Volume 32 | Supplement 1

Glasgow, Scotland, United Kingdom

June 10-13, 2023

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 bold in the contributor lists.

e-Posters EP01 Reproductive Genetics

EP01.001 Aspirin alleviates hypoxia-induced inhibitory effect of fibronectin on trophoblast invasion in preeclampsia

Meitsz Su 1

1National Cheng Kung University Hospital, Tainan, Taiwan

Background: Preeclampsia is a severe gestational hypertensive disorder that poses a major threat to maternal and fetal health. The pathoetiology is to have defects of trophoblast invasion and uterine spiral artery remodeling, which leads to a hypoxic placenta. Aspirin is an effective agent for preeclampsia prevention but the drug acting mechanism needs further investigation. Elevated Fibronectin (FN) expression in maternal circulation and placenta has been shown to be associated with preeclampsia, however, the role of FN in human pregnancy and its expression under hypoxia is unclear.

Methods and Results: In this study, FN was upregulated in the trophoblasts with decreasing cell invasiveness under hypoxia. By recombinant FN treatment, FN was shown to inhibit trophoblast invasiveness under hypoxia. Aspirin suppressed FN expression of trophoblasts and the FN-mediated effect on cell motility under hypoxia. FN activated signaling pathway of ERK, Akt and JNK, while aspirin induced Akt, JNK and p38 signaling in trophoblasts. By treating inhibitors of these signal molecules, aspirin was demonstrated to reverse FN-mediated cell motility through Akt and JNK signaling.

Conclusion: FN was upregulated but inhibited trophoblast invasion under hypoxia. Aspirin may exert its prevention effect from preeclampsia through suppressing FN expression and alleviating FN-mediated inhibitory effect on trophoblast invasion in early pregnancy.

Grants: National Science and Technology Council, Taiwan

Conflict of Interest: None declared.

EP01.003 Assessment of the clinical value of exome sequencing in infertile men with cryptorchidism

Anna-Grete Juchnewitsch 1, Kristjan Pomm2, Kristiina Lillepea1, Avirup Dutta1, Anu Valkna1, Galina Belova1, Viljo Kübarsepp3, Laura Kasak1, Margus Punab1;2;3, Maris Laan1

1University of Tartu, Institute of Biomedicine and Translational Medicine, Tartu, Estonia; 2Tartu University Hospital, Andrology Clinic, Tartu, Estonia; 3University of Tartu, Institute of Clinical Medicine, Tartu, Estonia

Background/Objectives: Cryptorchidism is a congenital malformation with at least one testicle missing from the scrotum. It occurs in 1-2% of newborn boys and is more prevalent in premature infants1. Variants in >20 genes have been described to be the causative factors of cryptorchidism, e.g. INSL32. If left untreated, undescended testicles can cause spermatogenic failure (SPGF) and/or testicular cancer later in life. In Estonia, about 8% of male infertility cases are caused by cryptorchidism3. We have hypothesized that a fraction of unexplained patients presenting congenital cryptorchidism and SPGF (<39 × 106 sperm/ejaculate) may be explained by unknown monogenic causes.

Methods/Results: ESTonian ANDrology (ESTAND) cohort recruited at the Andrology Clinic, Tartu University Hospital (AC-TUH) includes patients presenting idiopathic uni/bilateral cryptorchidism. To analyze monogenic causes of cryptorchidism, 151 patients were selected for whole-exome sequencing. All subjects have given their informed consent to perform genetic studies. In silico gene panel analysis of hypothesis-based candidate loci has been compiled and data analysis in progression. Pathogenicity will be evaluated based on the ACMG guidelines and patient interviews are performed to gather additional clinical data and identify potential pleiotropic effects.

Conclusions: The current study will contribute to clarify the genetic aetiology of monogenic cryptorchidism. As this condition is a frequent problem among men with reproductive failure, the outcome will have an impact on patient management and counselling.


1Kübarsepp et al., Andrology 10,303–309 (2022).

2Elamo et al., Best Pract Res Clin Endocrinol Metab 36,101619 (2022).

3Punab et al., Hum Reprod 32,18–31 (2017).

Grants: Estonian Research Council: PRG1021.

Conflict of Interest: None declared.

EP01.004 Preimplantation genetic testing in a woman carrier of double reciprocal translocation t(7;11) and t(8;13)

Dimitar Bakalov 1, Kalina Belemezova1;2, Radostina Raynova2;3, Slavena Davidova2, Mariela Hristova-Savova2, Tanya Milachich2, Atanas Shterev2, Ivanka Dimova1;2

1Medical University of Sofia, Sofia, Bulgaria; 2Medical complex Dr. Shterev, Sofia, Bulgaria; 3SBALAG Maichin Dom, Sofia, Bulgaria

Background: Cytogenetic analysis is still an invaluable instrument when diagnosing balanced chromosomal abnormalities, as those cannot be detected by microarray methods. Balanced reciprocal translocations are one of the most common structural rearrangements with a frequency of 0.16% to 0.20% (1:615-1:500), but at the same time, two or three independent two-way reciprocal or Robertsonian translocations are extremely rare to co-exist in the same carrier. Those are classified as complex chromosome rearrangements (CCRs). Those structural rearrangements are increasing the risk of producing unbalanced gametes even further than a single translocation and can be associated with infertility, pregnancy loss, or offspring abnormality.

Materials and methods: We present here a woman with repeated reproductive failures, performing cytogenetic analysis as a part of a routine examination. Preimplantation genetic testing for aneuploidy (PGT-A) was done further by next-generation sequencing.

Results: We discovered two separate reciprocal translocations in all cells analyzed in the patient – karyotype 46, XX,t(7;11)(p21;q23),t(8;13)(q24;p12). After assisted reproductive technique 3 embryos were available for trophectoderm biopsy at day 5. Two of them were aneuploid (45, XX,-7 and 48, XX, +7, +13) and one embryo was euploid.

