CG dinucleotides located in the promoter regions of MIR3621, MIR34C, MIR423 (increased methylation level), and MIR1178, MIRLET7A2, MIR548I3, MIR6854, MIR675, MIRLET7C, MIR99A (decreased methylation level) genes could alter their expression. Several targets of those miRNAs, e.g. NAP1L1 and EIF4B, were highly expressed in the retinal cell line. Over-representation analyses of miRNAs’ genes and their targets revealed enrichment in biological pathways related to eye structure and function, such as Wnt signaling, axon guidance, and insulin signaling.

Conclusions: Differential methylation of promoters of indicated miRNAs’ genes might influence their expression. Therefore, it may contribute to HM pathogenesis via the disrupted regulation of transcription of miRNAs’ target genes and biological pathways crucial for eye development and function.

Support: National Science Center in Poland (2019/35/N/NZ5/03150) to JS.

J. Swierkowska: None. S. Vishweswaraiah: None. J.A. Karolak: None. M. Mrugacz: None. U. Radhakrishna: None. M. Gajecka: None.

P02.035.C Genetics of Pain: Novel variants identified by the European Network on Inherited Sensory Neuropathies and Insensitivity to Pain (ENISNIP)

Annette Lischka 1, Katja Eggermann1, Arman Çakar2, Richard Boček3, Luca Bartesaghi4, Miriam Elbracht1, Thorsten Hornemann5, Jan Senderek6, Michaela Auer-Grumbach7, Yesim Parman2, Petra Laššuthová3, Ingo Kurth1

1Institute of Human Genetics, RWTH Aachen University Hospital, Aachen, Germany, 2Neurology Department, Neuromuscular Unit, Istanbul Faculty of Medicine, Istanbul University, Istanbul, Turkey, 3Department of Pediatric Neurology, 2nd Faculty of Medicine, Charles University, Prague, Czech Republic, 4Department of Neuroscience and Department of Clinical Neuroscience, Karolinska Institute, Stockholm, Sweden, 5Institute for Clinical Chemistry, University Hospital Zurich, Zurich, Switzerland, 6Friedrich-Baur-Institute at the Department of Neurology, University Hospital LMU Munich, Munich, Germany, 7Division of Orthopedics, Medical University of Vienna, Vienna, Austria.

Introduction: Mutations in approximately 20 genes lead to a monogenetic disorder of lack of pain perception. This includes clinical entities such as hereditary sensory and autonomic neuropathies (HSAN) and congenital insensitivity to pain (CIP). Clinically, the various disorders manifest themselves through repeated trauma and mutilation. Yet, small individual patient cohorts and the lack of standardized phenotype information hinder the complete elucidation of these genetic disorders.

Materials and Methods: The European Network on Inherited Sensory Neuropathies and Insensitivity to Pain (ENISNIP) was established by seven research centers and two patient advocacy organizations specialized on HSAN/CIP and accumulates the knowledge from clinicians, geneticists, basic scientists and patients. The exomes of 60 HSAN patients and, if available, unaffected family members were sequenced. Genetic and clinical data were shared and harmonized within the network.

Results: We identified 16 likely disease-causing novel variants in the following HSAN/CIP genes: ATL3, DST, FLVCR1, NGF, NTRK1, PRDM12, SCN9A, SPTLC2 and WNK1. All variants were rare or absent from control cohorts and none had previously been reported in the literature. If applicable, the pathogenicity was corroborated by segregation analyses within the families.

Conclusions: Through compiling the data within the ENISNIP network, here we report on 16 patients with novel pathogenic variants in known HSAN/CIP genes. In a next step, the data of genetically unsolved cases will be harmonized and re-evaluated. We will prospectively recruit and analyze additional patients to identify new disease-relevant genes.

A. Lischka: None. K. Eggermann: None. A. Çakar: None. R. Boček: None. L. Bartesaghi: None. M. Elbracht: None. T. Hornemann: None. J. Senderek: None. M. Auer-Grumbach: None. Y. Parman: None. P. Laššuthová: None. I. Kurth: None.

P02.036.A Genetics of Inherited Retinal Degenerations in Icelandic patients

Daniel A. Thorsteinsson 1, Vigdis Stefansdottir2, Sigridur Thorisdottir2, Thor Eysteinsson1, Jon J. Jonsson1,2

1Univ. of Iceland, Reykjavik, Iceland, 2Landspitali - National University Hospital of Iceland, Reykjavik, Iceland.

Introduction: The study objective was to delineate the genetics of inherited retinal degenerations (IRDs) in Iceland, a small nation of 364.000 and a genetic isolate. Benefits include delineating novel pathogenic genetic variants and defining genetically homogenous patients as potential investigative molecular therapy candidates.

Materials and Methods: The study sample comprised patients with IRD in Iceland ascertained through national centralized genetic and ophthalmological services at Landspitali, a national social support institute, and the Icelandic patient association. Information on patients’ disease, syndrome, and genetic testing was collected in a clinical registry. Variants were reevaluated according to ACMG/AMP guidelines.

Results: Overall, 140 IRD patients were identified (point prevalence of 1/2.600), of which 70 patients had a genetic evaluation where two-thirds had an identified genetic cause. Thirteen disease genes were found in patients with retinitis pigmentosa, with the RLBP1 gene most common (n = 4). The c.1073+5G>A variant in the PRPF31 gene was homozygous in two RP patients. All tested patients with X-linked retinoschisis (XLRS) had the same possibly unique RS1 pathogenic variant, c.441G>A (p.Trp147X).

Conclusion: Pathologic variants and genes for IRDs in Iceland did not resemble those described in ancestral North-Western European nations. Four variants were reclassified as likely pathogenic. One novel pathogenic variant defined a genetically homogenous XLRS patient group. Grants. Icelandic Student Innovation Fund no. 2107575389. The Richard P. Theodore and Dora Sigurjonsdottir Fund for improving scientific knowledge on blindness no. 2509962099.

D.A. Thorsteinsson: None. V. Stefansdottir: None. S. Thorisdottir: None. T. Eysteinsson: None. J.J. Jonsson: None.

P02.037.B The genetic testing landscape for inherited retinal diseases in the European region

Orla Galvin, Petia Stratieva, Avril Daly

Retina International, Dublin, Ireland.

While potential treatments emerge for Inherited Retinal Diseases (IRDs), the genetic characteristics of IRDs require a genetic diagnosis for inclusion in clinical trials. With a genetic diagnosis patients can take action on inheritance patterns and disease progression. To advocate for equitable, affordable, accessible and timely genetic testing for IRDs it was necessary to investigate the genetic testing landscape from a processes and systems perspective.Desktop research supplemented by survey of ophthalmic and/or genetic specialists across 18 countries was employed. Information was provided by: Medical Geneticists, Clinical Laboratory Geneticists, Ophthalmologists, Retinal Specialists.Genetic testing and counselling for IRDs vary substantially among countries from an awareness, accessibility and affordability perspective. Methods of genetic testing vary and include cerebral MRI, Sanger sequencing or Next Generation Sequencing, Whole Exome Sequencing or Whole Genome Sequencing, SNP array or MLPA. Affordability is a barrier for patients in countries without payment schemes (e.g., Poland) and where only targeted population is covered (e.g., Bulgaria). Research project parfticipation is in some regions an alternative, however limited, with patients often advised to send samples for examination abroad, or travel themselves for examination outside their country of origin.Huge disparity exists in the approach to genetic testing for IRDs. Greater awareness of genetic testing services is required among the health sector and eyecare professionals. A revised approach to the provision of genetic testing services is required to ensure equitable access, empower patients, improve access to clinical trials, therapy and delivery of care.Funded by an educational grant from the Allergan Foundation.

O. Galvin: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Significant; The Allergan Foundation. P. Stratieva: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Significant; The Allergan Foundation. A. Daly: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Significant; The Allergan Foundation.

P02.038.C Mutation spectrum of inherited retinal dystrophies in a cohort from the Basque Country (Spain)

María Rodríguez Hidalgo 1, Maitane Ezquerra-Inchausti1, Leire Escudero-Arrarás1, Araceli Lara-López1, Marta Galdós2, Maialen Aldazabal3, Gonzaga Garay-Aramburu3, Carlos Cruchaga4, Cristina Irigoyen5, Javier Ruiz-Ederra6,7

1IIS Biodonostia, San Sebastián, Spain, 2Cruces University Hospital, Bilbao, Spain, 3Araba University Hospital, Vitoria-Gasteiz, Spain, 4Washington University School of Medicine, St. Louis, MO, USA, 5Donostia University Hospital, San Sebastián, Spain, 6Miramoon Pharma-Biodonostia HRI, San Sebastián, Spain, 7RETICS OFTARED, Madrid, Spain.

Introduction: Inherited retinal dystrophies (IRD) are a heterogeneous group of diseases that mainly affect the retina, with more than 250 genes involved. The clinical and genetic heterogeneity complicates the identification of causative mutations. Here we present the results of genetic-molecular characterization in a cohort of Basque patients.

Materials and Methods: A retrospective study was carried out on 744 IRD affected individuals (from 266 unrelated families) using different molecular techniques, including gene panel, whole exome sequencing, and MLPA hybridation arrays.

Results: Overall, 50% (133/266) of the studied families were genetically characterized. 126 likely causative variants were identified. Most variants, 81, were missense/nosense; 28 small insertion/deletions, 12 affected splice regions, 4 involved copy number variations and 1 was a complex rearrangement. These variants were identified in 42 genes. The most recurrently mutated genes were USH2A, CERKL and RHO, in 21, 10 and 10 families respectively. Most frequent pathogenic variants were c.2276G>T (p.Cys759Phe) in USH2A, c.847C>T (p.Arg283Ter) in CERKL and c.3260C>T (p.Ser1087Leu) in SNRNP200, identified in 12, 10 and 8 families respectively.

Conclusions: Our study allowed us to characterize 50% of the families in our cohort, having important implications for genetic diagnosis and counselling to the Basque population. MRH is supported by a fellowship from Gobierno Vasco, Spain (Pre-2019-1-0325). Work supported by grants from the Instituto de Salud Carlos III y fondos FEDER (PI17/01413 and PI20/01186), from Gobierno Vasco (2018111062, MTVD19/BD/006, MTVD20/BD/002, ZL-2020/00780) and from the Foundation of Patients of Retinitis Pigmentosa (BEGISARE).

M. Rodríguez Hidalgo: None. M. Ezquerra-Inchausti: None. L. Escudero-Arrarás: None. A. Lara-López: None. M. Galdós: None. M. Aldazabal: None. G. Garay-Aramburu: None. C. Cruchaga: None. C. Irigoyen: None. J. Ruiz-Ederra: None.

P02.039.D RPE65-related retinal dystrophy: mutational and phenotypic spectrum in 45 affected patients

Rosario Lopez-Rodriguez 1, Esther Lantero1, Fiona Blanco-Kelly1, Almudena Avila-Fernandez1, Inmaculada Martin Merida1, Marta del Pozo-Valero1, Irene Perea-Romero1, Olga Zurita1, Belén Jiménez-Rolando2, Saoud Tahsin Swafiri1, Rosa Riveiro-Alvarez1, María José Trujillo-Tiebas1, Ester Carreño Salas2, Blanca García-Sandoval2, Marta Corton1, Carmen Ayuso1

1Department of Genetics & Genomics, Instituto de Investigación Sanitaria-Fundación Jiménez Díaz University Hospital- Universidad Autónoma de Madrid (IIS-FJD, UAM), Centre for Biomedical Network Research on Rare Diseases (CIBERER), Madrid, Spain, 2Department of Ophthalmology, Fundación Jiménez Díaz University Hospital (FJD), Madrid, Spain.

Introduction: Biallelic pathogenic RPE65 variants are related to a spectrum of clinically overlapping inherited retinal dystrophies (IRD). A better knowledge of the mutational spectrum and the phenotype-genotype correlation in RPE65-related IRD is needed.

Materials and Methods: Forty-five affected subjects from 27 unrelated families with RPE65-related IRD were included. Clinical evaluation consisted on self-reported ophthalmological history and objective ophthalmological examination. Patients’ genotype was classified accordingly to variant class (truncating or missense) or to variant location at different protein domains. Main phenotypic outcome was age at onset (AAO) of the symptomatic disease and a Kaplan-Meier analysis of disease symptom event-free survival was performed.

Results: Twenty-nine different RPE65 variants were identified in our cohort, 7 of them novel. Most frequent variants were p.(Ile98Hisfs*26), p.(Pro111Ser) and p.(Gly187Glu) accounting for the 24% of the detected alleles. Patients carrying two missense alleles showed a later disease onset than those with 1 or 2 truncating variants. While the 60% of patients carrying a missense/missense genotype presented symptoms before or at the first year of life, almost all patients with at least 1 truncating allele (91%) had an AAO ≤1 year (Log Rank test p < 0.05).

Conclusion: Our findings suggest an association between the type of the RPE65 carried variant and the AAO, providing useful data for clinical management of these patients.

Funding: CIBERER (06/07/0036), IIS-FJD BioBank (PT17/0015/0006), RAREGenomics-CM (CAM, B2017/BMD-3721), CAM (PEJD-2018/BMD-9544), ONCE, Ramon Areces Foundation, Conchita Rabago Foundation, the University Chair UAM-IIS-FJD of Genomic Medicine, ISCIII (PI16/00425, PI19/0321, FI17/00192 and CPII17/00006) and FEDER.

R. Lopez-Rodriguez: None. E. Lantero: None. F. Blanco-Kelly: None. A. Avila-Fernandez: None. I. Martin Merida: None. M. del Pozo-Valero: None. I. Perea-Romero: None. O. Zurita: None. B. Jiménez-Rolando: None. S.T. Swafiri: None. R. Riveiro-Alvarez: None. M.J. Trujillo-Tiebas: None. E. Carreño Salas: None. B. García-Sandoval: None. M. Corton: None. C. Ayuso: None.

P02.040.A Expanding genetic investigation in patients with isolated foveal hypoplasia

Lucia Tiberi 1, Camilla Rocca1, Angelica Pagliazzi2, Giovanna Traficante2, Giacomo Bacci3, Elisa Marziali3, Pina Fortunato3, Debora Vergani2,1, Daniela Formicola2, Viviana Palazzo2, Rosangela Artuso2, Samuela Landini2,1, Laura Giunti2,1, Elia Dirupo2, Sara Bargiacchi2

1University of Florence, Department of Experimental and Clinical Biomedical Sciences, Florence, Italy, 2Meyer Children’s hospital, Medical Genetics Unit, Florence, Italy, 3Meyer Children’s Hospital, Pediatric Ophthalmology Unit, Florence, Italy.

Foveal Hypoplasia is a rare ocular malformation that can be associated with variable ocular features such as congenital nystagmus, premature cataract, anterior segment dysgenesis and chiasmal misrouting, or either can be found in the context of aniridia, albinism or retinal dystrophy. To date, only PAX6, SLC38A8 and AHR are known to be associated with Isolated Foveal Hypoplasia (FVH), but few patients undergo extensive clinical and molecular investigations. In order to elucidate the genetic background of FVH, we performed WES in eight trios recruited at Meyer Children’s Hospital in Florence. The patients were phenotypized by ophthalmologist and geneticist and they all presented FVH with no clear iris transillumination defects. In five patients, WES revealed a combination of two in cis polymorphisms that are in trans with another TYR deleterious mutation: this triallelic genotype, already known to explain missing heritability in some OCA1B albinism patients, is likely to explain also part of FVH patients. One patient showed compound heterozygosity in TYR, with one frameshift and one rare missense variant. Another case presented compound heterozygous variants in OCA2. Only in one patient we couldn’t reach molecular diagnosis, although variants in TYR and OCA2 were found: nonetheless, no clear support of a possible digenic condition was found in the literature. Our results revealed that patients with FVH harbour mutations in genes classically associated to albinism, suggesting that FVH and OCA could be considered as part of the same spectrum. However, further studies in a larger cohort are necessary to better characterize genotype-phenotype correlation.

L. Tiberi: None. C. Rocca: None. A. Pagliazzi: None. G. Traficante: None. G. Bacci: None. E. Marziali: None. P. Fortunato: None. D. Vergani: None. D. Formicola: None. V. Palazzo: None. R. Artuso: None. S. Landini: None. L. Giunti: None. E. Dirupo: None. S. Bargiacchi: None.

P02.041.A Cone-related transcriptomic profiles in Keratoconus corneal epithelium

Katarzyna Jaskiewicz 1, Magdalena Maleszka-Kurpiel2,3, Malgorzata Rydzanicz4, Justyna A. Karolak5, Rafał Płoski4, Marzena Gajecka1,5

1Institute of Human Genetics, Polish Academy of Sciences, Poznan, Poland, 2Optegra Eye Health Care Clinic, Poznan, Poland, 3Chair of Ophthalmology and Optometry, Poznan University of Medical Sciences, Poznan, Poland, 4Department of Medical Genetics, Medical University of Warsaw, Warsaw, Poland, 5Chair and Department of Genetics and Pharmaceutical Microbiology, Poznan, Poland.

Introduction: Keratoconus (KTCN) is the most common corneal ectasia, affecting 1:2000 individuals worldwide, characterized by progressive thinning of the cornea leading to pathological cone formation. Since corneal epithelium (CE) thinning and CE thickness asymmetry constituting characteristic pattern were observed in KTCN, we aimed to analyze transcriptomic profiles of three distinct CE regions.

Materials and Methods: The 17 KTCN patients undergoing cross-linking procedure and 5 mild myopia patients undergoing refractive error correction (non-KTCN controls) were ascertained. The central, middle and periphery CE regions were separated from each obtained CE, based on CE thickness mapping. The RNA samples were extracted using RNA/DNA/Protein PurificationPlus MicroKit (NorgenBiotek). The NGS libraries were prepared using TruSeq Stranded TotalRNA LibraryPrep Gold (Illumina) and sequenced on Illumina NovaSeq6000 platform (100mln read pairs per sample). RNA-seq data was analyzed implementing previously established pipelines.

Results: We observed a characteristic doughnut pattern (thin cone center surrounded by thickened annulus) on each ectatic epithelial thickness map. The average CE thickness values of the three analyzed regions were found to be statistically different in KTCN patients. Those irregularities in epithelial thickness profiles in KTCN were reflected in transcriptomic profiles. The identified differentially expressed pathways (extracellular matrix organization, epithelial-mesenchymal transition) were consistent with previously reported as dysregulated in KTCN.

Conclusions: The phenotypic abnormalities in distinct KTCN CE regions are related to the identified transcriptomic alterations.

Support: The National Science Centre grant no.2018/31/B/NZ5/03280. Co-financed by the European Social Fund, Operational Programme Knowledge Education Development, project ‘International scholarship exchange of doctoral students and academic staff’, No.POWR.

K. Jaskiewicz: None. M. Maleszka-Kurpiel: None. M. Rydzanicz: None. J.A. Karolak: None. R. Płoski: None. M. Gajecka: None.

P02.042.C Assessing the potential of next-generation sequencing technologies to unravel the molecular spectrum of maculopathies

Marta del Pozo-Valero 1,2, Rosa Riveiro-Álvarez1,2, Inmaculada Martín-Mérida1,2, Fiona Blanco-Kelly1,2, Saoud Swafiri1,2, Isabel Lorda-Sanchez1,2, M Jose Trujillo-Tiebas1,2, Ester Carreño3, Belén Jiménez-Rolando3, Blanca García-Sandoval4,2, Marta Corton1,2, Almudena Avila-Fernandez1,2, Carmen Ayuso1,2

1Department of Genetics, IIS-FJD, UAM, Madrid, Spain, 2CIBERER, Madrid, Spain, 3Department of Ophthalmology, IIS-FJD, UAM, Madrid, Spain, 4Department of Opthalmology, IIS-FJD, UAM, Madrid, Spain.

Introduction: Inherited macular dystrophies (MD) comprise a heterogeneous group of disorders characterized by bilateral central visual loss and atrophy of the macula and underlying retinal pigment epithelium. The aim of this study is to assess the potential of next-generation sequencing (NGS) technologies to characterize MD patients.

Methods: The cohort was classified according to their suspected clinical diagnosis- Stargardt disease (STGD), cone and cone-rod dystrophy (CCRD) or other maculopathies (otherMD). Unsolved cases without NGS were re-studied mainly by exome sequencing.

Results: With the implementation of exome sequencing and the study of intronic regions of ABCA4, a total of 677 patients (65%) were characterized. The re-study of unsolved cases showed a characterization yield of 63%, including 75% of monoallelic STGD cases in which a second pathogenic variant was found. While most of the patients referred with STGD were characterized with ABCA4, most of patients with otherMD were unsolved. Other MD- related (BEST1, PROM1, PRPH2) but also unrelated (RHO, RPGR) genes were involved.

Conclusions: This study provides a genetic landscape of 1036 arMD families, giving a mutational spectrum of the genes involved in STGD, CCRD and otherMD groups of patients according to their suspected diagnosis. We demonstrate the increase of the characterization diagnostic yield after the implementation of exome sequencing regardless their diagnosis together with the analysis of the intronic region of ABCA4 in monoallelic STGD patients. This allows their genetic characterization, even when no clinical and familiar data are available, leading to their reclassification. Funding: ISCIII, CIBERER, ONCE, UniversityChairUAM-IIS-FJD, RAREGenomics-CM

M. del Pozo-Valero: None. R. Riveiro-Álvarez: None. I. Martín-Mérida: None. F. Blanco-Kelly: None. S. Swafiri: None. I. Lorda-Sanchez: None. M. Trujillo-Tiebas: None. E. Carreño: None. B. Jiménez-Rolando: None. B. García-Sandoval: None. M. Corton: None. A. Avila-Fernandez: None. C. Ayuso: None.

P02.044.A A burden of rare missense variants supports OTOG as a frequent gene in familial Meniere disease

Pablo Román-Naranjo 1, Paula Robles-Bolivar1, María del Carmen Moleón2, Andrés Soto-Varela3, Ismael Arán4, Juan Manuel Espinosa-Sánchez2, Juan Carlos Amor-Dorado5, Angel Batuecas-Caletrio6, Paz Perez-Vazquez7, Alvaro Gallego-Martinez1, Jose Antonio Lopez-Escamez1,2,8

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, 3Division of Otoneurology, Department of Otorhinolaryngology, Complexo Hospitalario Universitario, Santiago de Compostela, Spain, 4Department of Otolaryngology, Complexo Hospitalario de Pontevedra, Pontevedra, Spain, 5Department of Otolaryngology, Hospital Can Misses, Ibiza, Spain, 6Department of Otolaryngology, Hospital Universitario Salamanca, Instituto de Investigación Biomédica de Salamanca (IBSAL), Salamanca, Spain, 7Department of Otorhinolaryngology, Hospital Universitario de Cabueñes, Gijón, Spain, 8Department of Surgery, Division of Otolaryngology, Universidad de Granada, Granada, Spain.

Introduction: Meniere disease is a set of a rare inner ear disorder characterized by sensorineural hearing loss, vertigo, and tinnitus. Several genes, including FAM136A, DTNA, PRKCB or SEMA3D have been found in autosomal dominant familial Meniere’s disease (FMD). The aim of this study was to investigate 73 FMD patients (46 families) to search for new genes in FMD.

Materials and Methods: Rare variants were selected from patient exome sequencing data to perform a gene burden analysis. Allelic frequencies were compared with European and Spanish reference datasets. Only genes with at least 3 rare variants (MAF≤0.05) were retrieved.

Results: We observed an enrichment of rare missense variants in OTOG gene in FMD cases against either Non-Finnish European population from gnomAD (OR = 4.0(2.6-6.1), p = 4.6x10-9) or Spanish population (OR = 3.0(1.9-4.8), p = 1.2x10-5). Ten rare missense variants were identified in 15/46 (33%) families with MD in the OTOG gene. Six families with 2 or more shared variants were identified, suggesting compound heterozygous recessive inheritance (Table).

Conclusions: OTOG is a frequent gene in FMD and some families with shared variants showed an autosomal recessive inheritance consisting of compound heterozygous missense variants.

Funding: J.A.L.E. is partially funded by INT18/00031 from ISCIII. This study was funded by the Luxembourg National Research Fund INTER/ Mobility/17/11772209 Grant and EF-0247-2017 from Andalusian Health Government to J.A.L.E.

Table 7

P. Román-Naranjo: None. P. Robles-Bolivar: None. M. Moleón: None. A. Soto-Varela: None. I. Arán: None. J. Espinosa-Sánchez: None. J. Amor-Dorado: None. A. Batuecas-Caletrio: None. P. Perez-Vazquez: None. A. Gallego-Martinez: None. J. Lopez-Escamez: None.

P02.045.B Novel stopgain variant in SOX2 gene causing autosomal dominant type 3 syndromic microphthalmia

Florina Stoica 1, Adela Chirita-Emandi2, Andreea Ionescu2, Nicoleta Andreescu2, Maria Puiu2

1Ophthalmology Department, Emergency Clinical Municipal Hospital, Center of Genomic Medicine, University of Medicine and Pharmacy “Victor Babes”, Timisoara, Romania, 2Center of Genomic Medicine, University of Medicine and Pharmacy “Victor Babes”, Regional Center of Medical Genetics Timis, Clinical Emergency Hospital for Children “Louis Turcanu”, part of ERN ITHACA, Timisoara, Romania.

We aim to present a novel variant in SOX2 gene associated with a particular ocular phenotype in a child with AD type 3 syndromic microphthalmia.

Methods: The patient was evaluated by a multidisciplinary team. NGS used the TruSightOne Illumina gene panel (4813 genes), supported by our Ministry of Health.

Results: Ocular phenotypes in the 4 years old male patient included bilateral microphthalmia, retinal detachment, iris and chorioretinal coloboma, retinal dystrophy and severe visual impairment. The left eye presented high myopia and retinal pigment deposits. Head MRI showed optic nerve and chiasma hypoplasia. Other abnormalities were: mild intellectual disability, epilepsy, walking difficulty, leg bone pain, genu varrum, pes planus, and patent foramen ovale. He had normal male genitalia and height at 50th percentile for age. The endocrinologist monitors the child’s growth. NGS performed at 4 years of age detected a heterozygous stopgain variant NM_003106.3(SOX2):c.600C>A, (chromosome 3q26.33). This sequence change creates a premature translational stop signal NP_003097.1:p.(Tyr200Ter), classified as pathogenic.

Conclusions: Heterozygous pathogenic variants in SOX2 gene are associated with syndromic microphthalmia, including ocular and systemic abnormalities. Syndromic microphthalmia diagnosis was considered since patient’s birth, yet NGS was performed at 4 years, showing that access to molecular diagnosis is still limited in Romania. The novel pathogenic variant identified in the patient was associated with a particular phenotype specifically in regards to retinal pigment deposits, yet with normal male genitalia. The molecular diagnostic was important for the patient to receive specialized care, while the family to benefit from genetic counselling.

F. Stoica: None. A. Chirita-Emandi: None. A. Ionescu: None. N. Andreescu: None. M. Puiu: None.

P02.046.C The role of BMP3 in the development of myopia

Amy Findlay, Thibaud Boutin, Chloe Stanton, Ian Jackson, Veronique Vitart

MRC Human Genetics Unit, University of Edinburgh, Edinburgh, United Kingdom.

Our research focuses on functional follow up of GWAS hits using a variety of cell and animal models. A locus overlapping the BMP3 gene, significantly associated with retinal detachment risk and myopia, was further examined. Within the associated region, a BMP3 coding variant was prioritized as the candidate causal variant, alongside non-coding potentially regulatory variants. The BMP signalling pathway has long been implicated in the patterning and development of the eye, but BMP3’s role both within this pathway and during eye development and disease is unclear. The region of interest is conserved between mouse and human so producing animal models carrying the missense variant along with loss of function mutations was easily accomplished using CRISPR cas9 genome editing and comprehensive phenotyping was performed. Our unit is uniquely equipped to analyse eye phenotypes and using the Optical Coherence Tomography machine we are able to track disease progression throughout life and show that loss of function mice exhibit myopia. RNAseq analysis of loss of function cell lines has shown deregulation of a number of genes including BMP2, and 4, which have also been implicated in myopia. Future work is required to assess how much the mouse and human cell line results converge, but our results strengthen a role for a BMP pathway in myopia development.

A. Findlay: None. T. Boutin: None. C. Stanton: None. I. Jackson: None. V. Vitart: None.

P02.047.D Next-generation sequencing panels for hereditary hearing loss testing with approaches for difficult-to-sequence regions

Laura Sarantaus 1, Kimberly Gall2, Sari Tuupanen1, Marta Gandia1, Hatice Duzkale1, Inka Saarinen1, Johanna Sistonen1, Juha Koskenvuo1, Tero-Pekka Alastalo2

1Blueprint Genetics, a Quest Diagnostics Company, Espoo, Finland, 2Blueprint Genetics Inc, a Quest Diagnostics Company, Seattle, WA, USA.

Introduction: Hereditary hearing loss (HHL) is a genetically heterogeneous group of disorders. Determining the molecular etiology allows for personalised patient management and surveillance, and recurrence risk estimation for families. Comprehensive genetic testing in HHL using next-generation sequencing (NGS) is complicated owing to pseudogenes and segmental duplications affecting several key genes, such as STRC. A testing strategy that includes difficult-to-sequence regions is needed to maximise diagnostic yield.

Materials and Methods: To assess the diagnostic efficacy, we performed a retrospective review of test results from NGS panels performed for 934 de-identified patients tested consecutively for HHL at Blueprint Genetics, a CLIA-certified diagnostic laboratory. The analysis was boosted with clinically relevant deep intronic variants and a proprietary copy number variation (CNV) analysis method for exon-level CNVs. Variant interpretation followed ACMG guidelines. Most cases (86%, 799/934) were analysed using the Comprehensive Hearing Loss and Deafness Panel (239 genes), while a minority (14%, 135/934) were analysed using smaller panels.

Results: Molecular genetic diagnosis was established in 29.9% (279/934) of patients, distributed in 54 genes. The most commonly implicated genes were GJB2 (24.4%), STRC (9.7%), MYO15A (5.0%), and SLC26A4 (4.7%). CNVs were reported in 16.1% (45/279) of diagnosed patients; 38.5% of CNVs were intragenic. Tailored molecular testing approaches for difficult-to-sequence genes contributed to 12.9% (36/279) of diagnoses (confirmed with orthogonal methods).

Conclusions: These results emphasise the value of tailored approaches for difficult-to-sequence genes as well as the importance of including high-resolution CNV detection in enhancing the clinical utility of genetic testing using NGS panels for HHL.

L. Sarantaus: A. Employment (full or part-time); Significant; Blueprint Genetics. K. Gall: A. Employment (full or part-time); Significant; Blueprint Genetics Inc. S. Tuupanen: A. Employment (full or part-time); Significant; Blueprint Genetics. M. Gandia: A. Employment (full or part-time); Significant; Blueprint Genetics. H. Duzkale: A. Employment (full or part-time); Significant; Blueprint Genetics. I. Saarinen: A. Employment (full or part-time); Significant; Blueprint Genetics. J. Sistonen: A. Employment (full or part-time); Significant; Blueprint Genetics. J. Koskenvuo: A. Employment (full or part-time); Significant; Blueprint Genetics. T. Alastalo: A. Employment (full or part-time); Significant; Blueprint Genetics Inc.

P02.048.A North Carlina macular dystrophy: phenotypic variability and computational analysis of disease-implicated non-coding changes

David J. Green 1, Eva Lenassi1, Cerys S. Manning1, David McGaughey2, Vinod Sharma1, Graeme C. Black1, Jamie M. Ellingford1, Panagiotis I. Sergouniotis1

1University of Manchester, Manchester, United Kingdom, 2National Eye Institute, Bethesda, MD, USA.

Introduction: North Carolina macular dystrophy (NCMD) is an autosomal dominant, congenital disorder affecting the central retina. Here, we report clinical and genetic findings in three families segregating NCMD and use epigenomic datasets from human tissues to gain insights into the effect of NCMD-implicated variants.

Materials and Methods: Clinical assessment and genetic testing were performed. Publicly-available transcriptomic and epigenomic datasets were analyzed and the ‘Activity-by-Contact’ (ABC) method for scoring enhancer elements and linking them to target genes was used.

Results: A previously-described, heterozygous, non-coding variant upstream of the PRDM13 gene was detected in all six affected study participants (chr6:100,040,987G>C [GRCh37/hg19]). Inter- and intra-familial variability were observed; the visual acuity ranged from 0.0 to 1.6 LogMAR and fundoscopic findings ranged from visually insignificant, confluent, drusen-like macular deposits to coloboma-like macular lesions. Variable degrees of peripheral retinal spots (that were easily detected on widefield retinal imaging) were observed in all study subjects. Notably, a 6-year-old patient developed choroidal neovascularization and required treatment with intravitreal bevacizumab injections. Computational analysis of the five single nucleotide variants that are known to cause NCMD revealed that these non-coding changes lie within two putative enhancer elementspredicted to interact with PRDM13 in the developing human retina. PRDM13 was found to be expressed in the fetal retina, with highest expression in the amacrine precursor cell population.

Conclusions: We highlight the utility of widefield retinal imaging in individuals suspected to have NCMD and provide further evidence supporting the role of PRDM13 dysregulation in the pathogenesis of this condition.

D.J. Green: None. E. Lenassi: None. C.S. Manning: None. D. McGaughey: None. V. Sharma: None. G.C. Black: None. J.M. Ellingford: None. P.I. Sergouniotis: None.

P02.049.B Genotype phenotype correlations of 37 DFNB9 patients with auditory neuropathy and 17 new OTOF pathogenic variants

Sophie Achard1, Margaux SEREY-GAUT2, Laurence Jonard 3, Isabelle Rouillon1, Marine Parodi1, Natalie Loundon1, Elisa Rubinato2, Sandrine Marlin2

1Hopital Necker - Service d’ORL, Paris, France, 2Hopital Necker - Service de Génétique Clinique, Paris, France, 3Hopital Necker - Service de Génétique Moléculaire, Paris, France.

Auditory neuropathy represents 5-10% of child’s hearing loss. Pathogenic bi-allelic variations of OTOF result in autosomal recessive deafness DFNB9. We retrospectively studied the genotype-phenotype correlations of 37 cases from 30 families with pathogenic bi-allelic OTOF variations. Seventeen new pathogenic variants were identified. All patients had isolated auditory neuropathy. Hearing loss was pre-lingual in 78% of cases and profound in 70%. Hearing loss was progressive in 30%, fluctuating in 30% and temperature-sensitive in 22%. The diagnosis of auditory neuropathy was mainly based on the discordance of electrophysiological tests with acoustic otoemissions present (78%) and brainstem auditory evoked responses absent or desynchronized (81%). All patients with homozygous or compound heterozygous "loss of function" variants had congenital bilateral profound hearing loss, patients compound heterozygous for a “loss of function” variant and a missense variant had variable presentations. Those with two missense variants had a mild to severe hearing loss, which could be of secondary onset. 54% received cochlear implant rehabilitation, 16% of which were bilateral. Our study confirms a successful hearing rehabilitation with cochlear implants, with open word perception increasing from 0% before surgery to 80% at 8 years after implantation. However, cochlear implantation cannot be considered as a treatment (disturbance in noise, social difficulties and professional integration...). Gene therapy trials in mutant OTOF -/- adult mice have shown prolonged hearing rehabilitation, making it possible to consider a short-term therapeutic trial for this isolated congenital form of deafness (RHU AUDINNOVE 2019). This phenotype-genotype study is an essential prerequisite for the future therapeutic trial.

S. Achard: None. M. Serey-gaut: None. L. Jonard: None. I. Rouillon: None. M. Parodi: None. N. Loundon: None. E. Rubinato: None. S. Marlin: None.

P02.050.C A newborn with corneal clouding and a diagnosis of posterior polymorphous corneal dystrophy type 1 due to a duplication of the OVOL2 gene

Hester Y. Kroes 1, M B. Muijzer1, L Haer-Wigman2, E S. M. Voskuil-Kerkhof1, P M. van Hasselt1, R P. L. Wisse1

1University Medical Centre Utrecht, Utrecht, Netherlands, 2Radboud University Medical Centre, Nijmegen, Netherlands.

Introduction: PPCD1 was recently shown to result from activating mutations in the promotor region of OVOL2. We present a new pathogenic mechanism in line with this observation. Case report An infant boy presented with progressive corneal clouding at the Department of Ophthalmology. Pregnancy was unremarkable, family history negative. Ophthalmologic investigation revealed bilateral diffuse corneal clouding and otherwise normal results. Physical examination was normal. A diagnosis of autosomal recessive CHED (congenital hereditary endothelial dystrophy) was suspected.

Methods: High throughput DNA-analysis was performed in the index case and his parents. An in silico panel analysis of 424 genes associated with visual disorders was performed consisting of analysis of single nucleotide variants and copy number variants (CNVs), followed by SNP-array.

Results: No causative single nucleotide variants were detected in SLC4A11, OVOL2 or any other gene. CNV analysis and SNP array revealed a duplication of ~49 Kb in the chromosomal region 20p11.23 encompassing OVOL2, leading to a diagnosis of PPCD1. The duplication was absent in DNA from the parents.

Conclusion: We present the fifth index case of molecularly proven PPCD1. The underlying molecular abnormality, a duplication of the entire OVOL2 gene, suggests a gene dosage effect. So far, four PPCD1 families have been described, each with an activating variant in a distinct part of the promotor region of OVOL2. OVOL2 is a transcription factor acting as a repressor of ZEB1. Loss of function variants in ZEB1 cause PPCD3. This case seems to confirm that gain of function of OVOL2 causes PPCD1.

H.Y. Kroes: None. M.B. Muijzer: None. L. Haer-Wigman: None. E.S.M. Voskuil-Kerkhof: None. P.M. van Hasselt: None. R.P.L. Wisse: None.

P02.051.D Functional characterization of non-canonical splice variants in aniridia by minigenes and ex-vivo approaches

Alejandra Tamayo 1,2, María Tarilonte1, Jennifer Moya1, Patricia Ramos1, Saoud T Swafiri1,2, Fiona Blanco1,2, Cristina Villaverde1,2, Carmen Ayuso1,2, Marta Cortón1,2

1Instituto de Investigación Sanitaria Fundación Jiménez Díaz University Hospital, Madrid, Spain, 2Centre for Biomedical Network Research on Rare Diseases (CIBERER), Madrid, Spain.

Introduction: Mutations in PAX6 cause aniridia, a congenital disorder characterized by several structural eye anomalies. We describe the development of in-vitro and ex-vivo tools for the functional characterization of potentially spliceogenic variants and their impact on the canonical PAX6 splicing.

Materials and Methods: For minigene assays, a genomic segment encompassing the region of interest of PAX6 was cloned into expression vectors and variants were introduced by site-directed mutagenesis. In-vitro assays were performed by transient transfection into HEK293 and/or ARPE19 cell lines. Lymphoblastoid cell lines (LCL) were established by Epstein Barr virus-mediated transformation of blood lymphocytes from patients carrying splicing variants and control individuals. Total RNA was extracted and reversely transcribed. The splicing patterns of mRNA transcripts were compared by semiquantitative PCR and sequencing.

Results: We developed four minigene PAX6 constructs: i) exons 1 to 4, ii) exons 5a and 6, iii) exons 5 to 7 and iv) exons 8 to 11, respectively. To date, a total of 13 potentially spliceogenic variants were assayed and 11 of them showed aberrant splicing patterns. Expression analysis in 4 patient-derived LCL showed alterations on splicing patterns. Two out these 4 variants were also tested by minigene assays and the findings on splicing patterns showed a correlation between both approaches.

Conclusions: Minigene assays carrying multiple exons and LCL studies are useful strategies to gain insight into the pathogenicity of non-canonical splice PAX6 variants. These methods are easy-to-carry approaches for robust splice variant analysis and to help bring molecular diagnosis to aniridia patients.

A. Tamayo: None. M. Tarilonte: None. J. Moya: None. P. Ramos: None. S. Swafiri: None. F. Blanco: None. C. Villaverde: None. C. Ayuso: None. M. Cortón: None.

P02.052.A Clinical molecular genetic age characteristics of congenital aniridia in children

Natella Sukhanova 1, Rena Zinchenko2, L.A.Katargina.A.V.Marakhonov,T.A.Vasilieva,

1Federal State Budgetary Institution of Health’s Central Clinical Hospital of the Russian Academy of Sciences, Moscow, Russian Federation, 2Federal State Budgetary Scientific Institution “Research Centre for Medical Genetics, Moscow, Russian Federation.

Introduction: The relationship between the clinical features of the phenotype and the genotype confirmed by molecular genetic methods in children with aniridia expands our understanding of the clinical course of the disease and corrects possible treatment. Congenital aniridia (OMIM # 106210) is a monogenic hereditary pathology. The leading diagnostic signs are congenital absence of iris tissue, hypoplasia of fovea, accompanied by nystagmus.

Materials and Methods: The data of ophthalmological examination and molecular genetic analysis of 83 patients from 0 to 18 years old were analyzed. Mutations were previously identified in 56 patients as a result of sequencing of exons of the PAX6 gene. In 27 patients, various deletions of the 11p13 region were identified by MLPA.

Results: According to clinical signs, patients with congenital aniridia were distributed: the presence of complete or partial aniridia, nystagmus, keratopathy, cataracts and glaucoma. With nonsense mutations, the complete absence of iris tissue and the development of keratopathy observed in average age 5 ± 1 years. Patients with open reading frame shift mutations are significantly more likely to develop complicated cataract and glaucoma in average age 10 ± 2 years. Partial aniridia occurs with missense mutations significantly more often.

Conclusions: The identification of age-related characteristics depending on the detected mutations is of not only clinical, but also scientific interest, since it indicates one of the mechanisms of regulation of the PAX6 gene function in case of damage to the organ of vision.The research was partially supported by RSF grant №17-15-01051

N. Sukhanova: None. R. Zinchenko: None.

P02.053.B Minigene expression system for assess the effect of variants in the PAX6 gene on pre-mRNA splicing

Ksenia Davydenko, Alexandra Filatova, Mikhail Skoblov

Research Centre for Medical Genetics, Moscow, Russian Federation.

Introduction: Mutations in the PAX6 gene affect the normal development of the eye and lead to a spectrum of phenotypes, the most severe is aniridia. Today more than 600 mutations in PAX6 have been reported, about 100 of them were annotated as affecting splicing. Moreover, variants affecting splicing could masquerade as missense, nonsense or synonymous in databases. In order to establish the effect of reported mutations on splicing, a functional analysis is required.

Materials and methods: Bioinformatics analysis was performed using the SpliceAI (Illumina, USA) and MaxEntScan (MIT, USA) tools. The minigene expression system was used to evaluate the functional effect of SNVs on splicing.

Results: We created a system of 8 plasmids containing all 10 coding exons of the PAX6 gene. Plasmids were tested for correct splicing and resulted in normally spliced wild-type transcripts. For all 354 PAX6 SNVs from the HGMD, LOVD, and ClinVar databases we predicted the probability of influence on splicing. We selected 18 intronic SNVs outside ±1,2 position whose effect on splicing has not been confirmed experimentally and 20 exonic (synonymous, missense and nonsense) SNVs which actually could affect splicing. Functional analysis of these SNVs in the minigene expression system revealed a wide range of splicing defects.

Conclusion: We created and successfully tested a minigene expression system for assess the effect of variants in PAX6 gene on pre-mRNA splicing. This system can be used to establish the pathogenicity of splicing variants in the PAX6 gene, and to reclassify misclassified variants located in databases.

K. Davydenko: None. A. Filatova: None. M. Skoblov: None.

P02.054.C novel phenotype-genotype correlation with PEX6 gene in Saudi patients with heimler syndrome

Basamat Almoallem

Department of Ophthalmology, King Saud University, Riyadh, Saudi Arabia, Riyadh, Saudi Arabia.

Purpose: Peroxisome biogenesis disorders (PBDs; MIM# 601539) are heterogeneous disorders caused by defects in genes encoding proteins that are essential for peroxisomal matrix and membrane proteins. Heimler syndrome is one of the PBDs that is caused by mutations in PEX1 and PEX6 genes. In this study, we aimed to fully characterise the clinical and molecular aspects of two Saudi probands who were diagnosed early with Usher syndrome.

Method: We set up a comprehensive clinical and molecular genetic workflow including detailed ophthalmological and systemic assessments followed by genome-wide SNP microarrays analysis and targeted PEX6 gene screening by Sanger sequencing.

Results: Clinically: both probands appeared dysmorphic with long faces, high forehead, short nose, small low set ears, and full lips. Moreover, advanced inherited retinal dysfunction represented by waxy pallor optic disc, attenuated vessels, RPE mottling with intraretinal bony spicules pigmentations and central foveal atrophic changes in both eyes with bilateral sensory neural hearing loss and amelogenesis imperfecta. Genetically: an autozygous block was identified on chromosome 6p21.1 encompassing PEX6 gene using SNP microarrays. Novel PEX6 (NM_000287.3)c.290T>G (p.Val97Gly) was found and co-segregated with the phenotype in both families and found to be “likely pathogenic” according to ACMG guidelines.

Conclusion: Our study represents the first report of PEX6 associated Heimler syndrome in the middle east and considered to be the third in the literature so far. It highlights the importance of combining molecular diagnosis with the clinical findings to expand our knowledge of PEX6-related PBDs phenotype and the allelic spectrum for this gene.

B. Almoallem: None.

P02.055.D Investigation of transcriptional dysregulation events driving Posterior Polymorphous Corneal Dystrophy type 1

Nathaniel J. Hafford-Tear 1, Lubica Dudakova2, Stephen J. Tuft1,3, Amanda Sadan1, Nihar Bhattacharyya1, Christina Zarouchlioti1, Alison J. Hardcastle1, Petra Liskova2,4, Alice E. Davidson1

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

Purpose: Posterior Polymorphous Corneal Dystrophy (PPCD) is associated with a dysregulated cell state, induced by mutations that alter the levels of epithelial mesenchymal transition (EMT)-regulating transcription factors ZEB1, GRHL2 or OVOL2. Here we investigate the transcriptomic signature of dysregulation in PPCD type 1 (PPCD1) corneal endothelial cells (CECs) to identify biomarkers of the disease as potential therapeutic targets.

Methods: Primary CECs were cultured from five individuals affected with PPCD1 and five controls. All PPCD1 individuals were confirmed to harbour the same regulatory mutation in the OVOL2 promoter (NM_021220:c.−370T>C). Total RNA was extracted from each primary culture and paired-end Illumina RNA sequencing was performed. Transcriptomic analysis was performed using DESeq2 and IsoformSwitchAnalyzeR programs to investigate differential gene expression and splicing events in PPCD1 CECs compared to control CECs.

Results: Utilizing the EMT Gene Database, 279 EMT-associated genes were found to be highly dysregulated (p-value < .05; Log-2 fold change >1) in PPCD1 including CDH1, OVOL2, GRHL2, GATA6 and the epithelial splice regulator ESRP1. Alternative splicing in PPCD1 vs controls was further identified for ESRP1 targets, CD44 and FGFR2.

Conclusions: Our study detected aberrant upregulation of OVOL2 in PPCD1, supporting the hypothesis for pathogenic mechanism, and identified other EMT-associated genes and pathways that are significantly disrupted in PPCD1 CECs. Our data suggests that overexpression of the epithelial cell type-specific splicing regulator, ESRP1, induces aberrant splicing of CD44 and FGFR2. We hypothesise that this mechanism contributes to the ‘epithelialisation’ of the corneal endothelium observed in PPCD1 and may represent a target for future therapeutic interventions.

N.J. Hafford-Tear: None. L. Dudakova: None. S.J. Tuft: None. A. Sadan: None. N. Bhattacharyya: None. C. Zarouchlioti: None. A.J. Hardcastle: None. P. Liskova: None. A.E. Davidson: None.

P02.056.A Clinical-genetic correlation of the functional state of the retina in ABCA4-associated pathology

Vitaly V. Kadyshev 1, Andrei V. Marakhonov1, Inna V. Zolnikova1,2, Sergey I. Kutsev1, Rena A. Zinchenko1

1Research Centre for Medical Genetics, Moscow, Russian Federation, 2Helmholtz National Medical Research Center of Eye Diseases, Moscow, Russian Federation.

Aim: To set clinical and genetic correlations of retinal pathology by ABCA4 gene mutations, considering the functional state Material and methods: 15 Russian patients aged from 7 to 32 y.o. with inherited eye diseases ABCA4-associated. All patients besides ophthalmic examination, spectral-OCT, fundus autofluorescence, full-field electroretinogram (ERG), 30-Hz flicker ERG, macular chromatic ERG (MERG) to red stimulus, molecular genetic studies included Next Generation Sequencing and Sanger direct sequencing.

Results: In ABCA4-associated Stargardt disease (SD) genotype [p.L541P, p.A1038V] of «frequent» mutations was revealed in 9 patients, in 2 cases in was associated another «frequent» mutation p.G1961E. In 4 patients with genotype [p.L541P, p.A1038V] «severe» phenotype of Stargardt disease was found: with large defect of the ellipsoid zone, severely subnormal macular ERG (MERG) to red stimulus and subnormal 30 Hz flicker and full-field maximal ERG. In 2 cases with genotype [p.L541P, p.A1038V] and mutation p.G1961E was found «mild» phenotype. Nonrecordable MERG was found in 7 of 15 cases, 8 patients had subnormal. Subnormal 30 Hz flicker ERG was found in 8 patients with SD. Maximal ERG was reduced in 6 patients with SD.

Conclusions: The study made it possible to assess the effect of mutations in the ABCA4 gene not only on the structural changes but also on the functional state of the retina. The study expands the range of clinical and genetic features of hereditary ophthalmological pathology associated with the ABCA4 gene. Supported by RFBR (project № 19-015-00122 А), within the state task of the Ministry of education and science of Russia

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

P02.057.D Health-related quality of life amongst patients with retinal dystrophies

Deborah Schofield1, Joshua Kraindler 1, Rupendra Shrestha1, Sarah West1, Owen Tan1, Alan Ma2,3, Diana Jelovic2, John Grigg4,3,5, Robyn Jamieson2,4,3

1GenIMPACT: Centre for Economic Impacts of Genomic Medicine, Macquarie Business School, Macquarie University, Sydney, Australia, 2Eye Genetics Research Unit, Children’s Medical Research Institute, University of Sydney, Sydney, Australia, 3Sydney Children’s Hospitals Network, Sydney, Australia, 4Save Sight Institute, Sydney, Australia, 5Sydney Eye Hospital, Sydney, Australia.

Introduction: Retinal dystrophies (RD) are diseases leading to incurable blindness attributable to variations in more than 200 genes. There have been significant advances in gene therapies including Voretigene Neparvovec (VN), which was recently approved for intervention in RD due to variants in RPE65, by the Food and Drug Administration, and the National Health Service. Approval of publicly funded interventions often require cost-effectiveness analysis (quality adjusted life years (QALYs) gain). To our best knowledge, studies reporting health-related utility of RD - a key metric in computing QALYs - are scarce or have insufficient or indirect measures of QoL outcomes. We aim to present the first paper examining the health-related utility of RD using an established QoL measure - the Assessment of Quality of Life-8D (AQoL-8D).

Materials and methods: As part of the EPIC-Vision study, we collected patient (age≥18) reported AQoL-8D data as part of a customised survey. AQoL-8D measures functioning across eight dimensions: independent living, pain, senses, mental health, happiness, coping, relationships, self-worth. The EPIC-Vision survey also collected data on visual functioning (NEI-VFQ), and socioeconomic data.

Results: Average health-related utility for patients with RD was 0.6, significantly lower than the average population norm (0.8). Patient’s scores were relatively lower on independent living, mental health, relationship, self-worth, and senses. Regression analysis showed that patients with better visual functioning score had better health-related utility.

Conclusions: We reported for the first time, health-related utility for RD patients, which can be used to inform further studies on new interventions.Funding: NHMRC Partnership Grant (APP1116360).

D. Schofield: None. J. Kraindler: None. R. Shrestha: None. S. West: None. O. Tan: None. A. Ma: None. D. Jelovic: None. J. Grigg: None. R. Jamieson: None.

P02.058.C Genetic analysis of patients with retinal dystrophies in Croatia

Adriana Bobinec 1,2, Mijana Kero1,2, Ljubica Boban1,2, Nikolina Vidan Rogulj1, Ana-Maria Meašić1,2, Ivona Sansović1,2, Leona Morožin Pohovski1, Mirjana Bjeloš3,4, Mladen Bušić3,4, Ingeborg Barišić1,2

1Children’s Hospital Zagreb, Zagreb, Croatia, 2Department 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, 3University Department of Ophthalmology, University Clinical Hospital Sveti Duh, Zagreb, Croatia, 4Faculty of Dental Medicine and Health, Josip Juraj Strossmayer University of Osijek, Osijek, Croatia.

Introduction: Retinal dystrophies (RD) are a group of inherited retinal disorders characterized by progressive photoreceptors and pigment epithelial cells dysfunction causing severe visual loss and eventual blindness. Recently augmentation gene treatment has been approved for patients with biallelic mutations in the RPE65 gene known to cause pigmentary retinopathy type 20 and Leber congenital amaurosis 2. So far gene therapy in these patients provided improvement of functional vision without serious adverse reactions raising great interest for genetic testing of RD.

Materials and methods: Next-generation sequencing (NGS) was performed in 60 patients with a referral diagnosis of retinal dystrophy using Illumina TruSight One Kit.

Results: In 36 (60) patients different pathogenic variants were found in RP1, RP2, RHO, EYS, USH2A, BEST1, PROM1, C9, ABCA4, ADGRV1, BEST1, CEP290 genes. In two patients identified variants were classified as variants of unknown significance. In 22 patients NGS analysis did not reveal mutations in genes associated with RD.

Conclusion: Molecular genetic testing of retinal dystrophies has been successfully used to clarify clinical diagnoses and direct genetic counselling. NGS proved to be efficient and useful method for setting up the diagnosis in 63% of patients.This study was supported by CERRM, Republic of Croatia, and by the EU through ERDF, under grant agreement No. KK., project „Reproductive and Regenerative Medicine - Exploring New Platforms and Potentials”.

A. Bobinec: None. M. Kero: None. L. Boban: None. N. Vidan Rogulj: None. A. Meašić: None. I. Sansović: None. L. Morožin Pohovski: None. M. Bjeloš: None. M. Bušić: None. I. Barišić: None.

P02.059.D Implementation of the targeted retinal dystrophy panel in the routine genetic diagnostics of retinal disorders in Polish patients

Ewa Matczyńska 1,2, Przemysław Łyszkiewicz1, Anna Wąsowska1,2, Robert Szymańczak1, Ewa Suchecka1, Katarzyna Stradomska1, Marta Beć1, Maria Jędrzejowska1, Sławomir Teper2, Maciej Krawczyński3, Anna Boguszewska-Chachulska1

1Genomed S.A., Warsaw, Poland, 2Chair and Clinical Dept. of Ophthalmology, Medical University of Silesia, Katowice, Poland, 3Center of Medical Genetics Genesis Sp. z o.o., Poznań, Poland.

A targeted panel approach for genetic diagnosis of inherited retinal dystrophies (IRD) was developed, based on the results of the preceding NeuStemGen project (a cohort of Polish IRD patients analysed using WES). The implementation of the approach in routine diagnostics provided an opportunity to assess its performance along with strengths and weaknesses.