Conclusion: Double reciprocal translocations are very rare and include at least four breakpoints in different chromosomes. Chromosomal segregation is extremely complex, as both translocations produce 14 gametes separately, but the combinations could be 14 × 14 and only 4 gametes would produce a normal embryo (chance 1 to 51). PGT-A is the only way to choose the euploid embryo and our patient had the luck to have such an embryo for transfer.

Conflict of Interest: None declared.

EP01.005 Developing a candidate gene pipeline for monogenic causes of spermatogenic failure

Kristiina Lillepea 1, Anna-Grete Juchnewitsch1, Avirup Dutta1, Laura Kasak1, Anu Valkna1, Kristjan Pomm2, Margus Punab1;2;3, Maris Laan1

1University of Tartu, Institute of Biomedicine and Translational Medicine, Tartu, Estonia; 2Tartu University Hospital, Andrology Clinic, Tartu, Estonia; 3University of Tartu, Institute of Clinical Medicine, Tartu, Estonia

Background/Objectives: Worldwide, ~15% of couples face infertility and half of these cases are due to a male factor. In the current clinical pipeline, ~60% of cases with spermatogenic failure (SPGF) remain idiopathic1. Routine genetic testing is limited to chromosomal, CFTR, and AZF microdeletion analyses. Recently, exome sequencing (ES) has led to the discovery of many novel monogenic causes of male infertility phenotypes, pointing to substantial genetic etiology2. The ESTonian ANDrology (ESTAND) cohort recruited at the Andrology Clinic, Tartu University Hospital will be used to address the genetic etiology of male reproductive failure.

Methods/Results: 366 ESTAND participants with idiopathic SPGF were prioritized for ES to analyze potential monogenic causes of their condition. The study includes 184 non-obstructive azoospermia (no sperm in the ejaculate), 70 cryptozoospermia (<1 × 106 sperm/ejaculate), and 112 oligozoospermia (1–39 × 106) cases. A list of 159 candidate genes for SPGF was compiled based on the literature. Data analysis pipeline for variant prioritization was developed using population frequencies, known functional/medical consequences, and in silico predictions of candidate variants. The final pathogenicity assessment is based on the ACMG guidelines incorporated with extended phenotyping data and family health history to be gathered at follow-up interviews.

Conclusion: The developed pipeline will be used to assess potential monogenic causes of SPGF and to evaluate the added value of ES in improving the diagnostics and management of the reproductive and overall health of SPGF patients.


1Punab et al., (2017) Hum Reprod 32:18-31

2Laan et al., (2021) Br Med Bull 16;140:5-22

Grants: Estonian Research Council PRG1021.

Conflict of Interest: None declared.

EP01.008 Transfer of mosaic aneuploidy Embryo in a couple with balanced translocation

María Luz Bellido 1, Maria del Mar Del Aguila Garcia1, Antonio Miguel Poyatos-Andújar1

1Hospital Universitario Virgen de las Nieves, Granada, Spain

Background: The latest advances in genetics has allowed us to select and improve reproduction. These techniques tends to increase the diagnosis of embryos as mosaics and to overestimate or underestimate their true ability to develop a healthy baby.

Case: A woman with chromosomal formula: 46,XX,t(3;17)(p21.3;p13) who opted for PGS.

Methods: Biopsy of embryos at blastocyst stage and extraction of trophectoderm.

Amplification by Sureplex Amplification System, analysis results with BlueFuse Multi software (Illumina).

Karyotyping of amniocytes by conventional techniques.

Results: The table shows the results of the study embryos:


Chromosome endowment



Genomic amplification




Transfer recommendation















Not amplify

No diagnosis





-4q28.2q35.2 mos (25%)

Possible mosaic/balancing

After counseling




-2p25.3p11.2, -15





Complex aneuploidy



Karyotype amniocites: female, 46,XX,t(3;17)(p21.3;p13).

Delivery of a healthy baby at 39 weeks gestation.

Conclusion: the primary purpose of PGT-A is to detect aneuploidy, not the presence of mosaicism. Furthermore, a mosaic PGT-A result does not conclude the presence of mosaicism in the ICM.

Transfer of Mosaic embrios should be performed after genetic counseling of the couple and in the absence of normal balanced embryos. A prenatal study of amniotic fluid is indicated to determine possible chromosomal abnormalities. In this analysis we can also determine the presence of the balanced translocation by karyotyping. It is confirmed that the transfer of embryos with low mosaic aneuploidy can give rise to healthy offspring.

Conflict of Interest: None declared.

EP01.009 Genotyping for killer cell immunoglobulin-like receptors genes in women with recurrent implantation failure

Mariela Hristova-Savova 1, Petya Andreeva1, Ivelina Oprova1, Slavena Davidova1, Kalina Belemezova1, Atanas Shterev1, Ivanka Dimova2

1D-r Shterev Hospital, Sofia, Bulgaria; 2Medical University Sofia, Sofia, Bulgaria

Background: Killer Immunoglobulin-like Receptors (KIRs) family consists of 16 genes, encoding either inhibitory or activating receptors connected to the cytotoxic activity and cytokine secretion of natural killer cells. The great complexity of KIR genetic polymorphism is simplified into two haplotypes - A and B, which fundamentally differ in the presence of activating KIRs. Since activating KIRs are encoded only in haplotype B, an individual with two A haplotype (genotype AA) has only receptors that will transmit a strong inhibitory signal of uterine NK cells. The combination of the maternal KIR AA genotype with the paternal HLA- C2 allotype is responsible for the deleterious reproductive effect. The objective of our study was to determine the KIRs genotype in women with recurrent implantation failure (RIF) after assisted reproduction techniques and to make some correlation with specific clinical symptoms.

Materials and methods: Totally, 56 women with RIF were genotyped by PCR-SSP for activating and inhibitory KIR genes.

Results: The overall frequency of AA genotype was 34%. All inhibitory receptors’ genes had >90% frequency. Among the women with autoimmune thyroiditis (AT; n = 12), we detected significantly higher frequency for the activating 2DS1 (58.3% vs 25%) and lower frequency for 2DS2 (16.7% vs 52.5%). Clinical pregnancy was achieved in 16.7% of women with AA genotype after single embryo transfer (SET).

Conclusion: KIR genotyping is a valuable tool in decision making for SET and matching in oocyte donation. It is worthy to investigate the role of KIRs in the development of AT.

Conflict of Interest: None declared.

EP01.010 Single gene variants contribute to recurrent pregnancy loss – results from targeted next-generation sequencing

Karolina Matuszewska 1, Ewelina Bukowska-Olech2, Grzegorz Koczyk3, Delfina Popiel4, Adam Dawidziuk5, Barbara Więckowska6, Michał Piechota1, Marzena Wiśniewska1;2, Aleksander Jamsheer1;2, Anna Latos-Bieleńska1;2

1Centers for Medical Genetics GENESIS, Poznan, Poland; 2Poznan University of Medical Sciences, Department of Medical Genetics, Poznan, Poland; 3Institute of Plant Genetics, Polish Academy of Sciences, Biometrics and Bioinformatics Team, Poznan, Poland; 4Celon Pharma S.A., Research & Development Centre, Preclinical Development Department, Kazun Nowy, Poland; 5Łukasiewicz Research Network – Institute of Organisation and Management in Industry ORGMASZ, Warszawa, Poland; 6Poznan University of Medical Sciences, Department of Computer Science and Statistics, Poznan, Poland

Background: Recurrent pregnancy loss (RPL) occurs in 0,8-1,3% of couples attempting to be parents. Fetal chromosomal aberrations are the most common causes of miscarriage, identified in 50-70% of conception products. Among them, trisomy, monosomy, and polyploidy are present in 86% of noneuploid pregnancy losses, whereas submicroscopic chromosomal aberrations are detected in 8-16% of noneuploid pregnancy losses. We know a lot about the RPL etiology, but still, our knowledge is insufficient. RPL causes remain unexplained in more than 50% of cases.

Materials and methods: We have designed a custom next-generation sequencing gene panel encompassing 43 genes selected based on a medical literature review. The diagnostics were performed in trios – on the genomic DNA (gDNA) extracted from chorionic villi samples (with aCGH normal result) and gDNA isolated from peripheral blood leukocytes of 10 couples suffering from ≥3 pregnancy losses. We also tested 8 couples with no history of miscarriages and their healthy offspring.

Results: We found pathogenic or likely pathogenic variants within KIR2DL3, NLRP7, CYP1A1, GP6, THBD, KDR, and NOS3 genes in the experimental group. These variants were absent in the control groups.

Conclusion: Revealing new RPL candidate genes is crucial in understanding its causes. The results of our study may lead to further research aiming at identifying novel RPL molecular background and may result in genetic counselling improvement.

This work was supported by the grant from the Polish National Science Centre, Poland UMO-2017/27/N/NZ5/01061 to K.M.

Conflict of Interest: None declared.

EP01.013 Reporting on familial POI to study monogenic aetiology

Anu Valkna 1, Kristiina Rull1;2;3, Ülle Jakovlev4, Maris Laan1

1Tartu University, Institute of Biomedicine and Translational Medicine, Tartu, Estonia; 2Tartu University Hospital, Women’s Clinic, Tartu, Estonia; 3Tartu University, Institute of Clinical Medicine, Tartu, Estonia; 4East-Tallinn Central Hospital, Centre of Endocrinology, Tallinn, Estonia

Background/Objectives: Premature ovarian insufficiency (POI) refers to menopause before the age of 40 due to diminished ovarian reserve. Higher prevalence in a patient’s family compared to the general population (1%)1 suggests genetic aetiology of POI. The prevalence of self-reported familial POI is nearly four times higher than medically confirmed reports of familial POI (31 % vs 6.3%)2,3. The study aims to combine two strategies to pick up familial cases to assess the monogenic aetiology of idiopathic POI.

Methods: Patients with oligo/amenorrhea >4 months; age <40 years; FSH > 25 IU/l ≥2 times, were recruited at the Women’s Clinics of Tartu University Hospital and East-Tallinn Central Hospital, Estonia during August 2022 – January 2023. An in-depth interview was performed, and the familial cases were divided into two groups:

  • POI confirmed: proband’s family member fulfilled POI criteria confirmed by a managing clinician

  • POI suspected: diagnosis based solely on proband reports

Results: Twelve patients of the 39 total (31%) POI probands reported a family history of female infertility. Only three of them (8%) had a confirmed familial POI diagnosis.

Conclusion: Our results correlate with previous proband vs database reports of familial POI2,3. Heritability pattern is important to study monogenic aetiology. Given that proband-only reporting reveals a substantially greater frequency of familial POI, a more conservative approach should be advocated to prevent potential over-reporting.


1ESHRE. (2015).

2Vegetti, W. et al., Hum. Reprod. 13, 1796–1800 (1998).

3Verrilli, L. et. al Fertil. Steril. 119, 128–134 (2023).

Grants: PRG1021 (Estonian Research Agency)

Conflict of Interest: None declared.

EP01.015 The Patient with Azoospermia and Rare Gonosomal Anomaly

Dagmar Rašková 1, Jiří Horáček1, Inna Soldátová1, Monika Koudová1, David Stejskal1

1Gennet, Centre for Medical Genentics and Reproductive Medicine, Prague 7, Czech Republic

Background: The case report presents the sterile couple due to the partner’s azoospermia.

Methods: In the partner a deletion of the long arm of the Y chromosome in the AZFa, AZFb and AZFc regions was detected by QF-PCR method, the finding testified to the absence of the long arm of the Y chromosome. The partner’s karyotype from peripheral blood was 46, XX. The FISH examination revealed the Yp11.3 (SRY) locus translocated to one X chromosome (46,XX.ishder(X)t(X;Y)(p22.3;p11.3)(SRY+).