Polish patients with clinical symptoms of retinal dystrophies (N = 168) were subjected to NGS using a custom panel capturing coding regions of 270 IRD genes (RetNet), developed with the Roche NimbleGen SeqCap EZ. Bioinformatic analysis was performed with the GATKv4 and CoNVaDING (for CNV analysis), using the GRCh38 reference genome. Variant analysis and interpretation were completed according to ACMG guidelines using the in-house variant interpretation tool - BroVar.

Overall sequencing data metrics presented a satisfactory level of target coverage (mean coverage - 255x ± 55; fraction of target bases covered >20x - 0.996 ± 0.001) and high heterozygote SNP calling sensitivity (0.998 ± 0.0003), indicating near-optimal performance for SNPs and small indels, providing diagnosis confirmation for 77 patients. CNV analysis enabled finding causative variants for additional 9 patients (51% overall), however, CNV identification performed sub-optimal in several batches due to technical bias and a low number of samples per pool.

The targeted retinal panel approach has been successfully applied in routine IRD diagnostics of Polish patients, expanding the knowledge of IRD genetic background in the Polish population, simultaneously allowing for a cost-effective analysis (comparing to WES). Despite reliable coverage, identification of CNVs remains challenging. Further refinement of CNV and more complex variants’ calling is necessary.

E. Matczyńska: None. P. Łyszkiewicz: None. A. Wąsowska: None. R. Szymańczak: None. E. Suchecka: None. K. Stradomska: None. M. Beć: None. M. Jędrzejowska: None. S. Teper: None. M. Krawczyński: None. A. Boguszewska-Chachulska: None.

P02.061.B Metastasis suppressor 1 <MTSS1> as a novel candidate gene for inherited retinal dystrophy <IRD>

Solomon Merepa 1, Suzanne Broadgate1, Jing Yu1, Sumathi Sekaran1, Susan Downes1,2, Stephanie Halford1, United Kingdom Inherited Retinal Dystrophy Consortium (UKIRDC)

1Nuffield Laboratory of Ophthalmology, Department of Clinical Neuroscience, University of Oxford, Oxford, United Kingdom, 2Oxford Eye Hospital, John Radcliffe Hospital, Oxford University Hospitals NHS Foundation Trust, Oxford, United Kingdom.

Introduction: Over 260 genes underlying monogenic forms of inherited retinal dystrophies (IRDs) have been identified, enabling the development of treatment regimens such as gene therapy. However, there are still IRD cases whose underlying genetic causes are yet to be identified, prompting the need for continued disease-gene identification studies.

Methods: Two affected members of a family diagnosed with dominant retinitis pigmentosa (adRP) underwent whole-exome sequencing (WES). Various bioinformatics tools and gene sequencing databases including CADD and gnomAD were used to annotate and prioritise candidate variants. Segregation analysis was performed using Sanger sequencing. A variant segregating with the condition was characterised for expression, localisation and function in the mammalian retina using techniques including PCR, western immunoblotting, immunostaining, and site-directed mutagenesis.

Results: Of 22 shared dominant variants determined from WES, a mutation in the MTSS1 gene (c.624_628del (p.E213del)) segregated with disease in the family. The expression and function of MTSS1 in the mammalian retina has not been previously described. We report, for the first time, expression of MTSS1 in the human retina and using antibodies specific for Mtss1, we demonstrate immunostaining in layers of the mouse retina including the photoreceptors. Using mutant expression constructs, we are currently investigating if the mutation affects localisation and function of MTSS1 at the cellular level.

Conclusions: We provide here, for the first time, preliminary evidence linking mutation in the MTSS1 with dominant RP. Elucidating the specific role of MTSS1 in the mammalian retina could potentially inform therapeutic approaches for IRDs.

Funding: Retina UK, Fight for Sight, Commonwealth Scholarships Commission

S. Merepa: None. S. Broadgate: None. J. Yu: None. S. Sekaran: None. S. Downes: None. S. Halford: None.

P02.062.C First systematic molecular genetic analysis using NGS analysis of 120 Greek patients with retinal dystrophy

Smaragda Kamakari 1, Stavrenia Koukoula2, Vasiliki Kokkinou1, Lonnecke Haer-Wigman3, Ioannis Datseris4, Miltiadis Tsilimbaris5

1Ophthalmic Genetics Unit, Athens, Greece, 2Ophthalmica Institute of Ophthalmology and Microsurgery, Thessaloniki, Greece, 3Department of Human Genetics, Donders Centre for Neuroscience, Radbound University Nijmegen Medical Centre, Nijmegen, Netherlands, 4Department of Retinal Disorders, OMMA, Ophthalmological Institute of Athens, Athens, Greece, 5Department of Ophthalmology, University of Crete School of Medicine, Heraklion, Greece.

Purpose: Inherited Retinal Dystrophies (IRDs) are characterized by clinical variability and genetic heterogeneity. The aim of this study was to molecularly diagnose 120 Greek patients with different forms of IRDs.

Materials and Methods: 120 unrelated Greek patients were analyzed by Next Generation Sequencing (NGS), 13 and 107 of them using a 105 retinal and a 287 ophthalmic gene panel, respectively as described (Ellingford JM et al. J Med Genet 2016, Haer-Wigman L et al. Eur J Hum Genet. 2017). Additional analysis methods were used (Sanger, MLPA, array-CGH) in 6 cases.

Results: Potentially pathogenic mutations were detected in 47 retinal dystrophy genes including ABCA4, PRPF31, SPATA7, MERTK, FAM161A, CDHR1, USH2A, CNGB1, PROM1, RPGR and RP2 genes. The detection mutation rates were 46.15% (6/13) and 79.4% (85/107) for the 105 and 287 gene panels, respectively. These mutation rates were achieved using complementary methods in 6 cases. Final diagnoses included retinitis pigmentosa, Usher syndrome, cone-rod dystrophy and Leber congenital amaurosis and two rare cases of Knobloch and Oliver-McFarlane syndromes due to mutations in the COL18A1 and PNPLA6 genes, respectively.

Conclusions: This is the first systematic investigation of the molecular identity of 120 Greek patients with various subforms of IRDs by NGS and complementary methods leading to an overall mutation rate of 75.8%. A plethora of novel mutations was documented further expanding the genetic heterogeneity. The molecular identification established the complete diagnosis of the patients thus contributing to family making decision, prognosis and candidacy to current and future treatments.

S. Kamakari: None. S. Koukoula: None. V. Kokkinou: None. L. Haer-Wigman: None. I. Datseris: None. M. Tsilimbaris: None.

P02.064.A Whole locus sequencing identifies a prevalent founder deep intronic RPGRIP1 pathologic variant in the French Leber congenital amaurosis cohort

Isabelle Perrault 1, Sylvain Hanein2, Xavier Gérard1, Nelson Mounguengue1, Ryme Bouyakoub1, Mohammed Zarhate3, Cécile Fourrage4, Fabienne Jabot-Hanin4, Béatrice Bocquet5, Isabelle Meunier5, Xavier Zanlonghi6, Josseline Kaplan1, Jean-Michel Rozet1

1Laboratory of Genetics in Ophthalmology (LGO), INSERM UMR1163, Institute of Genetic Diseases, Imagine and Paris Descartes University, Paris, France, 2Translational Genetics, Institute of Genetic Diseases, Imagine and Paris Descartes University, INSERM UMR1163, PARIS, France, 3Genomics Platform, Institute of Genetic Diseases, Imagine and Paris Descartes University, Paris, France, 4Bioinformatic Platform. Institute of Genetic Diseases, Imagine and Paris Descartes University, Paris, France, 5Centre de Référence des Affections Sensorielles Génétiques, Institut des Neurosciences de Montpellier, CHU-Saint Eloi Montpellier, Montpellier, France, 6Eye Clinic Jules Verne, Nantes, France.

Leber congenital amaurosis (LCA) encompasses the earliest and most severe retinal dystrophies and can occur as a non-syndromic or a syndromic disease. Molecular diagnosis in LCA is of particular importance in clinical decision-making and patient care since it can provide ocular and extraocular prognostics and identify patients eligible to developing gene-specific therapies. Routine high-throughput molecular testing in LCA yields 70%-80% of genetic diagnosis. In this study, we aimed to investigate the non-coding regions of one non-syndromic LCA gene, RPGRIP1, in a series of six families displaying one single disease allele after a gene-panel screen of 722 LCA families which identified 26 biallelic RPGRIP1 families. Using trio-based high-throughput whole locus sequencing (WLS) for second disease alleles, we identified a founder deep intronic mutation (NM_020366.3:c.1468-128T>G) in 3/6 families. We employed Sanger sequencing to search for the pathologic variant in unresolved LCA cases (106/722) and identified three additional families (2 homozygous and 1 compound heterozygous with the c.930+77A>G deep intronic change). This makes the c.1468-128T>G the most frequent RPGRIP1 disease allele (8/60, 13%) in our cohort. Studying patient lymphoblasts, we show that the pathologic variant creates a donor splice-site and leads to the insertion of the pseudo-exon in the mRNA, which we were able to hamper using splice-switching antisense oligonucleotides (AONs), paving the way to therapies.This work was supported by grants from Retina France, UNADEV-AVIESAN ITMO MNP (R16073KS) and VISIO; J.-M.R. is member of ERN-EYE project (739534-‘ERN-EYE).

I. Perrault: None. S. Hanein: None. X. Gérard: None. N. Mounguengue: None. R. Bouyakoub: None. M. Zarhate: None. C. Fourrage: None. F. Jabot-Hanin: None. B. Bocquet: None. I. Meunier: None. X. Zanlonghi: None. J. Kaplan: None. J. Rozet: None.

P02.065.B Genetic study of Italian families affected by small fibre neuropathy identified variants in predisposing pain phenotype

Kaalindi Misra 1, Silvia Santoro1, Andrea Zauli1, Margherita Marchi2, Erika Salvi2, Raffaella Lombardi2, Daniele Cazzato2, Filippo Martinelli Boneschi3,4, Massimo Filippi5,6,7,8, Federica Esposito1,5, Giuseppe Pinter Lauria2,9

1Laboratory of Human Genetics of Neurological Disorders, IRCCS San Raffaele Scientific Institute, Milan, Italy, 2Neuroalgology Unit, Foundation IRCCS Carlo Besta Neurological Institute, Milan, Italy, 3Department of Biomedical Sciences for Health, University of Milan, Milan, Italy, 4Neurology Unit, Foundation IRCCS Cà Granda Ospedale Maggiore Policlinico, Milan, Italy, 5Neurology and Neurorehabilitation Unit, IRCCS San Raffaele Scientific Institute, Milan, Italy, 6Vita-Salute San Raffaele University, Milano, Italy, 7Neurophysiology Unit, IRCCS San Raffaele Scientific Institute, Milan, Italy, 8Neuroimaging Research Unit, Institute of Experimental Neurology, Division of Neuroscience, IRCCS San Raffaele Scientific Institute, Milan, Italy, 9Department of Biomedical and Clinical Sciences "Luigi Sacco", University of Milan, Milan, Italy.

Peripheral Neuropathy (PN) affects 2.4% of people and almost 50% of general population is known to have pain-related symptoms. Genetic studies in painful PN (PPN) revealed that Voltage Gated Sodium Channels (VGSCs) genes are involved in pain amplification. Here we aimed to broaden the genetic aspect of PPN by using whole exome sequencing (WES).

Six families with PPN were selected having at least one affected member, positive neurological examination and pain questionnaire result with numerical rating score >=4. Variants were filtered with manually curated gene panel, allele frequency (AF) and computational predictors. Segregation causative/protective models were applied according to pedigree and sharing models were applied after grouping probands of each family.

According to segregation causative and protective model, we found 129 and 112 variants respectively (AF<=10%) across families. Among genes shared between two families with causative approach, variants were observed in SCN9A, SV2C and DST, whereas protective variants in TRPM2 and LRP1. In shared model, we identified 21 variants and 53 genes shared across >=3 probands. Among shared genes with predicted high-impact variants in probands were observed in SCN9A, SCN7A, P2RY4, P2RX7, TRPV4 and TRPM1.

WES approach appears powerful in mutation detection and in revealing new genotype-phenotype association. In addition to VGSCs, other gene families including Transient Receptor Potential and Purinergic Receptor seem to play a role in pain modulation. The same approach will be replicated on new families already sequenced before proceeding with ad hoc functional experiments to deepen the role of genes in painful phenotype.

K. Misra: None. S. Santoro: None. A. Zauli: None. M. Marchi: None. E. Salvi: None. R. Lombardi: None. D. Cazzato: None. F.M. Boneschi: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Modest; Merck, Teva Pharmaceutical Industries, Italian Ministry of Health, Fondazione Italiana Sclerosi Multipla, Fondazione Cariplo. D. Speakers Bureau/Honoraria (speakers bureau, symposia, and expert witness); Modest; Medday, Biogen Idec, Merck-Serono, Excemed, Roche, Sanofi Genzyme, Teva Pharmaceutical Industries. F. Consultant/Advisory Board; Modest; Medday, Biogen Idec, Merck-Serono, Excemed, Roche, Sanofi Genzyme, Teva Pharmaceutical Industries. M. Filippi: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Significant; Roche, Biogen Idec, Merck-Serono, Novartis, Teva Pharmaceutical Industries. D. Speakers Bureau/Honoraria (speakers bureau, symposia, and expert witness); Modest; Bayer, Biogen Idec, Merck-Serono, Novartis, Roche, Sanofi Genzyme, Takeda, Teva Pharmaceutical Industries. F. Consultant/Advisory Board; Modest; Bayer, Biogen Idec, Merck-Serono, Novartis, Roche, Sanofi Genzyme, Takeda, Teva Pharmaceutical Industries. F. Esposito: D. Speakers Bureau/Honoraria (speakers bureau, symposia, and expert witness); Modest; Novartis, Sanofi Genzyme, Almirall, Merck-Serono. F. Consultant/Advisory Board; Modest; Novartis, Sanofi Genzyme, Almirall, Merck-Serono. G.P. Lauria: None.

P02.066.C Human knockouts of olfactory receptors genes and smell perception impairment in a large Italian cohort

Paola Tesolin 1, Maria Pina Concas2, Massimiliano Cocca2, Margherita Francescatto1, Agnese Luglio1, Beatrice Spedicati1, Agense Feresin1, Anna Morgan2, Paolo Gasparini2,1, Giorgia Girotto2,1

1University of Trieste, Trieste, Italy, 2Institute for Maternal and Child Health – IRCCS “Burlo Garofolo”, Trieste, Italy.

Genetic variations across Olfactory Receptors (OR) genes influence the diversity of odorant sensitivity among individuals. Nevertheless, there is still a lack of knowledge regarding the genetic bases of smell performance. Whole-genome sequencing and smell phenotypes data of 218 Italian individuals allowed the identification of 41 natural OR knockouts (KO) (i.e., genes carrying biallelic loss of function variants). In detail, the following steps have been performed:1. recursive partitioning analysis to evaluate the effect of OR-KO genes’ burden on the smell perception (measured by the number of errors in the Sniffin Sticks test),2. evaluation of the expression of these genes in human and mouse tissues using publicly available data,3. estimation of the presence of organ-related diseases in Human KO (HKO) individuals for OR expressed in non-olfactory tissues (Fisher test). Regarding (1), ageing and the burden of OR-KO led to a worsening of smell perception (p-value <0.05). In particular, the effect of the OR-KO burden was higher in younger individuals (aged≤57). With respect to (2), 33/41 OR genes have been detected in the human olfactory system (OS) and 27 in other tissues, while among the 60 putative ORs mouse homologues, 58 were expressed in the OS and 37 in other tissues. Finally, (3) 14 pathologies resulted in being more frequent in OR-HKO individuals (p-value < 0.01). Our work confirms the predominant role of age in worsening smell perception and highlights, for the first time, the role of the burden of OR-KO genes.

P. Tesolin: None. M. Concas: None. M. Cocca: None. M. Francescatto: None. A. Luglio: None. B. Spedicati: None. A. Feresin: None. A. Morgan: None. P. Gasparini: None. G. Girotto: None.

P02.067.D Insights into the pathogenesis of Stargardt disease associated with splicing mutation c.5714+5G&gtA in ABCA4 gene

Jana Sajovic 1, Andrej Meglič1, Zelia Corradi2,3, Mubeen Khan2,3,4, Frans F.M. Cremers2,3, Claire-Marie Dhaenens2,5, Martina Jarc Vidmar1, Damjan Glavač6, Aleš Maver7, Marija Volk7, Borut Peterlin7, Marko Hawlina1, Ana Fakin1

1Eye Hospital, University Medical Centre Ljubljana, Ljubljana, Slovenia, 2Department of Human Genetics, Radboud University Medical Center, Nijmegen, Netherlands, 3Donders Institute for Brain, Cognition and Behaviour, Radboud University, Nijmegen, Netherlands, 4Department of Pathology and Medical Biology, University Medical Center Groningen, Groningen, Netherlands, 5Univ. Lille, Inserm, CHU Lille, U1172-LilNCog-Lille Neuroscience & Cognition, Lille, France, 6Department of Molecular Genetics, Institute of Pathology, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia, 7Clinical Institute of Medical Genetics, University Medical Centre Ljubljana, Ljubljana, Slovenia.

Introduction: >1200 ABCA4 variants cause Stargardt disease making genotype-phenotype correlations difficult. Approximately 15% of Slovenian Stargardt patients harbour c.5714+5G>A which allowed phenotypic specification of this variant.

Methods: Sixteen patients with different genotypes were recruited. Group 1 harboured c.5714+5G>A in trans with a null mutation (N = 6) and group 2 had two null mutations (N = 10). Correlation between the degree of retinal pigment epithelium (RPE) atrophy (represented by the area of decreased autofluorescence on fundus imaging) and loss of photoreceptors (represented by outer nuclear layer (ONL) thickness on optical coherence tomography (OCT) and pattern electroretinography (PERG) amplitudes) was compared between groups. Effect of c.5714+5G>A on RNA splicing was analyzed using reverse transcription (RT)-PCR of mRNA isolated from patient-derived photoreceptor progenitor cells (PPCs).

Results: RT-PCR of mRNA from PPCs from a patient carrying c.4539+1G>T and c.5714+5G>A using primers in exons 38 and 44 showed a major normal product and minor exon 40 and exon 39/40 deletion products. Statistical analysis showed that for a similar RPE atrophy area, patients in Group 1 had significantly better structural (thicker ONL) and functional (higher PERG amplitudes) preservation of photoreceptors (multiple linear regression, p < 0.01). Preserved photoreceptors were seen above preserved RPE on OCT in Group 1.

Conclusions: Patients harbouring c.5714+5G>A exhibit distinctly different phenotype than patients carrying two null mutations, characterized by less photoreceptor loss at comparable degrees of RPE loss. We hypothesize that remaining ABCA4 function originating from the retained normally spliced product spares photoreceptors from early degeneration and shifts the pathogenesis to a primarily RPE disease.

J. Sajovic: None. A. Meglič: None. Z. Corradi: None. M. Khan: None. F. Cremers: None. C. Dhaenens: None. M. Jarc Vidmar: None. D. Glavač: None. A. Maver: None. M. Volk: None. B. Peterlin: None. M. Hawlina: None. A. Fakin: None.

P02.068.A Biallelic pathogenic variants in COL9A3 confirm autosomal recessive stickler syndrome

Aboulfazl Rad 1, Maryam Najafi2, Fatemeh Suri3, Stephen Loum1, Ehsan Ghayoor Karimiani4, David Murphy5, Mohammad Doosti4, Narsis Daftarian6, Paria Najarzadeh Torbati4, Afrooz Moghaddasi3, Hamid Ahmadieh3, Mohsen Rajati7, Narges Hashemi8, Barbara Vona1, Miriam Schmidts9

1Department of Otorhinolaryngology, Head and Neck Surgery, Tübingen, Germany, 23Pediatric Genetics Divison, Center for Pediatrics and Adolescent Medicine, University Hospital Freiburg, Freiburg University Faculty of Medicine, Mathildenstrasse 1, 79106 Freiburg, Germany, Freiburg, Germany, 3Ophthalmic Research Center, Research Institute for Ophthalmology and Vision Science, Shahid Beheshti University of Medical Sciences, Tehran, Iran, Tehran, Iran, Islamic Republic of, 4Department of Molecular Genetics, Next Generation Genetic Polyclinic, Mashhad 009851, Iran, Mashhad, Iran, Islamic Republic of, 5Department of Clinical and Movement Neurosciences, UCL Queen Square Institute of Neurology, University College London, United Kingdom, London, United Kingdom, 6Ocular Tissue Engineering Research Center, Research Institute for Ophthalmology and Vision Science, Shahid Beheshti University of Medical Sciences, Tehran, Iran, Tehran, Iran, Islamic Republic of, 7Associate professor of Otorhinolaryngology, Ghaem Hospital, Sinus and Surgical Endoscopic Research Center, Department of Otorhinolaryngology, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran, Mashhad, Iran, Islamic Republic of, 8Department of Pediatric Neurology, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran, Mashhad, Iran, Islamic Republic of, 9Pediatric Genetics Divison, Center for Pediatrics and Adolescent Medicine, University Hospital Freiburg, Freiburg University Faculty of Medicine, Mathildenstrasse 1, 79106 Freiburg, Germany, Freiburg, Germany.

Introduction: Stickler syndrome (STL) is a clinically and molecularly heterogeneous connective tissue disorder that includes ocular impairment, hearing loss, joint and craniofacial abnormalities. Pathogenic variants occurring in a variety of genes cause STL, mainly inherited in an autosomal dominant fashion. Autosomal recessive STL is ultra-rare with only a few cases reported to date, including three families with biallelic COL9A3 variants. Here, we report three unrelated families clinically diagnosed with STL who present novel biallelic loss of function variants in COL9A3.

Materials and Methods: Exome sequencing (ES) was employed to sequence the DNA of probands from three unrelated Iranian families. Conventional PCR and Sanger sequencing were performed to confirm segregation of the candidate variants in accessible family members.

Results: Our data revealed three novel loss of function variants in COL9A3 in three unrelated families. All affected individuals shared moderate- to high-myopia and moderate to severe sensorineural hearing loss. The other clinical observations including hypermobility, mild spondyloepiphyseal dysplasia, midface hypoplasia, depressed nasal bridge, anteverted nares, bifid uvula, cleft hard palate, and micrognathia were slightly different among cases. The parents in all three families did not have any STL-associated phenotypes.

Conclusions: Our report substantially expands the molecular genetic basis of autosomal recessive STL and confirms the clinical phenotypes of COL9A3-associated autosomal recessive STL.Keywords: Stickler syndrome, COL9A3, Autosomal recessive

A. Rad: None. M. Najafi: None. F. Suri: None. S. Loum: None. E. Ghayoor Karimiani: None. D. Murphy: None. M. Doosti: None. N. Daftarian: None. P. Najarzadeh Torbati: None. A. Moghaddasi: None. H. Ahmadieh: None. M. Rajati: None. N. Hashemi: None. B. Vona: None. M. Schmidts: None.

P02.069.B Clinical and molecular revaluation yield a 52% of characterization in syndromic retinal diseases

Irene Perea-Romero 1,2, Fiona Blanco-Kelly1,2, Iker Sanchez-Navarro1, Isabel Lorda-Sanchez1,2, Saoud Tahsin-Swafiri1,2, Almudena Avila-Fernandez1,2, Inmaculada Martin-Merida1,2, Maria Jose Trujillo-Tiebas1,2, Rosario Lopez-Rodriguez1,2, Ionut Florin Iancu1,2, Raquel Romero1,2, Mathieu Quinodoz3,4,5, Pablo Minguez1,2, Marta Corton1,2, Carlo Rivolta3,4,5, Carmen Ayuso1,2

1Health Research Institute-Fundación Jiménez Díaz University Hospital, Universidad Autónoma de Madrid (IIS-FJD, UAM), Madrid, Spain, 2Center for Biomedical Network Research on Rare Diseases (CIBERER), Instituto de Salud Carlos III, Madrid, Spain, 3Institute of Molecular and Clinical Ophthalmology Basel (IOB), Basel, Switzerland, 4Department of Ophthalmology, University of Basel, Basel, Switzerland, 5Department of Genetics and Genome Biology, University of Leicester, Leicester, United Kingdom.

Introduction: Syndromic retinal diseases (SRD) are a group of rare and complex inherited systemic disorders, characterized by a challenging molecular study and clinical management.

Materials and Methods: A cohort study was performed on 100 index cases with an a priori diagnosis of non-Usher SRD, using available clinical and familiar data and HPO (Human Phenotype Ontology) annotation, so every case was classified into 7 different clinical categories according to the most prominent symptoms. Over the years, diverse molecular and bioinformatic methods have been used. Accordingly, cases were classified into 3 subgroups depending on the previous molecular technique utilized, determining the new approach to perform.

Results: After the phenotypic classification, the most common SRD were ciliopathies (36%). A characterization rate of 52% was obtained, being 6 cases incompletely characterized with a gene that partially explained the phenotype. An increased characterization rate was achieved taking the naïve (67.4%) or the ciliopathies (66.7%) subgroups independently. Our results suggest that customized panels and clinical exome would be the first-tier approach in the naïve cases, whereas whole-exome sequencing would be in the re-studied and reanalysed. Due to the causative gene found, 27.3% of the completely characterized cases were reclassified into another clinical subgroup.

Conclusions. Our study provides an example of SRD in the daily clinical practice and the importance of a thorough clinical analysis and election of the molecular approach, in order to solve these complex cases and elucidate new possible phenotype-genotype associations.

Funding: ISCIII (PI16/00425; PI19/00321; FI17/00192), CIBERER (06/07/0036), University Chair UAM-IIS-FJD Genomic Medicine, RAREGENOMICS-CM

I. Perea-Romero: None. F. Blanco-Kelly: None. I. Sanchez-Navarro: None. I. Lorda-Sanchez: None. S. Tahsin-Swafiri: None. A. Avila-Fernandez: None. I. Martin-Merida: None. M.J. Trujillo-Tiebas: None. R. Lopez-Rodriguez: None. I.F. Iancu: None. R. Romero: None. M. Quinodoz: None. P. Minguez: None. M. Corton: None. C. Rivolta: None. C. Ayuso: None.

P02.070.C Could TGFBI-related corneal dystrophies be mimicked in zebrafish via CRISPR/Cas9-mediated hot spot arginine variations?

Fulya Yaylacioglu Tuncay 1, Beril Talim2, Pervin Rukiye Dinçer3

1Department of Medical Biology, Gulhane Medical Faculty, University of Health Sciences, Ankara, Turkey, 2Department of Pediatrics, Faculty of Medicine, Hacettepe University, Ankara, Turkey, 3Department of Medical Biology,Faculty of Medicine, Hacettepe University, Ankara, Turkey.

Introduction: TGFBI-related corneal dystrophies are characterized by hyaline or amyloid deposition in the cornea due to missense mutations of TGFBI. The protein product of this gene (TGFBIp) was shown in the cornea of zebrafish. Mutations of TGFBI affecting arginine residue on the 124th position were reported as one of the hot spots for this group of corneal dystrophies. This arginine residue was also conserved in zebrafish. Therefore, we aimed to make an in-frame change in zebrafish genome affecting this arginine residue by using CRISPR/Cas9 and then observe if any accumulation would occur in the cornea of adult zebrafish.

Materials and Methods: For the in-frame change in the target region nine injection groups were planned. The mixtures consisting of sgRNAs, Cas9 mRNA/protein with or without donor DNA were injected to one-cell stage embryos in each group (n = 100). T7 endonuclease assay, restriction fragment length polymorphism assay and Sanger sequencing were used for genotyping in the injection groups.

Results: The variation p. Ser115_Arg117delinsLeu (c. 347_353delinsT) was detected in one of the injection groups in F1 embryos. This variation deleted the target arginine in TGFBIp without causing frameshift. Three-months old and 1-year old heterozygote zebrafish were euthanized, and corneas were examined under the light microscope. No deposits could be detected with hematoxylin-eosin, Masson’s trichrome and congo red staining.

Conclusion: This is the first study in literature that succeeded to make an in-frame variation effecting the hot spot arginine residue of TGFBIp in zebrafish. However, this success could not result in deposit formation in zebrafish cornea.

F. Yaylacioglu Tuncay: None. B. Talim: None. P.R. Dinçer: None.

P02.071.D An USH2A founder mutation is the cause of Usher syndrome type 2 in Sardinia

Vincenzo Rallo 1,2, Rita Serra1,2, Maristella Steri2, Stefania Olla2, Michele Marongiu2, Edoardo Fiorillo2, Antonio Pinna1, Francesco Cucca1, Andrea Angius1,2

1University of Sassari, Sassari, Italy, 2Institute for Genetic and Biomedical Research (IRGB), Monserrato (Cagliari), Italy.

Introduction: Usher syndrome (USH) is the most common cause of combined sight and hearing loss, responsible for half of deaf-blindness cases. USH has divided into three types: USH1, characterized by severe bilateral hearing loss and early retinitis pigmentosa (RP); USH2, distinguished by moderate hearing loss and RP; USH3, showing progressive hearing loss, vestibular dysfunction and RP. USH is associated with 19 loci, with 16 causative genes identified. Here, we describe the clinical findings and molecular analysis of 3 USH-affected unrelated families living in the island of Sardinia (Italy).

Materials and Methods: We investigated all the members of 3 families in which 9 patients showed both sensorineural hearing loss and RP. All individuals underwent a complete ophthalmic examination and vestibular medical tests. Whole-genome sequencing data were available for genetic analysis.

Results: Clinical hypothesis indicated a suspected USH2 syndrome. We identified a single missense causal variant in the USH2A gene, in homozygous status in all patients and heterozygous in unaffected parents. Mutation-related haplotype reconstruction revealed a founder effect. To understand if this event was restricted to a geographical area or whole island, we analysed about 3500 Sardinians, revealing a frequency of about 2% heterozygotes distributed overall in Sardinia.

Conclusions: By using our approach, we were able to describe the first case of USH syndrome, its incidence and distribution in Sardinia. Thus, we are able to provide an attractive perspective for the feasibility of carrier status screening, possible genetic counselling at the base for prevention and early treatment strategies of this hereditary syndrome.

V. Rallo: None. R. Serra: None. M. Steri: None. S. Olla: None. M. Marongiu: None. E. Fiorillo: None. A. Pinna: None. F. Cucca: None. A. Angius: None.

P03 Internal Organs & Endocrinology (Lung, Kidney, Liver, Gastrointestinal)

P03.001.A Molecular analysis of PKD1 and PKD2 genes: a key for achieving a great diagnosis rate in autosomal dominant polycystic kidney disease

Elena Mesa-Rísquez, Alfonso Andújar, Isabel Sánchez-Guiu, Anna Baquero-Vaquer, Tania Otero-Rodríguez, María Dolores Ruiz, Roser Martínez-Rubio, Amparo Girós, María Sánchez-Ibáñez, Vanesa Felipe-Ponce, Dan Diego-Álvarez, Rosario Ferrer-Avargues, Uxía Esperón, Natalí Riva, Bárbara Masotto, Yolanda Moreno-Sáez, José Leonardo Díaz, Jaime García, Carmen Collado, María Dolores Oliver, Norma Aliaga, Angela Gaspar, Laura Cano, Ana Perpiñán, Nuria Serrano, Clara Casañ, Celia Buades-Gomis, Alejandro Romera-López, Mayte Gil-Borja, Sonia Santillán, Christian Martín Moya

Ascires Sistemas Genómicos, Paterna (Valencia), Spain.

Autosomal dominant polycystic kidney disease (ADPKD) is the most common inherited kidney disease, mainly caused by PKD1 and PKD2 genes. PKD1 gene analysis is technically complex due to its large size and the presence of several pseudogenes. The aim of this work is to present the diagnostic results of a ADPKD cohort.

143 ADPKD cases were included, of which 71.6% had family history of ADPKD. Patients were screened for PKD1 and PKD2 genes using Long Range PCR and Sanger sequencing or Nextera™ Technology for NGS. In some patients, a MLPA study was performed if a negative result was obtained from sequencing.

Genetic screening revealed 88 diagnosed patients and 55 undiagnosed, 27 of whom were carriers of Variants of Uncertain Significance (VUS) and the remaining 28 were negative. Disease-causing variants were either found in PKD1 (65 cases) or PKD2 (23 cases). Among the disease-causing variants in both genes, 25% of them were novel variants. Diagnostic variant effects were mainly nonsense (37.5%) and small deletions (26.14%). Other types of identified variants included small insertions (12.5%), missense (11.36%), splicing (7.95%), gross deletions (3.41%) and small indels (1.14%).

Our rate of 61.54% positive genetic diagnosis highlights the effectiveness of molecular genetic analysis as a powerful tool for achieving a diagnosis in ADPKD patients. NGS allows PKD1 and PKD2 simultaneous analysis, reducing processing time and costs. Extending the genetic analysis by exome directed panels to related genes as GANAB, DNAJB11 or performing cosegregation and functional studies of VUS could lead to a higher diagnostic rate.

E. Mesa-Rísquez: None. A. Andújar: None. I. Sánchez-Guiu: None. A. Baquero-Vaquer: None. T. Otero-Rodríguez: None. M.D. Ruiz: None. R. Martínez-Rubio: None. A. Girós: None. M. Sánchez-Ibáñez: None. V. Felipe-Ponce: None. D. Diego-Álvarez: None. R. Ferrer-Avargues: None. U. Esperón: None. N. Riva: None. B. Masotto: None. Y. Moreno-Sáez: None. J.L. Díaz: None. J. García: None. C. Collado: None. M.D. Oliver: None. N. Aliaga: None. A. Gaspar: None. L. Cano: None. A. Perpiñán: None. N. Serrano: None. C. Casañ: None. C. Buades-Gomis: None. A. Romera-López: None. M. Gil-Borja: None. S. Santillán: None. C.M. Moya: None.

P03.003.C The mild effect of the COL4A5 variant p.Gly624Asp in a group of 15 Polish patients with Alport syndrome

Paulina Halat-Wolska 1, Elżbieta Ciara1, Lukasz Obrycki2, Katarzyna Gadomska-Prokop2, Dorota Piekutowska-Abramczuk1, Joanna Kosińska3, Małgorzata Rydzanicz3, Piotr Stawiński3,4, Beata Chałupczyńska1, Kamila Frączak1, Marzena Gawlik1, Dorota Jurkiewicz1, Paweł Kowalski1, Magdalena Pelc1, Dorota Siestrzykowska1, Szymon Szyszkowski1, Dorota Wicher1, Ryszard Grenda2, Krystyna Chrzanowska1, Rafał Płoski3, Mieczysław Litwin2

1Department of Medical Genetics, The Children’s Memorial Health Institute, Warsaw, Poland, 2Department of Nephrology, The Children’s Memorial Health Institute, Warsaw, Poland, 3Department of Medical Genetics, Warsaw Medical University, Warsaw, Poland, 4Department of Genetics, Institute of Physiology and Pathology of Hearing, Warsaw, Poland.

Introduction: Alport syndrome (AS) is a clinically and genetically heterogeneous nephropathy caused by pathogenic variants in COL4A3, COL4A4 or COL4A5. About 85% of AS patients have X-linked inheritance, which causes more severe phenotype in males, whereas in females, penetrance depends on X-chromosome inactivation pattern. Originating in the Middle Ages COL4A5 variant c.1871G>A p.Gly624Asp is predominant in Central/East Europe and mostly gives mild AS symptoms.

Materials and Methods: Next-generation sequencing analysis of glomerulopathy and chronic kidney disease related genes panel was performed in Polish patients with suspected AS.

Results: In 65 patients clinical diagnosis was confirmed at the molecular level. The most frequent was X-linked AS caused by 32 different COL4A5 variants (75%). A recurrent COL4A5 variant p.Gly624Asp in 15 patients (nine males and six females) was identified. Additionally, four patients with this substitution had pathogenic variant in COL4A3, HNF1B or MYH9. The clinical course of patients with this genotype was mostly milder than observed in individuals with other COL4A5 variants. They presented only haematuria with or without proteinuria. Of these, two patients also had hearing impairment: male with only p.Gly624Asp variant and female with additional variant in COL4A3.

Conclusions: The results of this study broaden the genotypic spectrum of AS, which will facilitate future research on the genotype-phenotype correlations. Variant p.Gly624Asp in COL4A5 was predominant and accounted for 31% of X-linked AS in the study group. Observations of mild phenotype in our patients with this genotype relate to literature data. Partially supported: CMHI-M29/18

P. Halat-Wolska: None. E. Ciara: None. L. Obrycki: None. K. Gadomska-Prokop: None. D. Piekutowska-Abramczuk: None. J. Kosińska: None. M. Rydzanicz: None. P. Stawiński: None. B. Chałupczyńska: None. K. Frączak: None. M. Gawlik: None. D. Jurkiewicz: None. P. Kowalski: None. M. Pelc: None. D. Siestrzykowska: None. S. Szyszkowski: None. D. Wicher: None. R. Grenda: None. K. Chrzanowska: None. R. Płoski: None. M. Litwin: None.

P03.004.A Delineation of the phenotypic and genotypic spectrum of type-IV-collagen-related nephropathy - Alport syndrome and thin basement membrane nephropathy

Korbinian M. Riedhammer 1,2, Matthias C. Braunisch2, Jasmina Ćomić1, Adrian Lungu3, Jovana Putnik4, Gordana Miloševski-Lomić5, Michaela Gessner6, Nataša Stajić4, Ludwig Patzer7, Nora Emini8, Velibor Tasic8, Julia Hoefele1

1Institute of Human Genetics, Klinikum rechts der Isar, School of Medicine, Technical University of Munich, Munich, Germany, 2Department of Nephrology, Klinikum rechts der Isar, School of Medicine, Technical University of Munich, Munich, Germany, 3Pediatric Nephrology Department, Fundeni Clinical Institute, Bucharest, Romania, 4Institute for Mother and Child Health Care of Serbia "Dr Vukan Čupić", Department of Nephrology, Faculty of Medicine, University of Belgrade, Belgrade, Serbia, 5Department of Nephrology, University Children’s Hospital, Belgrade, Serbia, 6Department of General Pediatrics and Hematology/Oncology, Children’s University Hospital Tuebingen, Tuebingen, Germany, 7Children’s Hospital St. Elisabeth and St. Barbara, Halle (Saale), Germany, 8University Children’s Hospital, Medical School Skopje, Skopje, Macedonia, The Former Yugoslav Republic of.

Introduction: “Type-IV-collagen-related nephropathy” describes a spectrum of hereditary hematuric diseases comprising Alport syndrome (AS) and (milder) thin basement membrane nephropathy (TBMN). AS results from biallelic causative variants in COL4A3/4 (autosomal recessive AS) and hemizygous causative variants in COL4A5 (X-linked AS). Females with heterozygous causative variants in COL4A5 show a variable phenotype. Monoallelic causative variants in COL4A3/4 are identified in TBMN. The designation “autosomal dominant AS” for heterozygous carriers of causative variants in COL4A3/4 is contested.

Methods: 64 index patients, in whom exome sequencing had been performed, were assigned to a TBMN (18 cases) or AS (46 cases) subgroup based on clinical/histopathologic presentation and family history. Phenotypic and genotypic features were compared.

Results: Diagnostic yield of type-IV-collagen-related nephropathy classified as AS compared to TBMN was significantly different (65% vs. 28%; p = 0.01). One case, clinically classified as TBMN, had Dent disease genetically. The further solved TBMN cases carried heterozygous causative variants in COL4A3, COL4A4 or COL4A5 (female). Median age at first manifestation was significantly lower in AS compared to TBMN cases (5.5 years vs. 16.0 years; p = 0.001). TBMN cases had no extrarenal manifestations, in contrast to 28% of AS cases (p = 0.01). 39% of TBMN cases had a reported family history in contrast to 78% in AS cases (p = 0.006).

Conclusions: This study delineates the phenotypic and genotypic spectrum of type-IV-collagen-related nephropathy. Importantly, it takes the perspective of the clinician who has to integrate phenotypic and anamnestic data to assess the possibility of a hereditary disease.

K.M. Riedhammer: None. M.C. Braunisch: None. J. Ćomić: None. A. Lungu: None. J. Putnik: None. G. Miloševski-Lomić: None. M. Gessner: None. N. Stajić: None. L. Patzer: None. N. Emini: None. V. Tasic: None. J. Hoefele: None.

P03.005.A Systematic variant reinterpretation in patients with type-IV-collagen-related nephropathy (Alport syndrome/thin basement membrane nephropathy) reveals a high rate of ambiguous results

Korbinian M. Riedhammer1,2, Patrick Richthammer 1, Dominik S. Westphal1, Jasmina Ćomić1, Roman Günthner2, Matthias C. Braunisch2, Sabine Rath3, Anja K. Büscher4, Hanns-Georg Klein3, Stefanie Weber5, Julia Hoefele1

1Institute of Human Genetics, Klinikum rechts der Isar, Technical University of Munich, School of Medicine, Munich, Germany, 2Department of Nephrology, Klinikum rechts der Isar, Technical University of Munich, School of Medicine, Munich, Germany, 3Center for Human Genetics and Laboratory Diagnostics Dr. Klein, Dr. Rost and Colleagues, Martinsried, Germany, 4Pediatric Nephrology, Pediatrics II, University Children’s Hospital Essen, Essen, Germany, 5University Children’s Hospital, Marburg, Germany.

Introduction: Type-IV-collagen-related nephropathy covers a spectrum of hereditary hematuric diseases (thin basement membrane nephropathy, TBMN; Alport syndrome, AS). Whereas AS results from disease-causing variants in COL4A3-5 (autosomal recessive and X-linked AS), monoallelic disease-causing variants in COL4A3/4 are associated with TBMN.

Methods: Variants of 96 index cases reported between 2009 and 2014 with the clinical tentative diagnosis AS (77/96), TBMN (13/96) or unclear determination of AS/TBMN (6/96) were meticulously reinterpreted based on ACMG criteria and current amendments. Monoallelic (likely) pathogenic variants in COL4A3/4 in an AS case were not considered as diagnostic, as the designation “autosomal dominant AS” is contested. 6 cases had to be excluded from further analysis due to limited data.

Results: In total, 84 variants (allocated to 96 cases) and their genotypes have been reassessed (COL4A3: 21/84, COL4A4: 18/84, COL4A5: 45/84). 64/90 cases included in the further analysis could be classified as solved (original report: 83/90; p < 0.001, Fisher exact test). 26/90 were classified as “not solved“ (original report: 7/90), due to inconclusive results on variant (11/90), on genotype (12/90) or both variant and genotype level (3/90).

Conclusions: Transparent and coherent variant-/genotype-interpretation enables physicians to diagnose hereditary diseases and allows them to provide patients with well-founded information concerning prognosis and recurrence risk. This is especially true for type-IV-collagen-related nephropathy, covering an intricate phenotypic and genotypic spectrum. Since reassessment of variants/genotypes in this study showed a significant reduction in genetic diagnoses, reports on type-IV-collagen-related nephropathy obtained in the pre-ACMG era should be critically evaluated.

K.M. Riedhammer: None. P. Richthammer: None. D.S. Westphal: None. J. Ćomić: None. R. Günthner: None. M.C. Braunisch: None. S. Rath: None. A.K. Büscher: None. H. Klein: None. S. Weber: None. J. Hoefele: None.

P03.006.B Impact of human genetic variants on C-Reactive Protein levels and acute appendicitis

Isis Ricaño-Ponce 1, Toon Peeters1,2,3, Vasiliki Matzaraki1, Mihai Netea1,4, Inge Gyssens1,2,3, Vinod Kumar1,5

1Department of Internal Medicine and Radboud Center for Infectious Diseases, Radboud University Medical Center, NIJMEGEN, Netherlands, 2Department of Infectious Diseases & Immunity, Jessa Hospital, Hasselt, Belgium, 3Faculty of Medicine and Life Sciences, Hasselt University, Hasselt, Belgium, 4Human Genomics Laboratory, Craiova University of Medicine and Pharmacy, Craiova, Romania, 5University of Groningen, University Medical Center Groningen, Department of Genetics, Groningen, Netherlands.

Introduction: Acute appendicitis is one of the most common abdominal emergencies worldwide. Both environmental and genetic factors contribute to disease. C-Reactive protein is (CRP) is one of the most important biomarkers in the diagnosis of acute appendicitis. CRP-levels are significantly affected by genetic variation. However, it remains unclear whether such genetic variation is causally related to appendicitis risk. Therefore, in this study, we investigate the causal relationship between SNPs associated with circulating CRP-levels and the risk and severity of acute appendicitis.

Materials and Methods: we measured CRP-levels in serum of appendectomy patients (n = 325) with appendicitis severity categorised as complicated/uncomplicated. We also performed GWAS in the same group of patients. We performed intersection, colocalization of our GWAS results with appendicitis and CRP-associated loci from the Pan-UKBB cohort. We employed pathway enrichment analyses and functional-genomics approach to prioritise genes and pathways.

Results: We observed a significant enrichment of CRP-loci in SNPs associated to appendicitis and complicated appendicitis. Among these shared loci, we characterise two top loci at 1q41 and at 8p23.1. The HLX gene at 1q41 is involved in blood cells differentiation, liver and gut organogenesis. The locus at 8p23.1 affects multiple genes that are overexpressed in appendix tissue from appendicitis patients. Using a functional genomics approach we provide genetic evidence for the involvement of interferon signalling pathway in complicated appendicitis.

Conclusions: Our results suggest shared genetic mechanisms between appendicitis and CRP-levels. By identifying new pathways, our study identified genetic causes for severity of appendicitis.

Project funded by Limburg Clinical Research Program.

I. Ricaño-Ponce: None. T. Peeters: None. V. Matzaraki: None. M. Netea: None. I. Gyssens: None. V. Kumar: None.

P03.008.D Novel CTLA4 heterozygous mutation in a family with type 2 autoimmune polyendocrine syndrome

Federico Romani 1, Emanuele Micaglio1, Filippo Martinelli Boneschi2, Giorgio Nevio Casari3, Sara Benedetti3, Paola Carrera3, Carlo Pappone1

1IRCCS Policlinico San Donato, San Donato Milanese, Italy, 2IRCCS Ospedale Maggiore Policlinico, Milan, Italy, 3IRCCS San Raffaele Hospital, Milan, Italy.

Autoimmune polyendocrine syndromes are an emerging field in current medicine due to both diagnostic and therapeutic improvements. Although generally accepted as complex traits, many genes play a pivotal role in both pathogenesis and prognosis of autoimmune polyendocrine syndromes. Among those genes, CTLA4 has been described as very important for autoimmunity against endocrine tissues since 2004. We describe a family in which a heterozygous variant in CTLA4 gene segregates with a type 2 autoimmune polyendocrine overt phenotype. Both the proband and his sister are indeed affected by recurrent oral candidiasis, hypothyroidism, type 1 diabetes mellitus and Addison disease. Interestingly, in both patients the onset of clinical picture happened with a recurrent oral candidiasis, in absence of genetic mutation in MODY genes. Proband and sister carry a c.475G>C heterozygous mutation of CTLA4 gene, with unknown inheritance. The bioinformatic analysis suggested a pathogenic effect likely related to the absence of the described mutation in healthy people. This conclusion is supported by the segregation with the pathologic phenotype in the examined family. To the best of our knowledge, this is the first case of CTLA4 heterozygous mutation in a clinically confirmed family affected by type 2 autoimmune polyendocrine syndrome. Although proband’s parents were not available for the genetic analysis, this case highlights the growing relevance of genetic testing for this condition. Moreover, our data suggest a possible new genotype - phenotype correlation requiring further studies in order to increase the diagnostic yield of genetic testing in autoimmune polyendocrine syndromes.

F. Romani: None. E. Micaglio: None. F. Martinelli Boneschi: None. G.N. Casari: None. S. Benedetti: None. P. Carrera: None. C. Pappone: None.

P03.009.A Association of KEAP1 polymorphism with autoimmune thyroiditis

Cristina Robles Lázaro1,2, María Ovejero-Sánchez 3,2,4, Nerea Gestoso-Uzal2,3,4, Carlos Gutiérrez-Cerrajero2,3,4, Paloma Martín-Bejarano Soto2,3,4, Pamela Vázquez-Cárdenas3,2,4, Ana Belén Herrero2,3,4, Rogelio González-Sarmiento2,3,4

1Endocrinology Service, University Hospital of Salamanca, Salamanca, Spain, 2Molecular Medicine Unit, Department of Medicine, University of Salamanca, Salamanca, Spain, 3Institute of Biomedical Research (IBSAL), Salamanca, Spain, 4Institute of Molecular and Cellular Biology of Cancer (IBMCC), University of Salamanca-CSIC, Salamanca, Spain.

Introduction: Autoimmune thyroiditis is a chronic inflammatory process characterized by the presence of glandular lymphocytic infiltration and thyroid specific antibodies. Some studies suggest that oxidative stress could be involved in the development of this disease. Keap1-Nrf2 pathway is the major regulator of cytoprotective responses to oxidative and electrophilic stress. Thus, the main aim of this study is to determine if Single Nucleotide Polymorphisms (SNPs) in these genes could be associated with a higher risk of developing autoimmune thyroiditis.

Material and methods: 202 autoimmune thyroiditis patients and 340 healthy subjects without previous history of autoimmune disease were recruited. Genotyping of SNPs in Keap1 (rs1048290 and rs11545829) and NRF2 (rs2706110) was performed using TaqMan allelic discrimination assays. Odd ratios and 95% confidence intervals were estimated for each polymorphic variant to evaluate the association with risk of developing autoimmune thyroiditis, with p-values < 0.05 being considered statistically significant.

Results: The analysis of genotypic and allelic distribution of rs1048290 SNP in Keap1 gene and rs2706110 SNP in NRF2 gene did not show statistically significant differences between both groups of subjects. However, the heterozygous GA genotype of the Keap1 rs11545829 polymorphism was associated with increased risk of developing autoimmune thyroiditis. Also, the statistical analysis showed that G allele confers protection from this disease.

Conclusion: Our study suggests that the genotype AG in the polymorphism rs11545829 of Keap1 gene could increase the risk of developing autoimmune thyroiditis due to an alteration of the cellular response to oxidative stress.

This study was funded by FIS-FEDER: PI16/01920.

C. Robles Lázaro: None. M. Ovejero-Sánchez: None. N. Gestoso-Uzal: None. C. Gutiérrez-Cerrajero: None. P. Martín-Bejarano Soto: None. P. Vázquez-Cárdenas: None. A.B. Herrero: None. R. González-Sarmiento: None.

P03.010.B The use of high throughput sequencing for the identification of variants contributing to autosomal dominant polycystic kidney disease in the Maltese population

Maria Cini Masini 1, Francesca Borg Carbott1, Ritienne Attard1, Adrian Pleven1, Karen Cassar2, Roberta A. Callus3, Angela Borg Cauchi3, Valerie Said Conti4, Stephanie Bezzina Wettinger1, Rosienne Farrugia1

1Department of Applied Biomedical Science, Faculty of Health Sciences, University of Malta, L-Imsida, Malta, 2Department of Medicine, Faculty of Medicine and Surgery, University of Malta, L-Imsida, Malta, 3Renal Division, Department of Medicine, Mater Dei Hospital, L-Imsida, Malta, 4Department of Paediatrics, Mater Dei Hospital, L-Imsida, Malta.

Introduction: Autosomal dominant polycystic Kidney Disease (ADPKD) though rare, is the most common hereditary kidney disease. It is characterized by enlarged kidneys, bilateral formation and progressive expansion of renal cysts, as well as systemic manifestations from the progression of renal disease requiring renal replacement therapy or transplantation.

Materials and Methods: DNA samples were obtained from 16 recruited probands and 6 additional affected family members as part of the Malta NGS Project. We used Agilent SureSelectXT Targeted Enrichment gene panel and high throughput sequencing (HTS) on Illumina HiSeq4000 for 3 nuclear families. Whole exome sequencing was carried out on another 15 probands. HTS data was mapped to GRCh37 as paired-end libraries using NextGENe® software. After variant filtering and prioritization, variants were confirmed by Sanger sequencing using primers unique to PKD1 or PKD2.

Results: We identified 13 variants in PKD1; two frameshifts, seven missense changes (2 novel), and four nonsense mutations (2 novel). Each PKD1 variant was identified in a single family, and one family had 2 linked variants. A PKD2 splice site mutation, was identified in two unrelated probands. For two patients no causative variant was identified in known ADPKD candidate genes.

Conclusion: This study demonstrates that laborious long-range PCRs of PKD1 and PKD2 may be replaced by HTS and stringent data analysis reducing cost and analysis time.

Funding: The Malta NGS Project was supported by the R&I programme of 2012 through the Malta Council for Science and Technology. M. Cini Masini is funded through a Tertiary Education Scholarship Scheme.

M. Cini Masini: None. F. Borg Carbott: None. R. Attard: None. A. Pleven: None. K. Cassar: None. R.A. Callus: None. A. Borg Cauchi: None. V. Said Conti: None. S. Bezzina Wettinger: None. R. Farrugia: None.

P03.013.A Association between cortisol deficiency and life threatening arrhythmia

Afaf Alsubhi 1, Mohammed Aldubayee2, Wafaa Eyaid1

1Department of Pediatrics, Genetics division, King AbdulAziz Medical City, RIYADH, Saudi Arabia, 2Department of Pediatrics, King Abdullah Specialized Children’s Hospital (KASCH), RIYADH, Saudi Arabia.

A 5 days old full term baby girl was admitted to our pediatric intensive care unit with non ketotic hypoglycemia, abnormal movement along with bradycardia and progressive prolongation of the QT interval. The Whole exome sequencing showed likely pathogenic variant in the TBX19 gene that is associated with congenital isolated adrenocorticotropic hormone deficiency that was proven biochemically. She was treated with hydrocortisone and showed dramatic improvement of her QT interval length. After discharge, she lost follow up. She was readmitted again at the age of 16 months with similar symptoms that improved after restarting hydrocortisone therapy with improvement of the EKG findings and bradycardia. In the literature there is one case report of an adult patient with similar association between central cortisol deficiency and life-threatening arrhythmia.Hypoglycemia per se does not cause a prolonged QT interval; this indicates that cortisol deficiency might be the cause of this patient presentation.

A. Alsubhi: None. M. Aldubayee: None. W. Eyaid: None.

P03.014.B A novel homozygous missense variant in PTPN2 associated with early onset Crohn’s disease and growth failure

Tony Yammine, Alain Chebly, Nabiha Salem, Rita Esber, Mirna Souaid, Chantal Farra

Medical Genetics Unit, Beirut, Lebanon.

Crohn’s disease (CD) is a chronic idiopathic inflammatory bowel disease that can affect any part of the gastrointestinal tract. Major manifestations are diarrhea, abdominal pain, weight loss with or without fever. CD is a multifactorial disorder where genetic factors are important players. Several candidate loci or genes including PTPN2 (#MIM 176887) have been reportedly associated with CD. PTPN2 is a major contributor in controlling inflammatory signaling cascades and maintaining intestinal barrier functions. We conducted whole-exome sequencing in a 9-year-old Lebanese girl with a CD onset at 13 months and in both her asymptomatic parents. Analysis detected a novel homozygous variant in PTPN2: c.359C>T, p.(Ser120Leu) in the patient, while both her parents were heterozygous for the variant. c.359C>T is located in the protein tyrosine phosphatase domain within a highly conserved amino acid. It is classified as likely pathogenic according to the ACMG criteria, deleterious by SIFT and disease causing by Mutation Taster. In order to evaluate the hypothetical functional consequences of the identified variant, we performed a quantitative expression analysis of PTPN2 in blood tissues from the patient, her parents and two healthy controls. An almost absent PTPN2 expression was noted in the patient compared to her parents and the controls, suggesting a functional PTPN2 impairment caused by c.359C>T. This is the first time, a homozygous PTPN2 variant is associated with an early onset CD. Further reports with PTPN2 gene variants would enable a better delineation of the molecular basis of CD.

T. Yammine: A. Employment (full or part-time); Significant; Saint Joseph University. A. Chebly: A. Employment (full or part-time); Significant; Saint Joseph University. N. Salem: A. Employment (full or part-time); Significant; Saint Joseph University. R. Esber: A. Employment (full or part-time); Significant; Saint Joseph University. M. Souaid: A. Employment (full or part-time); Significant; Saint Joseph University. C. Farra: A. Employment (full or part-time); Significant; Saint Joseph University, Hotel Dieu de France.

P03.015.C Spectrum of CFTR variants in patients with cystic fibrosis revealed in Western Siberian Ugra region (Russia)

Maxim Donnikov 1,2, Vitaly Mescheryakov1, Dmitry Lozhkin2, Lev Kolbasin2, Andrey Glotov3, Oleg Glotov4, Irina Urvantseva5, Lyudmila Kovalenko1

1Medical Institute of Surgut State University, Surgut, Russian Federation, 2Medical Genetics Counseling Service of Diagnostics and Cardiovascular Surgery Center (Cardiology Clinic) of KHMAO-Ugra, Surgut, Russian Federation, 3Department of Genomic Medicine, D. O. Ott Research Institute of Obstetrics, Gynecology and Reproduction, Saint-Petersburg, Russian Federation, 4City Hospital 40, Saint-Petersburg, Russian Federation, 5Diagnostics and Cardiovascular Surgery Center (Cardiology Clinic) of KHMAO-Ugra, Surgut, Russian Federation.

Introduction: During the era of cystic fibrosis (CF) target therapy knowing population structure of CFTR variants is of immense need. Applying battery of molecular genetics tests in routine practice of regional genetics laboratory allowed to perform extensive analysis of CFTR variants in regional CF patients.

Materials and methods: gDNA obtained from 57 CF patients (1998-2020 yob) from regional CF registry by column extraction; HRMA performed using Bio-Rad software; MLPA and Sanger sequencing performed on GenomeLab GeXP (Beckman-Coulter); NGS performed on MiSeq using NimbleGen SeqCap_EZ_CysFib kit.

Results: Total of 117 CFTR alleles were revealed (see Table), among them 6 major regional mutations covering 69.2% of all alleles, with most frequent [delta]F508 variant. 28 variants of different types (mostly missense and nonsense) with frequency of 0.9% each (found only in 1 patient each) comprise 23.9% of all alleles. If classified by type of nucleotide sequence change, the spectrum contains 50.4% of ins/del without frameshift mutations ([delta]F508 as representative); 20.5% of missense variants (E92K); 9.4% nonsense (S466X); 9.4% ins/del with frameshift (1677delTA); 5.1% large rearrangements (dele2,3); 3.4% VUCS; 1.7% splicing defects.

Conclusions: If ranged, CFTR variants spectrum from regional CF cases resembles that of common Russian alleles for 5 major variants (except R1066C, listed in Table), while sharing only 2 major mutations with Europe ([delta]F508, N1303K). High prevalence of rare variants dictates the necessity of sequencing.

List of CFTR variants in CF patients from Ugra
  CFTR variant calling (HGVS; legacy) type of variant n = 117 %
1 c.1521_1523delCTT ([delta]F508) del w/o frameshift 57 48.7
2 c.54-5940_273+10250del21kb (CFTRdele2,3) large rearrangement 6 5.1
3 c.1545_1546delTA (1677delTA) del w/frameshift 6 5.1
4 c.274G>A (E92K) missense; splicing defect 5 4.3
5 c.3196C>T (R1066C) missense 4 3.4
6 c.3909C>G (N1303K) missense 3 2.6
7 c.412_413insACT (L138ins) ins w/o frameshift 2 1.7
8 c.1397C>G (S466X)* nonsense 2 1.7
9 c.1399C>T (R1070Q)* missense 2 1.7
10 c.3209G>A (L467F) VUCS 2 1.7
11-38 28 variants (each 0,9%) all 28 23.9

M. Donnikov: None. V. Mescheryakov: None. D. Lozhkin: None. L. Kolbasin: None. A. Glotov: None. O. Glotov: None. I. Urvantseva: None. L. Kovalenko: None.

P03.017.A Frequnecy of CFTR mutations in a Romanian cohort of individuals with cystic fibrosis

Georgiana Ilie 1, Viorica Radoi2,3, Andreea Ionescu4, Iuliana Chelu4, Cristina Dragomir4, Gratiela Chelu4, Mihaela Popa4, Bianca Basangiu4, Andra Perioc4, Oana Istrate4, Traian Grozescu2, Paul Iordache5, Lucian Pop3, Natalia Cucu6, Camil Laurentiu Bohiltea2,3, Radu-Ioan Ursu2,3,4

1”Carol Davila” University of Medicine and Pharmacy, Bucharest, Romania, 2”Carol Davila” University of Medicine and Pharmacy, Department of Medical Genetics, Bucharest, Romania, 3National Institute for Mother and Child Health ”Alessandrescu-Rusescu”, Bucharest, Romania, 4Synevo Romania, Department of Medical Genetics, Chiajna, Romania, 5”Carol Davila” University of Medicine and Pharmacy, Department of Epidemiology, Bucharest, Romania, 6University of Bucharest, Faculty of Biology, Department of Genetics, Bucharest, Romania.

Background: Cystic fibrosis is one the most common autosomal-recessive genetic disorders in the Caucasian population, caused by homozygous or compound heterozygous mutations in the CFTR gene (on the long arm of chromosome 7).

Material. Methods: The study group includes 74 patients with the clinical diagnosis of cystic fibrosis and 132 healthy relatives of patients with cystic fibrosis (carrier screening testing). The testing method was PCR using a panel including the most common 38 mutations in CFTR gene in the European population and the 5T-7T allele polymorphism.

Results: All the patients with the clinical diagnosis of cystic fibrosis were positive for mutations in the CFTR gene, in a homozygous or in a heterozigous compound state. Of all these pacients, 56.75% (42/74) had the frameshift deltaF508 mutation, followed by other common mutations in the studied group, such as the G551D, the R553X or the R1158X variants. 57 out of the total of 132 suspected healthy carriers (43.2%) were positive for CFTR mutations in a heterozygous status. The most prevalent mutation was in this case also the deltaF508 genetic variant. Rarer mutations, such as the W1282X and the c.711+1G>A variants, were detected in 1 patient each.

Conclusions: The results of the study are concordant with the reports in literature regarding the prevalence of CFTR mutations in the Eastern-European population. Genetic testing for CFTR mutations is important not only for the genetic diagnosis of the disease, but also for the identification of heterozygous carriers or individuals at risk of passing the disease on.

G. Ilie: None. V. Radoi: None. A. Ionescu: None. I. Chelu: None. C. Dragomir: None. G. Chelu: None. M. Popa: None. B. Basangiu: None. A. Perioc: None. O. Istrate: None. T. Grozescu: None. P. Iordache: None. L. Pop: None. N. Cucu: None. C. Bohiltea: None. R. Ursu: None.

P03.018.B Late diagnosisof cystic fibrosisin a 59-year-old patient

Elena Zhekaite 1, Elena Amelina2, Elena Kondratyeva1, Tagui Adyan1

1Research Centre for Medical Genetics, Moscow, Russian Federation, 2Scientific Research Institute of Pulmonology of the Federal Medical and Biological Agency of Russia, Moscow, Russian Federation.

Introduction: According to Russian CF Patient Registry 2018 the average age of CF patients in Russia was 12.8 ± 9.6 years, the average age of diagnosis - 3.1 ± 6.1 years (4.1month in Europe according to European Cystic Fibrosis Society Patient Registry 2016). Few patients in Russia are over 55 years of age.

Materials and methods: The patient had atypical clinical symptoms since childhood (chronic sinusitis mostly, polypotomy at the age of 17). For the last 15 years there were complaints of persistent cough, exacerbations of chronic bronchitis about 2 times a year. 3 years ago bronchiectasis were detected. Patient has no children, his profession - a worker at a steel plant. Physical examination revealed dyspnea, low BMI (19.2 kg/m2), severe bronchial obstruction (FVC 54%, FEV1 33%), bilateral bronchiectasis on CT.

Results: Sweat test (Nanoduct) was performed - conductivity 83mmol/l. DNA diagnostics in the gene СFTR identified 2 pathogenic variants - [c.1521_1523delCTT]; [c.413_415dupTAC].

Conclusions: Our clinical case shows that the patients with "mild" CFTR genotype can be diagnosed at any age, patients with bronchiectasis, chronic polysinusitis should be tested for CF.

E. Zhekaite: None. E. Amelina: None. E. Kondratyeva: None. T. Adyan: None.

P03.019.C Familial case of Cystic Fibrosis with genotype-phenotype correlations

Tinatin Tkemaladze1,2,3, Mariam Ghughunishvili 4,5, Eka Kvaratskhelia1,5, Elene Abzianidze1, Volha Skrahina3, Arndt Rolfs3,6,7

1Department of Molecular and Medical Genetics, Tbilisi State Medical University, Tbilisi, Georgia, 2G. Zhvania Pediatric Academic Clinic, Tbilisi State Medical University, Tbilisi, Georgia, 3Centogene GmbH, Rostock, Germany, 4G.Zhvania Pediatric Academic Clinic, Tbilisi State Medical University, Tbilisi, Georgia, 5Bakhutashvili Institute of Medical Biotechnology, Tbilisi State Medical University, Tbilisi, Georgia, 6University Rostock, Medical Faculty, Rostock, Germany, 7Arcensus GmbH, Rostock, Germany.

Introduction: Cystic Fibrosis (CF) is the most common recessive condition in Caucasians. Clinical expression of the disease is heterogeneous and no specific genotype-phenotype correlation had been observed. Certain CFTR variants are defined as “CF” alleles causing CF phenotype, and other alleles are defined as “risk factor” alleles, causing CBAVD, pancreatitis and atypical CF.

Case report: Here we describe a familial case of CF with three children: 11 yo and 8 yo siblings with classical CF phenotype had positive NBS and elevated sweat chloride test results. In younger 6 yo sibling NBS and sweat chloride test results were normal and she only had two episodes of airway infections and small weight.

Results: CFTR sequencing was performed for all three siblings: two older siblings with classical CF phenotype had 1677delTA and I1234V variants (both classified as “CF” alleles), whereas younger sibling had 1677delTA and L997F variants (classified as “CF” and “risk factor” alleles respectively). When the healthy parents’ samples were analyzed the father turned out to have one heterozygous 1677delTA allele, and the mother had two variants - L997F and I1234V (classified as “risk factor” and “CF” alleles respectively). Currently mother is 40 yo and she is completely healthy.

Conclusion: To date none of the above-mentioned combination of alleles detected in the siblings and the mother are described in the cftr2.org database. We propose that combination of “CF” allele and “risk factor” allele does not cause CF and proper classification of variants is crucial for correct interpretation and diagnosis of the condition.

T. Tkemaladze: None. M. Ghughunishvili: None. E. Kvaratskhelia: None. E. Abzianidze: None. V. Skrahina: None. A. Rolfs: None.

P03.020.D Unusual presentation of CF in an infant

Dario Portillo-Miño, Efren Esteban Cerón-Muñoz

Hospital Infantil Los Angeles, Pasto, Colombia.

Introduction: Cystic Fibrosis (CF) is the most common autosomal recessive, genetic condition in Caucasians, occurring in 1 of every 2,000 to 3,000 live births. Hepatic abnormalities in patients with CF have usually been reported in about 30-40% of the children. A multisystemic, progressive, and lethal disease is distinguished by the relationship of several organic components, mainly; pulmonary, pancreatic, and gastrointestinal.

Materials and methods: It is was the research in the clinic history, laboratory test, and molecular studies for the case. Besides, the search of the literature in PubMed, Google Scholar, Science Direct, LILACS, and Scielo. The case report is based on the CARE 2016 guideline.

Results: This case report attempts an approach to the clinical findings of hepatobiliary manifestations in CF. An infant was less than 1-month-old with an insidious clinical picture that debut with cholestasis and jaundice. The CFTR gene analysis results with mutation c.2353C>T (transition of cytosine by thiamin at position 2353 of cDNA), p.Arg785* (in the protein produces a change of arginine by stop codon), in a homozygous state, this change has been reported in the literature as a pathogenic change related to CF.

Discussion/Conclusion: CF is associated with liver involvement around 30%. In children, hepatobiliary symptoms occur at puberty when damage to the liver system is in advanced stages. The atypical presentation of CF with liver involvement is very rare and lethal in an infant. Understanding the different form of CF, it is essential for early diagnosis and to achieve integral management.

D. Portillo-Miño: None. E. Cerón-Muñoz: None.

P03.022.B Exome sequencing implicates heterozygous variant in DSTYK in functional urinary bladder disturbance

Clara Vidic 1, Marcin Zaniew2, Holger Thiele3, Janine Altmüller3, Heiko Reutter1,4, Alina C. Hilger1,5

1Institute of Human Genetics, University of Bonn, Bonn, Germany, 2Department of Pediatrics, University of Zielona Góra, Zielona Góra, Poland, 3Cologne Center for Genomics, University of Cologne, Cologne, Germany, 4Department of Neonatology and Pediatric Intensive Care, Children’s Hospital, University of Bonn, Bonn, Germany, 5Department of Pediatrics, Children’s Hospital, University of Bonn, Bonn, Germany.

Introduction: DSTYK encodes dual serine/threonine and tyrosine protein kinase. DSTYK has been associated with autosomal dominant congenital anomalies of the kidney and urinary tract and with autosomal-recessive hereditary spastic paraplegia. Here we report a father and his two dizygotic twin sons with a novel, heterozygous variant in DSTYK, all presenting with voiding dysfunction due to functional urinary bladder disturbance.

Materials and Methods: We performed whole exome sequencing of the family. All three presenting clinically with hesitancy, abnormal voiding pattern and night incontinence till adolescence. In the sons cystoscopy excluded urethral valves but showed hypertrophy of the bladder neck and trabeculated bladder. Additionally, both sons were diagnosed with epilepsy. Based on the pedigree, filtering of exome data focused on rare (MAF < 0.01%), autosomal-dominant variants, predicted to be deleterious, residing in highly conserved regions of the exome. Validation of prioritized variants was performed using Sanger sequencing.

Results: We identified a novel, heterozygous c.271C>A (p.Leu91Met) variant in DSTYK segregating with the disease. The amino acid is highly conserved. Rated deleterious by 3 different prediction programs (SIFT, PolyPhen, MutationTaster) and with a CADD score of 24.5, this variant was prioritized as likely disease causing.

Conclusion: To the best of our knowledge, we describe the first familial case with autosomal dominant inherited variants in DSTYK and specific functional bladder outlet obstruction and epilepsy. Acknowledgements: C.V. is supported by BONFOR grant O-149.0133. A.C.H. is supported by BONFOR grant O-149.0123.

C. Vidic: None. M. Zaniew: None. H. Thiele: None. J. Altmüller: None. H. Reutter: None. A.C. Hilger: None.

P03.024.D Polymorphisms of glutathione synthetase gene are associated with susceptibility to type 2 diabetes and hyperglycemia

Iuliia Azarova, Elena Klyosova, Ekaterina Shkurat, Alexey Polonikov

Kursk State Medical University, Kursk, Russian Federation.

Background: The present study investigated whether single nucleotide polymorphisms (SNPs) in glutathione synthetase (GSS) gene, antioxidant enzyme involved in glutathione metabolism, contribute to type 2 diabetes (T2D) susceptibility.

Methods: A total of 3198 unrelated Russian subjects including 1572 T2D patients and 1626 sex- and age matched healthy subjects were enrolled for the study. Three common tagSNPs such as rs13041792, rs1801310 and rs6088660 of GSS were genotyped using the MassArray System. Plasma glutathione and reactive oxygen species (ROS) levels of study participants were measured by fluorometric and colorimetric assays, respectively.

Results: We found that SNP rs13041792 at the GSS gene is associated with an increased risk of T2D (OR 1.24, 95%CI 1.06-1.44, P = 0.027). A GSS haplotype rs1801310G-rs6088660C-rs13041792A (OR 1.26, 95CI 1.08-1.47, Pglobal = 0.039) showed a significant association with T2D risk. Genotype rs13041792-A/A was associated with increased levels of fasting blood glucose (FBG) in entire group of T2D patients (P = 0.032) and diabetic males (P = 0.026). Meanwhile, genotype rs1801310-A/A was associated with decreased levels of total glutathione in plasma in diabetic females (P = 0.039). Genotype rs6088660-T/T showed an association with decreased levels of FBG in males (P = 0.007) and decreased levels of ROS in females (P = 0.039).

Conclusion: We found for the first time that genetic polymorphisms at GSS gene are associated with susceptibility to type 2 diabetes and related hyperglycemia through the mechanisms involving decreased antioxidant defense and increased production of reactive oxygen species. The study was supported by Russian Science Foundation (№20-15-00227).

I. Azarova: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Significant; Russian Science Foundation. E. Klyosova: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Significant; Russian Science Foundation. E. Shkurat: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Modest; Russian Science Foundation. A. Polonikov: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Significant; Russian Science Foundation.

P03.025.A Clinical utility of genetic testing in early-onset kidney disease: seven genes are the main players

Andrea Domingo Gallego 1, Marc Pybus1, Gemma Bullich2,1, Mónica Furlano1, Laia Ejarque-Vila1, Laura Lorente Grandoso1, Patricia Ruiz1, Gloria Fraga3, Mercedes López González4, Juan Alberto Piñero Fernández5, Lidia Rodríguez Peña5, Isabel Llano Rivas6, Raquel Sáez7, Anna Bujons-Tur1, Gema Ariceta4, Lluis Guirado1, Roser Torra1, Elisabet Ars1

1Fundació Puigvert, Barcelona, Spain, 2Centre Nacional d’Anàlisi Genòmica (CNAG)-Centre for Genomic Regulation (CRG), Barcelona, Spain, 3Hospital de la Santa Creu i Sant Pau, Barcelona, Spain, 4Hospital Vall d’Hebron, Barcelona, Spain, 5Hospital Universitario Virgen de la Arrixaca, Murcia, Spain, 6Hospital Universitario Cruces, Barakaldo-Bizkaia, Spain, 7Hospital Donostia, San Sebastian, Spain.

Background: Inherited kidney diseases are one of the leading causes of chronic kidney disease (CKD) that manifests before the age of 30 years. Precise clinical diagnosis of early-onset CKD is complicated due to the high phenotypic overlap, but genetic testing is a powerful diagnostic tool. We aimed to develop a genetic testing strategy to maximize the diagnostic yield for patients presenting with early-onset CKD and to determine the prevalence of the main causative genes.

Methods: We performed genetic testing of 460 patients with early-onset CKD of suspected monogenic cause using next-generation sequencing of a custom-designed kidney disease gene panel in addition to targeted screening for c.428dupC MUC1.

Results: We achieved a global diagnostic yield of 65% (300/460), which varied depending on the clinical diagnostic group. Among the 300 genetically diagnosed patients, the clinical diagnosis was confirmed in 77%, a specific diagnosis within a clinical diagnostic group was identified in 15%, and 7% of cases were reclassified. Of the 64 causative genes identified in our cohort, seven (COL4A3, COL4A4, COL4A5, HNF1B, PKD1, PKD2, and PKHD1) accounted for 66% (198/300) of the genetically diagnosed patients.

Conclusions: Two-thirds of patients with early-onset CKD in this cohort had a genetic cause. Just seven genes were responsible for the majority of diagnoses. Establishing a genetic diagnosis is crucial to define the precise etiology of CKD, which allows accurate genetic counseling and improved patient management.

Funding: Instituto de Salud Carlos III (ISCIII)/FEDER funds: PI15/01824, PI16/01998, PI18/00362, PI19/01633, RETIC REDINREN RD16/0009/0019, and Plataforma ISCIII Biobancos PT20/00196.

A. Domingo Gallego: None. M. Pybus: None. G. Bullich: None. M. Furlano: None. L. Ejarque-Vila: None. L. Lorente Grandoso: None. P. Ruiz: None. G. Fraga: None. M. López González: None. J. Piñero Fernández: None. L. Rodríguez Peña: None. I. Llano Rivas: None. R. Sáez: None. A. Bujons-Tur: None. G. Ariceta: None. L. Guirado: None. R. Torra: None. E. Ars: None.

P03.026.B iPSC as a genetic model for investigating NAFLD risk variants

Antonio Munoz 1, Yu-Lin Kuang1, Gilbert Nalula1, Elizabeth Theusch1, Gabriela Sanchez2, Carlos Iribarren2, Ronald M. Krauss1, Aras N. Mattis3, Marisa W. Medina1

1Department of Pediatrics, University of California San Francisco, San Francisco, CA, USA, 2Division of Research, Kaiser Permanente Northern California, Oakland, CA, USA, 3Department of Pathology, University of California San Francisco, San Francisco, CA, USA.

Introduction: Current cellular genetic models for Non-Alcoholic Fatty Liver Disease (NAFLD) have limitations that prevent the scalable investigation of gene variants. Missense variants rs738409 (I148M) in PNPLA3 (palatin like phospholipase domain containing protein 3) and rs58542926 (E167K) in TM6SF2 (transmembrane 6 superfamily member 2) increase hepatocyte intracellular lipid accumulation and are robustly associated with NAFLD. The goal of this study was to assess whether undifferentiated induced pluripotent stem cells (iPSCs) can be used to model the effect of these two NAFLD variants on intracellular lipid accumulation.

Materials and Methods: PNPLA3 and TM6SF2 expression were confirmed in iPSCs from individuals of European ancestry carrying PNPLA3 and/or TM6SF2 variant alleles (n = 10) and non-carriers (n = 10). Cells were exposed to oleate (0-100µM) or BSA control for 24hrs, and neutral lipids were stained with Nile red, visualized by fluorescence microscopy, and quantified by FACS.

Results: Oleate incubation led to a dose-dependent increase in intracellular lipids, rising 40 ± 18% (mean ± SD) in the oleate-treated cells compared to control-treated cells (p = 0.001, n = 20). Notably, the increase was greater in TM6SF2 and/or PNPLA3 variant carriers (48 ± 16%) compared to non-carriers (24 ± 8 %), p = 0.002. Even in BSA-treated cells, there was a trend of elevated lipid levels in carriers compared to non-carriers (p = 0.25).

Conclusion: Greater oleate-induced increases of intracellular lipids in iPSCs with the NAFLD risk alleles indicate that undifferentiated iPSCs are a reasonable, more efficient substitute for hepatocytes when studying cellular lipid accumulation and an informative cellular model for the identification and functionalization of NAFLD genetic risk variants. Funding Sources: P50GM115318, R01HL139902, P30DK026743

A. Munoz: None. Y. Kuang: None. G. Nalula: None. E. Theusch: None. G. Sanchez: None. C. Iribarren: None. R.M. Krauss: None. A.N. Mattis: None. M.W. Medina: None.

P03.028.D Genetic heterogeneity of megacystis-microcolon-intestinal hypoperistalsis syndrome

Martin Schwarz 1, Marcela Malíková1, Júlia Martinková1, Petra Cibulková2, Renáta Michalovská3, Markéta Havlovicová1, Milan Macek, jr1

1Department of Biology and Medical Genetics, Praha, Czech Republic, 2Agel Laboratories - Laboratoře AGEL a.s., Nový Jičín, Czech Republic, 3GHC Genetics s.r.o., Praha, Czech Republic.

Introduction: Megacystis-microcolon-intestinal hypoperistalsis syndrome (MMIHS; MIM 249210) is a rare multisystem syndrome where its name reflects most commonly affected organs. Although ACTG2, LMOD1, MYH11, MYL9, MYLK have been associated with the disease, 55% of all cases remain undiagnosed by genetic testing. Hereby, we report two additional cases with pathogenic variants in ACTG2, and a case where a pathogenic variant in KCNMA1 was found.

Materials and Methods: Next generation sequencing (NGS) was performed. Sanger sequencing was used to verify NGS results and determine the segregation of the presumably pathogenic variants in available first degree relatives.

Results: Two previously described de novo heterozygous pathogenic variants in the ACTG2 /(c.119G>A p.(Arg40His) and c.770G>A (p.Arg257His)/ where detected. A de novo heterozygous Class 4 variant c.1132G>A (p.Gly375Arg) was detected in the KCNMA1 gene.

Conclusions: Only 47 patients with ACTG2 related MMIHS have been reported thus far. We identified another two cases which will contribute to the better understanding of this heterogeneous disorder. Variants in KCNMA1 have also been associated with Liang-Wang multiple malformation syndrome (LIWAS; MIM:618729), where available literature is unfortunately sparse. Previously reported cases with LIWAS bearing the same variant, have among other pathognomonic features also megacystis and impaired bowel motility. Clinical presentation of LIWAS in the neonatal period could be similar to MMIHS with bowel and urinary bladder problems being dominant clinical signs. Therefore, LIWAS should be considered in the differential diagnosis of MMIHS, in particular during early childhood.

Supported by MH CZ - DRO, Motol University Hospital, Prague, Czech Republic 00064203.

M. Schwarz: None. M. Malíková: None. J. Martinková: None. P. Cibulková: None. R. Michalovská: None. M. Havlovicová: None. M. Macek, jr: None.

P03.030.B Discovery of novel miRNA markers for pituitary neuroendocrine tumour

Helvijs Niedra1, Raitis Peculis1, Ilze Konrade2,3, Inga Balcere2,3, Mihails Romanovs2,3, Liva Steina4, Janis Stukens4, Jelizaveta Sokolovska5, Janis Klovins1, Vita Rovite 1

1Latvian Biomedical Research and Study centre, Riga, Latvia, 2Riga East Clinical University Hospital, Riga, Latvia, 3Riga Stradins University, Riga, Latvia, 4Pauls Stradins Clinical University Hospital, Riga, Latvia, 5University of Latvia Faculty of Medicine, Riga, Latvia.

Introduction: Recently plasma miRNAs have been studied as biomarkers in various tumors including pituitary neuroendocrine tumors (PitNETs). The identification of PitNET derived miRNAs in plasma remains challenging due to tumor volume and miRNA dilution within blood. To our knowledge this is the first study that used the NGS approach to characterize circulating miRNAs in plasma acquired from Bilateral petrosal sinus sampling (BIPSS) - a gold standard in diagnosis of ACTH secreting PitNETs.

Materials and Methods: We sequenced plasma miRNA samples acquired from sinistral and dextral sides of sinus petrosus inferior and complementary plasma from peripheral blood (before CRH administration, 5 and 15 minutes after stimulation).

Results: The highest amount of differentially expressed miRNAs was observed 5 minutes after CRH stimulation (20 upregulated, 14 downregulated). The highest amount of ACTH was released in the sinistral side during the 5th minute after the stimulation by CRH. In the plasma of sinistral side at the 5th minute we identified two miRNAs: hsa-miR-7-5p and hsa-miR-375-3p that were highly upregulated compared to peripheral blood. Using clustering analysis, we were able to distinguish plasma samples acquired from BIPSS and blood plasma, indicating that the miRNA fraction characterized in this study has potentially PitNET borne origin.

Conclusions: Here we provide the first insight into the landscape of circulating miRNAs in close proximity to PitNET. The data indicates that ACTH secreting PitNET releases two circulating miRNAs upon stimulation with CRH (hsa-mir-7-5p, hsa-mir-375-3p) alongside with ACTH implying the further studies of these miRNA as diagnostic markers.

H. Niedra: None. R. Peculis: None. I. Konrade: None. I. Balcere: None. M. Romanovs: None. L. Steina: None. J. Stukens: None. J. Sokolovska: None. J. Klovins: None. V. Rovite: None.

P03.031.C GCK, HNF1A and HNF4A variant analysis in suspected MODY patients

Zivile Zemeckiene, Marius Sukys, Rimvydas Jonikas, Inga Nasvytiene, Inga Valanciute, Inga Poceviciene, Rasa Ugenskiene

The Hospital of Lithuanian University of Health Sciences Kauno Klinikos, Kaunas, Lithuania.

Introduction: Maturity-onset diabetes of the young (MODY) is an inherited form of diabetes mellitus which is caused by pathogenic variants in one of 14 known genes, mostly in GCK, HNF1A and HNF4A. A correct MODY diagnosis might change the treatment and reduce the risk of diabetic complications. In this study we analysed GCK, HNF1A and HNF4A genes in Lithuanian patients, consulted for MODY in The Hospital of Lithuanian University of Health Sciences Kauno Klinikos.

Materials and Methods: We performed Sanger sequencing of GCK, HNF1A and HNF4A genes for 77 adults (over 18 years) with suspected MODY during 2018-2020.

Results: For nine patients causative/likely causative variants were determined. Four patients had heterozygous variants previously reported as pathogenic (NM_000162.3(GCK):c.234C>G, NM_000162.3(GCK):c.703A>G (two patients), NM_000545.6(HNF1A):c.493T>C). The other variants were novel. Heterozygous GCK variant c.471_473del was detected in two patients from the same family with persistent hyperglycaemia. Variant causes deletion of amino acid (p.Glu157del) located in an important enzyme domain. Duplication of 19 nucleotides c.1745_1763dup in HNF1A was identified in patient with previously diagnosed diabetes. This variant creates frameshift with premature termination codon (p.Thr589ProfsTer66) which is known mechanism of MODY. Heterozygous HNF4A variant c.704G>T was found in two unrelated patients with previously diagnosed diabetes and family history. This variant changes amino acid (p.Arg235Ile) in the conservative ligand binding protein domain and might affect protein structure. All three novel variants were evaluated as likely pathogenic.

Conclusions: Six different pathogenic/likely pathogenic variants were determined in nine patients with suspected MODY diabetes. Three of the variants were novel.

Z. Zemeckiene: None. M. Sukys: None. R. Jonikas: None. I. Nasvytiene: None. I. Valanciute: None. I. Poceviciene: None. R. Ugenskiene: None.

P03.033.A Clinical description of a new type of mucopolysaccharidosis in Yakutia

Saina Novgorodova 1, Aytalina Sukhomyasova1, Elizabeth Gurinova2, Vera Argunova2, Lena Nikolaeva3, Nadezhda Maximova1

1Research Laboratory "Molecular Medicine and Human Genetics" of the Medical Institute NEFU, Yakutsk, Russian Federation, 2Republican hospital №1-National Center of Medicine, Yakutsk, Russian Federation, 3Republican Hospital No. 1-National Center of Medicine, Yakutsk, Russian Federation.

Since 2005, clinical symptoms associated with mucopolysaccharidosis-plus have been recorded in Yakut patients; previously undescribed storage disease with an autosomal-recessive type of inheritance was suspected [Gurinova E.E. 2014]. In 2017, a molecular genetic cause of a new rare autosomal-recessive syndrome in the Yakut and Turkish populations was identified, clinically similar to other types of mucopolysaccharidosis, but has other features of the course, such as congenital heart defects, renal and hematopoietic disorders leading to infant mortality [Kondo et al., 2017; Dursun et al., 2017; Vasilev et al., 2020]. The new disease was added into the McCusick international database under the OMIM number # 617303 and named mucopolysaccharidosis-plus syndrome (MPS-PS). In the Yakut and Turkish populations, the same mutation was found in the VPS33A gene (NM_022916.4: c.1492C> T, NP_075067.2: p.Arg498Trp, subsequently referred to as p.R498W)]. In this thesis, we present brief clinical features of 17 patients with MPS-PS in Yakutia. All children were born in Yakut families from young parents, not in consanguineous marriage. 9 children were born from the second pregnancy and later pregnancies. In 4 cases, pregnancy was complicated by the miscarriage threat; according to ultrasound data, no gestation pathology was detected. A characteristic feature of MPS-PS is early debut and early infant mortality, multisystem organ damage - lungs, kidneys (secondary nephrotic syndrome, severe proteinuria 2-3 g/day, nephromegaly), heart (septal heart disease, severe course), central nervous system and hematopoietic disorders (severe anemia requiring blood transfusion, coagulopathy with hemorrhagic syndrome), the activity of lysosomal hydrolases and urinary glucosaminoglycans test are uninformative.

S. Novgorodova: None. A. Sukhomyasova: None. E. Gurinova: None. V. Argunova: None. L. Nikolaeva: None. N. Maximova: None.

P03.035.C A clinical case of patient with cholestatic liver disease due to mutations of the MYO5B

Tamara Yurievna Lesnichenko 1, Natalia Alexandrovna Semenova2, Olga Nikolaevna Ivanova2, Elena Anatolievna Kamenec2, Ekaterina Yurievna Zakharova2

1Federal State Budgetary Educational Institution of Further Professional Education "Russian Medical Academy of Continuous Professional Education" of the Ministry of Healthcare of the Russian Federation, Moscow, Russian Federation, 2Federal State Budgetary Scientific Institution "Research Centre for Medical Genetics", Moscow, Russian Federation.

Introduction: Defects in MYO5B gene, on which bile salt export pump depends to localize to the canalicular hepatocellular membrane usually cause diarrhea 2, with microvillus atrophy (OMIM # 251850) but also may result in liver disease without enteropathy. It characterized by low-GGT-cholestasis variable severity, hepatomegaly, normal or mildly elevated transaminases.

Materials and Methods: We present a female patient at the age of 2y4m with isolated cholestasis. Her infantile period was normal. The disease manifested by high temperature, hepatomegaly, acholic stools. Serum clinical laboratory tests showed cholestasis with normal GGT activity, conjugated hyperbilirubinemia 30mmol/l, normal transaminase levels, increased serum alkaline phosphatase 587E/l, hypercholesterolemia 8,03mmol/l. Deficiency of lysosomal acid lipase was excluded by an enzyme test. NGS was used to analyze 52 genes responsible for hereditary diseases with cholestasis. Two nucleotide variants in MYO5B gene were detected.

Results: We identified a novel heterozygous frameshift duplication c.1604_1610dupGCCAGCA p.His537GlnfsTer28. Another heterozygous variant c.1201C>T (p.Arg401Cys rs761492029), revealed in the MYO5B gene, described as pathogenic. Then, we performed Sanger sequencing on the DNA extracted from the mother, the father and the sister of the proband to analyze the segregation of each genetic variant identified by NGS. Parents and older sibling are healthy and heterozygous for one or another mutant allele.

Conclusions: Thus, the findings support the disease-causing role of novel MYO5B mutation. Many patients with low-GGT-cholestasis remain genetically undiagnosed. Verification of the genetic aetiology of cholestasis allows selection of appropriate treatment strategies, determination of genetic risk and discussion of the necessity of prenatal testing.

T.Y. Lesnichenko: None. N.A. Semenova: None. O.N. Ivanova: None. E.A. Kamenec: None. E.Y. Zakharova: None.

P03.036.D Identification of gene variants in Indonesian Hirschsprung disease patients using whole exome sequencing

Gunadi 1, Alvin Santoso Kalim1, Marcellus1, Dyah Ayu Puspitarani1, Kristy Iskandar2, Galuh Dyah Nur Astuti3,4

1Pediatric Surgery Division, Department of Surgery/Genetics Working Group, Faculty of Medicine, Public Health and Nursing, Universitas Gadjah Mada/Dr. Sardjito Hospital, Yogyakarta, Indonesia, 2Department of Child Health/Genetics Working Group, Faculty of Medicine, Public Health and Nursing, Universitas Gadjah Mada/Dr. Sardjito Hospital, Yogyakarta, Indonesia, 3Department of Human Genetics, Radboud University Medical Center, Nijmegen, Netherlands, 4Division of Human Genetics, Center for Biomedical Research, Faculty of Medicine, Diponegoro University, Semarang, Indonesia.

Introduction: Hirschsprung’s disease (HSCR) is a complex genetic disorder with at least 24 genes have been identified for the pathogenesis of HSCR. However, they only contributed to 20% of HSCR cases. We aimed to further elucidate the genetic basis of HSCR in Indonesia.

Materials and Methods: Whole exome sequencing was performed in 20 sporadic isolated HSCR patients and four non-HSCR subjects as controls. We excluded variants presented in controls, followed by in silico prediction tools and population allele frequency databases to select rare variants. We determined the minor allele frequency (MAF) using gnomAD (MAF < 0.1%).

Results: We involved 11 (55%) males and 9 (45%) females. We identified several candidate genes, including PTPN13, UBR4, ECE1, GDNDF, ASTN1, CELSR3, XYLT1, SORL1 and FAT1. One of them, a novel frameshift variant in PTPN13 gene (c.5763delT; p.Tyr1921Ter) which may lead to loss of function on protein level. Moreover, we also identified compound heterozygous variants in MUTYH gene: the first variant, a known protein truncating variant associated with colorectal cancer, c.1354G>T; p.Glu452* and the second variant is a novel c.116C>T; p.Ala39Val variant. We also found co-mutation of UBR4 gene (c.5497G>A; p.Gly1833Arg) and novel variant of GDNF gene (c.349G>A; p.Gly117Ser) in one patient.

Conclusions: We identified several novel genes and variants that might contribute to the pathogenesis of HSCR. Our study further confirms the complex network during the enteric nervous system development and HSCR pathogenesis. This study was funded by the World Class Research grant (Ministry of Research and Technology/National Research and Innovation Agency, Indonesia

Gunadi: None. A. Kalim: None. .. Marcellus: None. D. Puspitarani: None. K. Iskandar: None. G. Astuti: None.

P03.037.A A case of familial brain-lung-thyroid syndrome due to a NKX2.1 run-on mutation

Federica Baldan1, Elena Cavaliere1,2, Anna J. Gortan1,2, Nadia Passon2, Dora Fabbro2, Dario Marin1,2, Myriam Carecchio3, Sara C. Credendino4, Rosa Gallo4, Paola Cogo1,2, Giuseppe Damante 1,2, Gabriella De Vita4

1University of Udine, Udine, Italy, 2Academic Hospital of Udine, Udine, Italy, 3University of Padua, Padua, Italy, 4University of Naples, Udine, Italy.

NKX2.1 is a homeobox gene encoding a tissue-specific transcription factor able to bind DNA by its homeodomain. NKX2.1 expression is fundamental for thyroid, lung and ventral forebrain development and correct maturation. Indeed, loss of function mutations of this gene cause the ‘brain-lung-thyroid’ syndrome (BLTs), which is characterized by various combinations of thyroid dysgenesis, infant respiratory distress syndrome, and hyperkinetic movement disorders, mainly chorea. Here, we describe a familial case of BLTs due to a NKX2.1 run-on mutation. The proband is an eleven-year-old boy who, at birth, suffered from respiratory distress associated to pulmonary hypertension. Delayed motor and speech language milestones were reported. Moreover, he suffered from frequent upper-respiratory infections, short stature due to subclinical hypothyroidism, sleep disturbance and learning difficulties. A neurologic assessment revealed generalized chorea unstable gait and mild hypotonia. The patient’s mother suffered of impaired gait and frequents falls, as well as learning disability and subclinical hypothyroidism. On examination, she presented with distal choreiform movements in the lower limbs. The patient’s aunt and grandmother reported gait imbalance and frequents falls during childhood; none of them presented with movement disorder. Genetic testing for the NKX2.1 gene was performed and a run-on mutation (c.1204dupT; p.*402Leuext*37) was identified: duplication of the thymine corresponding to the first base of the stop codon causes the changing of the stop codon in a leucine-coding codon. The resulting frameshift generated a tail of 36 additional amino acids at the protein C-terminus. Segregation analysis confirmed that all the affected relatives carried the same genetic variant.

F. Baldan: None. E. Cavaliere: None. A.J. Gortan: None. N. Passon: None. D. Fabbro: None. D. Marin: None. M. Carecchio: None. S.C. Credendino: None. R. Gallo: None. P. Cogo: None. G. Damante: None. G. De Vita: None.

P03.038.B Effects of growth hormone treatment in Noonan syndrome: correlations with genotype

Diana Miclea 1,2, Yline Capri2, Alain Verloes2

1"Iuliu Hatieganu" University of Medicine and Pharmacy, Cluj-Napoca, Cluj-Napoca, Romania, 2Department of Genetics, APHP- Robert Debré University Hospital, Paris, France.

Background: In Noonan syndrome(NS), the treatment with growth hormone(GH) has been carried out in the last decades, with reported effects on improvement of growth velocity and adult height, however, these data usually included patients with NS phenotype, only the last few years bringing data about GH treatment in genotyped NS patients.Aim.To evaluate the effect of GH treatment according to genotype in patients with NS, by a systematic review of the literature.

Methods: We evaluated auxological, hormonal and imagistic data before and under GH treatment in patients with genetically confirmed NS reported in literature between 2001 and 2020.

Results: We identified 21 studies, including data from 336 genotyped patients with NS. Of these, 243patients(72.3%) presented pathogenic variant for PTPN11gene, 17patients(5%) for SOS1, 15 patients(4.4%) for RAF1, 9patients(2.6%) for SHOC2, 8patients(2.3%) for KRAS, 5patients(1.4%) for BRAF and 2patients(0.5%) for each of the genes MEK1,SPRED1,HRAS,NRAS and RIT1. The age of GH treatment initiation was 8.3years. The mean period of treatment was of 6.7years. The height before GH treatment was -2.8SD and at the end of GH treatment was of -2.1SD, with a mean adult height of 165.6cm for males and 155.4cm for females. The target height was -0.6SD. It was not observed a difference on growth between the patient categories according to the genes.

Conclusion: This study is the first review about the pattern of growth under GH treatment in genotyped NS and even it was thought that this pattern is well known, we need more data to conclude the real effect on growth regarding each gene involved in NS.

D. Miclea: None. Y. Capri: None. A. Verloes: None.

P03.039.C Next generation sequencing in the diagnostic approach to autosomal dominant polycystic kidney disease

Deimante Brazdziunaite 1, Marius Miglinas2, Algirdas Utkus1

1Department of Human and Medical Genetics, Institute of Biomedical Sciences, Faculty of Medicine, Vilnius University, Vilnius, Lithuania, 2Center of Nephrology, Institute of Clinical Medicine, Faculty of Medicine, Vilnius University, Vilnius, Lithuania.

Introduction: Classically, autosomal dominant polycystic kidney disease (ADPKD) is presented as a common Mendelian disorder and represent the leading genetic causes of renal disease in adults. It is caused by mutations either in PKD1 or PKD2. However, next-generation sequencing (NGS) offers the opportunity to significantly improve the diagnostic yield in ADPKD. Body. Introduction of the first therapy approved for ADPKD - vasopressin V2-receptor antagonist (Tolvaptan) - brought more new attention to this disease. Using European Renal Association-European Dialysis and Transplant Association Registry, and population based studies, Willey et al. (2016) indicated that the ADPKD point prevalence in the EU is <5/10,000. Moreover, disease appears to be quite genetically heterogeneous. The PKD mutation database counts more than 1000 pathogenic variants in PKD1 and about 200 in PKD2 gene. According to various authors, about 8-10% of ADPKD cases do not have pathogenic variant in PKD1/2 genes. Possibility to apply extended diagnostics by NGS revealed previously unknown causes. Pathogenic variants in GANAB gene and PKD phenocopies due to variants in HNF1β, PKHD1, DNAJB11, or TSC1/2 genes are described. However, the list of novel phenocopies and potential candidate genes is not final yet. This brings questions whether clinical diagnostic criteria will be as specific and sensitive in distinguishing these new genetic forms and phenocopies of PKD.

Conclusion: Technological advances let us distinguish different aetiologies of apparently the same disease as well as expand our knowledge. Furthermore, in the era of individualised treatment the most accurate molecular diagnosis is required.

D. Brazdziunaite: None. M. Miglinas: None. A. Utkus: None.

P03.041.A Protein loosing enteropathy and lymphedema in a girl with a homozygous DCHS1 mutation

Tatiana V. Gabrusskaya1, Evgeny N. Suspitsin 1,2, Elizaveta Y. Lapina1, Elena A. Kornienko1

1St.-Petersburg State Pediatric Medical University, St.-Petersburg, Russian Federation, 2N.N. Petrov Institute of Oncology, St.-Petersburg, Russian Federation.

Introduction: Hennekam syndrome (HS) and van Maldergem syndrome (VMS) are two entities sharing some clinical features. While unusual facial gestalt and some degree of developmental delay is observed in both conditions intestinal lymphangiectasia and lymphedema have been considered exclusive for HS.

Materials and Methods: We present a case of 5 year-old girl of Syrian descent with severe protein loosing enteropathy (PLE) due to lymphangiectasia, lymphatic malformation in mediastinum, asymmetric lymphedema, facial dysmorphisms (flat nasal bridge, epicanthus, microtia), bilateral hearing loss and growth retardation. No parental consanguinity was reported. She was born preterm, had severe course of bronchopulmonary dysplasia and developed symptoms of PLE in the first year of life. The girl had hypoproteinemia, hypoalbuminemia, and elevated fecal alpha-1 antitrypsin. Right arm and left leg lymphedema became obvious by the age of 5. The patient was suspected to have Hennekam syndrome and subjected to clinical exome sequencing.

Results: No pathogenic variants in Hennekam syndrome-associated genes (CD55, FAT4, CCBE1 and ADAMTS3) have been found. Rare DCHS1 variant c.1954A>G (p.S652G) was detected in homozygous state; biallelic DCHS1 alterations have been previously described in some patients with van Maldergem syndrome type 1.

Conclusion: Since DCHS1 and FAT4 molecules form a receptor - ligand pair, we hypothesize that DCHS1 mutation may cause HS-like phenotype. We demonstrate that lymphatic pathology including PLE, lymphatic malformation and lymphedema can be a part of both conditions. This observation extends existing data on clinical overlap between VMS and HS.

The work was supported by Russian Science Foundation grant 20-45-01005

T.V. Gabrusskaya: None. E.N. Suspitsin: None. E.Y. Lapina: None. E.A. Kornienko: None.

P03.042.B Male pseudo hermaphroditism in a 54 years old woman

María Luz Bellido 1, Teresa de Haro1, Trinidad Gonzalez2, Matias Perez1

1Hospital Universitario Virgen de las Nieves, Granada, Spain, 2Hospital Universitario Clínico San Cecilio, Granada, Spain.

Introduction: Disorders of sex development (DSD) comprise a heterogeneous group of congenital conditions associated with atypical development of internal and external genitalia. Sex determination is governed by genes from SRY region at Y chromosome that initiates the male developmental program. However, there exist many other genes involve in this process.We describe the case of a 54-years-old woman with an incidental finding of absence of uterus, ovaries, prostate or seminal vesicles, after a TC scan performed for other pathologies.

Methods: GTG-banding karyotype was performed according to standard protocols. Twenty metaphases chromosomes were examined.Next-generation sequencing study was done by MiSeq analyzer from Illumina®.

Results: Karyotype result: male, 46,XY.Sequencing study finding:

Gene(localization) Variant Type Status dbSNP/HGMD Heritage Classification
SRD5A2(2p23.1) c.679C>T, (p.Arg227Ter) Nonsense Heterozygous rs121434248 / CM920642 AR Pathogenic
  c.344G>A, (p.Gly115Asp) Missense Heterozygous rs121434246 / CM920633 AR Probably Pathogenic

SRD5A2 codifies to 5-alpha-reductase type 2. This enzyme catalyzes the conversion of testosterone to dihydrotestosterone, which is essential for normal differentiation of the external male genitalia and virilization.

Conclusion: The final diagnosis was male pseudo-hermaphroditism due to 5-alpha-reductase type 2 deficiency (OMIM#264600). 46,XY patients show ambiguous genitalia at birth, including perineal hypospadias and a persistent urogenital sinus with a blind perineal vaginal orifice.DSD used to be diagnosed at birth or childhood by pediatricians or within the family, making our case especially unusual. The patients need an individualized management, especially for decisions related to sex of rearing, future intervention, hormone treatment and reproductive options.

M. Bellido: None. T. de Haro: None. T. Gonzalez: None. M. Perez: None.

P03.044.D The importance of NGS data reanalysis and further functional analysis: an example of impaired phosphate metabolism

Margarita Sharova 1, Svetlana Papizh2, Olga Levchenko1, Alexandra Filatova1, Andrey Marakhonov1, Anatoliy Tulpakov1, Mikhail Skoblov1

1Research Centre for Medical Genetics, Moscow, Russian Federation, 2Veltischev Research and Clinical Institute for Pediatrics of the Pirogov Russian National Research Medical University, Moscow, Russian Federation.

Introduction: The regulation of phosphate metabolism occurs mainly in kidneys through reabsorption of phosphate ions by the NaPi-IIa (SLC34A1) and NaPi-IIc (SLC34A3) transporters. Pathogenic variants in these genes are identified in patients with hereditary hypophosphatemic rickets with hypercalciuria or hypercalcemia, infantile 2.

Materials and methods: patients with a clinical diagnosis of hereditary hypophosphatemic rickets were referred for molecular genetic testing. Three patients underwent full-genome sequencing followed by reanalysis of genomic data; for one NGS panel data were reanalyzed. Experimental validation of the effect of variants on splicing was performed using minigene and RNA analysis.

Results: Primary NGS data analysis didn’t reveal causative variants for the phenotype or there was only one variant for autosomal recessive disease. Further investigation of data found in-frame deletion p.91_97del in SLC34A1 gene with global frequency 1,7% in two patients in compound heterozygous state, that previously was described as pathogenic. Intronic pathogenic deletion c.925+20_926-48del101was found in two brothers in compound heterozygous state. Also, VUS c.1449G>A in SLC34A1 gene was detected. Minigene assay showed that the variant leads to truncation of exon 13 by 34 nucleotides with frameshift and PTC formation. Another functional study by RT-PCR identified variant c.846G>A in SLC34A3 as splicing variant, leading to skipping of exon 8 without frameshift.

Conclusion: Reanalysis of genomic data is important not only over time, but also by other clinical bioinformaticians. Some pathogenic variants with high global frequency could be filtered out in the early stages of NGS analysis. Functional analysis allows to investigate the pathogenicity of VUS.

M. Sharova: None. S. Papizh: None. O. Levchenko: None. A. Filatova: None. A. Marakhonov: None. A. Tulpakov: None. M. Skoblov: None.

P03.045.A Contribution of a non-coding variant in autosomal recessive ACTG2-related visceral myopathy identified by whole-genome sequencing

Mari Mori 1,2, Kristen V. Truxal1,2, R. Tanner Hagelstrom3, Amanda Clause4, Vinay Prasad5,2, Kandamurugu Manickam1,2, Stephen G. Kaler1,2,6, Maria M. Alves7, Carlo Di Lorenzo8,2

1Division of Genetic and Genomic Medicine, Nationwide Children’s Hospital, Columbus, OH, USA, 2Department of Pediatrics, The Ohio State University, Columbus, OH, USA, 3Illumina Inc, San Diego, CA, USA, 4Nationwide Children’s Hospital, Columbus, OH, USA, 5Pathology & Laboratory Medicine, Nationwide Children’s Hospital, Columbus, OH, USA, 6Center for Gene Therapy, Abigail Wexner Research Institute, Nationwide Children’s Hospital, Columbus, OH, USA, 7Department of Clinical Genetics, Erasmus University Medical Center, Rotterdam, Netherlands, 8Division of Pediatric Gastroenterology, Hepatology and Nutrition, Nationwide Children’s Hospital, Columbus, OH, USA.

Introduction: Pediatric intestinal pseudo-obstruction (PIPO) is a heterogeneous condition characterized by impaired gastrointestinal propulsion and a broad clinical spectrum ranging from a severe form requiring parenteral nutrition to milder forms with some ability to tolerate oral nutrition. Various molecular bases underlying primary PIPO have been identified, of which ACTG2-related visceral myopathy is the most common for familial or sporadic (de novo) primary PIPO with autosomal dominant inheritance. However, a recent case report suggests that some mild ACTG2 variants may be associated with autosomal recessive inheritance. We present a family in which both parents have relatively mild gastrointestinal symptoms, and two sons have severe PIPO, consistent with autosomal recessive inheritance.

Results: Whole-genome sequencing (WGS) revealed a missense variant c.28G>A (p.Val10Met) in the ACTG2 gene in the mother and the sons, and deletion of ACTG2 exon 1 (non-coding) in the sons and presumably in the father.

Conclusions: Our case reinforces that monoallelic mild ACTG2 variants may underly mild primary PIPO, while biallelic mild variants can cause severe diseases. Prior molecular studies on PIPO in the literature were based on whole-exome sequencing or targeted Sanger sequencing, which may fail to detect deletion of the non-coding exon. Alterations in non-coding ACTG2 segments can be under-recognized causes of mild gastrointestinal symptoms and may explain some instances of inter-familial variability. WGS offers a more comprehensive genetic workup for severe primary or idiopathic PIPO because of such genetic heterogeneity. Notably, the absence of family history does not reliably exclude the presence of genetic causes in PIPO.

M. Mori: None. K.V. Truxal: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Significant; co-investigator, Abeona Therapeutics. R. Hagelstrom: None. A. Clause: A. Employment (full or part-time); Modest; Illumina, Inc.. E. Ownership Interest (stock, stock options, patent or other intellectual property); Modest; Illumina. V. Prasad: None. K. Manickam: None. S.G. Kaler: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Modest; Principal Investigator, NINDS (pending), Collaborator, NIGMS (pending), Sponsor, NINDS (pending). F. Consultant/Advisory Board; Modest; Vivet Therapeutics; Chairperson, Data Monitoring Committee. M.M. Alves: None. C. Di Lorenzo: F. Consultant/Advisory Board; Modest; Qol, Mahana, Innovative Health Solutions, Mallinckrodt, Allergan, Takeda.

P03.046.B Application of NGS sequencing for improved diagnosis in the pediatric nephrology setting

Radosveta Bozhilova 1, Olga Beltcheva1, Galia Zlatanova2, Kunka Kamenarova1, Kalina Mihova1, Felitsiya Shakova1, Dimitar Roussinov2, Maria Gaydarova2, Vanio Mitev1, Radka Kaneva1

1Molecular Medicine Center, Dpt. Medical Chemistry and Biochemistry, Medical University – Sofia, Sofia, Bulgaria, Sofia, Bulgaria, 2SBAL Pediatric Diseases, Nephrology and Hemodialysis Clinic, Department of Pediatrics, Medical University -Sofia, Sofia, Bulgaria, Sofia, Bulgaria.

Introduction: Nephrotic syndrome, resulting from pathological changes in the glomerular filter, is a common childhood disorder. While it usually resolves with corticosteroid treatment, 10-20% of the cases are resistant to therapy and often progress to end-stage renal disease. A monogenic cause of steroid resistant nephrotic syndrome (SRNS) is identified in up to half of the cases and more than 50 genes have been associated with the disease.

Materials and Methods: Nine SRNS patients, initially screened for NPHS1, NPHS2 and WT1 mutations, were recruited. Two children presented with extrarenal features - seizures and brain infarctions. We used TruSight One Sequencing Panel (Illumina) on MiSeq platform for identification of disease-causing variants and Sanger sequencing for confirming the mutations and establishing their origin. The pathogenicity of novel variants was evaluated based on the ACMG criteria.