Results: The finding testifies to Klinefelter syndrome with deletion of the long arms of the Y chromosome and translocation of the short arms of the Y chromosome to one of the X chromosomes.

Conclusion: This finding explains the partner’s azoospermia, but his phenotype is completely normal, without any symptoms of Klinefelter syndrome (only the level of FSH and LH is high and the level of testosterone is lower). The family solved the infertility by assisted reproduction with donated sperm.

Grant References: No

Conflict of Interest: Dagmar Rašková: None declared, Jiří Horáček Centre for Medical Genetics and Reproductive Medicine Gennet, Inna Soldátová: None declared, Monika Koudová: None declared, David Stejskal: None declared.

EP01.016 Exploring the clinical spectrum of disorders of sex development in the Moroccan population

MOHAMED HSSAINI 1, laila bouguenouch2, Sana Abourazzak2, Hicham Bekkari1

1Sidi Mohamed Ben Abdellah University, Fes, Morocco; 2Faculty of Medicine and Pharmacy Fez, Fès, Morocco

Background/Objectives: Disorders/Differences of Sex development (DSD) are defined as congenital conditions in which there is a discrepancy in the development of chromosomal and gonadal/genital sex. The aim of this retrospective descriptive study was to identify the clinical spectrum of DSD among patients in the Moroccan population over a 4-year period.

Methods: We reviewed the clinical records of 87 patients with DSD referred between January 2019 and December 2022 to pediatric endocrinology, medical genetics, pediatric genetics, and pediatric urology clinics.

Results: Of the 87 cases identified, 35 (40.22%) were classified as 46,XY DSD, 30 (34.48%) as 46,XX DSD, and 22 (25.28%) as sex chromosomal DSD. Positive consanguinity is reported at 40.22% of the cases. The most common clinical diagnoses among 46,XY DSD patients were androgen insensitivity syndrome, 5-alpha reductase deficiency and 3-beta-hydroxysteroid dehydrogenase deficiency, with several affected siblings within families. The most frequent reasons for consultation were hypospadias, amenorrhea, the micropenis and clitoral hypertrophy. Congenital adrenal hyperplasia accounted for 83.33% of the diagnoses retained in the 46,XX DSD patients. The most common presenting sign is acute dehydration followed by hypospadias. Among the 22 (25.28%) patients with sex chromosomal disorders, 14 (63.63%) had Turner syndrome, 6 (27.27%) had Klinefelter syndrome, 1 (4.54%) had 47,XXX syndrome, and 1 (4.54%) had mixed gonadal dysgenesis. Gender had been reassigned in 5 patients.

Conclusion: In summary, this study demonstrates the clinical diversity of DSD cases in Morocco. Further efforts are needed to improve awareness, screening, and management of DSD in the country, especially for consanguineous populations.

Conflict of Interest: None declared.

EP01.017 Evidences of oligogenic impact on the development of DSD features in a patient with a c.34G>C GATA4 mutation

Dmytro Sirokha 1, Vitalii Kalynovskyi2, Nataliya Zelinska3, Olexandra Gorodna1, Ludmila Livshits1

1Institute of Molecular Biology and Genetics, National Academy of Sciences of Ukraine, Kyiv, Ukraine; 2Taras Shevchenko Kyiv National University, Kyiv, Ukraine; 3Ukrainian Scientific and Practical Center for Endocrine Surgery, Transplantation of Endocrine Organs and Tissues, Kyiv, Ukraine

Background: Differences in sexual development (DSD) are an important class of rare human diseases involving numerous genes. Nevertheless, about half of 46,XY individuals remain genetically unsolved. To investigate disease-causing gene variants combination and genotype-phenotype correlation we analyzed 46,XY DSD patient and family members carriers of c.34G>C (p.Gly12Arg) in GATA4, which had not been previously described in DSD patients. Moreover, a GATA4 mutation c.34G>C was registered in ClinVar in a 46,XY person without DSD features. The aim of our study was to look for potential causative variants previously not implicated in DSD to analyze the oligogenic origin of described DSD phenotype.

Methods: Clinical, hormonal, and histological investigations as well as whole exome sequencing for 46,XY DSD patient were performed.

Results: 46,XY SRY+ patient had a female phenotype, with both gonads being dysgenetic and hypoplastic. Heterozygous missense mutation c.34G>C (p.Gly12Arg) in GATA4 gene (MAF = 0.0001752) was not previously identified as DSD-causing. Moreover, two rare hemizygous mutations: c.8212T>C (p.Ser2738Pro) in CFAP47 (MAF unknown) and c.1214G>A (p.Arg405His) in KIAA1210 (MAF = 0.008459) located on the X chromosome and involved in spermatogenesis were identified in our patient, but previously not described for DSD patients. Bioinformatic analysis revealed that all these variants are considered pathogenic.

Conclusion: Based on obtained results we hypothesize, that in GATA4 gene c.34G>C allele together with c.8212T>C in CFAP47 and c.1214G>A in KIAA1210 are resulting in oligogenic DSD features exclusively in 46,XY individuals.

Grant References: Molecular-Genetic Mechanisms of Human Disorders of Sexual Development, National Academy of Sciences of Ukraine [0121U110054].

Conflict of Interest: None declared.

EP01.020 Prevalence of thrombophilic polymorphism in women with recurrent pregnancy loss

Liliia Chorna1, Danuta Zastawna1, Yaryna Zahanyach1, Halyna Makukh 2, Yelyzaveta Poliakova2, Oksana Kolodiy3

1State Institution «Institute of Hereditary Pathology of National Academy of Medical Sciences of Ukraine», Diagnostics of hereditary pathology, Lviv, Ukraine; 2Lviv State Regional Clinical Perinatal Center, L’viv, Ukraine; 3Lviv clinical territorial medical association of obstetrics and gynecology, Women consultation, L’viv, Ukraine

Recurrent pregnancy loss (RPL) occurs in 1–3% of couples aiming at childbirth. Despite numerous scientific studies, the etiology remains uncertain in about 50% of cases. Inherited thrombophilia has been implicated, as a possible cause of RPL but results differ from study to study. Aim of this case-control study was to determine whether hereditary thrombophilia is more prevalent in women with RPL.