Results: Homozygous mutation in NOS1AP, recently found to be associated with SRNS, was identified in one case. An additional rare MYH11 variant may account for the brain infarctions in this child. A heterozygous frameshift mutation in CFHR5 and combinations of rare amino acid substitutions in known SRNS genes (ACTN4, LAMB2, etc.) accounted for three additional cases.

Conclusion: The application of NGS for screening of an extended gene panel allowed us to identify the genetic cause of the disease in approximately half of the SRNS patients recruited for the study. As expected, a wide variability of genes and mutation types were involved. A possible oligogenic inheritance was observed in some of the cases. Grant references: D-93/24.06.2020; D01-285/17.12.2019, MES, Bulgaria

R. Bozhilova: None. O. Beltcheva: None. G. Zlatanova: None. K. Kamenarova: None. K. Mihova: None. F. Shakova: None. D. Roussinov: None. M. Gaydarova: None. V. Mitev: None. R. Kaneva: None.

P03.047.C Functional consequences of rs1800629 TNF gene associated with asthma and tuberculosis development

Irina Zhalsanova, Elena Bragina, Nadezhda Babushkina, Nataliya Tarasenko, Maxim Freidin, Valery Puzyrev

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

Introduction: Tumor necrosis factor-alpha (TNF) is one of the proinflammatory cytokines involved in the various infectious and allergic diseases development. We studied the SNP(rs1800629) association located in the TNF gene promoter region with bronchial asthma (BA) and tuberculosis (TB) development and evaluated the functional consequences of the rs1800629 by analyzing the expression level in short-term cell cultures of peripheral blood mononuclear cells (PBMC) stimulated by inducers (lipopolysaccharide (LPS), interferon-gamma (IFN-γ)).

Methods: Genotyping was performed using qPCR among patients with BA, TB, and in the control group (n = 1231). TNF gene expression level was evaluated response to stimulation in PBMC from healthy donors, differentiated on genotypes rs1800629 (n = 17). Samples were incubated with stimuli (LPS, IFN-γ), and control samples without stimuli within overnight at +37°C. Target gene expression was analyzed by RT-PCR.

Results: Association analysis showed, AA genotype frequency in TB patients was higher than in patients with BA (p = 0.047) and the control group (p = 0.033). TNF gene expression level upon IFN-γ stimulation was 4,2 times higher compared to unstimulated PBMC (p < 0.05). The A allele (AA + GA) presence promoted a 1.5-fold decrease in TNF gene expression upon stimulation with IFN-γ compared to GG. TNF gene expression increased upon LPS stimulation, but no significantly.

Conclusions: Thus, differences in the TNF gene transcription pattern depending on the genotype and the stimulation mode. These features are important for understanding the genetic susceptibility to infectious and allergic diseases of individuals living in regions with different infectious load levels. The study was supported by the RFBR grant No.15-04-05852.

I. Zhalsanova: None. E. Bragina: None. N. Babushkina: None. N. Tarasenko: None. M. Freidin: None. V. Puzyrev: None.

P03.048.D Genome wide association study of type 2 diabetes complications in population of Latvia

Raitis Peculis 1, Monta Ustinova1, Raimonds Rescenko1, Vita Rovite1, Linda Zaharenko1, Ilze Elbere1, Laila Silamikele1, Ilze Konrade2,1, Jelizaveta Sokolovska3, Valdis Pirags3,1, Janis Klovins1

1Latvian Biomedical Research and Study Centre, Riga, Latvia, 2Faculty of Medicine, Riga Stradins University, Riga, Latvia, 3Faculty of Medicine, University of Latvia, Riga, Latvia.

Introduction: Complications of type 2 diabetes affect a significant proportion of patients and their prevalence, correlates with increased duration of diabetes. Yet, time of onset of complications is variable for different persons with type 2 diabetes suggesting an individual level predisposition and protection. Therefore, investigation of the genetic component of type 2 diabetes complications is warranted.

Materials and methods: We have assayed 601 type 2 diabetes patients with four common diabetes complications using Human GSA genotyping array. Genome wide association study was performed to investigate the genetic background of following phenotypes: diabetic neuropathy, diabetic nephropathy, macrovascular events, and ophthalmic complications. Genotype analysis was performed comparing diabetes patients with complication present to those without particular complication.

Results: Association analysis identified eight novel type 2 diabetes susceptibility loci with genome wide significance. Two SNPs, rs1132787 (GYPA) and rs522521 (LOC105371557) were associated with diabetic neuropathy, while seven (one shared with diabetic neuropathy) rs2477088 (PDE4DIP), rs522521 (LOC105371557), rs4852954 (NAT8), rs6032 (F5), rs6935464 (RPS6KA2), rs7236163 (ZNF519), and rs3095447 (CCDC146) (latter is also shared with ophthalmic complications) were showed significant association with macrovascular complications. The association results were adjusted for covariates (age, sex, BMI, diabetes duration, median HbA1c, and prescription drug use) and a genome-wide significant threshold of P < 5 × 10−8 was used.

Conclusions: Using the genome-wide genotyping approach this study identified ten novel associations with T2DM complications (at eight loci) and provides additional insight into genetic markers of these phenotypes. The study was supported by European Regional Development Fund Project No

R. Peculis: None. M. Ustinova: None. R. Rescenko: None. V. Rovite: None. L. Zaharenko: None. I. Elbere: None. L. Silamikele: None. I. Konrade: None. J. Sokolovska: None. V. Pirags: None. J. Klovins: None.

P04 Skeletal, Connective Tissue, Ectodermal and Skin Disorders

P04.001.B 6q13 microdeletion - mapping the clinical phenotype

Raquel Rodrigues, Oana Moldovan, Rosário Silveira-Santos, Ana B. Sousa, Ana Sousa

Hospital de Santa Maria, Lisboa, Portugal.

Introduction: Proximal 6q11q14 deletions are exceedingly rare, particularly the enclosed 6q13q14 microdeletions. Little is known about their phenotypic consequences, which include mild/moderate developmental delay/intellectual disability (DD/ID), minor dysmorphism, and connective tissue abnormalities. We describe a case of 6q13 microdeletion, among the smallest reported within this syndromic region, aiming to contribute to the genomic mapping of associated clinical features.

Methods: The proband, a 34yo male, was referred to the Genetics Department for mild ID and motor clumsiness. In childhood, he presented mild DD, language delay and learning difficulties. Additional features included strabismus, cryptorchidism, joint hypermobility, and abnormal foot position. A DNA sample was analyzed by array-CGH (180K CGX-HD). Segregation was investigated by FISH.

Results: Array-CGH revealed a 4.71Mb microdeletion in 6q13 (70917114_75625720; hg19), encompassing 17 OMIM genes. Parental studies confirmed this deletion occurred de novo.

Discussion: Previous reports proposed candidate genes for DD/ID within 6q11q4 region, namely KCNQ5, which is deleted in our patient. However, the chief candidate gene underlying learning difficulties and language delay was proposed to be HRT1B, which is not deleted in this case. DD/ID is unspecific and frequently features in contiguous deletion syndromes, probably contributed by several genes in the deleted interval. Conversely, connective tissue abnormalities are specific characteristics often observed in 6q11q14 microdeletions, including hypermobility and foot deformities. Thus far, the latter have been assigned to COL12A1 gene. However, COL12A1 is not deleted in our case, suggesting a key role for other genes, such as COL9A1 and COL19A1, in the etiology of these problems.

R. Rodrigues: None. O. Moldovan: None. R. Silveira-Santos: None. A.B. Sousa: None. A. Sousa: None.

P04.002.C A novel truncating variant in the FGD1 gene associated with Aarskog-Scott syndrome in a family previously diagnosed with Tel Hashomer camptodactyly

Lena Sagi-Dain1, Irena Kessel1, Amir Peleg1, Tamar Paperna2, Nina Ekhilevitch2, Alina Kurolap3, Hagit Baris Feldman3, Marina Bar-Shay 1

1Carmel Medical Center, Haifa, Israel, 2Rambam Medical Center, Haifa, Israel, 3Ichilov Medical Center, Tel Aviv, Israel.

Tel Hashomer camptodactyly syndrome is a genetic association, characterized by camptodactyly with muscular hypoplasia, skeletal dysplasia, and abnormal palmar creases. Currently, the genetic basis for this disorder is unknown, thus there is a possibility that this association may be contained within another genetic diagnosis. In this manuscript we present a family with a previous clinical diagnosis of Tel Hashomer camptodactyly syndrome. Whole exome sequencing revealed a novel hemizygous truncating variant c.269_270dup (p.Phe91Alafs*34) in the FGD1 gene (NM_004463.3) in all three symptomatic patients, congruous with a diagnosis of Aarskog-Scott syndrome. Our manuscript adds to the limited data on Aarskog-Scott syndrome, and emphasizes the importance of unbiased comprehensive genetic testing towards establishing a diagnosis for genetic associations.

L. Sagi-Dain: None. I. Kessel: None. A. Peleg: None. T. Paperna: None. N. Ekhilevitch: None. A. Kurolap: None. H. Baris Feldman: None. M. Bar-Shay: None.

P04.003.D Abdominal wall defects in the Czech Republic: Frequency and prenatal diagnostics

Antonin Sipek Sr 1,2,3, Vladimir Gregor1,2, Antonin Sipek Jr1,4,5, Jan Klaschka6,7, Marek Maly6,8, Jitka Jirova9

1Department of Medical Genetics, Thomayer University Hospital, Prague 4, Czech Republic, 2Department of Medical Genetics, Pronatal Sanatorium, Prague, Czech Republic, 3Department of Medical Genetics, Centre for Medical Genetics and Reproductive Medicine, Gennet, Prague, Czech Republic, 4Institute of Biology and Medical Genetics, 1st Faculty of Medicine, Charles University, Prague, Czech Republic, 5Institute of Medical Genetics, 3rd Faculty of Medicine, Charles University, 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 of Health Information and Statistics of the Czech Republic, Prague, Czech Republic.

Introduction: Omphalocele and Gastroschisis are the most important anomalies from the group of Abdominal Wall Defects (AWD). The main goal of our study was to evaluate the changes in the frequency of these anomalies in the Czech Republic and the overall effectiveness of their prenatal diagnostics.

Materials and Methods: We are using the official population data from the National Registry of Congenital Anomalies of the Czech Republic, which is run by the Institue of Health Information and Statistics of the Czech Republic. The registration process is country-wide and compulsory by the national law. We analyzed the numbers of omphalocele (Q792) and gastroschisis (Q793) cases in the newborns and prenatally diagnosed cases in the Czech Republic (1994-2018).

Results: The total incidence of omphalocele was 3.32 per 10.000 live births (1.39 in births and 1.93 in prenatally diagnosed cases). For gastroschisis, the total incidence was 3.09 (0.90 in births and 2.19 in prenatally diagnosed cases). The total frequency of both anomalies increased during the selected period in the Czech Republic, the increase is statically significant (p < 0.05). The average week of gestation at the time of positive prenatal diagnostics decreased significantly for both anomalies (P < 0.05).

Conclusions: The improvement of ultrasound prenatal diagnostics enabled the successful detection of AWDs in earlier gestation weeks. The overall incidence of both anomalies is however increasing in the Czech Republic, as more and more mothers prefer surgical intervention over the termination of the pregnancy.

Supported by the Ministry of Health of the Czech Republic, grant nr. AZV 17-29622A

A. Sipek Sr: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Modest; Ministry of Health of the Czech Republic, research grant: AZV 17-29622A. V. Gregor: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Modest; Ministry of Health of the Czech Republic, research grant: AZV 17-29622A. A. Sipek Jr: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Modest; Ministry of Health of the Czech Republic, research grant: TN, 00064190. J. Klaschka: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Modest; Ministry of Health of the Czech Republic, research grant: AZV 17-29622A. M. Maly: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Modest; Ministry of Health of the Czech Republic, research grant: TN, 00064190. J. Jirova: None.

P04.004.A Biallelic deep intronic variants c.1528-126A>G and c.5457+81T>A in TRIP11 are associated with achondrogenesis 1A

Priyanka Upadhyai, Radhakrishnan Periyasamy, Vishal S. Guleria, Shalini Nayak, Katta M. Girisha

Kasturba Medical College, Manipal Academy of Higher Education, Manipal, India.

Introduction: Biallelic loss of function variants in TRIP11 encoding for GMAP-210 cause lethal chondrodysplasia achondrogenesis type 1A (ACG1A). Complete depletion of TRIP11 impairs Golgi structure, vesicular transport and results in loss of IFT20 anchorage to the Golgi that is vital for ciliary trafficking and ciliogenesis. Here we report four affected foetuses, two each from two families, homozygous for putative pathogenic deep intronic variants in TRIP11.

Materials and Methods: Perinatal autopsy and radiological evaluation was performed, followed by exome and genome sequencing. Segregation analysis was performed by Sanger sequencing. Fibroblast cell-lines were available from an affected foetus. Transcript analysis was performed by qRT-PCR and RT-PCR respectively. Protein levels were ascertained by immunoblotting. The Golgi, ciliogenesis and autophagy were evaluated by immunostaining.

Results: The deep intronic variants c.1528-126A>G and c.5457+81T>A in TRIP11 were present in homozygous state in all affected foetuses and the parents were heterozygous carriers. This led to drastic depletion of TRIP11 mRNA, protein levels and produced severely compacted Golgi in fibroblasts. The c.5457+81T>A variant caused aberrant splicing and retention of 77 base-pairs of intron 18. We observed severe depletion of ciliated fibroblasts and reduction in cilia length, not reported so far in patient cells with TRIP11-related disorder. Moderate autophagic dysfunction was noted in affected fibroblasts, congruent with the role of IFT20 in modulating autophagy in primary cilia dependent manner in some cellular contexts.

Conclusion: Our findings illustrate how pathogenic variants occurring in deep intronic and putative regulatory regions of TRIP11 may impact its expression and activity leading to ACG1A.

P. Upadhyai: None. R. Periyasamy: None. V.S. Guleria: None. S. Nayak: None. K.M. Girisha: None.

P04.005.B Pre and post-natal achondroplasia, retrospective series of 64 consecutives cases with analyze of the diagnostic methods and timing issues

Genevieve Baujat, Roxana Borghese, Pascale Sonigo, Joana Bengoa, Caroline Michot, Elodie Millischer-Bellaiche, Sophie Rondeau, Beatrice Childs, Tania Attie-Bitach, Bettina Bessieres, Laurent Salomon, Yves Ville, Jean-Paul Bonnefont, Julie Steffann, Valerie Cormier-Daire

Necker Hospital, Paris, France.

The last years, diagnosis of achondroplasia benefited of advances in prenatal imaging, and in invasive and non-invasive molecular screening.

Objectives: To analyse diagnosis procedures and outcome on 64 consecutive confirmed cases of achondroplasia seen in the French Centre of Reference for Skeletal Dysplasia, between 2008 and 2016.MethodsDiagnosis stage/ age, analyse of the achondroplasia features by imaging (ultrasound (US), 3D-CT), molecular confirmation method, and pregnancy outcome were retrospectively determined.

Results: 64 cases of achondroplasia were included. The diagnosis was made during the pregnancy in 43 cases (67%, mean stage of 30 weeks). For the remaining 21 cases (33%), the diagnosis was performed at birth in all cases but one, diagnosed at 2 months. Among the eight foetuses with inherited ACH: 4 were diagnosed after early chorionic villus sampling, leading to termination of pregnancy (TOP) and 4 diagnosed after 26 weeks, by US.In the de novo prenatal 35 cases, mid-trimester US was normal in 80% of cases. The diagnosis was confirmed by the 3D-CT in all 17 cases when performed (52%), and/ or by molecular screening after amniocentesis (43%). The prenatal diagnosis led to TOP in 12 cases (34%), in a mean stage of 32 weeks. 66% of the foetuses diagnosed in prenatal went into the birth.

Conclusion: The systematic screening of the second term was normal in 80%. One third of the diagnosis led to TOP. The results confirm the late diagnosis of de novo achondroplasia during pregnancy, leading to major psychological and ethical issues for the parents.

G. Baujat: None. R. Borghese: None. P. Sonigo: None. J. Bengoa: None. C. Michot: None. E. Millischer-Bellaiche: None. S. Rondeau: None. B. Childs: None. T. Attie-Bitach: None. B. Bessieres: None. L. Salomon: None. Y. Ville: None. J. Bonnefont: None. J. Steffann: None. V. Cormier-Daire: None.

P04.007.D Shared Runs of Heterozygosity Mapping using whole genome sequencing reveals a complex structural variant in GSN causing novel cutaneous-visceral organ Gelsolin amyloidosis

Adam Jackson 1,2, Glenda Sobey3, Ashutosh Wechalekar4, Dorota Rowczenio4, . Genomics England Research Consortium5, Glenda Beaman2, Rob McMahon1, Unzela Khan1, Emma Miles1, Amy Goldman1, Simon Lovell6, Jill Clayton-Smith1,2, Siddharth Banka1,2

1Manchester Centre for Genomic Medicine, Manchester, United Kingdom, 2Division of Evolution and Genomic Sciences, School of Biological Sciences, Faculty of Biology, Medicine and Health, University of Manchester, Manchester, United Kingdom, 3National EDS Service, Sheffield Children’s NHS Foundation Trust, Sheffield, United Kingdom, 4National Amyloidosis Centre, University College London (Royal Free Campus), London, United Kingdom, 5Genomics England, London, United Kingdom, 6Division of Evolution and Genomic Sciences, School of Biological Sciences, University of Manchester, Manchester, United Kingdom.

Introduction: We report a multi-generational family of several individuals with cutis laxa, bowel perforation, cardiac rupture and clinical diagnosis of Ehlers-Danlos syndrome. Several histopathological, targeted sequencing studies failed to identify the genetic cause. Two affected individuals were detected with cutaneous amyloid, but proteomic studies failed to reveal its origin.

Methods and Results: We devised a novel Heterozygosity Mapping approach using srWGS data and identified a ~10.38Mb disease-haplotype on chromosome 9q33.1-q34.11. This region’s targeted analysis revealed an ~1kb deletion involving in-frame loss of exon 12 of GSN predicted to result in partial deletion and fusion of two gelsolin domains of the protein. DdPCR confirmed the variant’s segregation with the phenotype in extended family members (LOD score 4.52). Sanger sequencing revealed the variant to be a complex deletion-inversion-insertion event. Sequence analysis showed the variant to have likely originated from fork-stalling template-switching attributable to microhomology and formation of complex DNA structure due to multiple nested inverted and mirror repeats. Sequencing of mRNA and the amyloid protein is ongoing.

Conclusions: A complex structural variant (SV) involving exon 12 of GSN causes a novel form of potentially lethal ‘cutaneous-internal organ amyloidosis’ that is clinically, genetically and mechanistically distinct from Finnish-type amyloidosis. To our knowledge this is the first example of application of heterozygosity mapping to identify the cause of a Mendelian disorder, and of nested mirror and inverted repeats resulting in a germline SV in humans. This work highlights the value of WGS in resolving the cause of unsolved and novel disorders resulting from SVs.

A. Jackson: None. G. Sobey: None. A. Wechalekar: None. D. Rowczenio: None. .. Genomics England Research Consortium: None. G. Beaman: None. R. McMahon: None. U. Khan: None. E. Miles: None. A. Goldman: None. S. Lovell: None. J. Clayton-Smith: None. S. Banka: None.

P04.008.A Clinical features and molecular characterization of three Japanese patients with autosomal dominant Robinow syndrome caused by DVL3 variants

Kumiko Yanagi 1, Eriko Nishi2, Arisa Igarashi1, Maki Omata1, Yukimi Abe1, Nana Kobayashi1, Kazuhito Satou1, Kanako Ishii3, Nobuhiko Okamoto2, Yoichi Matsubara1, Tadashi Kaname1

1National Center for Child Health and Development, Setagaya, Japan, 2Osaka Women’s and Children’s Hospital, Osaka, Japan, 3Kyushu University, Fukuoka, Japan.

Autosomal dominant Robinow syndrome 3 (DRS3, OMIM 601368) is a rare skeletal dysplasia syndrome characterized by dysmorphic features resembling a fetal face, short stature, mesomelic limb shortening, vertebral and hand anomalies. Urogenital malformations are frequently reported. The causative gene, DVL3 (Disheveled 3, NM_004423) mapped on 3q27 encodes cytoplasmic phosphoprotein DVL3 known as a homologous to Drosophila dishvelled (dsh). Dsh relays Wnt signals from receptors to downstream effectors and roles on developmental processes, including segmentation and neuroblast specification. Only eight variants have been reported to date. All are splicing or frameshift variants with premature stop codons and predicted to result in a premature termination within Dhc-C domain of DVL3. Here we report three Japanese boys who were diagnosed with DRS3 by identification of heterozygous variants in DVL3. Two patients (Patient 1 and patient 3) were enrolled in the IRUD (Initiative on Rare and Undiagnosed Diseases) as undiagnosed patients. One patient (Patient 2) was initially suspected of having Aarskog syndrome (OMIM 305400), but was no pathogenic variants found in the FGD gene by direct sequencing. Whole exome sequencing and annotated variants filtering were performed in these patients. We identified two novel frameshift variants, c.1671_1686del:p.(Tyr558Alafs*105) in Patient 1 and c.1716-1722del:p.(Ser573Alafs*93) in Patient 2, and one novel in-frame deletion, c.1861_1884del:p.(Pro621_Ala628del) in Patient 3. The in-frame deletion lacks eight amino acid residues including two phosphorylation sites, which were evolutionary conserved. The deletion would affect DVL3 polymerization leading interrupt Wnt signaling. We are presenting clinical features in each patient.

K. Yanagi: None. E. Nishi: None. A. Igarashi: None. M. Omata: None. Y. Abe: None. N. Kobayashi: None. K. Satou: None. K. Ishii: None. N. Okamoto: None. Y. Matsubara: None. T. Kaname: None.

P04.009.B Defining the molecular pathology of autosomal recessive congenital icthyosis among a cohort of Egyptian patients

Khalda S. Amr, Nesma Mohamad Elaraby, Ghada Y. El-Kamah

National Research Centre, Cairo, Egypt.

Background Autosomal recessive congenital icthyosis (ARCI) are heterogeneous group of rare genodermatoses characterized by marked phenotypic and genotypic variability. Non syndromic ARCI are caused by mutations in more than a dozen of distinct genes. Molecular testing for ARCI in a clinical setting is not an easy task; some genes as (TGM1; 190195) on chromosome 14q11.2, are relatively common while mutations in other genes are rare. Aim: To identify the underlying molecular pathology in a cohort of 55 non syndromic ARCI Egyptian families with at least one affected sib, using next generation sequencing (NGS).

Methods: NGS analysis was performed through a panel of 14 ARCI genes.

Results: Genetic variants could be identified in 29 (52.7%) of the 55 studied non-syndromic ARCI families. All studied patients were descending from consanguineous families and most of the characterized mutations were found in a homozygous state. Characterized variants included; sixteen pathogenic, among which are five novel missense mutations and eleven previously described variants within the TGM1 gene. The five novel missense mutations are; (NM_000359.2) c.344T>A(Val115Asp), c.1165C>T(Arg389Cys), c.1423T>A(Cys475Ser), c.1762G>A(p.Ala588Thr), and c.1922G> (Cys641Val).

Conclusion: NGS panel analyses identified mutations within the TGM1 gene in 52.7% of studied families prioritizing it as a target gene for future molecular analyses among Egyptian ARCI patients. Limited resources prevented further whole exome analyses to help characterize the molecular pathology in the remaining almost 50% of our cohort as well as possible detection of other involved genes that might explain the marked heterogeneous phenotypes present within our patients.

K.S. Amr: None. N.M. Elaraby: None. G.Y. El-Kamah: None.

P04.011.D Bone mineralization inSATB2-associated syndrome

Anne Dittrich, Joyce Geelen, Jos Draaisma

Rradboudumc Amalia children’s hospital, Nijmegen, Netherlands.

Introduction: SATB2-associated syndrome, also called Glass syndrome, is caused by pathogenic variants or deletions/duplications affecting the special AT-rich sequence binding protein 2 (SATB2) gene. SATB2 is a transcriptional regulatory protein involved in craniofacial and central nervous system development. Glass syndrome is characterized by intellectual disability with severely limited speech, high palate and dentofacial abnormalities. In this case series we will focus on the role of SATB2 on bone metabolism

Materials and Methods: we collected the data on bone metabolism of all children followed at the Radboudumc Amalia children’s hospital.

Results: We have 4 children at follow-up with Glass syndrome. All children have signs of increased bone turnover. The oldest child had two long bone fractures at the age of 12 and low bone mineral density with a lumbar z-score of -3.5. For this reason therapy with intravenous bisphosphonate was started with good result. There were no additional fractures and the lumbar z-score increased to -1.9.

Conclusion: As shown, reduced bone mineralization in SATB2-associated syndrome due to high bone turnover starts at young age. In an earlier study the prevalence of decreased bone mineralization was reported to be 76% with 67% of patients had signs of increased bone turn-over. However, in a report with recommendations on the SATB2-associated syndrome, it is stated that one should consider osteopenia evaluation and optimize bone mineralization as needed. However, due to our experience, we would suggest that evaluation should be incorporated in the guideline, as preventive and therapeutic options are possible.

A. Dittrich: None. J. Geelen: None. J. Draaisma: None.

P04.012.A More than meets the eye: expanding and reviewing the clinical and mutational spectrum of brittle cornea syndrome

Tibbe Dhooge1, Tim Van Damme 1, Delfien Syx1, Laura Muiño Mosquera1,2, Sheela Nampoothiri3, Anil Radhakrishnan4, Pelin Ozlem Simsek-Kiper5, Gülen Eda Utine5, Maryse Bonduelle6, Isabelle Migeotte7, Osama Essawi1, Serdar Ceylaner8, Adila Al Kindy9, Brad Tinkle10, Sofie Symoens1, Fransiska Malfait1

1Center for Medical Genetics, Department of Biomolecular Medicine, Ghent University and Ghent University Hospital, Ghent, Belgium, 2Divison of Pediatric Cardiology, Department of Pediatrics, Ghent University Hospital, Ghent, Belgium, 3Department of Pediatric Genetics, Amrita Institute of Medical Sciences & Research Centre, Cochin, India, 4Department of Ophthalmology, Amrita Institute of Medical Sciences & Research Centre, Cochin, India, 5Department of Pediatric Genetics, Faculty of Medicine, Hacettepe University, Ankara, Turkey, 6Centre for Medical Genetics, Universitair Ziekenhuis Brussel, Brussels, Belgium, 7Center of Human Genetics, Université Libre de Bruxelles, Brussels, Belgium, 8Intergen Genetic Research Center, Ankara, Turkey, 9Department of Genetics, College of Medicine, Sultan Qaboos University, Muscat, Oman, 10Division of Medical Genetics, Peyton Manning Children’s Hospital, Indianapolis, IN, USA.

Brittle cornea syndrome (BCS) is a rare autosomal recessive disorder characterized by corneal thinning and fragility, leading to corneal rupture, the main hallmark of this disorder. Non-ocular symptoms include hearing loss, but also signs of connective tissue fragility, placing it in the Ehlers-Danlos syndrome (EDS) spectrum. It is caused by biallelic pathogenic variants in ZNF469 or PRDM5, which presumably encode transcription factors for extracellular matrix components. We report the clinical and molecular features of nine novel BCS families, four of which harbor variants in ZNF469 and five in PRDM5. We also performed a genotype and phenotype-oriented literature overview of all (N = 85) reported patients with ZNF469 (N = 53) and PRDM5 (N = 32) variants. Musculoskeletal findings may be the mean reason for referral, and often raise suspicion of another heritable connective tissue disorder such as kyphoscoliotic EDS, osteogenesis imperfecta or Marfan syndrome, especially when corneal rupture has not yet occurred. Our findings highlight the multisystemic nature of BCS and validate its inclusion in the EDS classification. Importantly, gene panels for heritable connective tissue disorders should include ZNF469 and PRDM5 to allow for timely diagnosis and appropriate preventive measures for this rare condition.

T. Dhooge: None. T. Van Damme: None. D. Syx: None. L. Muiño Mosquera: None. S. Nampoothiri: None. A. Radhakrishnan: None. P. Simsek-Kiper: None. G. Eda Utine: None. M. Bonduelle: None. I. Migeotte: None. O. Essawi: None. S. Ceylaner: None. A. Al Kindy: None. B. Tinkle: None. S. Symoens: None. F. Malfait: None.

P04.013.B Bilateral and symmetrical enchondromatosis lesions: clinical, radiologic and genetic findings

Pauline Marzin 1,2, Celine Huber2, Genevieve Baujat1,2, Caroline Michot1,2, Agnès Guichet3,4, Estelle Colin3,4, Michel Panuel5, Valérie Cormier-Daire1,2

1Service de Génétique clinique et Centre de référence pour les maladies osseuses constitutionnelles, AP-HP, Hôpital Necker-Enfants Malades, 75015 Paris, France, 2Université de Paris, Laboratory of Molecular and Physiopathological Bases of Osteochondrodysplasia, INSERM UMR 1163, Imagine Institute, 75015 Paris, France, 3Biochemistry and Genetics Department, University Hospital of Angers, Angers, France, 4MitoLab, UMR CNRS 6015-INSERM, MitoVasc Institute, University of Angers, Angers, France, 5Aix Marseille University, CNRS, EFS, ADES, Marseille, France.

Enchondromas are benign cartilaginous tumors, typically localized in the metaphyses. The most common enchondromatosis, Ollier disease and Maffucci syndrome related to IDH1 and IDH2, are characterized by asymmetrical lesions. Bilateral and/or symmetrical enchondromas can be seen in rarer enchondromatosis such as metachondromatosis, dyspondyloenchondromatosis, genochondromatosis and spondyloenchondrodysplasia (SPENCD, related to recessive variants in ACP5). Even though variants in PTPN11 and COL2A1 have been identified in metachondromatosis and dyspondyloenchondromatosis, respectively, molecular basis of bilateral and/or symmetrical enchondromas remain unknown. We performed whole exome sequencing analysis from blood samples in a cohort of 11 individuals from eight families presenting with bilateral and symmetrical enchondromas. We identified variants in known genes in three families of the eight (38%), including a mosaic variant in the IDH1 gene in two patients, with symmetrical enchondromas and a large homozygous deletion encompassing ACP5 in one patient with SPENCD. Variants of uncertain significant (VOUS) were identified in two other families, including a heterozygous nonsense variant in the PELI2 gene, encoding an interleukin-1 receptor-associated kinase (IRAK)-interacting proteins, in three affected members from the same family and a de novo heterozygous 1.5 Mb deletion of 12p12.1p11.22 in a patient with enchondroma and type E brachydactyly. No candidate variant was identified in three families including one family with recurrent sibs. These findings support the need of increasing the number of families tested and/or the analysis of affected tissues.

P. Marzin: None. C. Huber: None. G. Baujat: None. C. Michot: None. A. Guichet: None. E. Colin: None. M. Panuel: None. V. Cormier-Daire: None.

P04.014.C Sitting-to-standing height ratio is a sex-specific risk factor for chronic back pain

Maxim B. Freidin 1, Yakov A. Tsepilov2,3, Yurii S. Aulchenko2,3, Pradeep Suri4, Frances M. K. Williams1

1King’s College London, London, United Kingdom, 2Novosibirsk State University, Novosibirsk, Russian Federation, 3PolyOmica, ’s-Hertogenbosch, Netherlands, 4University of Washington, Seattle, WA, USA.

Background: Chronic back pain (CBP) is more common among females for reasons not fully understood. We hypothesized that sitting-to-standing height ratio, known to be different between the sexes, may contribute.

Methods: We used European individuals from UK Biobank (project #18219) comprising 222,361 males and 263,602 females to test this hypothesis. Logistic regression was used to assess the association of CBP with sitting height, standing height, and their ratio while adjusting for age, BMI, and job involving prolonged standing and heavy lifting. Mediation analysis and Mendelian randomization (MR) were applied to assess causality. Instruments for MR were selected from publicly available GWAS data for sitting-to-standing height ratio, ensuring non-overlap with chronic BP GWAS using UK Biobank.

Results: Both sitting and standing height exhibited a weak association of similar magnitude for CBP in both sexes (Table). However, sitting-to-standing height ratio was associated with greater odd of CBP in males (Table) but with lower odds in females (Table). Mediation analysis showed both direct and BMI-mediated risk effects of the ratio on CBP in males; while in females there was BMI-mediated risk effect and direct protective effect. MR supported a causal impact of sitting-to-standing height ratio on CBP risk in females, but not in males.

Conclusion: We provide evidence that different body proportions seen in men and women may contribute to the different risk of CBP between the sexes.

Odds ratios for anthropometric traits over chronic back pain
Trait Males Females
Standing height 1.013 (1.011-1.016) 1.011 (1.009-1.014)
Sitting height 1.030 (1.026-1.034) 1.013 (1.008-1.017)
Sitting-to-standing height ratio 14.373 (4.819-42.867) 0.139 (0.050-0.382)

M.B. Freidin: None. Y.A. Tsepilov: None. Y.S. Aulchenko: A. Employment (full or part-time); Modest; PolyOmica. P. Suri: None. F.M.K. Williams: None.

P04.016.A Transcriptomic diagnosis of a deep intronic CLCN7 mutation

Odelia Chorin 1, Naomi Yachelevich2, Khaled Mohamed3, Ilana Moscatelli4, John Pappas1, Kim Henriksen3, Gilad D. Evrony1,5

1Center for Human Genetics and Genomics, New York, NY, USA, 2Division of Clincal Genetic Services, Department of Pediatrics, New York, NY, USA, 3Nordic Biosciences Biomarkers and Research, Harlev, Denmark, 4Division of Molecular Medicine and Gene Therapy, Lund University, Lund, Sweden, 5Department of Pediatrics, and Department of Neuroscience & Physiology, New York University Grossman School of Medicine, New York, NY, USA.

Background Over half of children with rare genetic diseases remain undiagnosed despite maximal clinical evaluation and DNA‐based genetic testing. As part of an Undiagnosed Diseases Program applying transcriptome (RNA) sequencing to identify the causes of these unsolved cases, we studied a child with severe infantile osteopetrosis leading to cranial nerve palsies, bone deformities, and bone marrow failure, for whom whole‐genome sequencing was nondiagnostic.

Methods: We performed transcriptome (RNA) sequencing of whole blood followed by analysis of aberrant transcript isoforms and osteoclast functional studies.

Results: We identified a pathogenic deep intronic variant in CLCN7 creating an unexpected, frameshifting pseudoexon causing complete loss of function. Functional studies, including osteoclastogenesis and bone resorption assays, confirmed normal osteoclast differentiation but loss of osteoclast function.

Conclusion: This is the first report of a pathogenic deep intronic variant in CLCN7, and our approach provides a model for systematic identification of noncoding variants causing osteopetrosis—a disease for which molecular‐genetic diagnosis can be pivotal for potentially curative hematopoietic stem cell transplantation. Our work illustrates that cryptic splice variants may elude DNA‐only sequencing and supports broad first‐line use of transcriptome sequencing for children with undiagnosed diseases.

O. Chorin: None. N. Yachelevich: None. K. Mohamed: None. I. Moscatelli: None. J. Pappas: None. K. Henriksen: None. G.D. Evrony: None.

P04.017.B Two novel variants in MMP13 gene in a Czech family with metaphyseal anadysplasia type 1

Lucie Hrušková 1, Helena Paszeková1, Ivo Mařík2,3, Veronika Krulišová1, Daniela Zemková4, Anna Vážná5, Zděnka Vlčková1, Renata Michalovská1

1GHC Genetics, s.r.o., Prague, Czech Republic, 2Ambulant Centre for Defects of Locomotor Apparatus, Prague, Czech Republic, 3Faculty of Health Care Studies, West Bohemia University, Pilsen, Czech Republic, 4Motol University Hospital, Prague, Czech Republic, 5Department of Anthropology and Human Genetics, Faculty of Science, Charles University, Prague, Czech Republic.

Introduction: Metaphyseal anadysplasia type 1 is an autosomal dominant skeletal disease characterized by metaphyseal changes, epiphyseal dysplasia and rhizomelic shortening of limbs followed by regression of symptoms with later growth. Genetic origin is associated with pathogenic variants in MMP13 gene. Matrix metalloproteinase 13 encoded by this gene plays important roles in bone formation and growth.

Materials and Methods: We performed massive parallel sequencing of gDNA isolated from whole blood on NextSeq (Illumina) using panel Clinical Exome (CES) kit by Sophia Genetics, followed by Sanger sequencing and CNV analysis. This solution consists of 116,355 individually designed probes that span approx. 11 Mb of target regions covering more than 4,900 genes, with known mendelian/inherited disease-causing mutations.

Results: Molecular genetic analysis identified two previously unreported heterozygous cis-variants c.236A>C and c.251T>G in MMP13 in a Czech boy suspected with metaphyseal anadysplasia, type 1. Clinical-radiological examination showed rhizomelic shortness of stature, femoral bowing, abnormality of metaphyses, delayed ossification of carpal bones and patella. The same bone dysplasia occurs in the sister, mother, maternal aunt and maternal grandfather. Subsequent molecular genetic testing in these family members showed that the two variants described above in the MMP13 gene also occur in them and thus segregate with the occurrence of the disease in the family.

Conclusion: Our findings demonstrate the efficiency of exome sequencing approach in determination of molecular background in differential diagnosis not only of skeletal dysplasias. Further, our findings expand genotype spectrum of MMP13 associated disorders and offer precise diagnosis of metaphyseal anadysplasia type 1.

L. Hrušková: None. H. Paszeková: None. I. Mařík: None. V. Krulišová: None. D. Zemková: None. A. Vážná: None. Z. Vlčková: None. R. Michalovská: None.

P04.018.C Very early diagnosis of Cole-Carpenter syndrome: novel variant and maternal mosaicism

Daniele Guadagnolo 1, Gioia Mastromoro1, Enrica Marchionni1, Francesca Di Palma1, Claudia Cesario2, Mario Roggini3, Antonio Novelli2, Antonio Pizzuti1

1Department of Experimental Medicine, Sapienza University of Rome, Rome, Italy, 2Laboratory of Medical Genetics, Bambino Gesù Children’s Hospital, IRCCS, Rome, Italy, 3Department of Pediatrics, Sapienza University of Rome, Rome, Italy.

Introduction: Fetal abnormal head shape represents a significant challenge in prenatal diagnosis. It can be the initial presentation of several disorders, requiring multidisciplinary approaches.

Materials and methods: A 35-year-old woman was referred at 34 gestational weeks for unusual fetal head shape. Chromosomal molecular analysis, performed after amniocentesis for multiple soft markers, was normal. After caesarean delivery at 38 gestational weeks, the baby was in normal length and weight ranges, but macrocephaly, micrognathia and broad nasal bridge were noted. Total body X-rays and trio Clinical Exome Sequencing (CES) were requested.

Results: X-rays showed dolicocephaly, ossified metopic suture, widening of the bregmatic fontanella and of the sagittal and lambdoideal sutures, perisutural calcified spots. CES detected a novel heterozygous missense variant in P4HB (prolyl 4-hydroxylase subunit b; MIM*176790) in the proband (NM_000918.4:c.1375G>A;NP_000909.2:p.Gly459Arg;rs749458033). The same variant was found in heterozygosis with 34% mosaicism rate in the mother. Heterozygous P4HB mutations cause Cole-Carpenter syndrome 1 (MIM#112240). The variant was not described in literature, HGMD, ClinVar or DECIPHER. It is very rare (GnomADv2.1.1: 0.000016) and has high predicted deleteriousness. It is classified as Likely Pathogenic. Cole-Carpenter syndrome is characterized by dysmorphisms, wide sutures and fontanelles, short stature, proptosis, hydrocephalus, bone fragility, no intellectual disability. Somatic mosaicism for a pathogenic P4HB has already been described in the healthy father of an affected child.

Conclusion: Early molecular analysis for skull anomalies can guide subsequent clinical approaches but also yield complex results. We suspect that parental mosaicism might be more common than previously thought for P4HB variants.

D. Guadagnolo: None. G. Mastromoro: None. E. Marchionni: None. F. Di Palma: None. C. Cesario: None. M. Roggini: None. A. Novelli: None. A. Pizzuti: None.

P04.020.A hiPSC-derived epidermal keratinocytes from ichthyosis patients show altered expression of cornification markers

Dulce Lima Cunha1,2,3, Amanda Oram1, Robert Gruber4, Roswitha Plank1,2, Arno Lingenhel2, Manoj Kumar Gupta5, Janine Altmüller6, Peter Nürnberg6, Mariya Moosajee3, Matthias Schmuth4, Johannes Zschocke2, Tomo Šarić5, Katja Martina Eckl2,7, Hans Christian Hennies 1,2,6

1Department of Biological and Geographical Sciences, University of Huddersfield, Huddersfield, United Kingdom, 2Institute of Human Genetics, Medical University of Innsbruck, Innsbruck, Austria, 3Institute of Ophthalmology, UCL, London, United Kingdom, 4Department of Dermatology, Medical University of Innsbruck, Innsbruck, Austria, 5Center for Physiology and Pathophysiology, Institute for Neurophysiology, University Hospital Cologne, Cologne, Germany, 6Cologne Center for Genomics, University Hospital Cologne, Cologne, Germany, 7Department of Biology, Edge Hill University, Ormskirk, United Kingdom.

Introduction: Inherited ichthyoses represent a heterogeneous group of skin disorders characterised by impaired epidermal barrier function and disturbed cornification. Current knowledge about disease mechanisms has been uncovered mainly through mouse models or human organotypic models. However, most mouse lines suffer from severe epidermal barrier defects causing neonatal death and human keratinocytes have very limited proliferation ability in vitro.

Results: We have generated human induced pluripotent stem cells (hiPSCs) from patients with congenital ichthyosis, either non-syndromic autosomal recessive congenital ichthyosis (ARCI) or the ichthyosis syndrome trichothiodystrophy (TTD). hiPSCs were successfully differentiated into basal keratinocytes (hiPSC-bKs), with high expression of epidermal keratins 5 and 14. Terminal differentiation of hiPSC-bKs was induced and markers keratin 1 and involucirn were expressed. TTD1 hiPSC-bKs showed reduced expression of FLG and SPRR2B in line with previous reports and mouse models. ARCI hiPSC-bKs showed more severe defects, with downregulation of several genes involved in epidermal ceramide metabolism. Differences to findings in ARCI patients might point to the importance of specific mutations and an influence of patient treatment on their keratinocyte expression profiles.

Conclusions: Our results demonstrate the successful generation of hiPSC-based in vitro models mimicking the disease phenotypes, proving a valuable system for molecular investigations and drug development. Studies with organotypic models will be used to further characterize functional changes associated with ichthyosis. Patient-derived hiPSCs can be a long-term source for a variety of different cells and significantly contribute to the development of complex skin disease models, ultimately facilitating the translation of therapeutic approaches into clinical studies.

D. Lima Cunha: None. A. Oram: None. R. Gruber: None. R. Plank: None. A. Lingenhel: None. M. Gupta: None. J. Altmüller: None. P. Nürnberg: None. M. Moosajee: None. M. Schmuth: None. J. Zschocke: None. T. Šarić: None. K. Eckl: None. H. Hennies: None.

P04.021.B Four novel EFNB1 variants found through sequencing-based methods in female patients with craniofrontonasal syndrome

Ewelina Bukowska-Olech 1, Anna Jakubiuk-Tomaszuk2, Maria Jedrzejowska3, Ewa Obersztyn4, Pawel Gawlinski4, Michal Piechota5, Marta Bielska6, Aleksander Jamsheer1,5

1Department of Medical Genetics, Poznan University of Medical Sciences, Poznan, Poland, 2Department of Pediatric Neurology and Rehabilitation, Medical University of Bialystok, Bialystok, Poland, 3Department of Medical Genetics, The Children’s Memorial Health Institute, Warsaw, Poland, 4Department of Medical Genetics, Institute of Mother and Child, Warsaw, Poland, 5Centers for Medical Genetics GENESIS, Poznan, Poland, 6Department of Pediatrics, Hematology, Oncology and Diabetology, Medical University of Lodz, Lodz, Poland.

Background: Craniofrontonasal syndrome (CFNS) is a rare X-linked disorder that results from pathogenic variants in the EFNB1 gene. The syndrome inherits in a paradoxical manner and exceptionally presents greater severity of symptoms in heterozygous females than hemizygous males.

Materials and methods: We recruited five sporadic (patients 1-4) and one familial case (patients 5& 6), all of whom presented a spectrum of craniofacial abnormalities. Of note, we reported discordant phenotype in twin female patients 5& 6. We applied either targeted next-generation sequencing (NGS) of a custom gene panel or PCR and Sanger sequencing to achieve a molecular diagnosis. Besides, we run both zygosity analysis and X chromosome inactivation (XCI) assay for twin patients 5& 6.

Results: First, we reported three additional novel variants in the EFNB1 gene: p.(Trp12*), p.(Tyr73Metfs*86), p.(Glu210*), and one known: p.(Cys64Phe). Second, we confirmed the monozygosity of patients 5& 6. Finally, we showed random XCI in twin patient 5 (46% vs 54%), who presents milder phenotype in comparison to her twin sister (patient 6) having non-random XCI (84% vs 16%).

Conclusion: Our findings provide valuable molecular data that may be applied both in diagnostics and genetic counselling. We have described the intriguing differences of the clinical phenotype in the monozygotic twin patients 5& 6 harbouring an identical p.(Glu210*) variant and we have pointed to the presence of unusual phenomenon such as mildly affected females with CFNS.

This work was supported by the grants from the Polish National Science Centre, Poland: UMO-2016/23/N/NZ5/02577 to E.B.-O and UMO-2016/22/E/NZ5/00270 to A.J.

E. Bukowska-Olech: None. A. Jakubiuk-Tomaszuk: None. M. Jedrzejowska: None. E. Obersztyn: None. P. Gawlinski: None. M. Piechota: None. M. Bielska: None. A. Jamsheer: None.

P04.022.C Comprehensive genetic screening in patients with syndromic craniosynostosis

Trayan N. Delchev, Savina Hadjidekova, Hadil Kathom, Stoyan Bichev, Tsvetina Veleva, Iliana Boneva, Daniela Avdjieva-Tzavella

Medical University-Sofia, Sofia, Bulgaria.

Introduction: Syndromic craniosynostosis is a genetically determined premature ossification and closure of one or more of the cranial sutures. This may result in severe dysmorphism, increased intracranial pressure, seizures, visual and hearing defects, psychomotor delay and behavioural anomalies. Syndromic craniosynostosis is also commonly associated with additional malformations and dysmorphic features.

Materials and Methods: We present our comprehensive genetic investigation of 39 patients with syndromic craniosynostosis screened systematically with a combination of cytogenetic analysis, multiplex ligation-dependent probe amplification (MLPA) and array-based comparative genomic hybridisation (aCGH).

Results: Pathological findings were established in 15.3% (6/39) of the cases using aCGH, 8.33% (3/39) using MLPA and 2.85% (1/39) using conventional karyotyping. About 12.8% (5/39) of the patients with normal karyotype carried submicroscopic chromosomal rearrangements.

Conclusions: In our study array-based comparative genomic hybridisation had the highest detection rate compared to chromosome analysis and MLPA. „Gain of function“ variations and duplications were the predominant type of genetic defect we found. This suggests the leading role of those findings in the pathogenesis of syndromic craniosynostosis.

T.N. Delchev: None. S. Hadjidekova: None. H. Kathom: None. S. Bichev: None. T. Veleva: None. I. Boneva: None. D. Avdjieva-Tzavella: None.

P04.023.D Analysis of novel splice site variants in craniosynostosis causing genes

Angela Borst 1, Tillmann Schweitzer2, Denise Horn3, Erdmute Kunstmann1, Eva Klopocki1

1Institute of Human Genetics University of Wuerzburg, Würzburg, Germany, 2Department of Pediatric Neurosurgery University Hospital of Wuerzburg, Würzburg, Germany, 3Institute for Medical and Human Genetics Charité Universitätsmedizin Berlin, Berlin, Germany.

Congenital malformation of the skull are rare disease conditions, which may have severe impact on the life of patients. Craniosynostosis appears with a prevalence of 1 in 2500 newborns and represents a premature fusion of one or more cranial sutures. The disease can either occur as part of a syndrome or as non-syndromic, isolated craniosynostosis. The Craniosynostosis 3 (MIM 615314) phenotype, a molecular well-described form of isolated craniosynostosis, is caused by mutations in the TCF12 gene. In contrast to TCF12, mutations in the FGFR2 gene are multifaceted and are associated with diverse syndromes, including craniosynostosis syndromes, i.e. Apert (MIM 101200), Pfeiffer (MIM 101600) and Saethre-Chotzen (MIM 101400) syndromes.

The aim of our study was, to identify novel genetic variants in craniosynostosis associated genes by Next Generation Sequencing (NGS) using a specific craniosynostosis gene panel and to further analyse the detected genetic variants. In a proportion of cases changes in the splicing process of the corresponding gene were predicted in silico. To validate the consequences of the splice site variants on correct transcript splicing, we performed in vitro splice assays with mutation-matched minigene constructs in U2OS cells. In total, we investigated four potential splice site variants in TCF12 and FGFR2. In three patients with coronal synostosis, the variants lead to an aberrant splicing (exon skipping, inclusion of intronic sequence) of the TCF12 transcript. Taken together, we could validate novel splice site mutations in the TCF12 and FGFR2 genes and extend the mutational spectrum.

A. Borst: None. T. Schweitzer: None. D. Horn: None. E. Kunstmann: None. E. Klopocki: None.

P04.024.A TRAF3 and NBR1 both influence the effect of CYLD(Arg936X) mutation on NF-κB activity

Judit Danis 1,2,3, Evelyn Kelemen3,4, Neil Rajan5, Nikoletta Nagy1,3, Márta Széll1,3, Éva Ádám3

1ELKH-SZTE Dermatological Research Group, Szeged, Hungary, 2HCEMM-USZ Dermatological Research Group, Szeged, Hungary, 3Department of Medical Genetics, University of Szeged, Szeged, Hungary, 4Department of Dermatology and Allergology, University of Szeged, Szeged, Hungary, 5Institute of Genetic Medicine, Newcastle University, Newcastle upon Tyne, United Kingdom.

Recently the cases of a Hungarian and an Anglo-Saxon pedigrees has been presented, who are affected by CYLD cutaneous syndrome (syn: Brooke-Spiegler syndrome), carry the same disease-causing mutation (c.2806C>T, p.Arg936X) of the cylindromatosis (CYLD) gene but exhibit striking differences in their phenotypes. By whole exome sequencing, missense genetic variants of the TRAF3 and NBR1 genes were identified from affected family members of the Hungarian family, that are not present in the Anglo-Saxon family, suggesting their affected proteins (TRAF3 and NBR1) are putative phenotype-modifying factors. To clarify how wild type and mutant TRAF3 and NBR1 modify the effect of CYLD on NF-κB signal transduction pathway, an in vitro experimental system was set up. Our study revealed that the combined expression of mutant CYLD(Arg936X) both with TRAF3 and NBR1 caused increased NF-κB activity, regardless of the latter two proteins being wild type or mutant. We conclude that increased expression levels of these proteins further strengthen the effect of CYLD(Arg936X) mutation on the NF-κB activity in HEK293 cells and may explain the phenotype modifying effect of these genes in CYLD cutaneous syndrome.

Funding: NKFIH K128736, FK134355

J. Danis: None. E. Kelemen: None. N. Rajan: None. N. Nagy: None. M. Széll: None. É. Ádám: None.

P04.025.B Dedifferentiated liposarcoma: Case report

Fiorita G. L. Mundim 1,2, Wesley R. S. Liberato1, Reigson A. Dias1, Leticia Benevenutti1, Felipe R. C. Meira1, Katia M. T. C. Castro1, Pedro G. L. Mundim1, Nathalia T. Sousa1, Renan Reis2

1UNIFENAS, Alfenas/MG, Brazil, 2UNIVAS, Pouso Alegre/MG, Brazil.

Introduction: Dedifferentiated liposarcoma (DDL) is considered a well-differentiated form of histological (tumor) progression of liposarcoma. Around 15% of cases have the potential to metastasize and with a high rate of recurrence. Since DDL can morphologically mimic other sarcomas that have higher rates of metastasis, especially in the retroperitoneum and inguinal canal, the diagnosis and correct classification of DDL are essential to determine an adequate prognosis and treatment for the patient.

Materials and Methods: In the present case report, it is a female patient, 39 years old, residing in Pouso Alegre, Brazil, in October 2019. She started with pain in the left gluteal region associated with nodular lesion in the place with progressive growth. The biopsy showed a mesenchymal neoplasia. Subsequent immunohistochemical examination revealed a tumor compatible with a Dedifferentiated liposarcoma.

Results: DDL is a cellular and typically non-lipogenic sarcoma with significant pleomorphism. Molecularly characterized by a ring or a giant marker / rod chromosomes composed of material from 12q13-15. Most DDL also have gene amplification MDM2 and CDK4, which may be similar to other tumors (Intimal sarcoma, Parosteal osteosarcoma, low grade central osteosarcoma, etc.)

Conclusion: For the diagnosis and correct classification of the DDL, it is essential to carry out a cytogenetic study, Immunostaining for MDM2 and CDK4 or molecular testing for 12q13-15 amplification.

F.G.L. Mundim: None. W.R.S. Liberato: None. R.A. Dias: None. L. Benevenutti: None. F.R.C. Meira: None. K.M.T.C. Castro: None. P.G.L. Mundim: None. N.T. Sousa: None. R. Reis: None.

P04.027.D Targeted next generation sequencing of Hypohidrotic ectodermal dysplasia in Egyptian pedigrees

Hoda Abdalla Ahmed Radwan 1, Khalda Sayed Amr1, Eman Rabie1, Ghada El-Kamah1, Inas Sayed1, Mennat Mehrez1, Nehal Hassib1, Suher Zada2, Maha Abuzaid1, Mohamed Abdel-Kader1, Mostafa Mostafa1, Yasmine Mohsen1

1National Research Centre, Cairo, Egypt, 2American University, Cairo, Egypt.

Introduction: Ectodermal dysplasia (ED) defines a group of genetic disorders characterized by developmental defects of 2 or more structures of ectodermal origin. Pathogenesis is governed by defects in many genes involved in multiple developmental pathways. Hypohidrotic ectodermal dysplasia (HED) is the most common ED form characterized by hypodontia, hypohidrosis, hypotrichosis and in several instances dysmorphic features. Mutations in four genes: EDA, EDAR, EDARADD and WNT10A were reported to contribute to 90% of HED cases. The aim of our study is to assess the clinical and molecular profiles of HED in Egyptian patients.

Patients and Methods: Thirty-five HED patients descending from 32 unrelated pedigrees were clinically diagnosed based on thorough clinical and dental examination. Patients were tested using an NGS custom panel comprising the four aforementioned genes. Sanger sequencing was performed for variant confirmation and segregation analysis. Pathogenicity of variants was assessed according to American College of Medical Genetics (ACMG) guidelines as well as bioinformatics tools (e.g., Polyphen, SIFT, and Alamut).

Results: We identified 12 pathogenic mutations: six novel and six previously reported mutations. A molecular basis was identified in 16/32 pedigrees. EDA was the most frequent (13/16), followed by EDARADD (2/16) and EDAR (1/16). No variants were identified in the WNT10A gene.

Conclusion: The EDA, EDAR and EDARADD harbored the molecular pathology in 50% of the studied HED patients. That might be attributed to different ethnic backgrounds, and further analyses through exome sequencing of the uncharacterized patients might confirm rare genes involvement or identify new genes governing ED pathogenesis.

H.A.A. Radwan: None. K.S. Amr: None. E. Rabie: None. G. El-Kamah: None. I. Sayed: None. M. Mehrez: None. N. Hassib: None. S. Zada: None. M. Abuzaid: None. M. Abdel-Kader: None. M. Mostafa: None. Y. Mohsen: None.

P04.028.A Peds2Gene study: clinical and molecular delineation of a Spanish cohort of pediatric patients with Ehlers-Danlos Syndrome

Laura Plaza, Antonio Federico Martinez-Monseny, Dídac Casas, Asunción Vicente, Francesc Palau

Hospital Sant Joan de Deu, Barcelona, Spain.

Introduction: Ehlers-Danlos Syndrome (EDS) is a group of hereditary connective tissue disorders that manifests as skin hyperextensibility, hypermobility of joints and fragility of blood vessels. The hypermobility-type EDS (hEDS) is the most frequent and its molecular cause is unknown. Diagnosing EDS is challenging due to the high clinical variability and the lack of a diagnostic biomarker in paediatrics.

Materials and Methods: Prospective and retrospective observational study at a tertiary pediatric hospital including patients < 18 years at the date of diagnosis with EDS between 2012-2020. The data was collected reviewing clinical records.

Results: Thirty-five patients were included; hEDS (65.7%), classical (22.8%), vascular (5.7%), kyphoscoliotic (2,8%) and dermatosparaxis types (2.8%). Most of them were women (62.9%). Musculoskeletal symptoms were the commonest complication (91.4%), with chronic pain of joints being the most frequent feature (62.9%). Psychiatric disorders were present in 54.3% of patients, with the most frequent being anxiety and depression (22.8%). A higher Beighton score did not correlate with musculoskeletal, dermatologic, or other complications. Atrophic scarring and easy bruising were less frequent in hEDs patients (p < 0.000,p < 0.002). Chronic abdominal pain was significantly associated with migraine (p < 0.002). All the types have molecular confirmation except the hEDS, but we found some genes (TNXB,ELN,PIEZO2) that could be implicated, requiring future studies.

Conclusions: This is the first Spanish study focusing on pediatric EDS. We propose interesting and novel genotype-phenotype aspects. We provide detailed insights into the potential complications during childhood to help minimize the physical and emotional impact of the syndrome in patients and their families.

L. Plaza: None. A. Martinez-Monseny: None. D. Casas: None. A. Vicente: None. F. Palau: None.

P04.029.B New variant in TANGO1 gene in a patient with hypermobile type of Ehlers-Danlos syndrome

Anna Junkiert-Czarnecka, Maria Pilarska-Deltow, Aneta Bąk, Marta Heise, Olga Haus

Department of Clinical Genetics Collegium Medicum in Bydgoszcz, Bydgoszcz, Poland.

Introduction: Hypermobile Ehlers-Danlos syndrome (hEDS) is a non-inflammatory connective tissue disorder. It is perhaps the most common hereditary connective tissue disorder. hEDS unlike other types of EDS has no known genetic etiology. One of the dysfunctions in hEDS patients’ cells is the impaired transport of extracellular matrix proteins from cells to intercellular space. A protein taking part in this process is TANGO1 encoded by the TANGO1 gene. The aim of the study was a sequencing analysis of TANGO1 gene and an attempt to evaluate its potential role in the etiology of the hypermobile type of Ehlers-Danlos syndrome.

Methods: The study was carried out on a patient with a hypermobile type of EDS. It was a 48-year-old woman presented with: joints hypermobility, recurrent dislocations, whole body pain, easy bruising, striae, gothic palate and arachnodactyly. Her daughter had mild symptoms of hEDS. Sequencing analysis of TANGO1 was performed using the Sanger sequencing technique. For bioinformatic analysis, VarSome tool was applied.

Results: In studied patient variant c.5637_5638insA (p.Leu1880ThrfsTer6) in TANGO1 gene was detected. This variant was not described up to now in EDS patients, and according to VarSome it is pathogenic.

Conclusion: Analysis of the role of the TANGO1 gene in the etiology of hypermobile type of EDS is a new approach to evaluating the genetic background of this disorder. The impact of a pathogenic variant in TANGO1 on its function and resulting extracellular matrix condition need further investigations. This investigation was financed by a grant from National Science Centre Poland (2018/29/N/NZ5/00345).

A. Junkiert-Czarnecka: None. M. Pilarska-Deltow: None. A. Bąk: None. M. Heise: None. O. Haus: None.

P04.030.C Molecular spectrum in 23 Spanish families affected by hereditary multiple osteochondromas

M C. Martínez-Romero 1,2,3, A T. Serrano-Antón1,2,4, M J. Sánchez-Soler1,2,3, L Rodríguez-Peña1, M J. Ballesta-Martínez1,2,3, V López-González1,2,4, M Barreda-Sánchez1,3, M E. Pérez-Tomás1, P Carbonell-Meseguer5,2, C Salcedo-Cánovas6, E Guillén-Navarro1,2,4

1Sección de Genética Molecular (Centro de Bioquímica y Genética Clínica), Sección de Genética Médica (Servicio de Pediatría), Hospital Clínico Universitario Virgen de la Arrixaca. Instituto Murciano de Investigación Biosanitaria (IMIB-Arrixaca), El Palmar (MURCIA), Spain, 2CIBERER, Centro de Investigación Biomédica en Red de Enfermedades Raras, ISCIII, Madrid, Spain, 3Cátedra de Genética Médica. UCAM- Universidad Católica de Murcia, Murcia, Spain, 4Departamento de Pediatría. Facultad de Medicina. UMU-Universidad de Murcia, Murcia, Spain, 5Sección de Genética Molecular (Centro de Bioquímica y Genética Clínica), Sección de Genética Médica (Servicio de Pediatría). Hospital Clínico Universitario Virgen de la Arrixaca. Instituto Murciano de Investigación Biosanitaria (IMIB-Arrixaca), El Palmar (MURCIA), Spain, 6Servicio de Traumatología. CSUR ortopedia. Hospital Clínico Universitario Virgen de la Arrixaca, El Palmar (MURCIA), Spain.

Introduction: Osteochondroma represents the most common benign osseous tumour, 20-50%; in familiar occurrence two AD diseases are probable: hereditary multiple osteochondromas (HMO)(ORPHA:321) associated with variants in EXT1(MIM,#133700) or EXT2(MIM,#133701) genes and metachondromatosis (MC)(ORPHA:2499), caused in PTPN11(MIM,#156250). Both disorders have risk of malignant transformation (3-5%). Our goal is to find out the molecular spectrum in a cohort of Spanish patients with multiple osteochondroma.

Methodology: Coding and flanking exons of EXT1(NM_000127.2), EXT2(NM_207122.) and PTPN11(NM_002834.4) genes were performed by capture (SureSelectQXT®Agilent) and sequenced by Illumina platform. Minimum depth coverage was 20x. Copy Number Variation (CNV) were evaluated using DecoN algorism and MLPA-P215-B3-EXT-Kit.

Results: 23 index patients (12 females/11 males) with average age of 15 years were included. Distribution of variants by genes were EXT1(56.5%), EXT2(34.8%) and PTPN11(8.7%). De novo variants represented 56.5% cases versus 44.5% inherited. A total of 22 pathogenic variants (15 novel/7 reported in HGMD®2020.4) were identified, most of them privates (22/23) being only recurrent one variant in two cases (EXT1:c.936-11insTG). Type of variants: frameshift (45.4%), nonsense (27.3%), splicing (13.6%), CNV (9%) and missense (4.5%). Anatomical distribution referred of osteochondroma was mainly in long-bone extremities (65.2%) and hands (43.5%), nevertheless genotype-phenotype correlation by genes was not observed; in 7 patients (30.4%) osseous deformities required surgery but malignant degeneration was not notified.

Conclusion: -There is a broad allelic heterogeneity in EXT1 and EXT2 genes resulting with 15 novel variants identified. -Secondary variants in PTPN11 gene highlights the importance of expanding the study to other HMO genes for a more suitable personalized treatment.

M.C. Martínez-Romero: None. A.T. Serrano-Antón: None. M.J. Sánchez-Soler: None. L. Rodríguez-Peña: None. M.J. Ballesta-Martínez: None. V. López-González: None. M. Barreda-Sánchez: None. M.E. Pérez-Tomás: None. P. Carbonell-Meseguer: None. C. Salcedo-Cánovas: None. E. Guillén-Navarro: None.

P04.031.D Father and daughter with acromicric dysplasia

Cinzia Mautone 1, Mario Francesco Iasevoli1, Pamela Paglia1, Giuseppina Barra1, Dario Di Salvio2, Mariateresa Falco3, Carolina Mauro3, Emanuele Agolini4, Antonio Novelli4, Daniela Melis1,3

1Department of Medicine, Surgery and Dentistry "Scuola Medica Salernitana" University of Salerno, Baronissi (SA), Italy, 2Department of Maternal-Infant, Pediatric Section, University of Naples Federico II, Napoli, Italy, 3Pediatric Unit San Giovanni di Dio e Ruggi d’Aragona University Hospital, Salerno, Italy, 4Departmnent of Laboratories, Medical Genetic Unit Bambino Gesù Children’s Hospital, Roma, Italy.

Introduction: Mutations of FBN1 are responsible for different disorders including Acromicric dysplasia (characterized by short hands and feet, joint limitations), Geleophysic dysplasia (progressive heart involvement), and Weill-Marchesani syndrome (microspherophakia).Case report: We describe father and daughter with short stature with no other systemic involvement. Father was evaluated from first years of life for congenital hip dysplasia and short stature; he underwent GH therapy with poor results. His childhood X-rays showed shortness of long bones and hands’ phalanges and typical notches of II and V metacarpus. At physical examination both showed round face, well-defined eyebrows, bulbous nose with anteverted nostrils, disharmonic short stature with short limbs, relative macrocephaly, and brachydactyly; hand camptodactily was detected during childhood in the father and in the child at 4 years of age. No joint stiffness was recored during adulthood in the father. NGS panel of genes involved in skeletal dysplasias found an heterozygous variant (c.5285G>A) in FBN1 gene in both father and daughter, previously unreported; a different pathogenetic variant involving Glycine 1762 has been associated with both Acromicric and Geleophysical dysplasia. No cardiac or ocular involvement were present, thus a diagnosis of Acromicric dysplasia was proposed.

Conclusion: We report a familial case of Acromicric dysplasia, with a novel variant in FBN1 gene. Clinical features of both father and daughter overlap. The father underwent GH therapy in childhood, with poor results. The report could expand clinical features of this condition and help to define natural history of the disease.

C. Mautone: None. M. Iasevoli: None. P. Paglia: None. G. Barra: None. D. Di Salvio: None. M. Falco: None. C. Mauro: None. E. Agolini: None. A. Novelli: None. D. Melis: None.

P04.032.A Aarskog-Scott syndrome: case report and updated-review

Marta Spodenkiewicz 1, Gauthier Loron2,3,4, Céline Poirsier1, Francesco Laconi5, Marie Massier1, Perrine Venot2, Clémence Jacquin6, Lucas Hérissant1, Emilie Landais1, Martine Doco-Fenzy1,7,8

1Service de génétique, CHU de Reims, Reims, France, 2Service Pédiatrie B, CHU Reims, Reims, France, 3Faculté de médecine, Reims, France, 4CReSTIC/ EA3804, Reims, France, 5Service de chirurgie Pédiatrique, CHU de Reims, Reims, France, 6CHU Reims genetics Unit, Reims, France, 7SFR CAP SANTE, UFR de médecine, Reims, France, 8EA3801, Reims, France.

Aarskog-Scott syndrome (ASS) or facio-digito-genital dysplasia (OMIM#305400) is a rare X-linked inherited disorder caused by pathogenic variants in the FYVE, RhoGEF, and pleckstrin homology domain-containing protein 1 (FGD1) gene. This syndrome affects about 1 in 1,000,000 births. Affected patients present a broad clinical phenotype with dysmorphism, short stature, skeletal and urogenital anomalies.

The probant is a two years old boy with camptodactyly, dysmorphism, cryptorchidism and short stature. Exome sequencing found a hemizygous variant c.123del (NM_004463) in FGD1, inherited from his mother who presents camptodactyly and hypertelorism. FGD1 gene encodes the FGD1 protein, a guanine nucleotide exchange factors, able to activate Rho GTPase cell division cycle 42 (CDC42). Rho GTPase-CDC42 plays a role in control cytoskeleton-dependent membrane rearrangements, transcriptional activation, secretory membrane trafficking, extracellular matrix and cytoskeleton remodeling. Rho GTPases are involved in human diseases as developmental, hematological, autoinflammatory, autoimmune disorders. To our knowledge less than 60 damaging pathogenic variants in FGD1 were reported to date, and the variant of our patient was not yet reported.

The aim of this work is to report the case of our patient and his family with a highly variable phenotype and to review the literature of patients in order to update the spectrum of this rare disease.

M. Spodenkiewicz: None. G. Loron: None. C. Poirsier: None. F. Laconi: None. M. Massier: None. P. Venot: None. C. Jacquin: None. L. Hérissant: None. E. Landais: None. M. Doco-Fenzy: None.

P04.033.B Clinical and molecular study of a cohort of 79 patients with a pathogenic variation in GDF5 gene and review of the literature

William Dufour 1, Fabienne Escande1, Anne-Sophie Jourdain1, Odile Boute1, Anne Dieux1, Carherine Vincent-Delorme1, Muriel Holder2, Francesca Forzano3, Laurence Faivre4, Christel Thauvin4, Marion Gerard5, David Geneviève6, Marjolaine Willems6, Bertrand Isidor7, Chloé Quélin8, Sylvie Odent8, Valérie Cormier-Daire9, Geneviève Baujat9, Gilles Morin10, Lyse Ruaud11, Yline Capri11, Alice Goldenberg12, Yves Alembik13, Nicole Revençu14, Bianca Ethel Gutiérrez-Amavizca15, Nicolas Chassaing16, Fanny Laffargue17, Christine Francannet17, Tiffany Busa18, Véronique Paquis19, Sylvie Manouvrier1, Florence Petit1

1CHU de Lille, Lille, France, 2Guy’s and St Thomas’ NHS Foundation Trust, London, United Kingdom, 3Guy’s and St Thomas’ NHS Foundation Trust, London, France, 4CHU de Dijon, Dijon, France, 5CHU de Caen, Caen, France, 6CHU de Montpellier, Montpellier, France, 7CHU de Nantes, Nantes, France, 8CHU de Rennes, Rennes, France, 9Hôpital Necker-Enfants Malades, Paris, France, 10CHU Amiens-Picardie, Amiens, France, 11Hôpital Robert-Debré, Paris, France, 12CHU de Rouen, Rouen, France, 13CHU de Strasbourg, Strasbourg, France, 14Cliniques Universitaires Saint-Luc, Brussels, Belgium, 15Ciudad Juarez Autonomous University, Ciudad Juarez, Mexico, 16CHU de Toulouse, Toulouse, France, 17CHU de Clermont-Ferrand, Clermont-Ferrand, France, 18Hôpital Universitaire Timone Enfants, Marseille, France, 19CHU de Nice, Nice, France.

The GDF5 gene is mainly expressed in the joints during the embryonic period. It is known to be responsible for different bone pathologies according to its alteration mechanism: brachydactyly in case of mono-allelic loss of function; symphalangism and multiple synostoses in case of heterozygous gain of function; Grebe, Du Pan and Hunter-Thompson syndromes in case of bi-allelic loss of function; susceptibility to osteoarthritis and hip dysplasia in the presence of polymorphisms within its promoter. Through the numerous associated phenotypes, GDF5 illustrates the difficulties in interpreting genomic variations. We studied a large international cohort of 79 patients harbouring GDF5 variations in order to: 1) Describe precisely the clinical and molecular data of patients; 2) Identify rare phenotypes and 3) Demonstrate genotype-phenotype correlations. We observed that some features seems to have a higher frequency in our cohort compared to those of the literature, notably 2nd ray clinodactyly and Angel Shaped Phalango-Epiphyseal Dysplasia, joint laxity, various dental abnormalities, premature osteoathritis and hip disorders (mainly coxo-femoral dysplasia).

Table 1: Frequency of the different impairment in patients with brachydactyly type C
  cohort patients percentage literature patients percentage
Angel-Shaped Phalango-Epiphyseal Dysplasia 22/63 34.9% 5/27 18.5%
1st rays brachymetacarpy 49/63 77.8% 16/27 59.3%
2nd rays brachymesophalangy 51/63 81% 18/27 66.7%
3rd rays brachymesophalangy 36/63 57.1% 13/27 48.1%
5th rays brachymesophalangy 47/63 74.6% 19/27 70.4%
2nd rays brachybasophalangy 14/63 22.2% 8/27 29.6%
3rd rays brachybasophalangy 14/63 22.2% 10/27 37%
2nd rays clinodactyly 22/66 33.3% 17/30 56.7%
3rd rays clinodactyly 14/66 21.2% 11/30 36.7%
5th rays clinodactyly 43/66 65.2% 17/30 56.7%
polyphalangy 19/63 30.2% 9/27 33.3%
pseudoepiphyses 16/63 25.4% 6/27 22.2%
foot damage 20/49 40.8% 11/26 42.3%
height<-2SD 21/57 36.8% 7/19 36.8%
bone age delay 24/26 92.3% 7/8 87.5%
dental anomalies 19/55 34.5% 1/15 6.7%
hip anomalies 15/61 24.6% 3/19 15.8%
joint laxity 16/62 25.8% 2/17 11.8%
premature osteoarthrosis 6/33 18.2% 0/14 0%
asymptomatic 3/74 4.1% 4/33 12.1%

W. Dufour: None. F. Escande: None. A. Jourdain: None. O. Boute: None. A. Dieux: None. C. Vincent-Delorme: None. M. Holder: None. F. Forzano: None. L. Faivre: None. C. Thauvin: None. M. Gerard: None. D. Geneviève: None. M. Willems: None. B. Isidor: None. C. Quélin: None. S. Odent: None. V. Cormier-Daire: None. G. Baujat: None. G. Morin: None. L. Ruaud: None. Y. Capri: None. A. Goldenberg: None. Y. Alembik: None. N. Revençu: None. B.E. Gutiérrez-Amavizca: None. N. Chassaing: None. F. Laffargue: None. C. Francannet: None. T. Busa: None. V. Paquis: None. S. Manouvrier: None. F. Petit: None.

P04.034.C Porokeratosis of Mibelli in the Tunisian population: association of nonsense and synonymous variants

Haïfa El Mabrouk 1,2, Lobna Boussofara3,4, Hamza Chouk1,2, Nadia Gharyani Fétoui3,4, Ali Saad3,2, Mohamed Denguezli3,4, Dorra H’mida3,2

1Higher Institute of Biotechnology, Monastir, Tunisia, 2Laboratory of Human Cytogenetics, Molecular Genetics and Reproductive Biology, Farhat HACHED University Hospital, Sousse, Tunisia, 3Faculty of Medicine Ibn El Jazzar, Sousse, Tunisia, 4Department of Dermatology and Venerology, Farhat HACHED University Hospital, Sousse, Tunisia.

Introduction: Porokeratosis of Mibelli (PM) is a rare autosomal dominant genodermatosis linked to the MVK and PMVK genes. We aim to establish molecular evidence of PM in our Tunisian patients.

Material and methods: Direct sequencing was performed in a cohort of 8 patients with PM and 9 of their clinically healthy relatives from 2 unrelated families in central Tunisia.

Results: 7 patients and 2 healthy relatives presented a known pathogenic variant (PMVK c.412C> T; p.R138*), and 6 patients and 4 of their relatives presented a synonymous variant (PMVK c.147A>G; p.E49=).

Discussion: In the present work, we propose to report an association of the synonymous variant p.49Glu = in exon 2 to the pathogenic variant p.Arg138* at exon 4, both at the PMVK gene. This association seemed to modulate PM patients’ phenotype. Patients carrying the association present either moderate phenotypes; the number of lesions exceeded 10 covering approximately 5% of the body surface or severe phenotypes: in which affected body surface exceeds 10%, with lesions of the genitals and the face. When the synonymous variant is absent, patients present only a few annular plaques with reduced size, suggesting a mild clinical phenotype.

Conclusion: The association between the two variants lead us to believe that the synonymous variant could constitute a modifier of the PMVK gene.

H. El Mabrouk: None. L. Boussofara: None. H. Chouk: None. N. Gharyani Fétoui: None. A. Saad: None. M. Denguezli: None. D. H’mida: None.

P04.035.D GLI3-variants causing isolated polysyndactyly are not restricted to the gene’s last third

Henrike L. Sczakiel 1,2, Wiebke Hülsemann3, Angela T. Abad-Perez1, Sarina Schwartzmann1, Denise Horn1, Malte Spielmann4,5, Stefan Mundlos1,2, Martin A. Mensah1,6

1Charité - Universitätsmedizin Berlin, Institut für Medizinische Genetik und Humangenetik, Berlin, Germany, 2Max Planck Institute for Molecular Genetics, RG Development & Disease, Berlin, Germany, Berlin, Germany, 3Handchirurgie Kinderkrankenhaus Wilhelmstift, Hamburg, Germany, 4Institut für Humangenetik Lübeck, Universität zu Lübeck, Lübeck, Germany, 5Max Planck Institute for Molecular Genetics, Human Molecular Genomics Group, Berlin, Germany, 6Berlin Institute of Health, Berlin, Germany.

Introduction: Loss of function variants of GLI3 are associated with a variety of forms of polysyndactyly: Pallister-Hall-Syndrome (PHS), Greig-Cephalopolysyndactyly-Syndrome (GCPS) and isolated polysyndactyly. So far, mutations in the first and third third of the GLI3 gene have been associated with GCPS while mutations in the second third of GLI3 have been associated with PHS. Cases of isolated polysyndactyly were attributed to mutations in the third third of GLI3. Here we present more than 30 individuals from over 15 families with pre- and postaxial polysyndactyly in whom we could confirm GLI3 mutations.

Materials and Methods: Sanger sequencing of the GLI3 gene was performed in patients with clinical findings suggestive for a GLI3-associated polysyndactyly syndrome. Additionally, we searched the literature for previously reported cases of either PHS, GCPS or isolated polysyndactyly with confirmed mutations in the GLI3 gene.

Results: We tested over 80 individuals with polysyndactyly for GLI3 variants and detected a causing mutation in more than 15 families. The mutations spanned almost the entire coding region of the GLI3 gene (c.366 - c.4172) and were mostly amorphic. We observed no genotype-phenotype correlation within those cases. Overall, we found a high phenotypic variability within and across families. A close review of the literature revealed more cases of isolated polysyndactyly with mutations across nearly the entire coding region of GLI3.

Conclusions: We found that isolated polysyndactyly is a common phenotype of inherited as well as de novo GLI3 mutations and is not restricted to mutations in the last third of the GLI3 gene.

H.L. Sczakiel: None. W. Hülsemann: None. A.T. Abad-Perez: None. S. Schwartzmann: None. D. Horn: None. M. Spielmann: None. S. Mundlos: None. M.A. Mensah: None.

P04.037.B A novel c.671_682del NCSTN variant in a family with Hidradenitis Suppurativa

Nikolai P. Pace1, Dillon Mintoff2, Peter Bauer3, Isabella Borg 1,2

1University of Malta, Msida, Malta, 2Mater dei Hospital, Msida, Malta, 3Centogene GmbH, Rostock, Germany.

Introduction: Hidradenitis suppurativa (HS) is a chronic, suppurative condition of the pilosebaceous unit (PSU), the pathophysiology of which is still being elucidated. The disease is multifactorial, caused by both genetic and environmental factors. The potential role of underlying genetic variants, particularly within the γ-secretase complex has recently garnered significant research interest by the international HS community.

Methodology: The proband, a 21-year-old male and his 45-year-old mother had Hurley Stage IIIa HS and shared a follicular, Latent Class 2 phenotype. The maternal grandmother suffered from a mild form of axillary HS. In view of these findings, a familial form of HS with variable expressivity was suspected and molecular genetic studies were carried out.

Results: WES analysis on the proband revealed a novel heterozygous c.671_682del p. (Val224_Thr227del) variant in the Nicastrin (NCSTN) gene. This 12bp in-frame deletion lies in exon 6 of NCSTN, resulting in the loss of 4 amino acid residues. Targeted gene sequencing identified the same heterozygous variant in the affected mother. The NCSTN deletion was not identified in a local reference dataset comprising 60 ethnically matched whole exome sequences.

Conclusion: This result expands the mutational spectrum of the NCSTN gene. It is the first gene variant identified in HS Maltese patients. Further genetic studies will be carried out on all local HS patients to unravel the genetic aetiology of HS in the local population using a high throughput exome sequencing approach. We hope to establish genotype-phenotype associations which would lead to better clinical management and targeted treatment for HS.

N. Pace: None. D. Mintoff: None. P. Bauer: A. Employment (full or part-time); Modest; Centogene. I. Borg: None.

P04.038.C A missense mutation in VAV3 in a familial case of high bone mass

Núria Martínez-Gil 1, Diana Ovejero2, Natalia Garcia-Giralt2, Leonardo Mellibovsky2, Xavier Nogués2, Raquel Rabionet1, Daniel Grinberg1, Susanna Balcells1

1Department of Genetics, Microbiology and Statistics, Faculty of Biology, Universitat de Barcelona, CIBERER, IBUB, IRSJD, Barcelona, Spain, 2Musculoskeletal Research Group, IMIM (Hospital del Mar Medical Research Institute), Centro de Investigación Biomédica en Red en Fragilidad y Envejecimiento Saludable (CIBERFES), ISCIII, Barcelona, Spain.

Osteoporosis is the most common bone disease, characterized by a low bone mineral density (BMD) and increased risk of fracture. At the other end of the BMD spectrum, some individuals present strong, fracture-resistant, bones. Both osteoporosis and high bone mass (HBM) are heritable and their genetic architecture encompasses polygenic inheritance of common variants and some cases of monogenic highly penetrant variants in causal genes. We have investigated the genetics of a family presenting HBM segregating in an apparently Mendelian dominant pattern. Since polygenic causes and mutations in known HBM genes had been previously discarded, we searched for rare causal variants in novel genes by whole-exome sequencing in two affected and one non-affected family members. Rare coding variants fulfilling the inheritance pattern were further restricted to include only those genes that could be related to bone development, function or disease, leaving 8 variants. Of those, we highlight a missense variant in VAV3, a gene encoding a guanine-nucleotide-exchange factor with an important role in osteoclast activation and function. Although no previous cases of VAV3 mutations have been found in humans, Vav3 KO mice display dense bones, equivalent to the HBM phenotype present in our family. A second missense variant, which might play a secondary role, was found in SIK3. However, the mouse and human bone phenotypes associated with this gene do not fit with HBM. Future functional assessment of the VAV3 variant will likely confirm VAV3 as a novel HBM gene and a potential therapeutic target for osteoporosis. Funding: SAF2014‐56562‐R, SAF2016‐75948‐R.

N. Martínez-Gil: None. D. Ovejero: None. N. Garcia-Giralt: None. L. Mellibovsky: None. X. Nogués: D. Speakers Bureau/Honoraria (speakers bureau, symposia, and expert witness); Modest; Amgen, Lilly, UCB. F. Consultant/Advisory Board; Modest; UCB, Amgen. R. Rabionet: None. D. Grinberg: None. S. Balcells: None.

P04.039.D A new variant of c409delA in the EXT2 gene identified among Yakut families with hereditary multiple exostosis

Alexandra E. Yakovleva 1, Anastasia L. Danilova1, Diana A. Petukhova1, Polina I. Golikova1, Gavril D. Moskvitin1,2, Aitalina L. Sukhomyasova1,2, Nadezda R. Maksimova1

1Research Laboratory "Molecular Medicine and Human Genetics", M.K. Ammosov North-Eastern Federal University, Yakutsk, Russian Federation, 2Medical Genetic Center, Republican Hospital №1 - “National Medical Center”, Yakutsk, Russian Federation.

Hereditary multiple exostosis (HME) (OMIM 133700, OIMM 133701) is a genetically heterogeneous disease with an autosomal dominant mode of inheritance, characterized by the presence of multiple cartilaginous growths in the metaphyses of long bones.

Materials and Methods: We examined 27 unrelated probands with a clinical diagnosis of HME using mass parallel - massive for all EXT1 and EXT2 coding regions, followed by validation of the results by Sanger sequencing.

Results: Molecular genetic research revealed an unknown heterozygous frameshift variant c.409delA (p.Ile137fs) in the 2nd exon EXT2 among 8 unrelated Yakut patients, which was also confirmed by direct Sanger sequencing. The detected mutation was checked in the Human Gene Mutation Database (HGMD), Leiden Open Variation Database (LOVD), Genome Aggregation Database (gnomAD). This mutation predicted to be highly damaging on a protein function. Experiments were performed using the equipment of the Center for collective use of the North-Eastern Federal University.

Conclusions: As a result of this study, for the first time in the Republic of Sakha (Yakutia), a previously undescribed heterozygous frameshift variant c.409delA (p.Ile137fs) in the 2nd exon of the EXT2 gene. The study was supported by the Ministry of Science and Higher Education of the Russian Federation. (Project No. FSRG-2020-0014 "Genomics of Arctic: epidemiology, hereditary and pathology").

A.E. Yakovleva: None. A.L. Danilova: None. D.A. Petukhova: None. P.I. Golikova: None. G.D. Moskvitin: None. A.L. Sukhomyasova: None. N.R. Maksimova: None.

P04.041.B Aberrant binding of mutant HSP47 affects posttranslational modification of type I collagen and leads to osteogenesis imperfecta

Delfien Syx 1, Yoshihiro Ishikawa2,3, Jan Gebauer4, Sergei P. Boudko5, Brecht Guillemyn1, Tim Van Damme1, Sanne D’hondt1, Sofie Symoens1, Sheela Nampoothiri6, Douglas B. Gould2, Ulrich Baumann4, Hans Peter Bächinger3, Fransiska Malfait1

1Ghent University, Ghent, Belgium, 2UCSF School of Medicine, San Francisco, CA, USA, 3Oregon Health & Science University, Portland, OR, USA, 4University of Cologne, Cologne, Germany, 5Vanderbilt University Medical Center, Nashville, TN, USA, 6Amrita Institute of Medical Sciences and Research Center, Cochin, India.

Heat shock protein 47 (HSP47), encoded by the SERPINH1 gene, is a molecular chaperone essential for correct folding of collagens. We report a homozygous p.(R222S) substitution in HSP47 in a child with severe osteogenesis imperfecta (OI). The highly conserved p.R222 residue is located within the collagen-interacting surface and HSP47-R222S shows a significantly reduced affinity for type I collagen in binding assays. This altered interaction leads to posttranslational overmodification of type I procollagen, with increased glycosylation and/or hydroxylation of lysine and proline residues as shown by mass spectrometry. Since a normal intracellular folding and secretion rate of type I procollagen was observed, this overmodification cannot be explained by prolonged exposure of the procollagen molecules to modifying enzymes, as is commonly observed in other OI types. We found significant upregulation of several molecular chaperones and enzymes involved in procollagen modification and folding on Western blot and RT-qPCR and showed that an imbalance in binding of HSP47-R222S to unfolded type I collagen chains results in increased binding of other chaperones and modifying enzymes in a gelatin sepharose pulldown assay. In conclusion, our findings suggest a compensatory mechanism for aberrant HSP47-R222S binding, eventually leading to overmodification of type I (pro)collagen chains, thereby underscoring the importance of HSP47 for proper posttranslational modification and providing insights into the molecular pathomechanisms of the p.(R222S) alteration in HSP47, which leads to a severe OI phenotype. This work was supported by Research Foundation Flanders (Belgium), Ghent University, German Research Council, National Institutes of Health and Shriners Hospitals for Children.

D. Syx: None. Y. Ishikawa: None. J. Gebauer: None. S.P. Boudko: None. B. Guillemyn: None. T. Van Damme: None. S. D’hondt: None. S. Symoens: None. S. Nampoothiri: None. D.B. Gould: None. U. Baumann: None. H. Bächinger: None. F. Malfait: None.

P04.042.C Association of one-carbon metabolism-related genes and ichthyosis vulgaris manifestation in Eastern Ukraine

Olena M. Fedota1, Iurii O. Sadovnychenko 1,2, Halyna V. Makukh3, Lilia B. Chorna3, Larissa V. Roshcheniuk2,4, Vitalii M. Vorontsov4, Pavlo P. Ryzhko4

1V.N. Karazin Kharkiv National University, Kharkiv, Ukraine, 2Kharkiv National Medical University, Kharkiv, Ukraine, 3Institute of Hereditary Pathology of National Academy of Medical Sciences of Ukraine, Lviv, Ukraine, 4Regional Clinical Dispensary for Skin and Venereal Diseases no. 1, Kharkiv, Ukraine.

Introduction: The prevalence of ichthyosis vulgaris (IV) in Eastern Ukraine is 1:2557. The dermatosis is caused by FLG mutations R501X and 2282del4, but their penetrance in heterozygotes is incomplete. The purpose of the study was to analyze the effects of one-carbon metabolism-related genes on the IV clinical manifestation in FLG mutation carriers.

Materials and methods: The MTHFR C677T (rs1801133), MTHFR A1298C (rs1801131), MTR A2756G (rs1805087) and MTRR A66G (rs1801394) SNPs were analyzed by PCR-RFLP in 31 IV patients, 7 their non-IV first-degree relatives with FLG mutations and 150 healthy controls. Statistical analysis was performed using chi-square test and OR.

Results: In 2282del4/wt IV individuals, the distributions of wild-type, heterozygous and variant homozygous genotypes were: rs1801133 — 0.29:0.71:0.00; rs1801131 — 0.53:0.47:0.00; rs1805087 — 0.70:0.24:0.06; rs1801394 — 0.23:0.53:0.24. In this group the MTR 2756AA genotype and MTR 66GG genotype frequencies were 1.4-1.6 higher than in IV patients with the other FLG genotypes, the MTR 2756AA genotype frequency was 1.6 times higher than in healthy controls (p < 0.01). The association between IV and one-carbon metabolism-related genes was evaluated for single- and multilocus disease models. The strongest genotype-phenotype relationships were found for the genotypes: MTHFR 677CT — OR = 3.60 (95% CI 1.21−10.71, p = 0.032), MTHFR 677CT/MTHFR 1298AA+AC — OR = 4.39 (95% CI 1.47−13.14, p = 0.008), MTHFR 677CT/MTHFR 1298AA/MTRR 66AG — OR = 7.64 (95% CI 2.34−24.94, p = 0.001), MTHFR 677CT/MTHFR 1298AA/MTR 2756AA/MTRR 66AG — OR = 11.23 (95% CI 2.51−50.21, p = 0.002).

Conclusion: SNPs in one-carbon metabolism-related genes can be considered as modifiers of FLG 2282del4 mutation in IV clinical manifestation.

O.M. Fedota: None. I.O. Sadovnychenko: None. H.V. Makukh: None. L.B. Chorna: None. L.V. Roshcheniuk: None. V.M. Vorontsov: None. P.P. Ryzhko: None.

P04.044.D Incontinentia pigmenti and favism due to a large genomic deletion including IKBKG and G6PD

Maria Grossmann 1, Stephanie Spranger2, Snjezana Rendulic3, Andrea Bier1, Silke Reif1, Manuela Timmer1, Christian Jänecke1, Vivien Klaschka1, Jens Plaschke1, Stefan Krüger1

1Gemeinschaftspraxis für Humangenetik, Dresden, Germany, 2Praxis für Humangenetik, Bremen, Germany, 3Praxis für Humangenetik und Prävention, Stuttgart, Germany.

Incontinentia pigmenti (IP) is a genodermatosis in four stages including blistering, wart-like rash, swirling macular hyperpigmentation and linear hypopigmentation, and also affects hair, teeth, nails, eyes and brain. IP is caused by mutations in the IKBKG (NEMO) gene and inherited in an X-linked dominant manner with high penetrance. Affected females show a highly variable phenotype. IP is usually prenatally lethal in males. About 65% of females with IP have a recurrent ~11.7-kb deletion spanning exons 4 to 10 and ~8.6% have point mutations. Only seven other deletions were described to date.

Here we describe a 24-year-old female patient with typical skin lesions since birth. She showed neither neurological nor other symptoms of IP and suffered two miscarriages. Furthermore, the patient had intolerance of fava beans and some drugs, indicating X-linked dominant G6PD associated favism. Her mother and her sister were also affected of IP and favism.

Neither long-range (gap) PCR for detection of the recurrent exon 4 to 10 deletion, nor sequence analysis detected a pathogenic mutation. MLPA revealed a heterozygous deletion spanning the whole IKBKG gene as well as the 5’ adjacent entire G6PD gene. NC_000023.10:g.(153,744,322_153,759,773)_(153,793,401_153,798,250)del. This deletion is to our knowledge of yet undescribed extent. The only two patients with large IKBKG deletions involving the whole G6PD gene described so far by Fusco et al. did not show any clinical manifestations of G6PD deficiency in contrast to the patients described here.

In summary, this is the first description of a contiguous gene syndrome including Incontinentia pigmenti and favism.

M. Grossmann: None. S. Spranger: None. S. Rendulic: None. A. Bier: None. S. Reif: None. M. Timmer: None. C. Jänecke: None. V. Klaschka: None. J. Plaschke: None. S. Krüger: None.

P04.045.B The importance of extracutaneous organ involvement for the clinical severity and prognosis observed in incontinentia pigmenti caused by IKBKG mutations

Hwa Young Kim, Jung Min Ko, Hyun Beom Song, Kyu Han Kim, Jong Hee Chae, Man Jin Kim, Moon-Woo Seong

Seoul National University Hospital, Seoul, Korea, Republic of.

Incontinentia pigmenti (IP) is a rare X-linked skin disease caused by mutations of the IKBKG gene, which is required for activation of the nuclear factor-kappa B signaling pathway. Multiple systems can be affected with highly variable phenotypic expressivity. We aimed to clarify the clinical characteristics observed in molecularly-confirmed Korean IP patients. Medical records of 25 females confirmed as IP by molecular genetic analysis were retrospectively reviewed. A phenotypic score of extracutaneous manifestations was calculated to assess the disease severity. The IKBKG gene partial deletion or intragenic mutations were investigated by a long-range PCR, multiplex ligation-dependent probe amplification, and direct sequencing methods. Among 25 individuals, 18 (72%) were sporadic cases. All patients showed typical skin manifestations at inborn or during the neonatal period. Extracutaneous findings were noted in 17 (68%) cases; ocular manifestations (28%), neurological abnormalities (28%), hair abnormalities (20%), dental anomalies (12%), nail dystrophy (8%). The common IKBKG exon 4-10 deletion was observed in 20 (80%) patients. In addition, 5 intragenic sequence variants were identified, including 3 novel ones. The phenotype scores were highly variable from abnormal skin pigmentation only to one or more extracutaneous features, even though there was no meaningful significant difference for each clinical characteristic between the groups with sequence variants and common large deletion. Heterogeneity of extracutaneous manifestations and high incidence of sporadic cases were observed in our cohort with IP. Long-term monitoring with multidisciplinary management is essential for evaluating the clinical status, providing adequate genetic counseling and understanding genotype-phenotype correlation in IP.

H. Kim: None. J. Ko: None. H. Song: None. K. Kim: None. J. Chae: None. M. Kim: None. M. Seong: None.

P04.047.D Further insights in the FKBP14-related khyphoscoliotic Ehlers-Danlos syndrome: report of 3 unrelated individuals and 2 new pathogenic variants

Marlies Colman 1,2, Robin Vroman1,2, Tibbe Dhooge1,2, Zoë Malfait1,2, Biruté Burnyté3, Sheela Nampoothiri4, Delfien Syx1,2, Fransiska Malfait1,2

1Ghent University, Ghent, Belgium, 2Center for Medical Genetics, Ghent, Belgium, 3Vilnius University Hospital, Vilnius, Lithuania, 4Amrita Institute of Medical Sciences & Research Centre, Kerala, India.

The kyphoscoliotic type of Ehlers-Danlos syndrome (kEDS) is an autosomal recessive connective tissue disorder characterized by kyphoscoliosis, muscle hypotonia and generalized joint hypermobility. It results from deficiency of either lysyl hydroxylase 1 (LH1 encoded by PLOD1) or the peptidyl-prolyl cis-trans isomerase family FK506-binding protein 22kDa (FKBP22 encoded by FKBP14). FKBP22 acts as a molecular chaperone involved in the folding and quality control of collagen molecules (among which types III and VI collagen). Deficiency of FKBP22 may lead to accumulation of incorrectly folded collagen molecules in the endoplasmic reticulum (ER), causing premature interactions. We present the clinical manifestations of three non-related individuals with homozygous pathogenic FKBP14 variants: proband 1 (c.587A>G; p.(Asp196Gly)); proband 2 (c.362dupC; p.(Glu122Argfs*7)) and proband 3 (c.2T>G; p.(Met1?)); with experimental data of the variants found in probands 1 and 2. Both the c.587A>G; p.(Asp196Gly) and the c.362dupC; p.(Glu122Argfs*7) variant cause absence of FKBP22 protein. In addition, we found intracellular accumulation of types III and VI collagen and impaired fibroblast migration. Investigation of ER-stress related proteins (CHOP, XBP1, ATF6, (p)eIF2aplha, BIP) did not reveal any significant upregulation.

Conclusion: This study broadens the clinical and molecular spectrum of FKBP14-related kEDS with three non-related individuals and two new pathogenic variants. We showed delayed fibroblast migration and intracellular accumulation of type III and type VI collagen and we provide the first evidence of a homozygous pathogenic missense variant.M.C., D.S. and F.M. are a PhD candidate, a postdoctoral researcher and a senior clinical investigator respectively, of the Research Foundation Flanders, Belgium.

M. Colman: None. R. Vroman: None. T. Dhooge: None. Z. Malfait: None. B. Burnyté: None. S. Nampoothiri: None. D. Syx: None. F. Malfait: None.

P04.048.C Homozygosity for a previously unreported deletion in B3GAT3 causes linkeropathy - a clinical report describing an adult patient

Anneli C. S. Bolund, Bente Langdahl, Trine B. Laurberg, Michel B. Hellfritzsch, Hans Gjørup, Bjarne Møller-Madsen, Trine Ø. Nielsen, Stense Farholt, Pernille A. Gregersen

Aarhus University Hospital, Aarhus N, Denmark.

Background: Proteoglycans (PGs) are complex macromolecules consisting of a core protein and glycosaminoglycan (GAG) side chains. PGs are important for the constitution and functioning of the connective tissue. The normal composition of the GAG side chains defines the nature of the PGs and a wide range of biological events. Deficiencies of specific enzymes involved in the linkage of GAGs to the core protein, leads to a heterogeneous disease group called Linkeropathies. This is a group of multisystem conditions characterized by skeletal dysplasia and various extra-skeletal features. The conditions show variable severity and overlapping phenotypes. β-1,3-glucuronyltransferase 3, encoded by B3GAT3, is an enzyme involved in the linkage process to form functional PGs, and biallelic pathogenic variants in B3GAT3 hence leads to linkeropathy.

Case presentation: A now 22-year-old female patient, born of consanguineous parents, is presented. The disease history included congenital severe joint malalignment of elbows, hips, knees and feet, continuous hypermobility, severe kyphoscoliosis, osteoporosis with multiple fractures, congenital diaphragmatic hernia, and mild dysmorphic features. Whole exome sequencing was performed, and homozygosity for a novel in-frame deletion in B3GAT3, (c.61_63delCTC (p.(Leu21del))) was detected. Both unaffected parents (first double cousins) were heterozygous carriers.

Conclusion: This is the first report to describe homozygosity for an in-frame deletion in B3GAT3 (p.(Leu21del)) and the first adult phenotype described. Previously described cases of B3GAT3-deficiency were all children with phenotypes ranging from prenatal manifestation and early lethality to less severe. We suggest that this novel homozygous in-frame deletion in B3GAT3 causes a recessive form of linkeropathy.

A.C.S. Bolund: None. B. Langdahl: None. T.B. Laurberg: None. M.B. Hellfritzsch: None. H. Gjørup: None. B. Møller-Madsen: None. T.Ø. Nielsen: None. S. Farholt: None. P.A. Gregersen: None.

P04.049.B Lipoid proteinosis: A novel mutation in ECM1 gene in a Turkish patient

Elifcan Tasdelen 1, Isa An2

1Department of Medical Genetics, Sanliurfa Education and Research Hospital, Sanlıurfa, Turkey, 2Department of Dermatology, Sanliurfa Education and Research Hospital, Sanlıurfa, Turkey.

Background: Lipoid proteinosis (LP) (OMIM-247100), also known as Urbach-Wiethe disease caused by loss-of-function mutations in the ECM1 gene, is characterized by skin lesions such as yellow infiltrating papules, acneiform scars and verrucous hyperkeratosis. In most patients, hoarse voice is observed due to vocal cord thickening. All these findings occurs as a result of accumulation of hyaline-like material in the skin and mucosa. Furthermore, neuropsychiatric findings have been associated with intracranial calcifications observed in LP patients. Extracellular matrix protein-1 (ECM1) encoded by ECM1 gene is involved in differentiation of keratinocytes by binding to structural proteins, and angiogenesis. Case report: Here, we desribe 7-year-old girl, a child of consanguineous parents, with diffuse acneiform scar on the face, moniliform blepharosis on the eyelids, verrucous lesions on dorsum of the hands, hypopigmented patches on the arms and back and hoarse voice. In sequencing analysis of ECM1 gene, a novel homozygous NM_004425:c.1246 C>T(p.Arg416Ter) mutation in exon 8 causing premature stop codon was detected. This variant neither found in ExAC nor 1000G databases. The effect of the mutation on protein is predicted as damaging by in silico analysis.

Conclusion: Approximately sixty mutations associated with LP have been reported so far. Half of these mutations have been detected in exons 6 and 7 of the ECM1 gene. The p.Arg416Ter mutation is outside these hotspot regions and have not been reported previously. It explains the phenotype of our patient likely by causing nonsense mediated decay resulting in the non-functional ECM1 protein.

E. Tasdelen: None. I. An: None.

P04.050.C Novel variant in SMAD3 gene associated with Loeys-Dietz syndrome type 3

Albertas Valintėlis 1, Marius Šukys1, Eglė Ereminienė2, Inga Nasvytienė1, Lina Basel-Salmon3,4,5, Rasa Ugenskienė1

1Department Of Genetics And Molecular Medicine, Lithuanian University of Health Sciences, Kaunas, Lithuania, 2Department of Cardiology, Lithuanian University of Health Sciences, Kaunas, Lithuania, 3Raphael Recanati Genetics Institute, Rabin Medical Center, Beilinson Hospital, Petah Tikva, Israel, 4Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv, Israel, 5Laboratory of Immunology and Genetics, Felsenstein Medical Research Center, Petah Tikva, Israel.

Introduction: Loeys-Dietz syndrome (LDS) is a systemic connective tissue disorder characterized by vascular findings, skeletal abnormalities, craniofacial features and cutaneous findings which sometimes can be misdiagnosed as Marfan syndrome The diagnosis is established based on clinical findings and the identification of heterozygous pathogenic variant in one of the several reported genes.

Methods: We report a 41-year-old woman with a prior clinical diagnosis of Marfan syndrome (systemic score 8 and aortic root dilatation Z score 2.99). Additionally she had right vertebral artery tortuosity, a history of gastric hemorrhage from Dieulafoy’s ulcer, hand osteoarthritis, osteoporosis (repeated spontaneous metatarsal fractures), hypermobile joints (Beighton score 4), uvula aplasia, velvety and translucent skin with visible underlying veins. The patient also has primary thrombocythemia (with JAK2 pathogenic variant) and chronic tubotympanic suppurative otitis media. No affected relatives were documented.

Results: We performed cardiovascular gene panel analysis via Next generation sequencing: no FBN1 gene variant was found, but heterozygous gene SMAD3 variant NM_005902.4: c.1262G>A of unknown significance was detected. This variant is not found in control databases and was not reported in literature, in silico tools predict it as deleterious, UniProt database suggest that an important protein domain is affected. Variant segregation analysis was not performed as patient’s parents are deceased. As clinical features are more suggestive to Loeys Dietz type 3 syndrome and variant is predicted deleterious we determined the variant as likely pathogenic.

Conclusion: Genetic testing allowed us to establish a proper diagnosis and provide more extensive management plan for the patient.

A. Valintėlis: None. M. Šukys: None. E. Ereminienė: None. I. Nasvytienė: None. L. Basel-Salmon: None. R. Ugenskienė: None.

P04.051.D Four hypotrichosis families with pathogenic variants in the gene LSS presenting with and without neurodevelopmental phenotypes

Nicole Cesarato 1, Maria Wehner1, Mari Ghughunishvili2, Axel Schmidt1, Michael Lentze3, Cristina Has4, Matthias Geyer5, F. Buket Basmanav1, Regina C. Betz1

1Institute of Human Genetics, University of Bonn, Bonn, Germany, 2Givi Zhvania Academic Clinic of Pediatrics, Tbilisi State Medical University, Tbilisi, Georgia, 3Department of Pediatrics, University of Bonn, School of Medicine & University Hospital Bonn, Bonn, Germany, 4Department of Dermatology, University Hospital Freiburg, Freiburg, Germany, 5Institute of Structural Biology, University of Bonn, School of Medicine, Bonn, Germany.

Introduction: LSS, encoding for lanosterol synthase, has been associated with congenital cataract, autosomal recessive hypotrichosis simplex resp. congenital alopecia with or without neuroectodermal phenotypes including intellectual disability, epilepsy, microcephaly, genital abnormalities in males and dermatological symptoms. In this study, we report novel pathogenic variants found in LSS.

Materials and Methods: Direct Sanger sequencing of the LSS gene or Whole Exome Sequencing was performed on individuals affected with hypotrichosis and their family members. Protein structure modelling was used to reveal the consequences of the identified mutations.

Results: All affected individuals, originating from Iraq, Syria, Afghanistan and Georgia, suffered from mild hypotrichosis to severe alopecia. One patient presented also with a developmental speech disorder, learning difficulties and microcephaly. The following variants were found either in homozygous or in compound heterozygous state and co-segregated in these families: c.530G>A;p.(Arg177Gln), c.934C>T;p.(Arg312Trp), c.881G>T;p.(Arg294Leu), c.1702C>T;p.(Arg568Trp). The most interesting variant was the homozygous synonymous variant c.393G>A;p.(131Leu=), shown to activate a cryptic donor splice site, resulting in an in frame deletion of 11 amino acid residues (r.425-457del). Modelling of the mutant sites based on the crystal structure of LSS revealed that all mutations have consequences on the 3D protein structure.

Conclusions: It remains unclear which variants in LSS lead to an alopecia phenotype only, and which lead to accompanying severe neurodevelopmental and dermatological phenotypes. If significantly more patients with mutations in LSS will be reported, we may be able to develop a better understanding of this protein and allow predictions concerning the phenotypic outcomes of individual LSS mutations.

N. Cesarato: None. M. Wehner: None. M. Ghughunishvili: None. A. Schmidt: None. M. Lentze: None. C. Has: None. M. Geyer: None. F.B. Basmanav: None. R.C. Betz: None.

P04.052.A Searching for TGFB3 gene mutations among Polish patients with Marfan syndrome and related disorders

Maria Pilarska-Deltow 1, Marzena Skrzypczak-Zielińska2, Anna Junkiert-Czarnecka1, Aneta Bąk1, Marta Heise1, Olga Haus1

1Nicolaus Copernicus University, Torun, Poland, 2Institute of Human Genetics, Polish Academy of Sciences, Poznan, Poland.

Introduction: Marfan syndrome (MFS) is the best known congenital disease of connecting tissue. The classical symptoms of MFS include skeletal, cardiovascular and eye abnormalities. However, the variety and variable severity of them make the diagnosis difficult due to the similarities with Marfan-like syndromes, including Loeys-Dietz syndrome (LDS). According to the classification, there are six subtypes of LDS. Mutations in TGFB3 are responsible for LDS 5, which is less symptomatic and without molecular analysis can easily be confused with MFS.

Materials and Methods: Sanger sequencing was performed for 119 Polish patients with suspicion of Marfan or another Marfan-like syndrome.

Results: Sequencing of TGFB3 revealed three mutations. According to VarSome database, mutation c.412G>T (p.Ser138Ala), with the allele frequency 0.01% (ExAc) was predicted as benign. Mutation c.488G>A (p.Arg163Gln), with the allele frequency 0.004% (ExAc) was classified as a variant of uncertain significance (VUS) (VarSome). The frequency of the mutation c.1138C>T (p.Pro380Ser) is not available in the ExAc, according to in silico tools this is also VUS. In the case of this mutation (p.Pro380Ser) we confirmed the paternal inheritance of the mutation in the parental study.

Conclusions: Mutations were detected in patients in whom previous NGS examination of the common genes associated with Marfan-like syndromes did not reveal pathogenic variants. The results point at the need to study less obvious genes of marfanoid syndromes to improve their diagnostics, which may have implications for patient prognosis, as well as diagnosis and prevention in family members.The investigation was supported by Nicolaus Copernicus University grant WL174.

M. Pilarska-Deltow: None. M. Skrzypczak-Zielińska: None. A. Junkiert-Czarnecka: None. A. Bąk: None. M. Heise: None. O. Haus: None.

P04.053.B Targeted next generation sequencing substantially advances molecular diagnosis of Marfan and Marfan related disorders

Darina L. Kachakova 1, Kunka Kamenarova1, Kalina Mihova1, Ivo Kremensky1, Ivanka Dimova2, Vanyo Mitev1, Radka Kaneva1

1Laboratory of Genomic Diagnostics, Molecular Medicine Center, Department of Medical Chemistry and Biochemistry, Medical Faculty, Medical University of Sofia, Sofia, Bulgaria, 2Laboratory of Genomic Diagnostics, Molecular Medicine Center, Department of Medical Chemistry and Biochemistry, Medical Faculty, Medical University of Sofia, 2- Department of Medical Genetics, Medical Faculty, Medical University of Sofia, Sofia, Bulgaria.

Introduction: There are more than 200 heritable connective tissue disorders. Marfan syndrome (MFS) is a rare, autosomal-dominant connective tissue multisystem disorder. Significant clinical overlap with other systemic connective tissue diseases, has been documented.

Materials and methods: Eight patients with differential diagnosis Marfan and Marfan like syndromes and 1 patient with Ehlers-Danlos were directed for targeted next generation sequencing (NGS) in Molecular medicine center in the period 2019-2020 year. NGS was performed on MiSeq platform using TruSight one kit. Direct sequencing by Sanger was used in order to confirm estimated pathogenic variants.