57 females of unexplained RPL and 65 age-matched healthy controls were investigated for inherited thrombophilia. Detection of FGB 455G/A, FII 20210 G/A, FV 1691G/A, ITGA2 807С/Т, PAI-1 5G/4G, MTHFR 677С/Т loci was conducted by using RFLP- PCR method.

The frequency of the FGB 455 AA genotype and A allele was more prevalent in women with RPL (95% CI = 1.12-5.39, OR = 2.45, Р = 0.028). It was showed a significant higher frequency of the heterozygous Leiden mutation (FV 1691GA) than that of controls (16% vs 4%, OR = 4.31, 95% CI = 1.37-13.39, Р = 0.013). An increase in the ratio of homo- and heterozygous carriers of 4G allele of PAI-1 675 5G/4G loci in the group of women with RPL was revealed (95% CI = 1.13-3.14, OR = 1.9, Р = 0.015). Carriers of MTHFR 677TТ genotype and T allele were found to exhibit almost three-fold higher RPL risk (95% CI = 1.04-7.90, OR = 2.86, Р = 0.05). We did not find significant difference in other thrombophilic polymorphism.

The results showed that the genetic factors of thrombophilia: alleles 455A of FGB gene, 1691A of FV gene, 4G of PAI-1 gene and 677T of MTHFR gene might be involved in the etiology of RPL in west Ukrainian women.

Conflict of Interest: None declared.

EP01.021 Preliminary results of the first carrier screening study in the Romanian population

Diana Prepelita1;2, Andreea Tutulan-Cunita2, Anca Pavel2, Georgiana Lungu 2, VASILICA PLAIASU3, Ina Ofelia Focsa1;2, FLORINA MIHAELA NEDELEA4, Danai Stambouli2

1Carol Davila University of Medicine and Pharmacy, București, Romania; 2Cytogenomic Medical Laboratory, București, Romania; 3Institutul National pentru Sanatatea Mamei si Copilului “Alessandrescu-Rusescu”, București, Romania; 4Filantropia Clinical Hospital, București, Romania

Background/Objectives. Historically, carrier screening demonstrated to be efficient for preventing specific recessive diseases, found in general population or in a particular ethnic group. Nowadays it is possible to screen for hundreds of such conditions in one single test. Growing evidence shows the impact of extended carrier screening in the general population. The aim of our study was to investigate the carrier frequency in a Romanian cohort by a panel designed in-house.

Methods. A number of 150 genes were selected and included in the test, based on internationally recommended criteria such as high frequency in population, moderate/severe phenotype, well-defined phenotype and early onset of disease. Samples collected from 140 patients underwent next-generation sequencing (NGS). Sequencing data was analyzed using dedicated bioinformatic pipelines and variants were curated manually. Only likely pathogenic and pathogenic variants were taken into account.

Results. Out of the 140 patients included in the study, 108(77%) were carriers for at least one pathogenic/likely pathogenic variant. On average, an individual was a carrier for 1.34 pathogenic/likely pathogenic variants. Highest carrier rate was for 1 disease(34.2%) and 2 diseases(32.1%). The most frequent variants encountered were low penetrance variants in HFE (31.4%), followed by variants in ABCA4 (8%), GJB2 (7.8%), MEFV (5.7%), BTD (4.2%), CFTR (4.2%), PAH (4.2%), GBA (3.5%). These findings are in accordance to results described in literature.

Conclusion. Carrier screening proves to be an efficient tool to discover patients at risk for transmitting recessive disorders. Our study shows preliminary results of carrier frequency in Romania consistent with international data.

Conflict of Interest: None declared.

EP01.022 Reassessment of genetic imbalance in human blastocysts detected by PGT-A and PGT-SR

Andrei Tikhonov 1, Mikhail Krapivin1, Olga Efimova1, Yanina Sagurova1, Arina Golubeva2, Olga Chiryaeva1;3, Olga Malysheva1, Irina Mekina1, Evgeniia Komarova1, Dmitrii Staroverov2, Ekaterina Trusova2, Anna Pendina1;2

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

Background/Objectives: We aimed to verify PGT results in genetically imbalanced blastocysts.

Methods: PGT was performed by FISH at 3-4th day (n = 20), aCGH (n = 1) and NGS (n = 12) at 5th day. For verification of PGT results, blastocysts were separated into trophectoderm (TE) and inner cell mass (ICM). Then, aCGH was performed in TE and FISH was performed in both TE and ICM of each blastocyst.

Results: PGT results were confirmed in 13/33 (39.4%) blastocysts: monosomy 21, double monosomy 4 and 21, trisomies 8, 11, 16, 19, 22 initially revealed by PGT-A and chromosomal rearrangements initially revealed by PGT-SR. In the remaining 20/33 (60.6%) blastocysts, verification showed different results than PGT. Five blastocysts, classified as aneuploid by PGT-A using FISH, were verified as balanced (n = 2), as aneuploid but with another aneuploidy (n = 2) and as mosaic combining regular aneuploidy with mosaic aneuploidy of different chromosomes (n = 1). Seven blastocysts, classified as mosaic by PGT-A using FISH, were verified as balanced (n = 4), as regular aneuploid (n = 2), as mosaic with aneuploidy of a different chromosome (n = 1). Four blastocysts, classified as aneuploid by PGT-A using NGS, were verified as balanced (n = 2) and as mosaic aneuploid (n = 4). Four blastocysts, classified as mosaic aneuploid by PGT-A using NGS, were verified as balanced. In neither blastocyst discordance between TE and ICM was revealed during reassessment.

Conclusion: PGT results may not reflect genetic status of blastocyst which could be explained by different reasons including aneuploidy correction between 3 and 5th day and/or technical issues of PGT approaches.