Results: Genetic cause of the disease was estimated in 4 from 9 analyzed patients. In two patients FBN1 mutations were found (c.8051+2T>A and p.Arg516Ter). In the other patients the following pathogenic variants were detected: c.1877A>T (p.Lys626Met) in SKI and c.1642A>C (p.Ser548Arg) in SOS1 and thus clinical diagnoses were refined. In three patients we found VUS in the following genes: COL5A2, COL11A1, FLNB, NOTCH1, BAG3. In one patient with aortic aneurism a new likely pathogenic variant c.779delC (p.Pro260Hisfs*47) in FOXE3 was found. Pathogenic mutations in forkhead domain of this gene were previously linked to autosomal dominant thoracic aortic aneurism. Although the new variant lies outside this domain we suggest that is still possible to contribute to the disease.

Conclusion: Many connective tissue disorders have overlapping symptoms and targeted NGS sequencing contributes substantially to genetic diagnosis and refinement of the clinical diagnosis in some of the cases. This work was supported by infrastructural projects D01-285/2019; D01-395/2020 funded by MES.

D.L. Kachakova: None. K. Kamenarova: None. K. Mihova: None. I. Kremensky: None. I. Dimova: None. V. Mitev: None. R. Kaneva: None.

P04.054.C Four novel families expand the genotypic and phenotypic landscape of MESD-related Osteogenesis Imperfecta

Thao Tran1, Rachel Keller1, Brecht Guillemyn 2, Melanie Pepin1, Jane Corteville3, Samir Khatib4, Mohammad-Sadegh Fallah5, Sirous Zeinali5,6, Fransiska Malfait2, Sofie Symoens2, Paul Coucke2, Peter Witters7, Hamideh Bagherian4,5, Deborah Nickerson1, Michael Bamshad1, Jessica Chong1, University of Washington Center for Mendelian Genomics, Peter Byers1

1University of Washington, Seattle, WA, USA, 2Ghent University, Ghent, Belgium, 3Case Western Reserve University, Cleveland, OH, USA, 4GMDC Al Quds University, Al Quds, Palestinian Territory, 5Kawsar Human Genetics Research Center, Tehran, Iran, Islamic Republic of, 6Pasteur Institute of Iran, Tehran, Iran, Islamic Republic of, 7University Hospital Leuven, Leuven, Belgium.

The bone disorder osteogenesis imperfecta (OI) is genetically heterogeneous. Most affected individuals have an autosomal dominant disorder caused by heterozygous variants in either of the type I collagen genes (COL1A1 or COL1A2). Rare autosomal recessive forms of OI are associated with variants in genes that encode proteins involved in the modification of collagens, regulation of collagen expression, alteration of cellular differentiation along the bone lineage, and in collagen secretion. Recently, four different biallelic pathogenic variants in Mesoderm Development LRP Chaperone (MESD) were shown to cause a progressively deforming recessive type of OI, associated with recurrent fractures and oligodontia in five patients of five families. We report four additional patients from four independent families with biallelic variants in MESD: the earlier reported c.632dupA p.(Lys212Glufs*19) and c.676C>T p.(Arg226*) - which are associated with a severe form of OI - and one new pathogenic variant c.603-606delTAAA (p.Asn201Lysfs*15) which causes a neonatal lethal form of OI. MESD acts in the WNT signaling pathway where it is thought to play a role in the folding of the WNT co-receptors Low Density Lipoprotein Receptor-Related Proteins 5 and 6 (LRP5 and LRP6) and in chaperoning their transit to the cell surface. We hypothesize that in the absence of functional MESD, WNT signaling in osteogenic cells is blocked. Our report broadens the phenotypic and genetic spectrum of MESD-related OI, provides additional insight into the pathogenic pathways and underscores the necessity of MESD for normal WNT signaling and bone formation.

T. Tran: None. R. Keller: None. B. Guillemyn: None. M. Pepin: None. J. Corteville: None. S. Khatib: None. M. Fallah: None. S. Zeinali: None. F. Malfait: None. S. Symoens: None. P. Coucke: None. P. Witters: None. H. Bagherian: None. D. Nickerson: None. M. Bamshad: None. J. Chong: None. P. Byers: None.

P04.055.D Sixth family with confirmed metaphyseal dysplasia, Spahr type and a novel variant in MMP13: case report and review of the literature

André M. Travessa 1, Silvia Modamio-Høybjør2, Karen E. Heath2, Ana Berta Sousa1

1Medical Genetics Department, Department of Pediatrics, Hospital de Santa Maria, Centro Hospitalar Universitário Lisboa Norte, Lisbon, Portugal, 2Institute of Medical and Molecular Genetics (INGEMM), Hospital Universitario La Paz, Universidad Autonóma de Madrid, IdiPAZ, CIBERER, ISCIII, Skeletal Dysplasia Multidisciplinary Unit (UMDE), Hospital Universitario La Paz, and ERN-BOND, Madrid, Spain.

Introduction: Metaphyseal dysplasia, Spahr type (MDST, MIM#250400) is an ultra-rare metaphyseal dysplasia likely often mistaken as rickets. Only 16 patients from eight families have been reported, with molecular confirmation in five families (10 patients). We present a case of MDST and review the existing literature, highlighting the features that differentiate this from other metaphyseal dysplasias.

Case report: The proband is a 23-month-old gild referred for growth delay and genu varum. She was born at 33 weeks of gestation to healthy unrelated parents with no relevant family history. At birth, she had normal somatometry for gestational age. The neonatal period was unremarkable. Genu varum was noted at 6 months. Length crossed centiles downwards and was -2.75 SD at 19 months. She was otherwise healthy, and had a normal psychomotor development. The analytical evaluation of calcium and phosphate metabolism was normal, excluding rickets. However, the skeletal survey showed metaphyseal irregularities in the long bones, especially the lower limbs, compatible with a metaphyseal dysplasia. A customized skeletal dysplasia NGS panel was thus performed, and the pathogenic variant c.287_293del, p.(Cys96Phefs*19) in MMP13 gene was identified in apparent homozygosity, confirming the diagnosis of MDST.

Discussion and conclusions: We report the 11th patient (sixth family) with confirmed MDST, and review the existing literature. Patients with MDST present with postnatal mild short (or even normal) stature. Genu varum is milder and knee pain is more common as compared to other metaphyseal dysplasias. No extra-skeletal features or laboratory findings exist. NGS is the investigation of choice for MDST.

A.M. Travessa: None. S. Modamio-Høybjør: None. K.E. Heath: None. A.B. Sousa: None.

P04.057.B Clinical utility of a sponsored, no-cost skeletal dysplasia gene panel testing program: Results from 850 tests

Guillermo Seratti 1, Vikram Pansare1, Tiffany Yar Pang1, Emanuela Izzo1, William Mackenzie2, Cathleen Raggio3, Klane White4, Rebecca Truty5, Britt Johnson5, Swaroop Aradhya5

1BioMarin Pharmaceutical Inc, Novato, CA, USA, 2Nemours Alfred I. duPont Hospital for Children, Wilmington, DE, USA, 3Hospital for Special Surgery, Uniondale, NY, USA, 4Seattle Children’s Hospital, Seattle, WA, USA, 5Invitae, San Francisco, CA, USA.

Rare diseases, such as mucopolysaccharidosis (MPS) IVA (Morquio A syndrome) and MPS VI (Maroteaux-Lamy syndrome), are often misdiagnosed as other types of skeletal dysplasia (SD) or may go undiagnosed for extended periods, potentially resulting in delayed intervention and irreversible disease progression. Discover DysplasiasTM sponsored testing program offers a focused SD gene panel for US and Canadian patients, with the goal of helping facilitate timely diagnoses. Eligible patients must have one or more of: skeletal abnormalities suggestive of SD, short stature, disproportionate growth, dysmorphic facial features or other signs suggestive of SD. The program uses a panel with 109 genes associated with SD. Third-party reflex biochemical enzyme testing is offered for inconclusive MPS molecular results. Genetic counseling is provided for all patients as part of the program. A total of 850 US patients were tested under the program from December 2019-August 2020. Median age at testing was 7 years (range: 0-90). Initial symptom onset was noted prenatally for 22.7% and at birth for 32.9%. Median age at first sign among patients presenting after birth was 5 years. A genetic diagnosis was established in 210 patients (36 genes): a molecular diagnostic yield of 24.7%. One MPS VI patient and two MPS IVA patients were identified; the latter were confirmed by reflex enzyme testing. Use of a targeted gene panel testing program, with genetic counseling support and MPS reflex enzyme testing, has clinical utility in identifying the genetic etiology of MPS and SD, and may allow for disease-specific interventions and proactive treatment plans.

G. Seratti: A. Employment (full or part-time); Significant; BioMarin Pharmaceutical Inc. E. Ownership Interest (stock, stock options, patent or other intellectual property); Modest; BioMarin Pharmaceutical Inc. V. Pansare: A. Employment (full or part-time); Significant; BioMarin Pharmaceutical Inc. E. Ownership Interest (stock, stock options, patent or other intellectual property); Modest; BioMarin Pharmaceutical Inc. T.Y. Pang: A. Employment (full or part-time); Significant; BioMarin Pharmaceutical Inc. E. Ownership Interest (stock, stock options, patent or other intellectual property); Modest; BioMarin Pharmaceutical Inc. E. Izzo: A. Employment (full or part-time); Significant; BioMarin Pharmaceutical Inc. E. Ownership Interest (stock, stock options, patent or other intellectual property); Modest; BioMarin Pharmaceutical Inc. W. Mackenzie: D. Speakers Bureau/Honoraria (speakers bureau, symposia, and expert witness); Modest; BioMarin Pharmaceutical Inc. F. Consultant/Advisory Board; Modest; LPA, BioMarin Pharmaceutical Inc, MPS. Other; Modest; BioMarin Pharmaceutical Inc. C. Raggio: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Modest; BioMarin Pharmaceutical Inc, NextCure, OIF. D. Speakers Bureau/Honoraria (speakers bureau, symposia, and expert witness); Modest; BioMarin Pharmaceutical Inc, Alexion. F. Consultant/Advisory Board; Modest; OIF, EDS, SDMC, BioMarin Pharmaceutical Inc, Ascendis, Alexion, Mereo. K. White: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Modest; BioMarin Pharmaceutical Inc, Ultragenyx, Ascendis, Theracon. D. Speakers Bureau/Honoraria (speakers bureau, symposia, and expert witness); Modest; BioMarin Pharmaceutical Inc, Ultragenyx. F. Consultant/Advisory Board; Modest; National MPS Society, Little People of America, BioMarin Pharmaceutical. Other; Modest; UptoDate.com. R. Truty: A. Employment (full or part-time); Significant; Invitae. E. Ownership Interest (stock, stock options, patent or other intellectual property); Modest; Invitae. B. Johnson: A. Employment (full or part-time); Significant; Invitae. E. Ownership Interest (stock, stock options, patent or other intellectual property); Modest; Invitae. S. Aradhya: A. Employment (full or part-time); Significant; Invitae. E. Ownership Interest (stock, stock options, patent or other intellectual property); Modest; Invitae.

P04.058.C Exome sequencing combined with RNA sequencing clarifies the mode of inheritance of MYH3-associated spondylocarpotarsal syndrome: a case report

Marija Volk, Karin Writzl, Matevž Jus, Aleš Maver, Helena Jaklič, Borut Peterlin

Clinical institute of genomic medicine, UMC Ljubljana, Ljubljana, Slovenia.

Introduction: Pathogenic variants in the MYH3 gene have been associated with dominant and recessive Contractures, pterygia, and spondylocarpotarsal fusion syndrome, type 1 (CPSFS1). By combining exome and RNA sequencing, we provide evidence that MYH3:c.1581+1G>A variant, previously reported in association with apparently recessive CPSFS1 (PMID:29805041) is likely associated with a dominant predisposition to CPSFS1.

Materials and methods: A 14-month girl was referred to our institute with scoliosis, contractures, ptosis, short stature, bicuspid aortic valve and suspected skeletal dysplasia. Familial history revealed that the mother has short stature, scoliosis, contracture of Vth finger and ptosis, while maternal uncle and grandfather have short stature and scoliosis. We performed exome sequencing (ES) and cDNA sequencing of the targeted MYH3 region.

Results: ES revealed the presence of a heterozygous pathogenic variant MYH3(NM_002470.4):c.1581+1G>A in the proband. Because this variant was originally reported in association with recessive inheritance, it was not initially considered causative in heterozygous form. However, segregation analyses revealed that the similarly affected mother also carried the identified variant as affected proband. Expression analysis of mRNA MYH3 transcript showed retention of intron 15, which was predicted to lead to an in-frame insertion of 34 amino-acid sequences. This insertion possibly leads to a gain-of-function effect.

Conclusion: We demonstrate that MYH3 variant c.1581+1G>A is an in-frame insertion and might also be associated with the dominant type of CPSFS1. Our case highlights the utility of expression analysis, which is essential for the correct interpretation of splice-site variants and genetic counselling.

M. Volk: None. K. Writzl: None. M. Jus: None. A. Maver: None. H. Jaklič: None. B. Peterlin: None.

P04.059.D Myhre Syndrome: a rare cause of short stature and osteomuscular pain

Antonio Miguel Poyatos-Andújar 1, Susana García-Linares1, Margarita Martínez-Atienza2, Francisca Quintana-Luque1, Susana Pedrinaci-Rodríguez2, María Luz Bellido-Díaz2, Matías Pérez-Sánchez2

1Hospital Universitario Clínico San Cecilio, Granada, Spain, 2Hospital Universitario Virgen de las Nieves, Granada, Spain.

Introduction: Myhre syndrome is a rare autosomal dominant connective tissue disorder with multisystem involvement characterized by a clinical spectrum that includes growth retardation, skeletal anomalies, muscular hypertrophy, joint stiffness, facial dysmorphism, deafness, cardiovascular disease, learning and social challenges and abnormal sexual development. The molecularly confirmed cases have a de novo heterozygous gain-of-function mutation in SMAD4.

Material and Methods: We present a 20-years-old girl who was born after a dichorionic diamniotic twin pregnancy with increased nuchal translucency for this fetous. The patient present at birth low weight and increased pulmonary pressures, during her development present slow weight gain, muscular weakness, hearing loss, challenging behavior, scoliosis and menstrual dysfunction. All molecular and citomolecular studies performed during the infancy were normal. We performed a clinical exome sequencing (CES) analysis after clinical and familiar evaluations.

Results: We found the pathogenic variant c.1486C>T, p.Arg496Cys in the SMAD4 gene, this variant was determined as de novo with the segregation analysis.

Conclusions: In our case, the diagnosis was delayed beyond the second life decade, this illustrates the difficulty of correctly diagnosing patients with Myhre syndrome. The frequency of neoplasia in series of cases and endometrial cancer in patients with Myhre syndrome, raises the possibility of cancer susceptibility in these patients. It is important to alert clinicians to the possibility of detecting this syndrome for a correct treatment during infancy and surveillance during adult age.

A. Poyatos-Andújar: None. S. García-Linares: None. M. Martínez-Atienza: None. F. Quintana-Luque: None. S. Pedrinaci-Rodríguez: None. M. Bellido-Díaz: None. M. Pérez-Sánchez: None.

P04.060.A Large deletions of NF1: phenotypical description and parental origins of a severe condition

Laurence Pacot1,2, Dominique Vidaud1,2, Audrey Sabbagh3, Ingrid Laurendeau2, Audrey Briand-Suleau1,2, Audrey Coustier1, Théodora Maillard1, Cécile Barbance1, Patrick Nitschké4, Arnaud Jannic5, Salah Ferkal5, Béatrice Parfait1,2, Michel Vidaud1,2, members of the NF France Network, Pierre Wolkenstein5, Eric Pasmant 1,2

1Service de Génétique et Biologie Moléculaires, Hôpital Cochin, DMU BioPhyGen, AP-HP.Centre-Université de Paris, Paris, France, 2Institut Cochin, Inserm U1016 - CNRS UMR8104 - Université de Paris, CARPEM, Paris, France, 3UMR 261 MERIT, IRD, Université de Paris, Paris, France, 4Bioinformatics Platform, Imagine Institute, INSERM UMR 1163, Université de Paris, Paris, France, 5Département de Dermatologie, AP-HP and Université Paris 12, Hôpital Henri-Mondor, Créteil, France.

Background: Whole NF1 locus deletions are identified in 5-10% of patients affected by neurofibromatosis type 1 (NF1). Several studies have previously described particularly severe forms of the disease in NF1 patients with NF1 deletion. However, comprehensive descriptions of large cohorts are still missing to fully characterize this contiguous gene syndrome.

Methods: NF1 patients from a large French NF1 cohort were molecularly characterized using next-generation sequencing, microsatellites analysis, and MLPA between 2005 and 2020. NF1-deleted patients were enrolled and phenotypically characterized with a standardized questionnaire. Parental origin of de novo NF1 deletions was determined in thirty trios and three duos using four intragenic and three extragenic microsatellites in the NF1 locus.

Results: A deletion of the NF1 gene was detected in 4% (139/3479) of molecularly confirmed NF1 index cases. Patients ranged between 4 months and 69 years old, with a median at 21 years old. A comprehensive clinical assessment showed that 93% (116/126) of NF1-deleted patients fulfilled the NIH criteria for NF1. More than half has café-au-lait spots, skinfold freckling, Lisch nodules, neurofibromas, neurological abnormalities, and cognitive impairment or learning disabilities. Comparison with previously described “classic” NF1 cohorts showed a significantly higher proportion of symptomatic spinal neurofibromas, dysmorphism, learning disabilities, malignancies, and skeletal and cardiovascular abnormalities in the NF1-deleted group. Maternal origin of the deletion was confirmed in 25 cases.

Conclusions: We described the largest NF1-deleted cohort to date and confirmed a more severe phenotype in NF1-deleted patients with a predominant maternal origin of the deletions.

L. Pacot: None. D. Vidaud: None. A. Sabbagh: None. I. Laurendeau: None. A. Briand-Suleau: None. A. Coustier: None. T. Maillard: None. C. Barbance: None. P. Nitschké: None. A. Jannic: None. S. Ferkal: None. B. Parfait: None. M. Vidaud: None. P. Wolkenstein: None. E. Pasmant: None.

P04.061.B Identification of rare variants for nonsyndromic cleft lip with/without cleft palate in a cohort of multiplex families

Annika Scheer 1, Fabian Brand2, Julia Fazaal1, Nina Ishorst1, Peter M. Krawitz2, Elisabeth Mangold1, Kerstin U. Ludwig1

1Institute of Human Genetics, University of Bonn, School of Medicine & University Hospital Bonn, Bonn, Germany, 2Institute for Genomic Statistics and Bioinformatics, University Hospital Bonn, Bonn, Germany.

Nonsyndromic cleft with/without cleft palate (nsCL/P) are among the most common birth defects and have a multifactorial etiology. In the last years, genome wide association studies have identified around 40 common risk regions with small to moderate effect sizes, but the contribution of rare variants with higher effect sizes has been studied to a lesser extent. The aim of our study was to systematically identify potential causal rare variants in 55 individuals from 10 multiplex families affected with nsCL/P. Each of the families had at least three affected family members over at least two generations. Whole genome sequencing was performed using Illumina Truseq Nano DNA Library Prep Kit, and variant calling was performed according to an in-house pipeline on a family-wise basis. Overall, 7,435,742 single-nucleotide variants and 45,627 structural variants were identified in the entire cohort, representing a unique resource for the analysis of rare events. In our first analysis we focused on the protein-coding regions and are currently applying technical, pedigree-based and frequency filtering in each of the families. In family 1, this analysis together with subsequent annotation in the Variant Effect Predictor identified 66 candidate variants in 65 genes, one of which has been previously suggested as risk gene in an independent exome sequencing study (PLEKHA5, Cox et al. 2018). Based on single-cell expression data during mouse embryonic development, additional candidate genes (e.g. MPZL2, GPC6) were also identified. The analysis of the remaining nine families is currently ongoing, and results will be presented at the conference.

A. Scheer: None. F. Brand: None. J. Fazaal: None. N. Ishorst: None. P.M. Krawitz: None. E. Mangold: None. K.U. Ludwig: None.

P04.062.C Analysis of single-cell based expression variability between candidate genes for syndromic and non-syndromic forms of orofacial clefting

Anna Siewert 1, Julia Welzenbach1, Benedikt Reiz2, Elisabeth Mangold1, Henning Dickten2, Kerstin U. Ludwig1

1Institute of Human Genetics, University of Bonn, School of Medicine & University Hospital Bonn, Bonn, Germany, 2FASTGenomics, Comma Soft AG, Bonn, Germany.

Orofacial clefts (OFCs) are among the most common human birth defects. OFC can occur as an isolated phenotype, or as part of a more complex malformation syndrome. In the latter, additional tissues and organ systems show developmental differences beyond OFC. We here hypothesized that candidate genes involved in syndromic OFC-forms may be expressed in more cell types during embryonic development, compared to genes causing non-syndromic OFC. To study this, we re-analyzed recently published single-cell expression data from the Mouse Organogenesis Cell Atlas (Cao et. al 2019), covering the relevant time period for facial development from embryonic days E9.5-E13.5. We downloaded data from 100,000 sampled cells and performed data analyses using the R package Seurat (Stuart et. al 2019) and the analytical ecosystem FASTGenomics. Data was split by embryonic day to yield a developmental time frame. Candidate genes associated with non-syndromic OFCs were defined based on data from prior genome-wide association studies, while genes causing established OFC-syndromes (autosomal dominant (AD) and autosomal recessive) were retrieved from the literature. At each time point, we extracted the percentage of cell types expressing the respective gene and compared expression levels between the different groups. Our results show that on average, genes underlying AD forms of syndromic OFC are expressed in significantly more cell types during organogenesis compared to risk genes for non-syndromic OFC (P-values: 2.02x10-06 for E9.5; 1.44x10-04 (E10.5); 1.11x10-04 (E11.5); 0.002 (E12.5); 0.002 (E13.5)). This analysis will help to further understand the molecular pathways and etiological differences between syndromic and non-syndromic forms.

A. Siewert: None. J. Welzenbach: None. B. Reiz: A. Employment (full or part-time); Significant; Comma Soft AG. E. Mangold: None. H. Dickten: A. Employment (full or part-time); Significant; Comma Soft AG. K.U. Ludwig: None.

P04.063.D Search for exonic homozygous/compound heterozygous variants in affected sib-pairs identifies novel candidate genes for nonsyndromic cleft palate

Nina Ishorst 1, Helena Shkuro1, Julia Fazaal1, Ann Kathrin Hoebel1, Holger Thiele2, Teresa Kruse3, Michael J. Dixon4, Kerstin U. Ludwig1, Elisabeth Mangold1

1Institute of Human Genetics, University of Bonn, School of Medicine & University Hospital Bonn, Bonn, Germany, 2University of Cologne, Cologne Center for Genomics CCG, Cologne, Germany, 3Department of Orthodontics, University of Cologne, Cologne, Germany, 4Faculty of Biology, Medicine & Health, University of Manchester, Manchester, United Kingdom.

Nonsyndromic cleft palate only (nsCPO) belongs to the typical forms of orofacial clefts and is a common congenital malformation. nsCPO is a multifactorial disorder with environmental and genetic factors contributing to disease risk. Of note, the genetic contribution is rather high with an estimated heritability of >90%. To date, one common risk locus for nsCPO is confirmed, and nine have been reported recently but still await independent replication. Epidemiological observations and genetic studies suggest that high penetrance rare/low frequency variants contribute to nsCPO, which might be inherited in an autosomal-recessive manner. In this study, we focussed on detection of novel nsCPO candidate genes by identifying compound heterozygous/homozygous variants. To that end, we reanalyzed whole-exome sequencing data from six affected sib-pairs born to unaffected parents. After filtering for population frequency (MAF < 5%) and manual inspection of reads, we detected 169 putative compound heterozygous and 13 putative homozygous variants. We next prioritized these 182 variants/80 candidate genes based on (1) the functional effects at transcriptional level and in silico predictions, (2) intolerance for a certain class of rare variation, and (3) expression in mouse embryonic palatal shelves at embryonic day E13.5. This resulted in a list of the 32 most promising candidate genes, which include one compound heterozygous situation within GRHL3, which has been implicated in nsCPO, and novel genes encoding proteins involved in cell migratory processes, such as DDR2, a binding partner of the gene product of the well established clefting gene CDH1.

N. Ishorst: None. H. Shkuro: None. J. Fazaal: None. A.K. Hoebel: None. H. Thiele: None. T. Kruse: None. M.J. Dixon: None. K.U. Ludwig: None. E. Mangold: None.

P04.064.A Systematic analysis of non-coding de novo mutations from whole genome sequence data of triads with non-syndromic cleft lip with/without cleft palate

Hanna K. Zieger 1, Leonie Weinhold2, Axel Schmidt1, Manuel Holtgrewe3, Stefan A. Juranek4, Anna Siewert1, Frederic Thieme1, Fabian Brand5, Julia Welzenbach1, Dieter Beule3,6, Katrin Paeschke4, Peter M. Krawitz2, Kerstin U. Ludwig1

1Institute of Human Genetics, University of Bonn, School of Medicine & University Hospital Bonn, Bonn, Germany, 2Institute for Medical Biometry, Informatics and Epidemiology, University Hospital Bonn, Bonn, Germany, 3Core Unit Bioinformatics, Berlin Institute of Health, Berlin, Germany, 4Department of Oncology, Hematology and Rheumatology, University Hospital Bonn, Bonn, Germany, 5Institute for Genomic Statistics and Bioinformatics, University Hospital Bonn, Bonn, Germany, 6Max Delbrück Center for Molecular Medicine, Berlin, Germany.

Non-syndromic cleft lip with/without cleft palate (nsCL/P) is a common multifactorial disorder with strong genetic contribution. Here, we systematically investigate the contribution of de novo mutations (DNMs) to nsCL/P risk, using whole-genome sequence (WGS) data for 211 nsCL/P and 284 non-cleft reference trios from the Kids First Project. For the total set of 31,490 DNMs, overall comparison between cohorts did not show conclusive differences, neither in absolute numbers nor when weighted for different functional scores. However, we observed nominally significant accumulation of non-coding DNMs at bivalent TSS/enhancer chromatin states in nsCL/P during human embryonic face development at Carnegie Stage 15 (p = 0.0269), and a nominally significant enrichment of non-coding DNMs in topologically associating domains at two GWAS risk loci, i.e. 4q28.1 (7 cases, 0 controls, p = 0.0008) and 2p21PKDCC (7 cases, 2 controls, p = 0.0161). We finally used transcription factor (TF) binding information to identify TFs with potential key role in nsCL/P etiology. Based on position weight matrices, we predicted TF binding sites for 810 human TFs, and calculated changes of binding capacity at 28,773 DNM-sites. We observed a significant enrichment of DNM-hits for motif TFAP2A in nsCL/P, and identified ATF3, MSC and HES5/7 as potential TF candidates. Notably, for MSC and ATF3, this finding was supported by a strong quantitative effect on the predicted binding change, which for MSC is currently validated using in vitro assays. Our study provides novel insights into nsCL/P etiology and suggests a TF-based approach that can be used to annotate non-coding risk variants from WGS data.

H.K. Zieger: None. L. Weinhold: None. A. Schmidt: None. M. Holtgrewe: None. S.A. Juranek: None. A. Siewert: None. F. Thieme: None. F. Brand: None. J. Welzenbach: None. D. Beule: None. K. Paeschke: None. P.M. Krawitz: None. K.U. Ludwig: None.

P04.065.B Chondrocyte protein co-expression network analysis reveals a link between ECM mechanosensing and glucose metabolism in osteoarthritis

Aspasia Destouni 1, Konstantinos C. Tsolis2, Anastassios Economou2, Ioanna Papathanassiou3, Charalambos Balis1, Evanthia Mourmoura1, Aspasia Tsezou1,3

1Laboratory of Cytogenetics and Molecular Genetics, Faculty of Medicine, University of Thessaly, Larissa, Greece, 2Department of Microbiology and Immunology, Rega Institute for Medical Research, KULeuven, Leuven, Belgium, 3Department of Biology, Faculty of Medicine, University of Thessaly, Larissa, Greece.

Introduction: Knee osteoarthritis (OA) is the second most common structural OA disorder affecting approximately 22.9% of individuals 40 years and over globally. The only currently available effective treatment is total knee joint replacement, which is performed at the late stages of the disease. The lack of effective disease-modifying drugs and preventive tools highlights our incomplete understanding of the fundamental biological aspects of osteoarthritis.

Materials and methods: We performed label-free shotgun LC-MS in primary chondrocytes obtained from the articular cartilage of 10 OA and 6 healthy individuals. We implemented the statistical concepts of Weighted Gene Co-expression Network Analysis to reconstruct in an unbiased way the organisation of the chondrocyte proteome and to parse the shared chondrocyte protein interactome in disease associated modules.

Results: Chondrocyte proteome is parsed into functional modules with well characterized significance to the disease, such as core structural components of the cartilage ECM which form a meta-module with proteins mediating glycolysis. Meta-module protein abundance is reduced whilst proteins mediating focal adhesion and cytoskeletal dynamics are increased in OA. COL11A1 and TNC have significant associations with OA in the GWAS catalog and are members of the ECM module indicating that variants could be exerting a detrimental effect in the context of ECM mechanosensing-based regulation of chondrocyte metabolism.

Conclusions: Our systems analysis recapitulates hallmarks of OA and offers new insights into the modular structure of the protein interactome that is associated with OA chondrocyte biology. Grant: iStemTheOS, Grant No. MIS 5033630/ELKE5876 from the Hellenic Foundation for Research & Innovation.

A. Destouni: None. K.C. Tsolis: None. A. Economou: None. I. Papathanassiou: None. C. Balis: None. E. Mourmoura: None. A. Tsezou: None.

P04.066.C A family with Osteochondritis Dissecans and multiple fractures harboring COL9A2 and PLS3 Mutations

Estelle Arnaud Gouttenoire 1, Sophie Lang-Andrey2, Françoise Lemaire-Girard1, Jean Dudler2, Michael Morris1

1SYNLAB Switzerland, Department of Genetics, Lausanne, Switzerland, 2HFR Fribourg, Department of Rheumatology, Fribourg, Switzerland.

A 25-year-old woman consulted in our specialized genetic rheumatology clinic, with symptomatology evolving since the age of 11, with knee pain and episodes of joint blockage, MRI positive axial spondyloarthropathy and hyperlaxity. The diagnosis of bilateral osteochondritis dissecans (OCD) was made at the age of 22. She later developed ankle pain also related to OCD and spiral fracture of the tibia and proximal fibula, without trauma. The proband’s brother had multiple fractures and joint blockage since childhood, and 1 maternal uncle had joint blockage since childhood. The proband’s mother had pain in childhood and has been suffering from pain in her arms, knees and ankles for 1 year. The maternal grandmother has lumbar disc degeneration and hearing problem. NGS of a 251-gene skeletal dysplasia panel revealed that proband is double-heterozygote for COL9A2 c.186G>A and PLS3 c.827G>A, p.(Trp276*). PLS3 mutations cause X-linked osteoporosis with fractures. COL9A2 mutations have been associated with AD multiple epiphyseal dysplasia, characterized by early onset of pain associated with OCD in some patients, and also with intervertebral disc disease. Interestingly, these 2 variants segregate in the family. This approach confirms the importance of genetic consultation in familial rheumatological conditions and reveled the existence of 2 overlapping genetic connective tissue pathologies.

E. Arnaud Gouttenoire: None. S. Lang-Andrey: None. F. Lemaire-Girard: None. J. Dudler: None. M. Morris: None.

P04.067.D Atypical type VI Osteogenesis Imperfecta mouse models the intersection of IFITM5 and SERPINF1 pathways in patients

Gali Guterman Ram 1, Ghazal Hedjazi2, Chris Stephan3, Stéphane Blouin2, Victoria Schemenz4, Wolfgang Wagermaier4, Jochen Zwerina2, Peter Fratzl4, Kenneth M. Kozloff3, Nadja Fratzl-Zelman2, Joan C. Marini1

1Section on Heritable Disorders of Bone and Extracellular Matrix, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, USA, 2Ludwig Boltzmann Institute of Osteology at the Hanusch Hospital of OEGK and AUVA Trauma Centre Meidling, 1st Medical Department, Hanusch Hospital, Vienna, Austria, 3Department of Orthopaedic Surgery, University of Michigan, Ann Arbor, MI, USA, 4Max Planck Institute of Colloids and Interfaces, Dept. of Biomaterials, Potsdam, Germany.

Osteogenesis Imperfecta (OI) is a well-known skeletal dysplasia. Type V OI, caused by recurrent dominant mutation in IFITM5/BRIL, and type VI OI, caused by recessive null mutations in SERPINF1/PEDF, have distinct features. IFITM5-S40L mutations causes severe dominant atypical type-VI OI (aVI) with phenotype, bone histology and decreased cellular secretion of PEDF similar to type VI OI.

Our objective is understanding the pathways connecting IFITM5 and SERPINF1 in bone development.

We generated an Ifitm5 S42L knock-in mouse model. Newborn Ifitm5 S42L mice, heterozygous(HET) and homozygous(HMZ), are non-lethal, have flared rib cage, shoulder and knee dislocations. Radiographically, ≈50% HET mice exhibit fractures and 96% of HMZ incur fractures at multiple ages. Similar to patients, heterozygous males have normal PEDF level and increased serum ALP (p < 0.01). Mechanical testing of 2-month HET and HMZ showed reduced stiffness, yield and fracture load, with markedly increased brittleness. On µCT, HET mice have decreased Ct.Th, increased BV/TV, Tb.N. Whole-body DXA-aBMD was significantly decreased, with HMZ are more severe than HET(p < 0.01). qBEI of cortical bone revealed hypermineralization in 1-and 2-month HET, with increased CaMean, CaPeak(p < 0.05) and CaHigh(p < 0.0001, p = 0.0027 respectively) and increased density and decreased area(both p < 0.001) of osteocyte lacunar sections. Second harmonic generation florescence microscopy revealed HET bones contain mostly disordered matrix(p < 0.0001). Cultured calvarial and long bone osteoblasts exhibit differences in differentiation pattern, dependent on mating scheme, age and skeletal site.

Our murine model with physiologic levels of Ifitm5 S42L expression recapitulates patient phenotype and will be used to investigate mechanisms and pathways involving Ifitm5 and Serpinf1.

G. Guterman Ram: None. G. Hedjazi: None. C. Stephan: None. S. Blouin: None. V. Schemenz: None. W. Wagermaier: None. J. Zwerina: None. P. Fratzl: None. K.M. Kozloff: None. N. Fratzl-Zelman: None. J.C. Marini: None.

P04.068.A Study of OI patient osteoblasts to investigate phenotypic variability of dominant osteogenesis imperfecta

Milena Jovanovic, Apratim Mitra, Ryan Dale, Joan Marini

NICHD/NIH, Bethesda, MD, USA.

Osteogenesis Imperfecta (OI) is a heterogeneous bone disorder characterized by bone fractures, growth deficiency and skeletal defects. An important and unexplained feature of OI and many dominant disorders is phenotypic variability with the same mutation. We present the first comparative study of osteoblast differentiation from normal pediatric controls vs OI patients with phenotypic variability. We focused on COL1A1 mutations Gly352Ser and Gly589Ser. For each mutation we investigated osteoblasts in two unrelated patients, one with mild and one with severe phenotype. OB cell layer and secreted collagen were overmodified in all patients, shown by 3H steady-state assay, indicating delayed folding; however, quantitative analysis of hydroxylysines did not correlate with patient severity. Alizarin Red staining showed patient OB deposit significantly less mineral in vitro than controls (p < 0.05), with OB from severe patients depositing significantly less mineral than from mild patients (p < 0.05). RNA-Seq transcriptomics of differentiated osteoblasts showed proteasomal protein degradation, autophagy, and vesicle organization pathways upregulation vs controls, while protein translation was downregulated. OB from both severe patients have upregulation of pathways related to ubiquination vs controls. RNA-Seq is currently being validated by RT-qPCR and western blot and the functional significance of pathways investigated. This study will lead to novel insights into OI osteoblast differentiation, the respective roles of OB and matrix in phenotypic variability and the effect of substitution position along the collagen helix.

M. Jovanovic: None. A. Mitra: None. R. Dale: None. J. Marini: None.

P04.069.B Bone cell functions in PPIB knockout mouse model for type IX osteogenesis imperfecta are distinct from classical dominant OI

Ying Liu 1, Theresa Hefferan2, Nadja Fratzl-Zelman3, Joan Marini1

1Section on Heritable Disorders of Bone and Extracellular Matrix, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, USA, 2Biomaterials and Histomorphometry Core Laboratory, Mayo Clinic, Rochester, MN, USA, 3Ludwig Boltzmann Institute of Osteology at the Hanusch Hospital of OEGK and AUVA Trauma Centre Meidling, 1st Medical Department, Hanusch Hospital, Vienna, Austria.

Osteogenesis imperfecta (OI) is a collagen-related bone disorder, which is caused by either dominant mutations in collagen, or recessive defects in genes encoding collagen-interacting proteins. Cyclophilin B (CyPB), encoded by Ppib, functions as a procollagen prolyl 3-hydroxylation complex component (P3H1/CRTAP/CyPB) and independently as the major peptidyl-prolyl cis-trans isomerase (PPIase) catalyzing collagen folding. Mutations in Ppib cause recessive type IX OI. We reported previously that Ppib-/- mice have abnormal type I collagen post-translational modification and crosslinks. This study focuses on Ppib-/- bone cell functions, utilizing histomorphometry and qBEI analysis of femoral tissue, RT-PCR and alizarin red staining of osteoblasts, and osteoclast differentiation. Histomorphometry of Ppib-/- femora reveals reduced trabecular thickness (p < 0.05), trabecular number (p < 0.01), bone volume (p < 0.001), and cortical thickness (p < 0.0001). Distinct from high turnover in dominant OI, Ppib-/- osteoblast number and surface are significantly decreased (p < 0.01; p < 0.05). Osteoclast number does not differ between KO and WT bone, as well as in vitro osteoclast differentiation. Ppib-/- femora show reduced osteoid volume (p < 0.05), MAR (p < 0.0001) and BFR/BS (p < 0.01) vs WT. Ppib-/- osteoblasts reveal elevated matrix mineralization (p < 0.001), consistent with increased expression of late osteoblast differentiation markers Sost, Mepe, Phex, Dmp1, vs WT. qBEI analysis yields increased CaMean, CaPeak (both p < 0.001) and CaHigh values (p = 0.014) in femoral midshaft cortical bone of KO vs WT. Cyclophilin B/Ppib KO mice, modelling type IX OI, have bone cell functions distinct from classical dominant OI. PPIB KO bone has a low turnover cellular pattern with decreased osteoblast number and bone formation, increased mineralization, and normal osteoclast numbers.

Y. Liu: None. T. Hefferan: None. N. Fratzl-Zelman: None. J. Marini: None.

P04.070.C Genome sequencing discloses a homozygous noncoding deletion of 72kb upstream of SNX10 in autosomal recessive osteopetrosis

Gandham SL Bhavani 1, Prajna Udupa1, Neethukrishna Kausthubham1, Sandip Bartakke2, Katta M. Girisha1

1Department of Medical genetics, Kasturba Medical College, Manipal, Manipal Academy of Higher Education, Manipal, India, 2Department of Clinical Hematology, Aditya Birla Memorial Hospital, Pune, India.

Introduction: Autosomal recessive osteopetrosis (ARO) is a rare genetic disorder of bone resorption caused by defective osteoclasts resulting in increased bone density. Other characteristic features of this condition are macrocephaly, visual impairment, splenomegaly, and bone marrow failure. The incidence of this disorder is 1 in every 250,000 live births with onset ranging from neonatal stage to adulthood. Eight genes are associated with autosomal recessive osteopetrosis. Pathogenic variants in SNX10 (sorting nexin 10) contribute to ARO in 4% of cases. SNX10 helps in osteoclast differentiation by RANKL-stimulation.

Materials and methods: We ascertained a four-year old boy born to consanguineously married couple. Detailed clinical evaluation and skeletal survey were done. We performed exome sequencing followed by genome sequencing to identify the genetic etiology.

Results: Proband exhibited macrocephaly, visual impairment, pectus carinatum and hepatosplenomegaly. Increased bone density, sandwich vertebrae and bone-in-bone appearance were observed on radiographs. Exome sequencing was non-diagnostic. Whole genome sequencing identified a large homozygous indel, g.26263639_26335652delinsCAA, which includes ~72kb deletion along with three-nucleotides insertion in the SNX10 gene. This deletion encompasses noncoding exon 1, 5’ untranslated region, the promoter region, and upstream of SNX10. Parents are carriers of this deletion.

Conclusion: The novel ~72kb indel in noncoding region of SNX10 is the likely cause of autosomal recessive osteopetrosis in our patient. We hereby report the second large homozygous indel in SNX10 resulting in autosomal recessive osteopetrosis.

G.S. Bhavani: None. P. Udupa: None. N. Kausthubham: None. S. Bartakke: None. K.M. Girisha: None.

P04.071.D The use of bisphosphonates to treat osteoporosis in patients with Lysinuric Protein Intolerance

Jos Draaisma, Joyce Geelen, Maaike de Vries, Anne Dittrich

Radboudumc Amalia children’s hospital, Nijmegen, Netherlands.

Background: Lysinuric Protein Intolerance (LPI) is an autosomal metabolic disorder. Patients present with failure to thrive, cytopenia, acute encephalopathy or developmental disability. Long term complications includes also low bone mineral density. In general the treatment is focused on the prevention of hyperammonemia. The aim of this report is to propose the use of bisphosphonates for osteoporosis in patients with LPI.

Methods: Clinical description of a patient and review of literature.

Results: A 8-year old girl was born to non-consanguineous parents. She had a normal course until the age of 6 years. Since than she had multiple fractures including multiple vertebral fractures at different occasions due to mild trauma. Further investigation led to the genetically confirmed diagnosis LPI. The lumbar Z-score was -3.7. She was treated with intravenous pamidronate and supplemental calcium and vitamin D. No further fractures occurred. After one year the z-score increased to -1.9, after two year -1.3.

Discussion: In a cohort study performed in France 80% of the patients with LPI was diagnosed with osteopenia. In a series of 29 patients in Finland, 69% of patients had one or more fractures, mostly in childhood. The exact mechanism of the osteoporosis in LPI is still not fully understood. The normal initial therapy of patients with LPI (a protein-restricted diet and supplemental L-citrulline) does not change the signs of low bone mineral density. Bisphosphonates can be used to treat osteoporosis in patients with LPI.

J. Draaisma: None. J. Geelen: None. M. de Vries: None. A. Dittrich: None.

P04.072.A Severe osteosclerosis and early poor outcome in a patient with TCIRG1 mutation

Elena Sukarova-Angelovska, Marina Krstevska-Konstantinova, Natasa Alulovska, Svetlana Kocheva

Pediatric Clinic, Skopje, Macedonia, The Former Yugoslav Republic of.

Osteopetrosis is a heterogeneous group of disorders of bone metabolism triggered by defective osteoclast function. Increased bone density leads to worsening of many body functions. Among all known genetic causes, loss of function mutation in TCIRG1 gene is responsible for the disease in 70% of the cases. Impaired function of α3 subunit V-ATPase leads to inappropriate extracellular acidification, osteoclast proliferation and unossified osteoid matrix. We present a male neonate that admitted our clinic shortly after birth due to the suspicion of sepsis. Episodes of multifocal osteomyelitis occurred successively. He had several dysmorphic features-macrocephaly, protruded forehead, prominent eyes, receding mandible, large ears. Several painful swellings were present due to the multiple bone fissures. X-ray series confirm sclerosis of all bones, appearance of dense bone formation, sandwich vertebrae, widened growth plate and calvarial bone thickening. The mutation of TCIRG1 was found (G 2415A). Development of side effects occurred within the first several months (metabolic acidosis, hepatosplenomegalia, anemia, skeletal deformities; blindness and deafness). He developed hydrocephalus and consecutive seizures at 9 months and died at the age of 11 months due to respiratory failure. Although the mutations in TCIRG1 gene are frequent cause of AR osteopetrosis, there is a wide heterogeneity of clinical presentation - from moderate to malignant, primarily due to the different mutation and consecutive acidification error. Genotype-phenotype studies are limited; moreover, the same mutation could give rise to different phenotypes. Further investigation is needed in order to elucidate all contributing mechanisms that can indicate the severity of the disease.

E. Sukarova-Angelovska: None. M. Krstevska-Konstantinova: None. N. Alulovska: None. S. Kocheva: None.

P04.074.C Periodontal (formerly type VIII) Ehlers-Danlos syndrome: description of 12 novel cases and expansion of the clinical phenotype

Salima EL CHEHADEH 1,2, Anne LEGRAND3,4, Corinne STOETZEL2, Véronique GEOFFROY2, Clarisse BILLON3,4, Salma ADHAM4, Xavier JEUNEMAITRE3,4, Roland JAUSSAUD5, Jean Muller2,6, Elise SCHAEFER1, Karelle BENISTAN7, Sébastien GAERTNER8,9, Agnès BLOCH-ZUPAN10,11,12, Marie-Cécile MANIERE10,11,12, Catherine PETIT10,11,13, Anne-Claire Bursztejn14,15, Laurence BAL16, Anthony REYRE17, Tiffany BUSA18, Hélène DOLLFUS1,2, Dan LIPSKER14,19

1Service de Génétique Médicale, Institut de Génétique Médicale d’Alsace (IGMA), Hôpitaux Universitaires de Strasbourg, Hôpital de Hautepierre, Strasbourg, France, 2Laboratoire de Génétique Médicale, UMRS_1112, Institut de Génétique Médicale d’Alsace (IGMA), Université de Strasbourg et INSERM, Strasbourg, France, 3Université de Paris, INSERM, U970, Paris Centre de Recherche Cardiovasculaire, Paris, France, 4Centre de Référence des Maladies Vasculaires Rares, AP-HP, Hôpital européen Georges Pompidou, Paris, France, 5Département de médecine interne immunologie clinique, CHRU de Nancy et université de Lorraine, Nancy, France, 6Laboratoire de diagnostic génétique, Nouvel hôpital civil, Hôpitaux Universitaires de, Strasbourg, France, 7Centre de référence des syndromes d’Ehlers-Danlos non vasculaires, Hôpital Raymond Poincaré, Garches, Assistance Publique Hôpitaux de Paris, Garches, France, 8Service des Maladies Vasculaires - Hypertension Artérielle, Hôpitaux Universitaires de Strasbourg, Strasbourg, France, 9INSERM, UMR 1260, Regenerative Nanomedicine, FMTS, Strasbourg, France, 10Université de Strasbourg, Faculté de Chirurgie Dentaire, Strasbourg, France, 11Hôpitaux Universitaires de Strasbourg, Pôle de Médecine et Chirurgie Bucco-Dentaires, Hôpital Civil, Centre de référence des maladies rares orales et dentaires, O-Rares, Filière Santé Maladies rares TETE COU, European Reference Network ERN CRANIO, Strasbourg, France, 12Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), INSERM U1258, CNRS-UMR7104, Université de Strasbourg, Illkirch-Graffenstaden, France, 13INSERM, UMR 1260 ‘Osteoarticular and Dental Regenerative Nanomedicine’, Faculté de Médecine, Fédération de Médecine Translationnelle de Strasbourg (FMTS), Strasbourg, France, 14Clinique Dermatologique, Hôpitaux Universitaires de Strasbourg, Strasbourg, France, 15Service de dermatologie, hôpitaux de Brabois, CHU de Nancy, Vandœuvre-lès-Nancy, France, 16Timone Aortic Center, Timone Hospital, APHM ; Department of Vascular Surgery, Timone Hospital, APHM, Marseille, France, 17Service de neuroradiologie interventionnelle, CHU de Marseille, Hôpital La Timone, Marseille, France, 18Département de génétique médicale, CHU de Marseille, Hôpital La Timone, Marseille, France, 19Faculté de Médecine, Université de Strasbourg, Strasbourg, France.

Periodontal Ehlers-Danlos syndrome (pEDS) is a rare condition caused by autosomal dominant pathogenic variants in the C1R and C1S genes, encoding subunits C1R and C1S of the first component of the classical complement pathway. It is predominantly characterized by early-onset severe periodontitis with premature tooth loss, pretibial hyperpigmentation and skin fragility. Rare arterial complications have been reported, but venous insufficiency is rarely described. Here we report twelve novel patients carrying either de novo (4/12) or inherited (8/12) heterozygous pathogenic variants in C1R and C1S, in order to characterize their clinical phenotype, with a focus on the vascular features. All presented with typical pEDS clinical signs including severe periodontitis (except the youngest patient aged 3 years) with complete tooth loss in three patients before the age of 30. Three patients and one relative also displayed widespread venous insufficiency leading to chronic varicose leg ulcers. One patient suffered an intracranial aneurysm with vascular complications in 3 relatives including thoracic and abdominal aortic aneurism and dissection and intracranial aneurysm rupture. This work highlights the importance of early diagnosis of pEDS to direct appropriate dental care and precise the genetic counselling. It also confirms that vascular complications are possible, although they are not frequent, which leads us to propose to carry out a first complete vascular evaluation after the diagnosis. Larger case series are however needed to precise the frequency of these vascular complications and to improve our understanding of the link between complement pathway activation and connective tissue alterations observed in these patients.

S. El chehadeh: None. A. Legrand: None. C. Stoetzel: None. V. Geoffroy: None. C. Billon: None. S. Adham: None. X. Jeunemaitre: None. R. Jaussaud: None. J. Muller: None. E. Schaefer: None. K. Benistan: None. S. Gaertner: None. A. Bloch-zupan: None. M. Maniere: None. C. Petit: None. A. Bursztejn: None. L. Bal: None. A. Reyre: None. T. Busa: None. H. Dollfus: None. D. Lipsker: None.

P04.075.D Comprehensive analysis of the genetic determinants of proportionate short stature by targeted NGS

Angel Campos-Barros 1,2, Elena Vallespín1,2, Karen E. Heath1,2, Angela Del Pozo1,2, Mario Solís1, Isabel González-Casado3, Paula Casano4, Isabel R. Martínez-Orga1, María Fernández-Elvira1, Victoria E. Fernández-Montaño1

1INGEMM, IdiPAZ, Hospital Universitario La Paz, Madrid, Spain, 2CIBER de Enfermedades Raras (U753), ISCIII, Madrid, Spain, 3Dept. of Pediatric Endocrinology, Hospital Universitario La Paz, Madrid, Spain, 4Dept. of Pediatric Endocrinology, Hospital Sant Joan de Déu, Barcelona, Spain.

Proportionate short stature is a common condition (3% of general population) with a heterogeneous genetic etiology. Despite the advances in the understanding of pathophysiological growth regulation, the underlying genetic causes are still underdiagnosed. Aim and subjects: Molecular genetic screening of a cohort of 201 pediatric cases (86 females and 115 males) with proportionate short stature (height< -2.0 SDS) by targeted NGS.

Methods: Genomic DNA samples were analyzed with a custom panel (Seq-Cap-EZ; Nimblegen, Roche) including 331 known genes implicated in the etiology of syndromic and non-syndromic proportionate short stature. Variant prioritization was based on sequence quality assessment (Q>30); coverage (mean >90x; %bp>20x >80%); population frequency (MAF <1% in gnomAD controls), variant effect (missense, nonsense, frameshift, splicing effect) and in silico pathogenicity prediction (CADD_1.4 score >20).

Results and discussion: 97 potentially deleterious variants were identified in a total of 84 (41.8%) index cases, of which, 35 (57.3%) in genes involved in GH-IGF1 signaling (i.e. GHRH, GHRHR, GHR, IGF1R, PTPN11, IGF2R), and 26 (26.8%) in genes regulating IGF1 bioavailability (IGF1, IGFALS, IGFBP4, PAPPA2, PAPPA). The remaining variants were in genes: i) associated with syndromic forms of proportional short stature, KDM6A (Kabuki-syndrome; n = 3), NFKB2 (DAVID syndrome, n = 3), CUL7 (3M-syndrome, n = 3), and BTK (n = 1); ii) implicated in pituitary morphogenesis, PROKR2 (n = 5), IHH (n = 5); iii) extracellular matrix genes, ACAN (n = 11); or iiii) in genes encoding paracrine factors of the growth plate, NPPC; (n = 2) and NPR2 (n = 3). Targeted NGS analysis significantly improves the diagnostic rate of proportionate short stature. Grants PI 12/00649; 18/00402 (ISCIII); ENDOSCREEN S2010/BMD-2396

A. Campos-Barros: None. E. Vallespín: None. K.E. Heath: None. A. Del Pozo: None. M. Solís: None. I. González-Casado: None. P. Casano: None. I.R. Martínez-Orga: None. M. Fernández-Elvira: None. V.E. Fernández-Montaño: None.

P04.076.A Plasma inorganic pyrophosphate levels correlate to specific phenotypes and variant archetypes of ABCC6 in pseudoxanthoma elasticum patients

Matthias Van Gils 1,2, Justin Depauw1, Shana Verschuere1,2, Lukas Nollet1,2, Olivier M. Vanakker1,2

1Center for Medical Genetics Ghent, Ghent, Belgium, 2Department of Biomolecular Medicine, Ghent University, Ghent, Belgium.

Introduction: Pseudoxanthoma elasticum (PXE) is an ectopic mineralization disease caused by biallelic ABCC6 mutations. Decreased inorganic pyrophosphate (PPi) levels have been linked to ectopic mineralization in patients. However, whether correlations exist between PPi levels and the phenotype or ABCC6 genotype is unknown.

Methods: PPi levels of 128 blood samples (92 [62 patients], 22 [21 carriers] and 14 [14 controls]) were measured. ABCC6 pathogenic variants (C3-C5) were considered. PXE phenotypes were assessed using the Phenodex scoring (PS) system at initial patient sampling. Two-fold statistical analysis was performed: bulk analysis and single/first sample analysis. Correlations between PPi, age, sex and PS were investigated. Based on mutation archetypes (erroneous mRNA [D], nonsense [N] and missense [M] variants) and increasingly stringent variant selection genotype-PPi correlations were assessed.

Results: Patients and carriers had lowered PPi levels (respectively ±51% and ±78% of controls; P < 0.001) with sample range overlap between all groups. Sex (Bulk: P = 0.010; Single: P = 0.060) and age (Bulk: P = 0.005, r = 0.288, Single: P = 0.015, r = 307) significantly affected PPi levels only in patients. A significant inverse correlation between PPi levels and cardiac PS scores was identified (P = 0.041, r = -0.263) alongside a weak inverse association between PPi and vascular PS (P = 0.09). Significant differences in PPi levels between D+M and N+M genotypes were found in bulk analysis only (P < 0.05).

Conclusions: PPi is reduced in patients and carriers but overlap between groups suggests other pro-mineralization factors are also relevant in at least some patients. Correlations between PPi and cardio(vascular) phenotypes suggest PPi has promise as a biomarker; genotype-correlations however require further confirmation.

M. Van Gils: None. J. Depauw: None. S. Verschuere: None. L. Nollet: None. O.M. Vanakker: None.

P04.077.B lncRNA gene variants SPRR2C rs2291979 and LOC105375120 rs4724102 are associated with psoriasis

Laimutis Kucinskas 1, Migle Anilionyte1, Vesta Kucinskiene2, Skaidra Valiukeviciene2

1Institute of Biological System and Genetic Research, LUHS, Kaunas, Lithuania, 2Department of Skin and Venereal Diseases, Lithuanian University of Health Sciences (LUHS), Kaunas, Lithuania.