Grant References: Supported by RSF22-75-00125.

Conflict of Interest: Andrei Tikhonov Part-time, RSF 22-75-00125 (principal investigator), Mikhail Krapivin Part-time, RSF 22-75-00125, Olga Efimova Part-time, Yanina Sagurova Full, Arina Golubeva: None declared, Olga Chiryaeva Part-time, Olga Malysheva Part-time, Irina Mekina: None declared, Evgeniia Komarova: None declared, Dmitrii Staroverov: None declared, Ekaterina Trusova: None declared, Anna Pendina: None declared.

EP01.023 Expanded carrier screening in gamete donors

Jan Diblik 1, Monika Koudová1, Martina Bittoova1, Michala Hrabíková1, David Stejskal1

1GENNET - Center of Medical genetics and reproductive medicine, Prague, Czech Republic

Background: Carrier screening for recessive diseases is a standard part of the examination of gamete donors for assisted reproduction. Czech Society of Medical Genetics and Genomics currently recommends examination of 3 genes: CFTR, GJB2 and SMN1 (group 1), we have added DHCR7, ATM and NBN (group 2) and genes on X chromosome: AR, COL4A5, GLA, IL2RG, MTM1, OTC, DMD (group 3).

Methods: GENNET CarrierTest© uses custom designed NGS panel for detection of mutations in 72 genes associated with autosomal recessive and X-linked disorders.

In total 32,890 samples were analysed. The limited set of 13 genes was examined in 4,618 donors, data from the remaining 59 genes were stored. The full CarrierTest© was performed in 23,654 patients (couples with infertility, recurrent pregnancy loss or family history of inherited disorders).

Results: In the group 1 mutations were detected in 364 (7.9%) donors and 2972 (10.5%) patients, group 2: 192 (4.2%) donors and 1116 (3.9%) patients, group 3: 9 (0.2%) donors and 71 (0.3%) patients.

Of the examined potential donors, a total of 546 (11.8%) had at least one finding and were excluded, this is 3.9% more than with the basic examination. 5215 (18.4%) patients had mutations in at least one of remaining 59 genes, that would require genetic matching if they need a donor.

Conclusion: The expansion of carrier testing in donors makes it possible to further reduce the risks of recessive diseases. New rules should allow the donation of gametes even to carriers, if the recipient’s examination and matching is ensured.

Conflict of Interest: Jan Diblik Gennet, Monika Koudová Gennet, Martina Bittoova Gennet, Michala Hrabíková Gennet, David Stejskal Gennet.

EP01.024 Investigating lncRNA expression patterns in human oocytes from patients with polycystic ovaries

Mehmet Aktan1, Hakan Aytaçoğlu 1, Burcu Ozbakir2;3, Pınar Tülay1;3

1Near East University, Medical Genetics, Nicosia (North), Cyprus; 2Near East University, Obstetrics And Gynecology, Nicosia (North), Cyprus; 3Near East University, DESAM Research Institute, Nicosia (North), Cyprus

Background/Objectives: Polycystic ovary syndrome (PCOS) is a common premenopausal female disorder characterized by excess androgen and ovarian abnormalities. PCOS may have an effect on the levels of gene expression in different tissues, including ovarian and possibly oocyte samples. Gene expression levels are controlled by multiple mechanisms, including methylation and non-coding RNAs. Thus, in this study, the expression levels of long non-coding RNAs (lncRNAs) in human oocytes obtained from patients with polycystic ovaries and patients without polycystic ovaries were investigated.

Methods: Thirteen metaphase II stage oocytes were obtained from individuals who applied to the Near East Hospital’s IVF Clinic. The expression levels of three lncRNAs targeting the CYP11A1 gene were investigated in oocyte samples. The real-time polymerase chain reaction was used to collect expression data for each oocyte.

Results: Three lncRNAs (RP11-573D15.8, RP11-156E8.1, and lnc-CYP11A1-1) targeting CYP11A1 gene were shown to be expressed in human metaphase II stage oocytes. There was no statistically significant (p > 0.05) change in the expression levels of these lncRNAs in oocyte samples obtained from patients with polycystic ovaries compared to patients without polycystic ovaries.

Conclusion: The expression of CYP11A1, which was previously shown to be upregulated in oocytes obtained from patients with polycystic ovaries, is not implied to be regulated by RP11-573D15.8, RP11-156E8.1, or lnc-CYP11A1-1 since there was no difference in the expression levels of these lncRNAs in two groups investigated. Different target lncRNA expression will be investigated to elucidate the regulation mechanism of up-regulated CYP11A1 expression level in human oocytes obtained from patients with polycystic ovaries.

Conflict of Interest: None declared.

EP01.025 Genome-wide association study meta-analysis supports association between MUC1 and ectopic pregnancy

Natàlia Pujol Gualdo 1;2, Reedik Mägi1, Triin Laisk1

1Estonian Genome Centre, Institute of Genomics, University of Tartu, Tartu, Estonia; 2Research Unit of Clinical Medicine, Department of Obstetrics and Gynecology, Oulu, Finland


Ectopic pregnancy is an important cause of maternal morbidity and mortality worldwide. To better understand the genetic risk factors underlying this pregnancy complication, we conduct a GWAS meta-analysis and identify two genome-wide significant loci on chromosomes 1 (rs4971091, p = 5.32 × 10−9) and 10 (rs11598956, p = 2.41 × 10−8). Follow-up analyses propose MUC1, an epithelial glycoprotein with an important role in barrier function, as the most likely candidate for the association on chromosome 1. We also characterise the phenotypic and genetic correlations with other phenotypes, identifying a genetic correlation with smoking and diseases of the (genito)urinary and gastrointestinal system, and phenotypic correlations with various reproductive health diagnoses, reflecting the previously known epidemiological associations.