The aim: to determine the significance of SNV of genes SPRR2C rs2291979, LOC105375120 rs4724102 and LINC01698 rs75193730 in the genotypes of psoriasis and control group persons and evaluate associations between genotype and clinical signs of psoriasis. Patients and Methods. Ninety five samples from psoriasis patients’ group and 77 samples from a control group of healthy people were examined. Genomic DNA was extracted from peripheral blood leukocytes by DNA salting out procedure. Genotyping was performed by real time polymerase chain reaction and data were analysed by statistical analysis program IBM SPSS statistics.

Results: SPRR2C SNV rs2291979 frequency of A allele was 7,9 % compare to 1,3 % of control group (p = 0,011). LOC105375120 genotype G/G frequncy was 57,9 % in patient group compare with 28,6 % control group (p < 0,001) and allele G frequency was 77,4 % in patient group (p < 0,00001). No statistically significant difference was found between LINC01698 SNV rs75193730 genotypes and alleles.

Conclusions: Statisticaly significant higer frequency was determinated of SNV SPRR2C rs2291979 A allele and LOC105375120 G/G genotype and G allele compare with control group. No statistically significant associations of clinical signs and alleles and genotypes of genes SNV SPRR2C rs2291979, LOC105375120 rs4724102 and LINC01698 rs75193730 were determinated.

L. Kucinskas: None. M. Anilionyte: None. V. Kucinskiene: None. S. Valiukeviciene: None.

P04.078.C The HRAS-RIN1 signaling axis controls integrin trafficking in keratinocytes and its dysregulation contributes to the epidermal manifestation in Costello Syndrome

Theresa Nauth, Laura Isabel Brandenstein, Verena Rickassel, Georg Rosenberger

University Medical Center Hamburg-Eppendorf, Institute of Human Genetics, Hamburg, Germany.

Germline missense mutations in the HRAS gene cause Costello syndrome (CS), a rare developmental disorder characterized by a typical facial gestalt, postnatal growth deficiency, intellectual disability, predisposition to malignancies as well as skeletal, cardiac and dermatological abnormalities. The molecular pathophysiology caused by heterozygous HRAS gain-of-function mutations have been analyzed in various tissues and cell types. However, up to date the molecular basis for cutaneous manifestations in CS is largely unknown. To study epidermal pathobiology, we generated permanent human keratinocyte cells (HaCaT) stably expressing wild type HRASWT or CS-associated HRASGly12Ser and screened for keratinocyte-specific HRAS binding partners by affinity purification and quantitative mass spectrometry. We identified and verified RIN1 (Ras and Rab interactor 1) as most important interaction partner of active HRAS variants in keratinocytes. By its dual function as an activator of ABL1/2 and RAB5A, RIN1 is involved in cytoskeletal remodeling and endosomal sorting of cell surface receptors, such as integrins. FACS-based integrin endocytosis assays showed aberrant integrin trafficking in keratinocytes expressing HRASGly12Ser and application of primaquine revealed integrin recycling to be essentially affected. By immunocytochemistry, we detected an increase of intracellular vesicular β1 integrin which strongly co-localized with RAB5, RAB21 and EEA1. The altered bioavailability of integrins in keratinocytes expressing HRASGly12Ser was associated with impaired cell spreading. Our data demonstrate that CS-associated HRASGly12Ser interferes with RIN1-RAB5 mediated endosomal sorting of integrins and, thus, adhesion-dependent processes. We conclude that dysregulation of receptor trafficking and cell adhesion are relevant in the pathobiology of CS.

T. Nauth: None. L.I. Brandenstein: None. V. Rickassel: None. G. Rosenberger: None.

P04.079.D IRAK2 is associated with rheumatoid arthritis susceptibility

Rim Sghiri 1, Hana BenHassine2, Asma Boumiza2, Khadija Baccouche3, Foued Slama2, Adel Almogren1, Zahid Shakoor1, Elyes Bouajina3, Ramzi Zemni2

1King Saud University, Riyadh, Saudi Arabia, 2Faculte de Medecine de Sousse, Sousse, Tunisia, 3Hopital Farhat Hached de Sousse, Sousse, Tunisia.

Objectives: Investigate the association of the single nucleotide polymorphisms of interleukin-1 receptor-associated kinase 2 (IRAK2) rs3844283 and rs708035 with rheumatoid arthritis (RA).

Patients and methods: IRAK2 rs3844283 and rs708035 genotyping was determined by mutagenically separated PCR in a cohort of 222 (30 men, 192 women, mean age 49 years) adult RA patients and 224 matched controls.

Results: IRAK2 rs3844283 C allele was detected in 66% of RA patients and 74% of controls. The CC genotype was the most frequent genotype in both RA patients (45.5%) and the controls (56.3%). The G allele was found to be associated with RA susceptibility (OR = 1.47, 95% CI = 1.10-1.96, p = 0.008). The GG genotype was found to be associated with RA in the co-dominant and the dominant models (OR = 2.03, 95% CI = 1.08-3.81, p = 0.042 and OR = 1.54, 95% CI = 1.06-2.23, p = 0.023, respectively). IRAK2 rs708035 was found not to be in the Hardy-Weinberg equilibrium. The hyperfunctional IRAK2 rs708035 A allele was more frequent in RA patients than in controls (69.9 versus 62.2%, respectively, p = 0.015). Moreover, IRAK2 rs708035 and IRAK2 rs3844283 were in linkage disequilibrium and the GA haplotype was significantly more frequent in RA patients than in controls (p = 0.034).

Conclusion: This study for the first time ever reports the association of IRAK2 rs3844283, IRAK2 rs708035, and the corresponding haplotypes with RA. Functional studies are recommended to elucidate the risk posed by the GA haplotype for the development of RA.

R. Sghiri: None. H. BenHassine: None. A. Boumiza: None. K. Baccouche: None. F. Slama: None. A. Almogren: None. Z. Shakoor: None. E. Bouajina: None. R. Zemni: None.

P04.080.A A TRAF6 genetic variant is associated with low bone mineral density in rheumatoid arthritis

Rim Sghiri1, Hana BenHassine 2, Najla El Amri3, Khadija Baccouche3, Foued Slama2, Zahid Shakoor4, Adel Almogren4, Elyes Bouajina3, Ramzi Zemni2

1king Saud University, Riyadh, Saudi Arabia, 2Faculte de Medecine de Sousse, Sousse, Tunisia, 3Hopital Farhat Hached de Sousse, Sousse, Tunisia, 4King Saud University, Riyadh, Saudi Arabia.

Objectives: This study was aimed at investigating the association of the single nucleotide polymorphism of tumor necrosis factor receptor associated factor 6 (TRAF6), rs540386, with low bone mineral density (BMD) among patients with rheumatoid arthritis (RA).

Patients and Methods: TRAF6 rs540386 genotyping was performed by mutagenically separated PCR in a cohort of 188 (23 men, 165 women, median age, 56.2 years) adult RA patients and 224 age and gender-matched controls. BMD was measured using dual-energy X-ray absorptiometry (DXA) (Lunar Prodigy advance scans, GE Healthcare, USA).

Results: Among the RA patients, 64 (55 women, 9 men) had low BMD comprising of 57 patients with osteoporosis and 7 with osteopenia. Whereas TRAF6 rs540386 was not associated with RA susceptibility, it was however found to be a risk factor for reduced lumbar spine Z-score in the recessive model (OR = 3.34, 95% CI = (1.01-11.00), p = 0.038). This association was confirmed further in the multivariate logistic regression analysis taking into account several potential confounding factors (OR = 3.34 (1.01-11.00), p = 0.048). In addition, mean total femur Z-score was found to be reduced in TT patients when compared to CC + CT patients (- 1.30 ± 1.32 versus - 0.60 ± 1.05, p = 0.034). No association between TRAF6 rs540386 and local bone damage was observed.

Conclusions: This study for the first time ever demonstrated an association between a genetic variant of TRAF6 and low BMD among patients with RA. Further investigations are needed to elucidate the exact role of this variant.

R. Sghiri: None. H. BenHassine: None. N. El Amri: None. K. Baccouche: None. F. Slama: None. Z. Shakoor: None. A. Almogren: None. E. Bouajina: None. R. Zemni: None.

P04.081.B Association between a functional polymorphism within the IL-17RC gene and idiopathic scoliosis in Bulgarian population

Svetla Nikolova 1, Milka Dikova2, Alexandre Loukanov3

1Sofia University, Sofia, Bulgaria, 2Medical University-Sofia, Sofia, Bulgaria, 3Saitama University, Saitama, Japan.

Introduction: Several genome wide association studies suggested the common IL-17RC gene variants could take part in the pathogenesis of idiopathic scoliosis. The present case-control study investigated the association between a functional polymorphism, IL-17RC*rs708567 (G/A), and idiopathic scoliosis in a Bulgarian population sample.

Materials and Methods: The association study was performed on 127 patients and 254 controls after obtaining written informed consent. The mean Cobb angle was 53.8° ± 21.2. The mean age of patients was 11.2 ± 2.9 years. The cases were divided into subgroups based on the age of onset, sex, family history, and progression. The genotyping was carried out by TaqMan Real-Time PCR method. The statistical analysis was performed by Pearson’s Chi-squared test and Fisher’s Exact Test with p-value less than 0.05 as statistically significant.

Results: The frequencies of the variant A allele and AA genotype in the total group of patients and in the subgroup of patients with Cobb angle above 40° were significantly higher than that in the controls (p < 0.05). In addition, this case-control study revealed statistically significant association between IL-17RC*rs708567 (G/A) and primary scoliosis in males, females, adolescents, familial and sporadic cases.

Conclusions: The results confirmed previously reported associations between a common variant within IL-17RC and idiopathic scoliosis in Caucasian and Asian population groups and suggested that the molecular marker rs708567 is an independent predisposing and modifying factor of idiopathic scoliosis in different subgroups of Bulgarian patients.This work was supported by MEXT/JSPS KAKENHI T20K05260 and Jikoshunyu Kyoinhaibun-keihi T5452 funded by Saitama University.

S. Nikolova: None. M. Dikova: None. A. Loukanov: None.

P04.082.C Genotype-phenotype correlation of aberrations at 7q21.2-q21.3 locus in patients affected with isolated or syndromic form of split-hand/foot malformation

Anna Sowińska-Seidler 1, Magdalena Socha1, Anna Materna-Kiryluk1,2, Aleksander Jamsheer1,2

1Department of Medical Genetics, Poznan University of Medical Sciences, Poznań, Poland, 2Centers for Medical Genetics GENESIS, Poznań, Poland.

Split-hand/foot malformation (SHFM) refers to the group of congenital limb malformations characterized by the absence or hypoplasia of the central rays of the autopods. Eight loci are associated with this clinically and genetically heterogeneous disorder. SHFM type 1 (SHFM1) maps to 7q21.2-q21.3 and occurs in an isolated form, associated with other abnormalities, or as a part of a congenital anomaly syndrome. In most cases SHFM1 results from deletions encompassing the DLX5/DLX6 genes or their regulatory elements. Herein, we report on five new index cases harboring 7q21.2-q21.3 rearrangements and perform a genotype-phenotype correlation for these individuals and two previously published families of Polish origin affected with SHFM1. Chromosome analysis including conventional GTG banding and array-based comparative genomic hybridization (aCGH) were applied to identify the causative aberrations. Balanced translocations: t(7;12)(q21.2;q21.3) and t(7;10)(q21.2;q22.2) were identified in the most severely affected patient diagnosed with EEC and developmental delay, and in a patient with bilateral ectrodactyly of the hands and feet and hearing loss, respectively. Two other sporadic patients affected with isolated ectrodactyly of the feet carried microdeletions spanning less than 200 kb encompassing the limb-specific enhancers within DYNC1I1. Also, a 4.5 Mb deletion of the 7q21.2-q21.3 region was identified in a sporadic patient diagnosed with EEC syndrome. We present the spectrum of abnormalities in SHFM1 cases that depends on the 7q21.2-q21.3 aberrations breakpoints, deletion size and its gene/regulatory elements content. This work was supported by the grants from the Polish National Science Centre UMO-2016/21/D/NZ5/00064 to A.S-S., UMO-2016/23/N/NZ2/02362 to M.S. and UMO-2016/22/E/NZ5/00270 to A.J.

A. Sowińska-Seidler: None. M. Socha: None. A. Materna-Kiryluk: None. A. Jamsheer: None.

P04.083.D SHFM3 caused by a duplication involving BTRC but not POLL and with possible modifier variants in FRAS1 and C2CD3

Gökhan Nalbant 1, Aslıhan Tolun2

1Department of Biostatistics and Bioinformatics, Institute of Health Sciences, Acibadem University, Istanbul, Turkey, 2Department of Molecular Biology and Genetics, Istanbul Technical University, Istanbul, Turkey.

Introduction: Split-hand/foot malformation (SHFM) involves the central rays, the phenotype is very diverse, and six loci are known. SHFM3 (10q24) displays dominant inheritance. Causative duplication includes two genes; BTRC is involved in Wnt signalling cascade by regulating β-catenin levels in limb development and POLL in base excision repair. Whether both genes contribute to the condition has not been elucidated. The disorder varies in severity even among relatives. We investigated a Turkish family afflicted with SHFM3.

Materials and Methods: Exome sequencing for unaffected mother, affected father and two affected daughters was performed to find the causal variant. SNP genotypes were used to detect the disease gene locus and to investigate for any deletion or duplication linked to the malformation.

Results: Linkage analysis revealed a single locus at 10q24.32. All 7 affected individuals investigated carried a maximal 264 kb heterozygous duplication. Very rare FRAS1:p.E296K and C2CD3:p.A2041V were found in daughters but not father.

Conclusions: We present the smallest duplication causing SHFM3 and involving BTRC gene only, indicating that POLL does not contribute to the condition. Candidate variants detected could be the genetic factors that aggravate the malformation in siblings and are being investigated for possible relations to pathways regulated by BTRC. Both FRAS1- and C2CD3-deficit phenotypes include autopod malformations. We will also search for modifiers in the second-cousin. Modifier variants could give a clue about why this disorder exhibits variable severity among kin. Supported by the Research Fund of the Istanbul Technical University (2021-42537).

G. Nalbant: None. A. Tolun: None.

P04.084.A Is Xlαs associated with short stature?

Arrate Pereda 1, Yerai Vado1,2, Karen E. Heath3,4,5, Jesus Pozo-Roman6,7,8, Maria Angeles Santos-Mata9, Elena Artola10, Guiomar Perez de Nanclares1

1Rare Diseases Research Group, Molecular (Epi)Genetics Laboratory, BioAraba Health Research Institute, OSI Araba, Araba University Hospital-Txagorritxu, Vitoria-Gasteiz, Araba, Spain, 2NanoBioCel Research Group, Laboratory of Pharmacy and Pharmaceutical Technology, Faculty of Pharmacy, University of the Basque Country UPV/EHU, Vitoria-Gasteiz, Araba, Spain, 3Institute of Medical and Molecular Genetics (INGEMM), La Paz University Hospital, Autonomous University of Madrid, IdiPAZ, Madrid, Spain, 4Skeletal dysplasia Multidisciplinary Unit (UMDE), La Paz University Hospital, Madrid, Spain, 5CIBERER, ISCIII, Madrid, Spain, 6Departments of Pediatrics and Pediatric Endocrinology, Hospital Infantil Niño Jesús University Hospital, Autonomous University of Madrid, Madrid, Spain, 7La Princesa Research Institute, Madrid, Spain, 8CIBEROBN, Institute of Health Carlos III, Madrid, Spain, 9Pediatric Endocrinology Unit, Paediatric Service of the University Hospital de Jerez, Jerez de la Frontera, Cádiz, Spain, 10Paediatric Department, Donostia University, Donostia, Gipuzkoa, Spain.

Introduction: GNAS, located on 20q13.2, is a highly complex imprinted locus from which at least four transcripts (NESP55, GNAS, XL-GNAS and A/B) are generated and, some of them, in an allele specific way. It is well known that heterozygous GNAS alterations in either allele are associated with growth impairment, pre- and postnatally. But less is known about the effect of genetic alterations at XLαs (eXtra Large Gsα), encoded by and alternative exon 1 of the paternally expressed long form of Gsα.

Patients and Methods: Three independent families in which the probands (P) were referred for short stature (SS) (P1) and pseudopseudohypoparathyroidism (PPHP) or PTH-related protein signalling disorder type 2 (iPPSD2) (P2,P3) were studied by NGS-custom panels. Variant confirmation and cosegregation studies were carried out via Sanger sequencing. Parental origin of the allele was tested by allele-specific RT-PCR amplification, and sequencing.

Results: Three different heterozygous variants of uncertain significance (VUS) were identified in the XLαs exon (Table 1). Familial studies suggested cosegregation when the variant was present on the paternal allele.

Conclusion: We describe the first three families in whom XLαs specific exon variants on the paternal allele seem to be associated with short stature and brachydactyly (BD). Additional studies are required. Funding: ESPE RU Grant 2020; ISCIII, Spanish Ministry of Economy and Competitiveness, co-financed by the European Regional Development Fund (PI20/00950); University Basque Country UPV/EHU (PIF17/29).

Table 12

A. Pereda: None. Y. Vado: None. K.E. Heath: None. J. Pozo-Roman: None. M. Santos-Mata: None. E. Artola: None. G. Perez de Nanclares: None.

P04.085.B Monogenic variants in short stature: use and efficiency of targeted NGS panel in 300 patients

Caroline Michot 1, Pauline Marzin1, Geneviève Baujat1, Coralie Haudry2, Anne-Laure Tourre2, Julie Steffann2, Sophie Rondeau2, Valérie Cormier-Daire1

1Reference Center for skeletal dysplasia, INSERM UMR1163, Paris Descartes-Sorbonne Paris Cité University, Imagine Institute, Necker-Enfants Malades Hospital, Paris, France, 2Molecular Genetic Unit, Paris Descartes-Sorbonne Paris Cité University, Necker-Enfants Malades Hospital, Paris, France.

Short stature is a frequent reason for paediatric consultations. After clinical, endocrinological and radiological explorations, even patients shorter than -2,5 SD remain without diagnosis. Next generation sequencing (NGS) has improved the ability to offer testings for heterogeneous conditions. We report the analysis of 300 patients with short stature.

A targeted NGS panel was designed to sequence 150 skeletal dysplasia genes. Amplicons were captured by a custom Sure-Select kit (Agilent) and sequenced on HiSeq (Illumina). Annotation and analysis of variants were realized by a homemade interface (PolyWeb). Results were discussed with the physicians of the reference center for Skeletal Dysplasias.

Pathogenic variants were identified in 48 patients (16%) and variants of unknown significance (VOUS) in 18 cases (6%), involving 8 different genes. Pathogenic variants were identified in: NPR2 (n = 16), ACAN (n = 11), FGFR3 (n = 8), IHH (n = 8) and SHOX (n = 5). Patients had neither body disproportion, nor Madelung deformation. X-rays showed no or minor abnormalities, such as bone age anomalies, slightly curved radius or stocky long bones. VOUS variants involved the same genes, and also COL9A2 (n = 1), COL11A1 (n = 2) and COL11A2 (n = 2). These last variants were classified as class 3, as patients displayed no epiphyseal dysplasia.

Targeted NGS allowed molecular elucidation in 16 % of patients with proportionate short stature and minor skeletal features. These results highlight the impact of monogenic variants in short stature patients, involving a few number of genes, and the efficiency of targeted NGS in this indication. Molecular elucidation is important for the assessment of therapeutic options for these patients.

C. Michot: None. P. Marzin: None. G. Baujat: None. C. Haudry: None. A. Tourre: None. J. Steffann: None. S. Rondeau: None. V. Cormier-Daire: None.

P04.086.C Identification and tissue-specific characterization of novel SHOX-regulated genes highlights SOX family members among other genes

Sandra Hoffmann 1, Ralph Roeth1, Sabrina Diebold2, Jasmin Gogel1, Steffen Just2, Gudrun A. Rappold1

1Institute of Human Genetics, Heidelberg, Germany, 2Clinic for Internal Medicine II, Molecular Cardiology, Ulm, Germany.

SHOX-deficiency causes a spectrum of clinical phenotypes related to skeletal dysplasia and short stature including Léri Weill dyschondrosteosis, Langer mesomelic dysplasia, Turner syndrome, as well as idiopathic short and tall stature. SHOX controls chondrocyte proliferation and differentiation, bone maturation, as well as cellular growth arrest and apoptosis via transcriptional regulation of its direct target genes NPPB, FGFR3, and CTGF. However, our understanding of SHOX-related pathways is still incomplete. To elucidate the underlying molecular mechanisms and to better understand the broad phenotypic spectrum of SHOX-deficiency, we analyzed differentially expressed genes in human fibroblasts (NHDF), where SHOX is expressed at detectable level. Twenty-three putative target genes were selected for further validation by whole-body and tissue-specific characterization (head, heart and pectoral fins) in developing shox-deficient zebrafish embryos. Physiological relevance was confirmed for the majority of these genes in pectoral fins and network-based analysis showed that 20/23 genes act in a common network. Interestingly, several sox family members (sox5, 6, 8 and 18) were shown to be significantly deregulated in shox-deficient pectoral fins among other genes including nppa, nppc; cdkn1a, cdkn1ca, cyp26b1, cyp26c1, highlighting the important role of gene family members in shox-related growth disorders. Our results provide novel insights into the genetic pathways and molecular events leading to the clinical manifestation of SHOX-deficiency.

S. Hoffmann: None. R. Roeth: None. S. Diebold: None. J. Gogel: None. S. Just: None. G.A. Rappold: None.

P04.087.D Exome analysis of prenatal and postnatal cases referred with skeletal dysplasia

Andrea Haworth 1, Tessa Homfray2, Suzanne Drury1, Rand Dubis1, Meriel McEntagart2, Katrina Tatton-Brown2, Helen Savage1, John Filby1, Nicholas J. Lench1, Louisa Ive1, Eva Serra1, Natalie Trump1, Nayana Lahiri2, Janna Kenny2, Frances Elmslie2, Phil Ostrowski2, Esther Dempsey2, John Short2, Charlene Crosby2, Christine M. Hall2, Sahar Mansour2

1Congenica Limited, Cambridge, United Kingdom, 2St George’s Hospital NHS Trust, London, United Kingdom.

Skeletal dysplasias (SD) are rare disorders representing approximately 5% of all congenital anomalies. They are highly heterogeneous and clinical findings are often non-specific, so accurate diagnosis often relies on expert interpretation of radiological findings. Until recently treatments have been limited but development of new therapeutics has highlighted the need to provide accurate and timely diagnosis. We describe the genomic and phenotypic findings of SD cases referred to clinical genetics over an approximately 2.5-year period. All cases underwent exome sequencing (ES) as part of a service provided by Congenica and the South West Thames Regional Genetics Service, London. 53 cases were referred with a clinical diagnosis of possible SD and a molecular diagnosis was obtained in 49% (26/53). A higher diagnostic yield was observed in prenatal (64%, 16/25) rather than postnatal cases (35.7%, 10/28). Causal variants were identified in 26 genes. Variant types identified included those impacting both coding and non-coding regions (5’UTR) and CNVs. The molecular diagnosis was not always concordant with the suspected clinical diagnosis, particularly in a prenatal setting and the advantages of undertaking analysis of all skeletal dysplasia genes in these cases is illustrated in two cases (SOX9 and GNPTAB). Accurate molecular diagnosis has directly influenced patient management, including 2 cases with spondylocarpotarsal synostosis where surveillance was initiated and 1 case of prenatal hypophosphatasia where diagnosis provided appropriate management of pregnancy and access to therapeutic intervention. Furthermore, in one family the identification of the underlying molecular cause has expanded the phenotypic spectrum of disease (cartilage-hair hypoplasia).

A. Haworth: A. Employment (full or part-time); Significant; Congenica Limited. T. Homfray: None. S. Drury: A. Employment (full or part-time); Significant; Congenica Limited. R. Dubis: A. Employment (full or part-time); Significant; Congenica Limited. M. McEntagart: None. K. Tatton-Brown: None. H. Savage: A. Employment (full or part-time); Significant; Congenica Limited. J. Filby: A. Employment (full or part-time); Significant; Congenica Limited. N.J. Lench: A. Employment (full or part-time); Significant; Congenica Limited. E. Ownership Interest (stock, stock options, patent or other intellectual property); Significant; Congenica Limited. L. Ive: None. E. Serra: A. Employment (full or part-time); Significant; Congenica Limited. N. Trump: A. Employment (full or part-time); Significant; Congenica Limited. N. Lahiri: None. J. Kenny: None. F. Elmslie: None. P. Ostrowski: None. E. Dempsey: None. J. Short: None. C. Crosby: None. C.M. Hall: None. S. Mansour: None.

P04.088.A What are key parameters for obtaining the most likely clinical diagnosis from the wide phenotypic spectrum of skeletal dysplasia in patients with previously identified disease-causing gene variant

Marija Mijovic 1, Bojan Bukva2,3, Jelena Ruml Stojanovic1, Aleksandra Miletic1, Brankica Bosankic1, Hristina Petrovic1, Goran Cuturilo1,3

1University Children’s Hospital, Department of Clinical Genetics, Belgrade, Serbia, 2University Children’s Hospital, Department of Orthopedic Surgery, Belgrade, Serbia, 3Faculty of Medicine, University of Belgrade, Belgrade, Serbia.

Introduction: It is known that alteration of several genes associated with skeletal disorders could lead to multiple, highly variable phenotypes. Aim of present study was to establish the crucial clinical and/or genetic parameters for obtaining the diagnosis in three patients with previously unclassified skeletal dysplasia in whom the causal gene variant was identified.

Materials and Methods: Genetic testing was performed using whole exome sequencing (WES). Detailed analysis of genotype-phenotype correlation was performed by the team of geneticist and child orthopedist. Contribution of following parameters was assessed: specific radiological and orthopedic signs; existence of associated disorders; adult phenotype in familial cases; phenotype of the patient with the same variant from the literature if available, and gene localization of the identified variant.

Results: Case 1: Infant with severe nonfamilial skeletal dysplasia, with congenital arthrogryposis and multiple associated problems. WES result: TRPV4: heterozygous pathogenic missense variant c.806G>A, p.Arg269His. Clinical diagnosis: Metatropic dysplasia. Case 2: Child with familial skeletal dysplasia with short stature and coxa vara. WES result: COL2A1: heterozygous pathogenic missense variant c.1681G>A, p.Gly561Ser. Clinical diagnosis: Spondyloepiphyseal dysplasia with metatarsal shortening (Czech dysplasia). Case 3: Young child with familial skeletal dysplasia with short stature, joint laxity and other skeletal involvement. WES result: COMP: heterozygous likely pathogenic missense variant c.1293C>A, p.Asp431Glu. Clinical diagnosis: Multiple epiphyseal dysplasia (Fairbank type).

Conclusion: Multidisciplinary approach could facilitate the establishment of the most likely clinical diagnosis in patients with skeletal disorders with previously identified causative gene variant. Key parameters for obtaining the clinical diagnosis differ from gene to gene.

M. Mijovic: None. B. Bukva: None. J. Ruml Stojanovic: None. A. Miletic: None. B. Bosankic: None. H. Petrovic: None. G. Cuturilo: None.

P04.089.B A previously unreported pathogenic variant in TAB2 in a patient with polyvalvular heart dystrophy, short stature and dysmorphism

Nis Brix 1,2, Rikke Christensen1, Susanne W. Grotkjær3, Dorte G. Nielsen4, Kasper J. Kyng5, Pernille A. Gregersen1,6

1Department of Clinical Genetics, Aarhus University Hospital, Aarhus N, Denmark, 2Department of Public Health, Aarhus University, Aarhus, Denmark, 3Department of Pediatrics, Herning Hospital, Herning, Denmark, 4Department of Cardiology, Aarhus University Hospital, Aarhus N, Denmark, 5Department of Pediatrics and adolescence, Aarhus University Hospital, Aarhus N, Denmark, 6Centre for Rare Diseases, Department of Pediatrics and Adolescence, Aarhus University Hospital, Aarhus, Denmark.

Introduction: TGFβ-activated kinase 1-binding protein 2 (TAB2) encodes a scaffold protein that forms the TAK1-TAB complex involved in proliferation, differentiation and myocardial homeostasis. Pathogenic variants are associated with autosomal dominant congenital heart defects (OMIM #614980). We describe a 5-year-old girl and her mother with a previously unreported frameshift variant in TAB2 identified by whole exome sequencing (WES). Clinical report: The girl was born by elective caesarean section (gestational age 39+0, birth weight 2594g). She was followed for growth retardation, and at 4.5 years, she was -3SD for height and weight. She had mild insufficient dysplastic atrioventricular valves and joint hypermobility. She was dysmorphic with fifth finger clinodactyly, pes planus, mild hypertelorism, epicanthal folds, mild midface hypoplasia, frontal bossing, bulbous nasal tip and broad mouth. The mother has short stature (-2.5SD), a dysplastic mitral valve with mild insufficiency, and has previously been treated for supraventricular tachycardia. Several other family members on the mother’s side have short stature and variable cardiac abnormalities.

Methods and Results: Using WES performed on DNA from the patient and both parents, we analysed data as a trio with focus on maternally inherited variants and found heterozygosity for a previously unreported maternally inherited class 4 variant (ACMG guidelines) inTAB2 (NM_015093.5:c.[604_605dup];[=], p.(Pro203Hisfs*41)). Segregation analysis in the family is ongoing.

Discussion: Our findings, especially the precise clinical description of an adult person (the mother) and the extensive pedigree, add to the phenotypic spectrum of a TAB2-related systemic connective tissue disorder with polyvalvular dystrophy, short stature and dysmorphism.

N. Brix: None. R. Christensen: None. S.W. Grotkjær: None. D.G. Nielsen: None. K.J. Kyng: None. P.A. Gregersen: None.

P04.090.C Evidence that ciliary genes contribute to non-syndromic familial tall stature

Birgit Weiss1, Birgit Eberle1, Ralph Röth1, Christiaan de Bruin2, Julian C. Lui3, Katrin Hinderhofer1, Jeffrey Baron3, Jan M. Wit2, Gudrun A. Rappold 1

1Institute of Human Genetics, Heidelberg, Germany, 2Leiden University Medical Center, Leiden, Netherlands, 3National Institute of health, Bethesda, MD, USA.

Human growth is a complex trait. A considerable number of gene defects have been shown to cause short stature, but there are only few examples of genetic causes of non-syndromic tall stature. Besides rare variants with large effects and common risk alleles with small effect size, oligogenic effects may contribute to this phenotype. Exome sequencing was carried out in a tall male (height 3.5 SDS) and his parents. Filtered damaging variants with high CADD scores were validated by Sanger sequencing in the trio and three other affected and one unaffected family members. Network analysis was carried out to assess links between the candidates, and the transcriptome of murine growth plate was analyzed by microarray as well as RNA Seq. Heterozygous gene variants in CEP104, CROCC, NEK1, TOM1L2 and TSTD2 predicted as damaging were found to be shared between the four tall family members. Three of the five genes (CEP104, CROCC and NEK1) belong to the ciliary gene family. All genes are expressed in mouse growth plate. Pathway and network analysis indicated close functional connections. Together, these data expand the spectrum of genes with a role in linear growth and tall stature phenotypes.

B. Weiss: None. B. Eberle: None. R. Röth: None. C. de Bruin: None. J.C. Lui: None. K. Hinderhofer: None. J. Baron: None. J.M. Wit: None. G.A. Rappold: None.

P04.091.D Compound heterozygous mutations in ERCC2 are associated with trichothiodystrophy type 1

Fahrettin Duymus, Deniz Esin, Busra Goksel Tulgar, Nadir Kocak, Tulin Cora

Selcuk University, Konya, Turkey.

A 9-month-old male patient was admitted to our clinic with sparse hair, developmental delay and recurrent infections. He was the first child of child of non-consanguineous Turkish parents. In the patient’s anamnesis, it was learned that there was a rash on her skin at birth.İchthyosis and mild scaling were reported on the scalp, trunk, and lower extremities of the patient from 1 month of age. In the evaluation of the patient, there were short brittle hair, dry skin, dystrophic tooth structure, and hyperkeratosis in the feet. Whole exome sequencing analysis was performed from the patient who had ichthyosis and frequent infections.ERCC2:c.2164C>T and ERCC2:c.1867dup heterozygous mutations were detected. The clinical findings of the patient were also compatible with trichothiodystrophy (TTD). TTD is a rare genetic disease characterized by brittle hair, ichthyosis, and recurrent infections. Molecular genetic evaluation is very useful in the diagnosis of TTD. ERCC2: c.2164C> T mutation was previously reported as compound heterozygous with pathogenic p. (Arg616Pro) variant in a patient diagnosed with TTD in 1994 by Broughton et al. To the best of our knowledge the ERCC2: c.1867dup variant has not been previously reported in the literature and is a frame shift-type variant. it has never been described before that the compound heterozygosity of these mutations causes photosensitive TTD. He was diagnosed to have photosensitive TTD type 1 and his mother and father are the carriers. Genetic counseling was given to the family of the patient. A schedule was prepared for regular clinic follow-up of the patient.

F. Duymus: None. D. Esin: None. B. Goksel Tulgar: None. N. Kocak: None. T. Cora: None.

P04.093.B An exceptional biallelic N-terminal frameshift mutation in ZMPSTE24 leads to non-lethal progeria due to utilization of a downstream alternative start codon

Lukas Kaufmann 1, Jasmin Blatterer1, Erich Schaflinger1, Aiman S. Khan2, Lisa Auinger1, Benjamin Tatrai1, Sumra W. Abbasi3, Muhammad Z. Ali2, Ansar A. Abbasi4, Ali Al Kaissi5, Klaus Wagner1, Muzammil A. Khan2, Christian Windpassinger1

1Diagnostic & Research Institute of Human Genetics, Graz, Austria, 2Gomal Centre of Biochemistry and Biotechnology, D. I. Khan, Pakistan, 3NUMS Department of Biological Sciences, Rawalpindi, Pakistan, 4Department of Zoology, Mirpur, Pakistan, 5Ludwig Boltzmann Institute of Osteology, Vienna, Austria.

Introduction: Mandibuloacral dysplasia with type B lipodystrophy (MADB) is a rare autosomal recessive disorder of premature aging, associated with severe abnormalities in bone, skin and adipose tissue. The main underlying genetic cause of MADB are homozygous or compound heterozygous missense mutations in the ZMPSTE24-gene. Biallelic loss-of-function mutations in ZMPSTE24, however, have been associated with lethal restrictive dermopathy (RD), which leads to death within the first weeks of life. In the present study, a large consanguineous Pakistani family segregating MADB was recruited for molecular investigation in order to identify the underlying genetic cause.

Materials and Methods: The disease was diagnosed through clinical features of MADB, assisted by radiologic and biochemical tests. For genetic analysis whole exome sequencing (WES) was performed, followed by homozygosity mapping and variant annotation using VarSeq™ v2.2 (Golden Helix, Inc.).

Results: Whole exome sequencing revealed a novel N-terminal homozygous frameshift mutation NM_005857:c.28_29insA, p.(Leu10Tyrfs*37) in ZMPSTE24 segregating with the disease phenotype. Remarkably, the potential loss-of-function mutation results in a non-lethal phenotype in our patients. A more in-depth analysis of the mutation revealed that the observed one base pair insertion creates a novel downstream in-frame start codon.

Conclusions: To our knowledge, this is the first report of a biallelic loss-of-function mutation not implicated in lethal RD. We propose a rescue mechanism involving the usage of a gained in-frame downstream start codon, which could potentially avoid frameshift and lead to residual enzymatic function. These findings might be crucial for the interpretation of far N-terminal mutations in complex genotype-phenotype correlations.

L. Kaufmann: None. J. Blatterer: None. E. Schaflinger: None. A.S. Khan: None. L. Auinger: None. B. Tatrai: None. S.W. Abbasi: None. M.Z. Ali: None. A.A. Abbasi: None. A. Al Kaissi: None. K. Wagner: None. M.A. Khan: None. C. Windpassinger: None.

P05 Cardiovascular Disorders

P05.001.C Segregation of rs897543876 in BMP4 gene in family with nonsyndromic aortic dilatation

Zuzana Capkova 1,2, Jana Petrkova1,2, Pavlina Capkova1,2, Lucie Punova1,2, Martin Prochazka1,2, Josef Srovnal1,3

1Department of Medical Genetics, University Hospital Olomouc, Olomouc, Czech Republic, 2Department of Medical Genetics, Faculty of Medicine and Dentistry, Palacký University Olomouc, Olomouc, Czech Republic, 3Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacký University Olomouc, Olomouc, Czech Republic.

Background: Aortic dilatation is the most common pathological involvement of the thoracic aorta. This aberration can arise by various causes such as congenital connective tissue disorder (CCTD), injury or inflammation and is accompanied by bicuspid aortic valve, arterial hypertension and atherosclerosis. CCTD were associated with increase of aortic dilatation but genetics of nonsyndromic aortic dilatation is still unknown. Case report: Here we report family with aortic dilatation. Proband was genetically tested for CCTD in his 61 years. Aortic dilatation was observed and his echocardiography showed bicuspid aortic valve. Hypertension was treated in this patient. His brother had aortic dilatation and aortic insufficiency. Daughter of proband was observed for anxiety. Daughter and son of proband were without aortic dilatation.

Method and result: Proband was consulted by geneticist and biological materials was collected with his informed consent to genetic testing. Marfan syndrome was excluded by sequencing of FBN1. Also DiGeorge syndrome was excluded by multiplex ligation-dependent probe amplification (MLPA). Moreover MLPA revealed heterozygously deleted one probe in BMP4. Sequencing of probe area showed unknown variant rs897543876 (NM_001202.6:c.-144C>T). Brother and daughter of proband also carried rs897543876.

Discussion and conclusion: rs897543876 is unknown variant which appears in 0.012 %-0.014 % worldwide and predicted as variant with moderate pathogenicity according to prediction tools. The variant is located in 5´UTR and may downregulated expression of BMP4. However, future studies are necessary for confirmation of rs897543876 pathogenic role. Acknowledgment: MH CZ—DRO FNOL 00098892, IGA UP LF_2020_007, IGA UP LF_2020_018, TACR TN01000013, No. CZ.02.1.01/0.0/0.0/16_019/0000868, LM2018132, A-C-G-T, CZ.02.1.01/0.0/0.0/16_026/0008448.

Z. Capkova: None. J. Petrkova: None. P. Capkova: None. L. Punova: None. M. Prochazka: None. J. Srovnal: None.

P05.002.D Malignant ventricular arrhythmia in a Czech representative cohort of sudden cardiac death (SCD) victims and cardiac arrest survivors (CAS) aged 1 - 65 years: results of a candidate gene panel-based sequencing strategy

Pavel Votypka 1, Petra Peldova1, Stepanka Pohlova-Kucerova2, Marketa Kulvajtova3, Petr Peichl4, Marek Sramko4, Andrea Gregorova5, Terezia Tavacova6, Jana Petrkova7, Martin Dobias8, Petr Tomasek9, Vlastimil Vancura10, Milan Macek Sr.1, Milan Macek Jr.1, Jan Janousek11, Josef Kautzner4, Alice Krebsova4

1Department of Biology and Medical Genetics, 2nd Faculty of Medicine, Charles University and Motol University Hospital, Prague, Czech Republic, 2Department of Forensic Medicine, University Hospital Hradec Kralove, Hradec Kralove, Czech Republic, 3Institute for Forensic Medicine, 3rd Faculty of Medicine, Charles University, Prague, Czech Republic, 4Institute for Clinical and Experimental Medicine, Prague, Czech Republic, 5Department of Biology and Medical Genetics, Faculty Hospital Ostrava, Ostrava, Czech Republic, 6Children´s Heart Center, Faculty Hospital Motol, Prague, Czech Republic, 71st Department of Internal Medicine - Cardiology and Laboratory of Cardiogenomics, Faculty Hospital Olomouc and Palacky University, Olomouc, Czech Republic, 811Institute of Forensic Science and Medical Law, Faculty Hospital Olomouc and Palacký University, Olomouc, Czech Republic, 9Department of Forensic Medicine, University Hospital Bulovka., 2nd Faculty of Medicine, Charles University, Prague, Czech Republic, 10Department of Cardiology, Faculty Hospital Pilsen, Pilsen, Czech Republic, 11Children´s Heart Centre, 2nd Faculty of Medicine, Charles University and Motol University Hospital, Prague, Czech Republic.

Introduction: Hereditary cardiomyopathy and arrhythmic syndromes are associated with an increased risk of malignant ventricular arrhythmia leading to SCD or cardiac arrest. Genetic testing confirms clinical / autopsy diagnosis and enables stratified care.

Patients and Methods: Altogether 166 CAS (71 female and 97 males) and 76 SCD victims (25 female and 51males) were analysed. In the CAS group 103 cases have dg. of idiopathic ventricular arrhythmia (iVF), 34 arrhythmogenic cardiomyopathy (ACM; of which 27 right -/ 7 left predominant) and 2 have hypertrophic cardiomyopathy (HCM). The SCD group comprises 11 cases with post mortem dg. of hypertrophic - (HCM) /dilated- (DCM), 16 arrhythmic cardiomyopathy (ACM), 20 sudden arrhythmic death (SADS) and 18 sudden unexplained death cases (SUD). Massively parallel sequencing (MiSeq platform; Illumina.com) utilised a custom-made panel with 100 candidate genes (SOPHiA Genetics; Switzerland). Presence of Class 4-5 variants was validated by Sanger sequencing and via family segregation analyses.

Results: The cumulative detection rate of Class 4-5 variants in CAS was in 49/166 (30 %) and in SCD victims 18/76 (22 %). The most commonly affected gene was PKP2 (12/67 variant-positive cases), followed by KCNH2 (8/67), RYR2 (7/67), TTN (7/67) and SCN5A (6/67).

Conclusion: In a representative Czech cohort, cardiac arrest could be frequently explained by a molecular cause. Malignant arrhythmias in CAS and SCD victims are mainly due to pathogenic variants in genes associated with ACM, LQT2, CPVT and LQT3. Supported by Ministry of Health of the Czech Republic, grant Nr. NV18-02-00237.

P. Votypka: None. P. Peldova: None. S. Pohlova-Kucerova: None. M. Kulvajtova: None. P. Peichl: None. M. Sramko: None. A. Gregorova: None. T. Tavacova: None. J. Petrkova: None. M. Dobias: None. P. Tomasek: None. V. Vancura: None. M. Macek Sr.: None. M. Macek Jr.: None. J. Janousek: None. J. Kautzner: None. A. Krebsova: None.

P05.004.B Analysis of the effect of periodontopathogens on the molecular mechanisms of atherosclerosis. Systematic review

Talia Yolanda Marroquin 1, Sandra Guauque-Olarte2

1Universidad Cooperativa de Colombia, Pasto, Colombia, 2Universidad Cooperativa de Colombia, Medellin, Colombia.

Introduction: The role periodontal pathogens in atherosclerosis has not been fully understood. The aim of this systematic review was to describe the effect of periodontopathogens on the molecular mechanisms involved in atherosclerosis.

Materials and methods: This systematic review followed the Cochrane and PRISMA guidelines. Screened databases were PubMed, Science Direct, and Lilacs. Original studies published from January 2015 to August 2020 about periodontitis and atherosclerosis were included. A tool for quality assessment, based on STROBE checklist, was applied.

Results: A total of 722 records were collected, 33 papers were selected for full-text reading. Porphyromonas gingivalis,Tannerella forsythia, Fusobacterium nucleatum, and Treponema denticola, were able to disseminate from the oral cavity and invade atherosclerotic plaque in humans. In mice sera, these bacteria significantly increased the expression of 25 genes or proteins belonging to apoptosis, TLR signalling, TGF-beta, inflammation, and angiogenesis pathways. Ten papers reported that P. gingivalis and Eikenella corrodens significantly increased the expression of 42 biomarkers associated with atherosclerosis development and progression pathways such as apoptosis, cytochrome-c, inflammation, entdothelin, B cell activation, vascular endothelial growth factor, cell adhesion, and toll like receptor pathways, in HAECs, HUVECs, and AoSMCs. Filifactor alocis significantly increased angiogenesis-related biomarkers. P. gingivalis significantly decreased the expression biomarkers associated with antiapoptotic mechanisms in HCAECs, HASMSs, HUVECs.

Conclusions: Data-analysis revealed the significant effect of some periodontopathogens on proatherogenic mechanisms. Network analysis will be performed on this data. It is important to study the effect of other periodonthopathogens in atherogenesis. SGO received funding by CONADI and TYM by Minciencias.

T.Y. Marroquin: None. S. Guauque-Olarte: None.

P05.005.C Somatic mosaicism of human coronary artery cells in atherosclerosis

Aleksei Zarubin, Anton Markov, Maria Nazarenko

Research Institute of Medical Genetics, Tomsk National Research Medical Center, Tomsk, Russian Federation.

Introduction: Accumulation of somatic mutations is usually associated with aging, and age-associated diseases, e.g., atherosclerosis. We assessed the scale and cell specificity of this phenomenon in the human coronary arteries affected by atherosclerosis using single cell RNA sequencing (scRNA-seq) data.

Materials and Methods: We used public dataset (PRJNA544957). Briefly, coronary arteries from four explanted heart affected by atherosclerosis were dissociated and used for scRNA-seq. Sample datasets were pooled for clustering and cell type determination. Single cells were genotyped using cellSNP-lite. Total cell genotype served as reference for every sample separately. We counted the number of cells with somatic mutations in each cluster.

Results: The maximal proportion of mosaic cells was specific for plasma cells (53.9%). Somatic variants were found to a greater extent in genes of HLA receptor family and cytochrome b-245. Smooth muscle cells were the second cluster in terms of somatic mosaicism frequency (13.5%). They also contained alterations in HLA genes, but also in the genes of metallothionein and apolipoprotein D. Somatic heterogeneity of macrophages was found in actin and tubulin genes, in addition to genes of previously described cell types. At the same time, the proportion of cells containing two or more somatic variants among smooth muscle cells was significantly less (0.3%) than in plasma cells (30%).

Conclusion: We have discovered somatic mosaicism in coronary artery cells affected by atherosclerosis. Different cell types have different levels of mosaicism. At the same time, there are genetic variants both shared by all cells and specific for individual cell types.

A. Zarubin: None. A. Markov: None. M. Nazarenko: None.

P05.006.D A Polygenic Risk Score for Atrial Fibrillation in a Hispanic/Latino Population

Brandon Chalazan 1, Christina Lee2, Valerie Morrill2, Sara Darbar3, Aylin Ornelas-Loredo3, Arvind Sridhar4, Martha Daviglus3, Patrick Ellinor2, Dawood Darbar3

1University of British Columbia, Vancouver, BC, Canada, 2Broad Institute, Boston, MA, USA, 3University of Illinois, Chicago, IL, USA, 4University of Illinois at Chicago, Chicago, IL, USA.

Background: Previous genetic studies for atrial fibrillation (AF) have identified common and rare variants associated with AF. Recently, polygenic risk scores (PRSs) have been developed for AF almost exclusively in patients of European ancestry. Here, we performed a candidate single nucleotide polymorphism (SNP) on a large cohort of Hispanic/Latino individuals determine the combined impact of genetic variants.

Purpose: To generate a PRS to estimate the risk of AF in a Hispanic/Latino cohort.

Methods: We prospectively enrolled 713 participants from the UIC AF Registry and UIC Cohort of Patients, Family and Friends. The cohort was genotyped for the top 9 known AF risk alleles and a PRS was developed using these SNPs. The cohort was stratified into quartiles of low: 0-20%; intermediate: 20-80%; and high: 80-100% genetic risk.

Results: The study cohort consisted of 625 Hispanic/Latino subjects with a mean age 46.0 ± 13.5 years and 57% were male. Patients in the intermediate and high genetic risk group were associated with a 2.50-fold (odds ratio [OR] 2.50; 95% confidence interval [CI]: 0.92-6.81; P = 0.07) and 2.70-fold (OR 2.70; 95% [CI]: 1.13-6.43; P = 0.02) increase in AF risk compared to the low risk group.

Conclusions: We show that a PRS is capable of predicting and risk stratifying AF patients in Hispanic/Latinos populations. Further studies will explore the impact of additional AF loci on risk stratification and determine if PRSs can be integrated into clinical practice.

B. Chalazan: None. C. Lee: None. V. Morrill: None. S. Darbar: None. A. Ornelas-Loredo: None. A. Sridhar: None. M. Daviglus: None. P. Ellinor: None. D. Darbar: None.

P05.007.A Mendelian randomization suggests a causal effect of abdominal obesity on postprandial lipemia

Malene Revsbech Christiansen 1, Torben Hansen1, Thorkild I. A. Sørensen1,2, Niels Grarup1, Mario García Ureña1, Dmitrii Borisevich1, Jean-Michel Oppert3, José Alfredo Martínez Hernandéz4,5, Ellen Blaak6, Tuomas Oskari Kilpeläinen1

1Novo Nordisk Foundation Center for Basic Metabolic Research, Copenhagen N, Denmark, 2Department of Public Health, Section of Epidemiology, University of Copenhagen, Copenhagen, Denmark, 3Nutrition Department, Pitie-Salpêtrière Hospital, Assistance Publique Hôpitaux de Paris, Sorbonne University, Paris, France, 4Department of Nutrition, Food Sciences, and Physiology, Center for Nutrition Research, University of Navarra, Pamplona, Spain, 5Centro de Investigación Biomédica en Red Fisiopatología de la Obesidad y Nutrición, Instituto of Health Carlos III, Madrid, Spain, 6Department of Human Biology, NUTRIM School of Nutrition and Translational Research in Metabolism, Maastricht University, Maastricht, Netherlands.

Introduction: High postprandial lipemia is associated with an increased risk of cardiovascular disease, independent of fasting lipid levels. Abdominal and gluteofemoral fat handle lipoproteins differently, which could affect postprandial lipemia and thus cardiovascular risk. We aimed to study the causal influences of body fat distribution on postprandial lipemia after a high fat meal, using Mendelian randomization.

Materials and methods: A total of 764 adults with obesity from seven European countries consumed a liquid high fat meal. Postprandial concentrations of triglycerides, glycerol, free fatty acids, and the cholesterol component of remnant-like particles (RLP), LDL and HDL were measured for 3 hours. Waist-hip ratio adjusted for BMI (WHRadjBMI) was instrumented using a genetic score of 442 independent WHRadjBMI-associated genetic variants. Two-stage least squares regression analyses were used to assess causal associations of WHRadjBMI with postprandial lipid levels. Linear regression analyses were used to assess associations of waist circumference and hip circumference adjusted for BMI (WCadjBMI and HCadjBMI) with postprandial lipid levels.

Results: Instrumental variable analyses suggested that WHRadjBMI is causally associated with higher postprandial lipemia after a high fat meal, including higher postprandial levels of triglyceride (P = 0.044) and RLP cholesterol (P = 0.020). WCadjBMI and HCadjBMI showed directionally opposite effects: WCadjBMI was associated with higher levels of triglyceride and RLP cholesterol and HCadjBMI with lower levels (P for difference = 1.67x10-5 and 5.64x10-4, respectively).

Conclusion: Our results suggest that higher abdominal fat deposition is causally associated with higher postprandial lipemia. Furthermore, gluteofemoral fat deposition may show the opposite effect.

Funding: MRC (NNF17SA0031406), TOK (NNF20OC0063707), DB (NNF17CC0026760)

M.R. Christiansen: None. T. Hansen: None. T.I.A. Sørensen: None. N. Grarup: None. M.G. Ureña: None. D. Borisevich: None. J. Oppert: None. J. Martínez Hernandéz: None. E. Blaak: None. T.O. Kilpeläinen: None.

P05.008.B A FAIR Brugada Syndrome data repository to facilitate cardiogenetic research

Marta M. Tébar Ruiz 1,2, Dorien Daneels1,3, Gudrun Pappaert4, Elyssa Cannaerts1, Thomy de Ravel1, Carlo de Asmundis4, Juan Sieira4, Ann Nowé2,5, Pedro Brugrada4, Catharina Olsen1,2,3, Tom Lenaerts2,5,6, Sonia Van Dooren1,2,3

1Centre for Medical Genetics, research group Reproduction and Genetics, research cluster Reproduction, Genetics and Regenerative Medicine, Vrije Universiteit Brussel (VUB) and Universitair Ziekenhuis Brussel (UZ Brussel), Brussels, Belgium, 2Interuniversity Institute of Bioinformatics in Brussels (IB)2, Brussels, Belgium, 3Brussels Interuniversity Genomics High Throughput core (BRIGHTcore), VUB-ULB, Brussels, Belgium, 4Heart Rhythm Management Centre, Vrije Universiteit Brussel (VUB) and Universitair Ziekenhuis Brussel (UZ Brussel), Brussels, Belgium, 5Vrije Universiteit Brussel, Artificial Intelligence Lab Brussels, Brussels, Belgium, 6Machine Learning Group, Université Libre de Bruxelles (ULB), Brussels, Belgium.

Introduction: Brugada Syndrome (BS) is a rare cardiac inheritable disorder associated with a high-risk of sudden cardiac death, which may be the first symptom, making essential its detection in pre-symptomatic individuals. Drug-induced ECG monitoring and genetic testing provide powerful early-warning systems. However, in approximately 70% of the cases no causative variant(s) can be identified. In addition, comparisons across multiple centres are currently difficult as so far, no registry with clinical information and causative genetic variants has been made available to the research community. In this study we provide a first structured repository for BS, following FAIR data principles and data protection regulations, in order to facilitate comparisons and further clinical, genetic and AI-driven research.

Methods and results: Universitair Ziekenhuis Brussel (UZB) has collected data for BS families since 1992. We manually curated clinical data of patients who consented further research (157 individuals). In addition, we (re-)classified variants for mutations found in the BS and primary cardiac arrhythmia associated genes using Hofman (PMID: 23963746) and ACMG classification guidelines (PMID: 25741868), to highlight differences in classification algorithms and validate the used methodology.

Conclusions: Combining clinical and genetic data is essential to find phenotype-genotype correlations, especially in cases like BS where different causative variants may lead to different severity grades. This new UZB BS repository is intended to boost the integration of data analysis coming from different groups, ensuring a better understanding and diagnosis of BS patients.This initiative is supported by IMAGica IRP project grant of the Vrije Universiteit Brussel.

M.M. Tébar Ruiz: None. D. Daneels: None. G. Pappaert: None. E. Cannaerts: None. T. de Ravel: None. C. de Asmundis: None. J. Sieira: None. A. Nowé: None. P. Brugrada: None. C. Olsen: None. T. Lenaerts: None. S. Van Dooren: None.

P05.009.C Mutations in structural genes in Brugada Syndrome

Marianna Farnè 1, Rita Selvatici1, Cristina Balla2, Mauro Biffi3, Elia De Maria4, Cecilia Trabanelli1, Alice Margutti1, Matteo Bertini2, Alessandra Ferlini1, Francesca Gualandi1

1Unit of Medical Genetics, Deparment of Medical Sciences, University of Ferrara, Ferrara, Italy, 2Cardiology Department, University Hospital Sant’Anna of Ferrara, Ferrara, Italy, 3Department of Cardiology, University Hospital of Bologna Sant’Orsola-Malpighi, Bologna, Italy, 4Cardiology Unit, Carpi Ramazzini Hospital, Carpi (Modena), Italy.

Introduction: Brugada syndrome (BrS) is a hereditary disease with high allelic/genetic heterogeneity and associated with the risk of ventricular arrhythmias and sudden cardiac death. This clinical entity is classically described as an arrhythmic condition occurring in a structurally normal heart, but this assumption has been recently contradicted by several observations, which suggest a link between BrS and structural cardiomyopathies.

Materials and methods: We studied a cohort of patients with spontaneous or drug-induced type 1 EKG pattern, variably associated with symptoms and a positive family history by a Next Generation Sequencing panel approach, including both channelopathies and cardiomyopathies genes.

Results: We identified in eleven subjects (13.8%) likely pathogenic/pathogenic variants in genes associated with arrhythmogenic (AC) or hypertrophic (HCM) cardiomyopathy. Four mutations were identified in the two major HCM genes: missense changes p. Arg783His and p.Val1213Met in MYH7, a frameshifting p.Lys1065Glnfs*12 and a splicing c.1458-1G>A variations in MYBPC3. All of these mutations are known being associated to HCM. Nevertheless, only the patient carrying the MYBPC3 splicing mutation showed clinical evidence of structural cardiomyopathy.

Conclusions: Our study showed genotypic overlap between BrS and structural cardiomyopathies, including HCM. This observation supports Brugada type 1 ECG pattern as a possible early sign of an occult structural heart disease, with implications in clinical management and genetic counselling.

M. Farnè: None. R. Selvatici: None. C. Balla: None. M. Biffi: None. E. De Maria: None. C. Trabanelli: None. A. Margutti: None. M. Bertini: None. A. Ferlini: None. F. Gualandi: None.

P05.010.D Congenital heart defects in Noonan syndrome and PTPN11 muta□onCongenital heart defects in Noonan syndrome and PTPN11 mutation

Laura Claudia Popa 1, Nicoleta Andreescu1,2, Simona Farcas2, Adela Chirita-Emandi1,2, Maria Puiu1,2

1Louis Turcanu Children Hospital, Timisoara, Romania, 2Victor Babes University, Timisoara, Romania.

Background: Noonan syndrome is a common autosomal dominant RASopathy disorder clinically characterized by facial dysmorphism, congenital heart disease, short stature and molecularly characterized by mutations in most common genes: PTPN11, SOS1, RAF1, RIT1, KRAS. Mutations in the PTPN11 are the most frequent causes of Noonan syndrome also the determining factor of the most serious heart defects with high risk of morbidity. Aim: The aim of this study is to identify the congenital heart defects associated with PTPN11 mutations in children from our hospital in conjunction with the literature reports.

Materials and method: A number of sixteen pediatric patients, diagnosed with Noonan syndrome were clinically and genetically investigated at Timisoara Genomic Medicine Centre. The molecular testing was performed on MiSeq Illumina platform using Illumina TruSight Cardio Sequencing Panel kit. From sixteen patients’ cases for which a mutation was identified, there were twelve in PTPN11 and four in SOS1. In this presentation we focus only on the heterozygous mutations found in PTPN11.

Results: In the remaining twelve cases, pulmonary valve stenosis was found in seven cases; atrial septal defect was identified in 3 cases, aortic coarctation, aortic regurgitation and hypertrophic cardiomyopathy were each identified in two cases, pulmonary regurgitation, heart failure, aortic dysplasia, ventricular septal defect and coronary sinus dilatation were each identified in one case.

Conclusion: The high incidence of cardiac abnormalities suggests that proper cardiac evaluation is important in genetic counseling, case management, and possibly in the identification of the responsible gene.

Key words: cardiac defects, Noonan syndrome, PTPN 11.

L.C. Popa: None. N. Andreescu: None. S. Farcas: None. A. Chirita-Emandi: None. M. Puiu: None.

P05.011.A Limitations in the interpretation of variants of uncertain significance in cardiomyopathies

Clara Herrero-Forte 1,2, María Fenollar-Cortés1,2, Carmen Cotarelo-Pérez1,2, Victoria Cañadas-Godoy3,2, María Alejandra Restrepo-Córdoba4,2, Laura Daganzo-Sierro1,2, Emma Soengas-Gonda1,2, Raluca Oancea-Ionescu1,2

1Unidad de Genética Clínica. Servicio de Análisis Clínicos. Instituto de Medicina del Laboratorio, Madrid, Spain, 2Instituto de Investigación Clínico San Carlos (IdISSC). Hospital Clínico San Carlos, Madrid, Spain, 3Consulta de Cardiopatías Familiares/Unidad de Arritmias. Servicio de Cardiología., Madrid, Spain, 4Unidad de Insuficiencia Cardiaca y Cardiopatías Familiares. Servicio de Cardiología., Madrid, Spain.

Introduction: Next Generation Sequencing (NGS) in cardiomyopathies has improved the diagnostic yield. However, detecting variants of uncertain significance (VUS) could be a problem because those with high probability of pathogenicity require additional studies in order to determine their clinical significance.

Materials and Methods: NGS analysis using virtual panel of 50 genes for hypertrophic cardiomyopathy (HCM) or dilated cardiomyopathy (DCM) was performed in 102 patients. Sanger sequencing for cosegregation analysis was able in only 6 of 17 VUS with high probability of pathogenicity. Alpha-galactosidase and lyso-GL-3 levels were measured to confirm Fabry disease. Current scientific evidence was reviewed.

Results: 61 patients with HCM: 15 Pathogenic or likely pathogenic variants (25%) and only 9 VUS were considered with high probability of pathogenicity (15%). 41 patients with DCM: 5 Pathogenic or likely pathogenic variants (12%) and only 8 VUS were considered with high probability of pathogenicity (20%). Cosegregation analysis didn´t allow to confirm the pathogenicity of the 6 VUS with high probability of pathogenicity. An indirect functional study of Fabry disease confirmed the pathogenicity in one case. One VUS with high probability of pathogenicity has been reclassified to likely benign due to a new scientific evidence published by other authors.

Conclusions: Cosegregation studies could be useless in those families with small number of affected relatives. The genetic characteristics of cardiomyopathies, mainly incomplete penetrance, also contributes to a poor performance of the cosegregation studies. Functional studies are more suitable for reclassification of VUS, although they are not usually accessible in healthcare practice.

C. Herrero-Forte: None. M. Fenollar-Cortés: None. C. Cotarelo-Pérez: None. V. Cañadas-Godoy: None. M.A. Restrepo-Córdoba: None. L. Daganzo-Sierro: None. E. Soengas-Gonda: None. R. Oancea-Ionescu: None.

P05.012.B OBSCN as a candidate gene for apical hypertrophic cardiomyopathy

Busra Unal 1,2, Nihat Bugra Agaoglu1,2, Ozlem Akgun Dogan3,2, Levent Doganay1,2,4, Mehmet Agirbasli5

1Department of Clinical Genetics, Umraniye Teaching and Research Hospital, University of Health Sciences, Istanbul, Turkey, 2GLAB (Genomic Laboratory), Health Directorate of Istanbul, İstanbul, Turkey, 3Department of Pediatric Genetics, Umraniye Teaching and Research Hospital, University of Health Sciences, Istanbul, Turkey, 4Department of Gastroenterology and Hepatology, Umraniye Teaching and Research Hospital, University of Health Sciences, İstanbul, Turkey, 5Department of Cardiology, Goztepe Teaching and Research Hospital, Istanbul Medeniyet University, Istanbul, Turkey.

Apical hypertrophic cardiomyopathy(AHCM) involving the distal portion of the left ventricle is a relatively rare form of hypertrophic cardiomyopathy(HCM) with genetic heterogeneity. Recent molecular genetics studies have shown that pathogenic mutations in genes coding sarcomere/Z-band components, including titin/connectin and its associate proteins, plays a role in the etiology. Among these genes, OBSCN stands out as a strong candidate and encodes the obscurin, a protein associated with titin/connectin. We present a 58 years old female patient with a likely pathogenic(LP) variant in the OBSCN in clinical exome analysis(CES) to elucidate the etiology of AHCM. Next-generation sequencing was performed on the NextSeq500 platform using the virtual panel for Apical hypertrophic cardiomyopathy(AHCM). We reported a heterozygous LP variant on OBSCN by attributing PVS1, PM2 criteria. (NM_001098623 GRCh37:hg19: Chr1:228560464 exon 94 c.21989_22002del p. Lys7330Argfs*8).This variation creates a frameshift and causes an amino acid substitution of lysine to arginine at codon 7330(PVS1). This variant is not present in population databases(PM2). There weren`t any other pathogenic or likely pathogenic variants associated with HCM. In their experimental study, Borisov et al. demonstrated that obscurin activity varies and is an important mediator during myocardial hypertrophy. Although this is the first case report in the literature of an LP variation in OBSCN in an apical HCM patient, new patients and functional studies are needed to show its association with the disease.

B. Unal: None. N.B. Agaoglu: None. O. Akgun Dogan: None. L. Doganay: None. M. Agirbasli: None.

P05.013.C A novel missense mutation of TNNI3K associated with cardiac conduction disease

Ilyas Ahmad 1,2, Stephanie Tennstedt1,2, Shafaq Ramzan3,1, Shahid Mahmood Baig3, Jeanette Erdmann1,2

1Institute for Cardiogenetics, University of Luebeck, Luebeck, Germany, 2DZHK (German Centre for Cardiovascular Research) partner site Hamburg / Kiel / Luebeck, Luebeck, Germany, 3National Institute for Biotechnology and Genetic Engineering (NIBGE), Faisalabad, Pakistan.

Cardiac conduction disease (CCD), which causes altered electrical impulse propagation in the heart, is a serious life-threatening condition with high morbidity and mortality rates. In majority of CCD, the synchronous activity of impulse-generating nodes and impulse-conduction is undermined for the normal heartbeat. CCD exhibit genetic and clinical heterogeneity with diverse underlying pathomechanisms. In this study, we investigated a consanguineous Pakistani family having four affected individuals with heart arrhythmia followed by ventricular septal defect in only one individual. We applied whole exome sequencing (WES) and co-segregation analysis on DNA samples from all available individuals which revealed a novel recessive biallelic mutation (NM_015978.2: c.1531T>C: p.S511P) in the highly conserved kinase domain in cardiac troponin I-interacting kinase (TNNI3K) gene. The missense variant was absent from ethnicity matched healthy controls and available public databases. The mutated residue is highly conserved, and its substitution is predicted to be pathogenic by in silico prediction methods. Furthermore, molecular dynamics simulation studies of the dimer suggests that p.S511P has an indirect effect on the orientation of the monomers facing each other, in the direction of a more energetically unfavorable orientation. Since the orientation of the monomers with respect to each other plays a crucial role in the function of the protein, therefore, we hypothesize that TNNI3K-S511P might negatively affect the kinase activity of the protein. In conclusion, this study reports a novel recessive mutation p.S511P in TNNI3K and expands the spectrum of TNNI3K protein in the pathogenicity of CCD.

Keywords: CCD, TNNI3K

I. Ahmad: None. S. Tennstedt: None. S. Ramzan: None. S.M. Baig: None. J. Erdmann: None.

P05.015.A Diagnostic yield of WES-based gene panels in patients with congenital structural heart defects - a multicenter study

Marrit M. Hitzert1, Rosalie L. Neijzen 2, Dennis Dooijes2, Yvonne J. Vos1, Rosa L. E. van Loon2, Wilhelmina S. Kerstjens-Frederikse1

1University Medical Center Groningen, Groningen, Netherlands, 2University Medical Center Utrecht, Utrecht, Netherlands.

Introduction: Congenital heart defects (CHD) affect 1% of live births. A monogenetic cause can be identified in 5% to 10% of patients. WES-based gene panels are widely used, while there is no consensus on which genes to include and which patients could benefit most. We evaluated the diagnostic yield of WES-based gene panels in CHD patients.

Patients: We retrospectively evaluated the results from WES-based gene panels in CHD patients in the Netherlands at the University Medical Center Groningen (UMCG) and the University Medical Center Utrecht (UMCU). Inclusion lasted from 2013 (UMCU) and 2017 (UMCG) up until April 2020. We collected clinical data including phenotype and family history of CHD.

Results: A total of 328 patients were included with most patients having a left-sided (n = 117; 35.7%) or conotruncal heart defect (n = 94; 28.7%). In 3/117 and 8/94 patients a (likely) pathogenic variant was demonstrated. Total diagnostic yield was comparable between the UMCG and the UMCU (6.2% and 6.0%, respectively) and was higher in syndromic than non-syndromic patients (12.5% vs. 5%; P = 0.038).

Conclusions: Our diagnostic yield of CHD gene panels as used in clinical practice (6%) is comparable to the previously reported yield in study settings (5% to 10%), and is highest in syndromic patients.

Cardiac phenotype Familial Y/N Syndromic Y/N Gene DNA variant P/LP
Left atrial isomerism, PAH N Y DNAH11 c.5778+1G>A P
    DNAI1 c.48+2dupT P
Right atrial isomerism, right-sided aortic arch, AVSD, (sub)valvular PS Y Y GDF1 c.681C>A, p.(Cys227*) P
    GDF1 c.681C>A, p.(Cys227*) P
Dextrocardia, right isomerism, univentricular heart, AVSD, TAPVR N Y PKD1L1 c.2027C>T, p.(Pro676Leu) LP
    PKD1L1 c.5728C>T, p.(Arg1910Trp) LP
TGA, VSD N N GDF1 c.681C>A, p.(Cys227*) P
Truncus arteriosus, VSD N N NKX2-6 c.455dupA, p.(Gln153Alafs*?) P
    NKX2-6 c.455dupA, p.(Gln153Alafs*?) P
    PLD1 c.2191A>T, p.(Arg731*) P
    CEP290 c.133_136delCAAG, p.(Gln45Lysfs*3) P
Truncus arteriosus, interruption of the aortic arch N ? GAT6 c.1417_1426del, p.(Lys473Glyfs*8) P
Tetralogy of Fallot N N FLT4 c.89C>T, p.(Pro30Leu) LP
    GDF1 c.681C>A, p.(Cys227*) P
PS, VSD Y Y EP300 c.3739T>C, p.(Cys1247Arg) LP
Truncus arteriosus N N CRELD1 c.959delA, p.(Gln320Argfs*25) LP
Tetralogy of Fallot N Y GLI3 c.642delC, p.(Met215*) LP
    CRELD1 c.1103T>A, p.(Leu368*) LP
Truncus arteriosus N N HAND1 c.410_411delinsA, p.(Arg137fs) P
PS, VSD N N JAG1 c.1278del, p.(Cys427fs) P
AVSD Y Y MYH11 c.4882A>C, p.(Lys1628Gln) LP
BAV, TAAD Y N GDF1 c.681C>A, p.(Cys227*) P
BAV N N FOXC2 c.1402dupG, p.(Glu468Glyfs*?) LP
HLHS, BAV Y Y PTPN11 c.1505C>T, p.(Ser502Leu) P
HLHS N Y PTPN11 c.179G>C, p.(Gly60Ala) LP
ASD Y Y ACTC1 c.904T>A, p.(Ser302Thr) LP
HLHS, incomplete AVSD, primum septum defect, narrower AoV, hypoplasia aortic arc with COA Y Y PTPN11 c.1529A>C, p.(Gln510Pro) P
HLHS with mitral valve atresia, hypoplastic aortic arch, PDA ASD N N NOTCH1 c.3177del, p.(Val1060fs) P

M.M. Hitzert: None. R.L. Neijzen: None. D. Dooijes: None. Y.J. Vos: None. R.L.E. van Loon: None. W.S. Kerstjens-Frederikse: None.

P05.017.C CHDbase: a genetic knowledge base for congenital heart disease

Weizhen Zhou1, Wenke Li1, Ruby W Wang2, Huayan Shen 1, Wen Chen1, Qingyi Zeng1, Hao Wang1, Meng Yuan1, Ziyi Zeng1, Jinhui Cui1, Fred Y Ye2, Zhou Zhou1

1Chinese Academy of Medical Sciences, Fuwai Hospital, Beijing, China, 2School of Information Management, Nanjing University, Nanjing, China.

Introduction: Congenital heart disease (CHD) is the most common birth defect, affecting nearly 1 per 100 newborns. CHD covers broad structural heart malformations and shows a complex genetic basis. Yet, the susceptibility genes with different strengths of evidence are scattered in literature without professional data integration and comprehensive analyses. The whole picture of CHD genetics and the genotype-phenotype correlations are unclear.

Method: A total of 1,150 publications were systemically reviewed. Metadata for each study including sample features, experimental design, and statistical results were collected and functional annotations of genes and mutations were performed. The patterns of CHD genes across various types were analyzed and a database was constructed.

Result: We integrated 1,139 genes, 1,022 copy number variations and structural variations, 2,641 single-nucleotide variations or small insertions/deletions and 36 linkage regions associated with CHD from 1,150 publications. We extracted a core network of 164 genes using k-core decomposition based on the protein-protein network and revealed the tissue and developmental stage expression patterns, as well as critical biological pathways underlying CHD. Additionally, we refined CHD subtypes using genotype-phenotype correlations and six subtypes were proposed to be more genetically homogeneous. A MySQL-based online database (http://chddb.fwgenetics.org/) was developed to share with the CHD research community.

Conclusion: This genetic database of CHD presented a clear big picture of CHD genetics, and the atlas of CHD genes and subtyping provided important implications for mechanism research and clinical diagnostics and treatment.This work was supported by the CAMS Initiative for Innovative Medicine (2016-I2M-1-016)

W. Zhou: None. W. Li: None. R. Wang: None. H. Shen: None. W. Chen: None. Q. Zeng: None. H. Wang: None. M. Yuan: None. Z. Zeng: None. J. Cui: None. F. Ye: None. Z. Zhou: None.

P05.018.D Mutation burden in patients with small unrepaired atrial septal defects

Anne Kathrine Møller Nielsen 1, Camilla Nyboe2, Anne Sif Lund Ovesen2, Sebastian Udholm2, Malthe Mølgård Larsen3, Vibeke Hjprtdal1, Lars Allan Larsen3

1Rigshopitalet, Copenhagen, Denmark, 2Aarhus University Hospital, Aarhus, Denmark, 3University of Copenhagen, Copenhagen, Denmark.

Introduction: In a recent nationwide cohort study, we have discovered that patients living with an atrial septal defect (ASD) have a shorter life expectancy, increased risk of atrial fibrillation, pneumonia, and psychiatric issues compared to the general population. The pathophysiological mechanisms are still unknown. The objective of this study is to investigate if this group of patients is burdened by mutations in genes associated with ASD.

Methods: We included 147 patients with an unrepaired ASD. We curated a list of ASD candidate genes, and patients were analyzed for genetic variants in these genes, using targeted next generation sequencing. We filtered for protein altering variants (PAVs) with minor allele frequency (MAF) <0.01 and predicted pathogenicity using the Combined Annotation Dependent Depletion (CADD) method. The number of rare and pathogenic variants in cases were compared with variants identified in 33,370 controls of European ancestry.

Results: We identified 384 rare (MAF < 0.01) PAVs in 59 genes. ASD patients were significantly enriched for very rare (MAF < 0.0001) PAVs compared to controls (P = 0.0001). The frequency of PAVs with CADD score ≥ 30 was significantly higher in ASD patients, compared to controls (P = 0.0042).

Conclusions: Patients with a small, unrepaired ASD were enriched for rare PAVs within 59 ASD candidate disease genes. Our results suggest that recurrence risk may be increased for this group of patients and warrants further investigations. Funding: This work was supported by Novo Nordic Foundation (Grant no. NNFSA170030576), Brd. Hartmanns Fond, Kong Christian den Tiendes Fond, Dagmar Marshalls Fond and Eva & Henry Frænkels Mindefond.

A.K.M. Nielsen: None. C. Nyboe: None. A.S.L. Ovesen: None. S. Udholm: None. M.M. Larsen: None. V. Hjprtdal: None. L.A. Larsen: None.

P05.019.A Role of xenobiotic biotransformation genes in genetic predisposition of congenital heart diseases

Anna Tsepokina, Svetlana Shmulevich, Anastasia Ponasenko, Andrey Shabaldin

Research Institute for Complex Issues of Cardiovascular Diseases, Kemerovo, Russian Federation.

Introduction: Congenital heart diseases are one of the most common multi-factorial fetal abnormalities caused by a complex of endo- and exogenous factors. A number of studies have shown a significant role of genes encoding enzymes involved in different phases of xenobiotic metabolism in the congenital heart diseases pathogenesis.

Material and Methods: In the presented research, 131 children with congenital heart diseases and 101 women having children with this pathology were included in the study group. In control group, 103 healthy children and their mothers were included. Single-nucleotide polymorphisms in the xenobiotic biotransformation genes CYP1A1 (rs1048943), CYP1A2 (rs762551), GSTP1 (rs6591256, rs1871042 and rs17593068) were detected by the real-time polymerase chain reaction. Gene-gene interactions were determined using the Multifactor Dimensionality Reduction method.

Results: The frequency distribution of alleles and genotypes in all studied groups were corresponded to the theoretically expected according to Hardy-Weinberg equilibrium. Comparative analysis of polymorphisms of CYP1A1 (rs1048943), CYP1A2 (rs762551), and GSTP1 (rs6591256, rs1871042 and rs1793068) genes in the study and control groups of women and their children did not show statistically significant differences between groups. We obtained no difference in the frequency of CYP1A1, CYP1A2 and GSTP1 between the study and control groups. At the same time, the genetic combinations GSTP1 (rs6591256)—GSTP1 (rs1871042) and GSTP1 (rs6591256)—GSTP1 (rs1871042)—CYP1A1 (rs1048943) in women; and GSTP1 (rs1793068)—GSTP1 (rs6591256)—GSTP1 (rs1871042)—CYP1A1 (rs1048943)—CYP1A2 (rs762551) in children contribute to the pathogenesis of congenital heart diseases. This study was supported by basic research project №0546-2019-0002.

A. Tsepokina: None. S. Shmulevich: None. A. Ponasenko: None. A. Shabaldin: None.

P05.020.B Prediction of coronary artery disease: Do risk factor genetic risk scores add value?

Julia Ramírez 1, Stefan van Duijvenboden2, William J. Young1, Andrew Tinker1, Pier D. Lambiase2, Michele Orini2, Patricia B. Munroe1

1William Harvey Research Institute, London, United Kingdom, 2University College London, London, United Kingdom.

Introduction: Coronary artery disease (CAD) is heritable and has a polygenic architecture. Current genetic risk scores (GRS) for CAD predict CAD independent from traditional risk factors, however these scores may not capture all genetically pre-determined risk. We hypothesised that a score including additional GRSs for multiple cardiovascular risk factors, explaining additional biology underlying CAD, may improve CAD prediction.

Methods: We used data from 52,254 participants in UK-Biobank without a previous diagnosis of cardiovascular disease. In a training subset (50%, N = 26,127), we identified the risk factors significantly associated with CAD using Multivariable Cox regression to build three scores, Sc1 including sex, age and traditional risk factors, Sc2 including Sc1 and a GRS for CAD, and Sc3 including Sc2, and 26 additional GRSs for traditional and electrocardiogram risk factors.

Results: In an independent test subset (50%, N = 26,127) for CAD, Sc2 had a higher area under the curve (AUC) than Sc1 (0.757 versus 0.735, P = 7.4 x 10-14). The hazard ratios (HRs) (95% confidence interval [CI]) for individuals in the top versus bottom 20% of the distribution were 17.2 (10.7 - 27.7) versus 14.9 (9.3 - 23.7). Adding the two GRSs that remained independently associated with CAD, GRSs for low-density lipoprotein and for triglycerides, did not significantly improve risk stratification (AUC of 0.759, P = 7.0 x 10-2, HR [CI] of 16.6 [10.4 - 26.4]).

Conclusion: Our results do not support additional benefit for CAD prediction by adding several independent GRSs in addition to a CAD GRS alone.

J. Ramírez: None. S. van Duijvenboden: None. W.J. Young: None. A. Tinker: None. P.D. Lambiase: None. M. Orini: None. P.B. Munroe: None.

P05.021.C Clinical impact of re-evaluating genes and variants implicated in dilated cardiomyopathy

Sophie L.V.M. Stroeks L. V. M. Stroeks1, Debby M. E. I. Hellebrekers1, Ingrid P. C. Krapels 1, Godelieve R. F. Claes1, Upsana Tayal2, Els K. Vanhoutte1, Artur van den Wijngaard1, Michiel T. H. M. Henkens1, James S. Ware2, Stephane R. B. Heymans1, Han G. Brunner1, Job A. J. Verdonschot1

1Maastricht University Medical Center, Maastricht, Netherlands, 2Imperial College London, London, United Kingdom.

Purpose: Accurate interpretation of variants detected in dilated cardiomyopathy (DCM) is crucial for patient care but has proven challenging. We applied a set of proposed refined ACMG/AMP criteria for DCM, re-classified all detected variants in robust genes, and associated these results to patients´ phenotype.

Methods: The study included 902 DCM probands from the Maastricht Cardiomyopathy Registry, who underwent genetic testing. Two gene-panel sizes (extended n = 48; and robust panel n = 14) and two standards of variant classification (standard versus the proposed refined ACMG/AMP criteria) were applied to compare genetic yield.

Results: A pathogenic variant was found in 17.8% of patients, and a variant of uncertain significance (VUS) was found in 32.8% of patients when using method 1 (extended panel (n = 48)+standard ACMG/AMP), compared to respectively 16.9% and 12.9% when using method 2 (robust panel (n = 14)+standard ACMG/AMP), and respectively 14% and 14.5% using method 3 (robust panel (n = 14)+refined ACMG/AMP). Patients with pathogenic variants had significantly lower event-free survival compared to genotype-negative DCM patients.

Conclusion: Stringent gene selection for DCM genetic testing reduced the number of VUSs whilst retaining ability to detect similar pathogenic variants. The number of genes on diagnostic panels should be limited to genes which have the highest signal to noise ratio.

S.L.V.M. Stroeks: None. D.M.E.I. Hellebrekers: None. I.P.C. Krapels: None. G.R.F. Claes: None. U. Tayal: None. E.K. Vanhoutte: None. A. van den Wijngaard: None. M.T.H.M. Henkens: None. J.S. Ware: None. S.R.B. Heymans: None. H.G. Brunner: None. J.A.J. Verdonschot: None.

P05.022.D Experience of an Italian reference laboratory for a rare disease: Hereditary Haemorragic Telangiectasia

Carla Olivieri 1, Fabio Pagella2,3, Sara Plumitallo1, Uroš Hladnik4, Elisabetta Buscarini5, Elina Matti3, Anna Sbalchiero1, Guido Manfredi5, Giuseppe Spinozzi3, Elisabetta De Sando6, Sara Ugolini3, Cesare Danesino1

1General Biology and Medical Genetics Unit, Department of Molecular Medicine, University of Pavia, Pavia, Italy, 2Department of Otorhinolaryngology, University of Pavia, Pavia, Italy, 3Department of Otorhinolaryngology, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy, 4Unità di Genetica Istituto per le malattie rare "Mauro Baschirotto" - B.I.R.D. Foundation o.n.l.u.s., Costozza di Longare (VI), Italy, 5UOC Gastroenterologia-Centro di riferimento HHT, ASST Ospedale Maggiore di Crema, Crema (CR), Italy, 6Clinica Pediatrica, Fondazione IRCCS Policlinico San Matteoi, Pavia, Italy.

Introduction: Hereditary Haemorragic Teleangiectasia (HHT) is an autosomal dominant disorder affecting 1:5000-8000 individuals worldwide. Mucocutaneous telangiectases and Arteriovenous malformations in internal organs (mostly lungs, liver and central nervous system) are the disease hallmarks. HHT is caused by pathogenetic variants in ENG, ACVRL1, SMAD4 and GDF2, belonging to the TGFβ/BMPs pathway. We report the experience of our research laboratory in the last five years (2015-2020), focusing on mutation analysis.

Materials and Methods: Patients’ samples were collected by HHT reference centres in Pavia and Crema (CR). Index cases’ samples were analysed by NGS sequencing panel of the four HHT causative genes and MLPA; the molecular investigation in patients’ relatives was performed by Sanger sequencing.

Results: We collected 334 patients’ samples; 99/334 were index cases. Results are summarized in the table below.

  Subjects ACVRL1 ENG SMAD4 Not Found In progress Unaffected
Index case 99 39 (39.4%) 26 (26.3%) 1 (1%) 27 (27.3%) 6 (6%) -
Patient’s relatives 234 86 60 - - 8 80
Total patients 333 125 86 1 28 13 80

Conclusions: Our data confirm that HHT is mostly underdiagnosed; however, the presence of a reference center enhances the quality of genetic and clinical results. Moreover, we also corroborate the previous observation that in our country ACVRL1 is the major HHT gene. Not found subjects can harbor variants in intronic or regulatory regions rather than in novel genes. However, we are collecting WES data to re-analyse these cases. Grant: CO: Italian Ministry of Education, University and Research to the DMM-University of Pavia “Dipartimenti di Eccellenza (2018-2022)”

C. Olivieri: None. F. Pagella: None. S. Plumitallo: None. U. Hladnik: None. E. Buscarini: None. E. Matti: None. A. Sbalchiero: None. G. Manfredi: None. G. Spinozzi: None. E. De Sando: None. S. Ugolini: None. C. Danesino: None.

P05.023.A Hereditary Haemorrhagic Telangiectasia: evidence of a common ancestor in 19 families from Northern Italy

Anna Sbalchiero 1, Yasmin Abu Hweij1, Tommaso Mazza2, Elisabetta Buscarini3, Fabio Pagella4,5, Elina Matti4, Guido Manfredi3, Giuseppe Spinozzi4, Sara Ugolini4, Carla Olivieri1

1General Biology and Medical Genetics Unit, Department of Molecular Medicine, University of Pavia, Pavia, Italy, 2Unit of Bioinformatics, Fondazione IRCCS Casa Sollievo della Sofferenza, San Giovanni Rotondo (FG), Italy, 3UOC Gastroenterologia-Centro di riferimento HHT, ASST Ospedale Maggiore di Crema, Crema (CR), Italy, 4Department of Otorhinolaryngology, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy, 5Department of Otorhinolaryngology, University of Pavia, Pavia, Italy.

Introduction: Hereditary Haemorrhagic Telangiectasia (HHT) is an autosomal dominant vascular disorder affecting 1:5000-8000 individuals worldwide. The genes associated with HHT (ENG, ACVRL1, SMAD4, GDF2) belong to the TGF-β signalling pathway. Evidence for a “Founder Effect” was demonstrated only few times in the HHT literature. We found 19 HHT unrelated families, coming from the region around Bergamo (Northern Italy) and sharing the same pathogenetic variant: the ACVRL1 in-frame deletion c.289_294del (p.H97-N98). Here we suggest the presence of a common ancestor in whom this variant arose and date its origin about 200 years ago.

Materials and Methods: We analysed 88 subjects from 19 families: 66 disease-variant carriers and 22 unaffected. We used eight microsatellite markers spanning 3.7Mb surrounding the ACVRL1 locus. After haplotype reconstruction, the pathogenetic variant’s age estimation was carried out with the DMLE+2.3 software package.

Results: We observed a common disease haplotype in 16/19 families. Three families showed evidence of recombination around the ACVRL1 locus. Subsequent analyses suggested that the mutation occurred about 8 (95% credible set: 6-11) generations ago, corresponding to about 203 (165-265) years ago.

Conclusions: We hypothesise for the first time a “founder effect” for a HHT pathogenetic variant in Italy. This information is also useful to notify the Bergamo Local Health Authority of a higher incidence of a rare, underdiagnosed disease. This will increase HHT patients’ awareness and offer them better clinical care and genetic diagnosis. Grant: CO: Italian Ministry of Education, University and Research to the DMM-University of Pavia “Dipartimenti di Eccellenza (2018-2022)”

A. Sbalchiero: None. Y. Abu Hweij: None. T. Mazza: None. E. Buscarini: None. F. Pagella: None. E. Matti: None. G. Manfredi: None. G. Spinozzi: None. S. Ugolini: None. C. Olivieri: None.

P05.024.B Exosomal microRNA biosignature related with hypertension-associated kidney disease

Ana Ortega 1, Javier Perez-Hernandez2, Olga Martinez-Arroyo1, Angela L. Riffo-Ramos3, Belen Sanchez-Garcia1, Daniel Perez-Gil4, Fernando Martinez5, Felipe Chaves1, Josep Redon1, Raquel Cortes1

1Biomedical Research Institute Clinic Hospital - INCLIVA, Valencia, Spain, 2INSERM, U1016, I, Cochin Institute, Paris, France, 3Universidad de La Frontera, Temuco, Chile, 4Genomics England, Dawson Hall, Charterhouse Square, London, United Kingdom, 5Medical Research Institute La Fe Hospital, Valencia, Spain.

Introduction: Urinary albumin excretion (UAE) is a marker of cardiovascular risk and renal damage in hypertension. MicroRNAs (miRNAs) packaged into exosomes function as paracrine effectors in cell communication and the kidney is not exempt. This study aimed to state an exosomal miRNA signature related to hypertension-associated kidney disease.

Material and Methods: Exosome samples from patients with hypertension with/without UAE were isolated and characterized. Three unique small RNA libraries from each subject were prepared (total plasma, urinary, and plasma-derived exosomes) for next-generation sequencing profiling. Differentially expressed miRNAs were over-represented in KEGG pathways, and selected miRNAs were validated by RT-qPCR in a confirmation cohort.

Results: A signature of 29 dysregulated circulating miRNAs was identified in UAE hypertensive subjects, regulating 21 pathways. Moreover, changes in the levels of 4 exosomes-miRNAs were validated in a confirmation cohort and found associated with albuminuria. In particular, miR-26a, major regulator of TGF-β signaling, was found downregulated in both type of exosomes when compared with healthy controls and to hypertension normoalbuminurics (P < 0.01). Similarly, decreased miR-26a levels were found in podocyte-derived exosomes after TGF-β stress.

Conclusions: Our results revealed an exosomes miRNA signature associated to albuminuria in hypertension. In particular, exosomes miR-26a seemed to play a key role in the regulation of TGF-β, a relevant effector in podocyte damage. These findings support the use of exosomes miRNAs as biomarkers of cardiovascular risk progression and therapeutic tools in early kidney damage. Funding from Carlos III Health Institute (PI12/02615, PI19/01796 PI18/01405, CD18/00166 and with ERDF.

A. Ortega: None. J. Perez-Hernandez: None. O. Martinez-Arroyo: None. A.L. Riffo-Ramos: None. B. Sanchez-Garcia: None. D. Perez-Gil: None. F. Martinez: None. F. Chaves: None. J. Redon: None. R. Cortes: None.

P05.025.C Identification of a novel TPM1 variant causing hypertrophic cardiomyopathy in an Indian family

Prabodh Kumar 1, Ganesh Paramasivam2, Tom Devasia2, Mukund Prabhu2, Maneesh K. Rai3, Prakashini K4, Sandeep Mallya5, Dinesh Reghunathan1, Krishnananda Nayak6, Rajasekhar Moka1

1Department of Cell and Molecular Biology, Manipal School of Life Sciences, Manipal Academy of Higher Education, Manipal, India, 2Department of Cardiology, Kasturba Medical College, Manipal Academy of Higher Education, Manipal, India, 3Department of Cardiology, Kasturba Medical College, Manipal Academy of Higher Education, Mangalore, India, 4Department of Radiodiagnosis and Imaging, Kasturba Medical College, Manipal Academy of Higher Education, Manipal, India, 5Department of Bioinformatics, School of Life Sciences, Manipal Academy of Higher Education, Manipal, India, 6Department of Cardiovascular Technology, Manipal College of Health Profession, Manipal Academy of Higher Education, Manipal, India.

Hypertrophic cardiomyopathy (HCM) is an inherited cardiovascular disorder characterized by unexplained left ventricular hypertrophy. It affects about 20 million people globally, and its prevalence is estimated to be more than 1 in 500 in the general population. We report a case of an Indian family with clinically defined HCM. Genetic analysis was performed using a targeted amplicon panel containing 28 genes on a semiconductor next-generation sequencer. An autosomal dominant; heterozygous mutation in the TPM1 gene (α-tropomyosin), NM_001018005.2:c.203A>G, p.Gln68Arg, was identified and validated by Sanger sequencing in all affected members of the family but was absent in the unaffected family member. This variant was not reported in the literature in HCM cases nor described in the general population databases. We did not detect any other known pathogenic or likely pathogenic variants in other genes. α-tropomyosin is an α-helical coiled-coil dimer that spans the actin filament’s length as a co-polymer. It plays a critical role in sarcomeric contractile regulation by stabilizing the actin filaments and interacting with other actin-binding proteins. HCM causing TPM1 mutations act in a dominant-negative, poison polypeptide mechanism, altering this delicate balance and increasing the myofilament’s calcium sensitivity causing hypercontraction and hypertrophy. Our finding adds to the mutational spectrum of TPM1, which is an uncommon cause of HCM. Identification of novel variants or genes causing HCM can unravel the clinical and genetic heterogeneity in HCM. This work was supported by DST-SERB grant (EEQ/2019/000477), CSIR-SRF grant (09/1165(0010)/2019-EMR-I), TIFAC and DST-FIST.

P. Kumar: None. G. Paramasivam: None. T. Devasia: None. M. Prabhu: None. M.K. Rai: None. P. K: None. S. Mallya: None. D. Reghunathan: None. K. Nayak: None. R. Moka: None.

P05.026.D Comprehensive genetic study in a young patient with cardiac interventricular septal hypertrophy

Tsenka Boneva 1, Kalina Belemezova2, Ivan Gruev3, Ivanka Dimova4

1UMHAT Alexandrovska, Cardiology clinic, Sofia, Bulgaria, 2Genetic Laboratory, SAGBAL “ Dr Shterev”, Sofia, Bulgaria, 3Multidisciplinary Transport Hospital "Tsar Boris III" – Sofia, Sofia, Bulgaria, 4Department of Medical genetics, Medical University Sofia, Sofia, Bulgaria.

An isolated hypertrophy of the basal segment of the interventricular septum protruding into the outflow tract of the left ventricle may be difficult to distinguish from genetic hypertrophic cardiomyopathy (HCM). Because of potential life-threatening complications associated with a diagnosis of HCM, careful investigations of patients are needed, including genetic analysis. We report here a 16-years old patient, diagnosed with septal hypertrophy (>11 mm thickness) and slight tricuspid valve regurgitation. There was family history for heart-related mortality from both paternal and maternal side. Next generation sequencing was performed to analyze point mutations and deletions/ duplications in 125 genes associated with Arrhythmia, Cardiomyopathies and Sudden Cardiac Death. We detected variants of unknown significance (VOUS) with the population frequency of less than 0.1% in 4 genes - listed at the Table below.

Gene Genetic variant OMIM disease Inheritance
BAG3 c.1673C>T (p.Ala558Val) Dominant dilated cardiomyopathy, Myofibrillary myopathy 6, Charcot-Marie-Tooth type 2 Paternal
DSP c.5062G>A (p.Ala1688Thr) Autosomal dominant arrhythmogenic right ventricular cardiomyopathy, dilated cardiomyopathy with sparse hair, keratoderma and agenesis of the teeth, autosomal recessive DC Maternal
JUP c.1379G>A (p.Arg460His) Autosomal dominant arrhythmogenic right ventricular cardiomyopathy and autosomal recessive Naxos disease Maternal
SCN10A c.724T>A (p.Ser242Thr) Autosomal-dominant neuropathy and Brugada syndrome Paternal

All VOUS were inherited from clinically healthy parents - 2 from the mother and 2 from the father. This points to a probable polygenic etiology of the disease, in which the severity of the disease cannot be accurately determined. Regular assessment of cardiac function is recommended, along with decreasing in high intensity sport activity.

T. Boneva: None. K. Belemezova: None. I. Gruev: None. I. Dimova: None.

P05.027.A Molecular genetic testing for hypertrophic and dilated cardiomyopathy in inherited cardiovascular condition genetics service: lessons from a Thai cohort

Objoon Trachoo, Teerapat Yingchoncharoen, Tawai Ngernsritrakul, Nareenart Iemwimangsa, Bhakbhoom Panthan, Sommon Klumsathien, Sasima Srisukh, Anucha Mukdadilok, Sitthakom Phusanti, Angkana Charoenyingwattana, Takol Chareonsirisutthigul, Wasun Chantratita, Tarinee Tangcharoen

Faculty of Medicine Ramathibodi Hospital, Bangkok, Thailand.

Introduction: Hypertrophic cardiomyopathy (HCM) and idiopathic dilated cardiomyopathy (DCM) are the most common referral in Inherited Cardiovascular Condition (ICC) Genetics Service. Several issues have to be discussed with patients and families during genetic consultation session, including the option for genetic testing and cardiovascular surveillance in family members.

Materials and Methods: Next-Generation Sequencing data of all patients affected by HCM and idiopathic DCM in ICC clinic were analysed using target gene panel to classify variant pathogenicity. All subjects were asked to contact their asymptomatic first-degree relatives for genetic counselling about their risk and to initiate cardiovascular surveillance.

Results: Sixty subjects (30-HCM and 30-DCM) were enrolled during 1 January 2014 – 31 December 2020. Molecular detection frequency was 53.33% (33.33% pathogenic/likely pathogenic, 20% VUS) for HCM and 20% (10% pathogenic/likely pathogenic, 10% VUS) for DCM. The most prevalent gene attributing to HCM was MYBPC3 (30%). The others were identified in one of these genes-ACTN2, MYL2, MYH7, TNNI3, TPM1, TTR and VCL (3.33% each). Amongst DCM, the variants were detected in TTN truncating variant (6.67%), MYH7 (6.67%), MYBPC3 (3.33%) and SCN5A (3.33%). Following clinical surveillance in family members, the detection frequency of new pre-symptomatic cases was 9.09% for HCM and 7.14% for DCM.

Conclusions: In our cohort, MYBPC3 was the most prevalent gene related to HCM. Amongst idiopathic DCM, TTN and MYH7 were the most common. Additionally, our genetics service was able to detect new cases approximately 1/10 of asymptomatic family members. Grants: Thailand Research Fund and Thailand Centre of Excellence of Life Science.

O. Trachoo: A. Employment (full or part-time); Modest; Samitivej Srinakarin Hospital, Phyathai 2 Hospital, Bumrungrad Hospital, Jetanin IVF, Panthupark Genetics Clinic. A. Employment (full or part-time); Significant; Samitivej Sukhumvit Hospital. C. Other Research Support (supplies, equipment, receipt of drugs or other in-kind support); Significant; 3billion, Inc. D. Speakers Bureau/Honoraria (speakers bureau, symposia, and expert witness); Modest; PTT Public Company Limited. E. Ownership Interest (stock, stock options, patent or other intellectual property); Significant; Genome Star, Co., Ltd., Leader Medical Genetics and Genomics, Co., Ltd., Sofiva Genomics Bangkok, Co., Ltd.. F. Consultant/Advisory Board; Modest; Biomolecular Laboratory, Co., Ltd., Vejthani Hospital. F. Consultant/Advisory Board; Significant; Thonburi Bumrungmuang Hospital. T. Yingchoncharoen: A. Employment (full or part-time); Modest; Bumrungrad Hospital. T. Ngernsritrakul: None. N. Iemwimangsa: None. B. Panthan: A. Employment (full or part-time); Modest; Genome Star, Co., Ltd.. S. Klumsathien: None. S. Srisukh: None. A. Mukdadilok: None. S. Phusanti: None. A. Charoenyingwattana: E. Ownership Interest (stock, stock options, patent or other intellectual property); Modest; Leader Medical Genetics and Genomics, Co., Ltd. T. Chareonsirisutthigul: A. Employment (full or part-time); Modest; Panthupark Genetics Clinic. W. Chantratita: None. T. Tangcharoen: None.

P05.028.B If double is the trouble, the triple is undoable: a fatal association of Hypertrophic Cardiomyopathy (MYH7 pArg719Trp), Heterozygous Familial Hypercholesterolemia (LDLR pGlu343Lys) and SARS CoV-2 infection

Nicola Marziliano 1,2, Alessandro Medoro2, Roberto Ottaviano1, Donatella Mignogna2, Mariano Intrieri2, Giuseppe Giuliani1

1ASST-Rhodense, Rho, Italy, 2University of Molise, Department of Medicine and Health Sciences, Campobasso, Italy.

Introduction: Hypertrophic Cardiomyopathy (HCM; MIM #192600) and Heterozygous Familial Hypercholesterolemia (HeFH; MIM #144010) are the most common genetic cardiovascular disorders. Both HCM and HeFH can lead to severe heart failure and sudden cardiac death. In this report, we describe a case of a young man who suddenly died after a fatal arrythmia and additionally resulted positive for SARS-CoV-2 virus with high titer in myocardium.

Methods and Results: The proband is a young man (32-year-old) who suddenly died during physical exercise. Autopsy findings showed increased wall thickness of interventricular septum (IVS; 18 mm) and left posterior wall (LPW; 20 mm) with patchy myocardial disarray. Non-obstructive diffuse coronary artery disease (CAD) was also observed. Furthermore, the presence of pulmonary thromboembolism with lymphocytic myocarditis prompted the search of cardiotropic viruses within the myocardium. Real-Time PCR (RT-PCR) tested positive for a high concentration of SARS-CoV-2. Molecular autopsy identified two genetic variants classified as pathogenic in the MYH7 (p.Arg719Trp) and LDLR (p.Glu343Lys) genes. Co-segregation analysis via Sanger sequencing within the family (N = 22) showed that LDLR mutation was maternally inherited, while MYH7 genetic lesion was de novo.

Conclusion: Electrical remodeling associated with a genetic substrate and a concomitant presence of diffuse CAD and SARS-CoV-2-induced myocarditis might trigger a fatal arrhythmia. This is of paramount importance for the first- or second-degree relatives in which the identification of the pathogenic substrate, that renders them vulnerable to an increased risk for life-threatening cardiac events, including sudden death, might prompt for clinical and tailored treatments.

N. Marziliano: None. A. Medoro: None. R. Ottaviano: None. D. Mignogna: None. M. Intrieri: None. G. Giuliani: None.

P05.029.C ‘Sequencing of cardiomyopathy genes in patients with hypertrophic cardiomyopathy reveals enrichment for rare variants in the genes for arrhythmogenic right ventricular cardiomyopathy’

Ramil Rinatovich Salakhov 1, Maria V. Golubenko1, Alexey A. Zarubin1, Elena N. Pavlyukova2, Alexander F. Kanev2, Oleg S. Glotov3, Diana A. Alaverdian3, Viktoriia V. Tsay3, Nail R. Valiakhmetov1, Maria S. Nazarenko1

1Research Institute of Medical Genetics, Tomsk, Russian Federation, 2Cardiology Research Institute, Tomsk, Russian Federation, 3City Hospital No. 40, Saint Petersburg, Russian Federation.

Introduction: Hypertrophic cardiomyopathy (HCM) is caused presumably by mutations in the cardiac sarcomere genes. However, mutations in other genes can be causal, and some modifying loci are suggested. In some genes, mutations can lead to different phenotypes, and more than one mutation can be identified in the patient.

Materials and methods: We studied 46 cardiomyopathy genes with NGS panel “TruSight Cardiomyopathy” (Illumina) in 12 patients with HCM. The effects of the identified variants were assessed according the ACMG guideline.

Results: 6 pathogenic / probably pathogenic variations in the cardiac sarcomere genes (MYH7, MYBPC3, TNNT2, ACTNA, MYOZ2) were found, including 3 novel variants. One patient had pathogenic variant in the gene for Danon disease (LAMP2). In addition, one patient had premature termination variant in the PKP2, and the patient with the TNNT2 mutation had additional frameshift variant in the DSC2. Mutations in these genes cause arrhythmogenic right ventricular cardiomyopathy (ARVС), while our patients had clear HCM phenotype. Moreover, we identified several rare (<1% in GnomAD) nonsynonymous variants which were estimated as benign, likely benign or of uncertain significance. In particular, more than one patient had these rare variants in the LDB3, DSP, TMEM43 genes which are known as genes for ARVD and/or dilated cardiomyopathy.

Conclusion: Accumulation of rare variants in the ARVC genes has been found in patients with HCM that requires further investigation. These results highlight the idea of genetic continuum for different cardiomyopathy phenotypes.

This study was supported by Grant of the President of Russian Federation МК-1093.2020.7.

R.R. Salakhov: None. M.V. Golubenko: None. A.A. Zarubin: None. E.N. Pavlyukova: None. A.F. Kanev: None. O.S. Glotov: None. D.A. Alaverdian: None. V.V. Tsay: None. N.R. Valiakhmetov: None. M.S. Nazarenko: None.

P05.030.D MYH7 p.(Arg1712Gln) is a founder pathogenic variant in an international large cohort of hypertrophic cardiomyopathy patients

Luisa Marsili 1,2,3, Francesco Russo2, Flavie Ader4,5,6, Marie-Line Bichon7,8, Laurence Faivre9, Arjan C. Houweling2, Bertrand Isidor7,8, Ronald H. Lekanne Deprez2, Benoit Mazel9, Sandra Mercier7,8, Gilles Millat10,11,12, Jan G. Post13, Pascale Richard4,5,14, Irma van de Beek2, Alexa M. C. Vermeer2, Ludolf G. Boven15, Jan D. H. Jongbloed15, Peter van Tintelen2,13

1Univ. Lille, Lille, France, 2Department of Clinical Genetics, Amsterdam UMC, University of Amsterdam, Amsterdam, Netherlands, 3CHU Lille, Clinique de Génétique, Lille, France, 4HU Pitié Salpêtrière-Charles-Foix, DMU BioGem, Service de Biochimie Métabolique, UF de cardiogénétique et myogénétique moléculaire et cellullaire, Paris, France, 5Sorbonne Université, UMRS1166 Equipe 1, Paris, France, 6UFR Pharmacie, Faculté de Santé, Université de Paris, Paris, France, 7CHU de Nantes, Service de Génétique Médicale, Nantes, France, 8L’institut du thorax, INSERM, CNRS, UNIV Nantes, Nantes, France, 9Centre de génétique et FHU TRANSLAD, CHU Dijon, Dijon, France, 10Laboratoire de Cardiogénétique Moléculaire, Centre de Biologie et Pathologie Est, Hospices Civils de Lyon, Lyon, France, 11Institut NeuroMyoGène, CNRS UMR 5310, INSERM U1217, Université Claude Bernard Lyon 1, Lyon, France, 12Université de Lyon, Lyon, France, 13Department of Genetics, University of Utrecht, University Medical Center, Utrecht, Netherlands, 14APHP, Centre de référence pour les maladies cardiaques héréditaires, Hôpitaux Universitaires Pitié-Salpêtrière, Paris, France, 15University of Groningen, University Medical Center Groningen, Department of Genetics, Groningen, Netherlands.

Introduction: The MYH7 c.5135G>A p.(Arg1712Gln) variant has been identified in several hypertrophic cardiomyopathy (HCM) patients worldwide and it is classified as likely pathogenic on ClinVar. Using data from a large international cohort, we delineate its associated phenotype, gain more evidence for its pathogenicity, and evaluate its founder effect.

Materials and Methods: We retrospectively collected clinical and genetic data of 22 probands and 74 family members. To determine the founder status, haplotypes were reconstructed for 42 patients.

Results: Fifty-three individuals carried the MYH7 p.(Arg1712Gln) variant, 38 (72%) were diagnosed with HCM. The mean age of HCM diagnosis was 48.8 years (SD 18.1; range 8-74). The clinical presentation ranged from asymptomatic left ventricular hypertrophy to arrhythmias (atrial fibrillation as well as malignant ventricular arrhythmias). Aborted sudden cardiac death (SCD) leading to HCM diagnosis occurred in one proband at the age of 68, and family history of SCD was reported by five (39%) probands. No heart failure death nor heart transplant were reported. Women had a generally later-onset disease with 14% of female carriers diagnosed with HCM at age 50, compared with 54% of male carriers, and penetrance reaching 95% and 92% at age 70 in men and women, respectively. The disease was fully penetrant at age 75 in both sexes. Haplotype analysis showed a founder origin in the majority of patients.

Conclusions: Our data showed that MYH7 p.(Arg1712Gln) is a pathogenic founder variant in HCM and that cardiac screening should be pursued after the seventh decade in healthy carriers, especially women.

L. Marsili: None. F. Russo: None. F. Ader: None. M. Bichon: None. L. Faivre: None. A.C. Houweling: None. B. Isidor: None. R.H. Lekanne Deprez: None. B. Mazel: None. S. Mercier: None. G. Millat: None. J.G. Post: None. P. Richard: None. I. van de Beek: None. A.M.C. Vermeer: None. L.G. Boven: None. J.D.H. Jongbloed: None. P. van Tintelen: None.

P05.031.A A new perspective in the study of genetic basis of hypertrophic cardiomyopathy

Elena V. Filatova 1, Natalia S. Krylova2, Ivan Vlasov1, Maria Maslova2, Natalia Poteshkina2, Petr A. Slominsky1, Maria Shadrina1

1Institute of Molecular Genetics of National Research Centre «Kurchatov Institute», Moscow, Russian Federation, 2Pirogov Russian National Research Medical University, Moscow, Russian Federation.

Introduction: It is now generally accepted that the exact genetic cause of hypertrophic cardiomyopathy (HCM) is unknown in at least 25% of hereditary cases. To date, a causal relationship with the development of HCM has been established for 10 genes; for another 20 genes, there are data on individual clinical cases indicating such a relationship. However, the full range of pathogenic variants, leading to the development of HCM, has not yet been described. In this regard, the aim of our study was to search for new genes, pathogenic variants in which may be potentially involved in the pathogenesis of HCM.

Materials and Methods: The study included 98 non-related patients with HCM. NGS of exons of 4800 genes associated with the development of various diseases was carried out, and bioinformatic analysis was performed to predict the potential pathogenicity of variants.

Results: The analysis identified 73 potentially pathogenic variants in 43 genes, for which a connection with the development of HCM was not previously shown, but which are involved in the development of other pathologies of the cardiovascular system. Most of the genes identified are involved in the functioning of the heart in general and the sarcomere in particular.

Conclusion: Thus, we were able to identify new variants and genes that may lead to the development of HCM or may be involved in the pathogenesis of the disease. This work was supported by the Russian Foundation for Basic Research (grants no. 19-015-00343) and the Russian Science Foundation (grants no. 21-75-20120).

E.V. Filatova: A. Employment (full or part-time); Modest; Institute of Molecular Genetics of National Research Centre «Kurchatov Institute». B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Significant; Russian Foundation for Basic Research, Russian Science Foundation. N.S. Krylova: None. I. Vlasov: A. Employment (full or part-time); Modest; Institute of Molecular Genetics of National Research Centre «Kurchatov Institute». M. Maslova: None. N. Poteshkina: None. P.A. Slominsky: A. Employment (full or part-time); Significant; Institute of Molecular Genetics of National Research Centre «Kurchatov Institute». B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Significant; Russian Science Foundation. M. Shadrina: A. Employment (full or part-time); Modest; Institute of Molecular Genetics of National Research Centre «Kurchatov Institute».

P05.032.B The expression level of cytokine genes in the cases of native heart valves in infectious endocarditis

Maxim Sinitsky, Anna Tsepokina, Maria Khutornaya, Anastasia Ponasenko