Funding Statement

NPG was supported by MATER Marie Sklodowska-Curie which received funding from the European Union’s Horizon 2020 research and innovation program under grant agreement No. 813707. This study was funded by European Union through the European Regional Development Fund Project No. 2014-2020.4.01.15-0012 GENTRANSMED. Computations were performed in the High-Performance Computing Center of University of Tartu. We want to acknowledge the participants of the Estonian Biobank, and participants and investigators of the FinnGen study. The writing of this paper was supported by the writing retreats organised by the Institute of Genomics, University of Tartu.

Conflict of Interest: None declared.

EP01.026 FMR4 as a potential blood biomarker for fragile X-associated primary ovarian insufficiency

Laia Rodriguez-Revenga 1;2;3, Ines Agusti1, Maria Isabel Alvarez1;2;3, Robin Wijngaard4, Aina Boirras1, Dolors Manau1

1Hospital Clinic Barcelona, Barcelona, Spain; 2CIBERER, Barcelona, Spain; 3FUNDACIÓ DE RECERCA CLÍNIC BARCELONA-INSTITUT D’INVESTIGACIONS BIOMÈDIQUES AUGUST PI I SUNYER., Barcelona, Spain; 4Radboud University, Nijmegen, Netherlands

Background/Objectives: Female FMR1 premutation carriers are at risk for developing fragile X-associated primary ovarian insufficiency (FXPOI), a condition characterized by amenorrhea before age 40 years. Not all FMR1 premutation women suffer from FXPOI and nowadays there are no biomarkers that can predict the occurrence. Long non-coding RNAs (lncRNAs) comprise a group of regulatory transcripts. Previously, we described a significant association between FXPOI and high expression levels of FMR4 (FMR1-derived lncRNA), suggesting a potential role of FMR4 as a possible biomarker for FXPOI (Alvarez-Mora el al., 2022). A limitation in the study design was that it was exploratory. Herein, we further examined the role of FMR4 as biomarker to assess the risk of developing FXPOI, by characterizing young FMR1 permutation female carriers who have not been diagnosed as FXPOI.

Methods: Serum anti-Müllerian hormone (AMH) level and antral follicle count (AFC) were used to assess woman’s ovarian reserve. FMR4 transcript level was evaluated in total RNA extracted from peripheral blood by digital droplet PCR.

Results: A negative association was found between AMH, AFC and FMR4 (R2 = 0.2 for AMH and R2 = 0.4 for AFC), suggesting that FMR4 might help as an additional marker predicting ovarian reserve.

Conclusion: FMR4 might help to better assess the risk of FMR1 premutation women of developing FXPOI.

Grants: This study was supported by Fundación Merck Salud (19-FE-011) and Instituto de Salud Carlos III (ISCIII), (through the project PI21/01085), co-funded by the European Union. The CIBER de Enfermedades Raras is an initiative of the Instituto de Salud Carlos III.

Conflict of Interest: None declared.

EP01.027 Cases of newborns with CF after IVF in Bulgaria

Nadezhda Yaneva 1, Guergana Petrova2, Irena Bradinova1, Alexey Savov1

1National Genetic Laboratory, University Hospital of Obstetrics and Gynecology “Maichin Dom”, Medical University, Sofia, Obstetrics and Gynecology, Sofia, Bulgaria; 2Pediatric cilinic, UMHAT “Alexandrovska”, Sofia;, Medical University of Sofia, Pediatric, Sofia, Bulgaria

Cystic fibrosis (CF) is inherited condition with a progressive chronic clinical course and commonly premature death. The percentage of CF carriage in the general population in Bulgaria is 1 of 33 persons. Screening for CFTR mutations of donors for IVF procedures is not routine practice in Bulgaria. In the last years we have had several cases of newborns with CF after IVF. The purpose of this study was to assess the frequency of occurrence of cystic fibrosis in cases born after IVF.

We report 5 children conceived through IVF genetically and clinically confirmed with CF after birth. Four families had to use donor oocyte. In one case parent’s own gametes were used. The methodology for mutation detection includes Sanger sequencing, MLPA analysis and NGS.

In Bulgaria there are currently 248 alive confirmed CF patients. Five of them are born after IVF. All children were diagnosed in the last 5 years. The age of diagnosis was predominantly in the first year of life, since there is no known family history for CF for one child despite classical failure to thrive and nasal polyposis the diagnosis was confirmed late at the age of 9 years.

At the birth incidence of CF in Bulgaria was estimated using epidemiological approaches more than 20 years ago (1 in 3600 live births). Considering the carrier frequency with the many ethical and social issues that can arise we strongly advocate every IVF center to test its donors for CF carriage especially for those diagnosed with CBAVD.

Conflict of Interest: None declared.

EP01.028 In vitro Effects of Platelet-Rich Plasma on Spermatogenesis in NOA cases

O. Sena AYDOS 1, Sunay Zenginer1;2, Tulin Ozkan1, nazila farhangzad1;2, Asuman Sunguroglu1, Kaan Aydos3

1Ankara University, Faculty of Medicine, Department of Medical Biology, Ankara, Turkey; 2Ankara University, Institute of Health Sciences, Ankara, Turkey; 3Ankara University, Faculty of Medicine, Department of Urology, Ankara, Turkey

Azoospermia is a condition that affects 1% of the male population.While approximately 15% of them develop due to hypogonadotropic hypogonadism or obstructive causes and there is a chance for treatment, the fertility of the remaining cases will only be possible by obtaining sperm from the testicles with microTESE.In about half of them, mature spermatozoa are not found.In such cases, different methods have been tried for the maturation of the progenitor germ cells in the testis with different techniques.

Platelet-rich plasma(PRP) is a blood-derived product enriched with platelet density.In experimental studies, the use of PRP in the proliferation and differentiation of sperm cells has been suggested as an effective treatment method. The aim of the study is to determine the effects on spermatogenic cells and their contribution to differentiation with pre-meiotic, meiotic and post-meiotic markers by culturing testicular tissue samples obtained by microTESE from non-obstructive azoospermic(NOA) patients with PRP in appropriate culture medium conditions.

Testicular tissue samples obtained by microTESE in 4 cases with spermatozoa in the testicles and in 10 infertile men diagnosed with NOA were cultured in vitro in PRP supplemented medium.Expression of pre-meiotic(PLZF,VASA),meiotic(SYCP3,CREM),post-meiotic(PRM2,ACR) genes as biomarkers in spermatogenesis were evaluated by RT-PCR.

The decrease in VASA and CREM expressions was found to be statistically significant when compared with the controls in the group without PRP(p < 0.05).Our results indicate that patients diagnosed with NOA may benefit more from PRP treatment, and PRP can provide positive contributions to infertility treatment in NOA cases.

This study was supported by Ankara University(BAP)(Project No:TYL-2022-2421)

Conflict of Interest: O. Sena AYDOS This study was supported by Ankara University Scientific Research Projects Coordination Unit (BAP) (Project No: TYL-2022-2421), Sunay Zenginer: None declared, Tulin Ozkan: None declared, nazila farhangzad: None declared, Asuman Sunguroglu: None declared, Kaan Aydos: None declared.

EP01.029 Inferring genetic architecture of preeclampsia using integrative analysis of genome and transcriptome data

Ekaterina Trifonova 1;2, Maria Gavrilenko1, Viktoria Serebrova1, Anastasia Babovskaya1, Tatyana Gabidulina2, Aleksei Zarubin1, Stepanov Vadim1

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

Preeclampsia (PE) is the main cause of maternal and perinatal morbidity and one of the most common pregnancy complication associated with placental pathology. The cause of PE has not yet been fully defined. We used an integrative approach to identify new genetic markers of PE. The genes for study were selected by genome-wide gene expression analysis of the placental tissue in normal and PE pregnancies. The 83 tagging single-nucleotide polymorphisms (SNPs) in placental genes were chose to conduct genotyping based on a case-control study consisting of 551 PE cases and 712 controls from Russian, Buryat and Yakut populations. The joint effects of SNPs on the PE risk were assessed by haplotypes and structure of linkage disequilibrium analysis. Machine learning methods were used to evaluate the association between SNPs and PE. We discovered new candidate genes associated with PE at the genomic and transcriptome levels. We found common and population-specific associations of SNPs with PE. Allelic variations in CCSAP, CORO2A, NDRG1, PLIN2, SIGLEC6 and ZNF175 genes are associated with PE in all ethnic groups. Moreover, SNPs of the RAC2, GPT2 and BHLHE40-AS1 genes are associated with PE in Yakut women only. Association with PE was found for rs6818337 of ANKRD37 gene just for Buryats. Epistatic interactions of the studied genes were revealed in all ethnic groups via MDR analysis. Overall, this study supports the hypothesis that genes identified as differentially expressed in placental tissue in case of PE and normal pregnancy indicate a genetic association with PE at the genome level.

Conflict of Interest: None declared.

EP01.030 The relationship between ovarian insufficiency and MTR and MTRR genetic polymorphisms

Ivelina Oprova 1, Petya Andreeva1, Kalina Belemezova2, Mariela Hristova-Savova2, Martin Georgiev3, Ivanka Dimova4

1Shterev Hospital, IVF unit, Sofia, Bulgaria; 2Shterev Hospital, Genetic department, Sofia, Bulgaria; 3Medical University-Sofia, Sofia, Bulgaria; 4Medical University-Sofia, Genetic department, Sofia, Bulgaria

Background: Premature ovarian insufficiency (POI) is the early depletion of the ovarian reserve affecting 1–2% of women < 40 years of age. It has been demonstrated that female reproduction and gene polymorphisms of the enzymes in the one-carbon metabolic pathway are related. Although there is insufficient data for patients with POI, women who carry these associated gene variations. The aim of this study was to investigate whether there was a causal predisposition between MTRR A66G and MTR A2756G polymorphism in women with POI and world controls.

Methods: In this study MTR A2756G and MTRR A66G genotypes, containing SNPs rs1805087 and rs1801394 were determined in all the participants by Real-time polymerase chain reaction.

Results: The prevalence of MTR A2756G and MTRR A66G polymorphisms was determined in 27 POI patients and 2504 healthy women – the World control group. The genotype distribution of MTR A2756G was identical between POI patients and controls. We discovered nearly twice as many MTRR A66G genotypes in the POI group as in the world control group, but without significance (29.63% and 15.73% respectively, p = 0.3).


Allele frequency

Heterozygous frequency

Homozygous frequency



World controls


World controls


World controls


MTR A2756G rs1805087








MTRR A66G rs1801394







**P = 0.3*NS

Conclusion: The POI’s etiology does not appear to be related to MTR A2756G genotype gene polymorphisms. The trend of higher MTRR A66G genotypes in homozygous frequency in the POI group should be investigated further.

Conflict of Interest: None declared.

EP01.031 Thrombophilia gene mutations, infertility and in vitro fertilization

Ketevan Kartvelishvili 1, Nino Pirtskhelani2;3, Natia Popkhadze4, Nino Kochiashvili2

1National Forensics Bureau, Biology Department, T’bilisi, Georgia; 1National Forensics Bureau, Biology Department, T’bilisi, Georgia; 3Tbilisi State Medical University, Molecular and Medical Genetics, T’bilisi, Georgia; 4Invitro Life, T’bilisi, Georgia

Background: Thrombophilia genes are involved not only in coagulation and fibrinolysis, but also in fertilization, fetal development and tissue remodeling, as well as recurrent implantation failure, recurrent pregnancy loss and congenital abnormalities. The aim of our study was to evaluate incidence of inherited thrombophilia in women with unexplained infertility, which underwent IVF, or were preparing for IVF and association of thrombophilia with IVF failure.

Methods: 60 unrelated Georgian women, with unexplained infertility, undergoing IVF cycles, were genotyped by multiplex PCR for simultaneous detection of six genetic risk factors associated with thrombophilia (Factor V (G1691A; H1299R), Prothrombin (G20210A), MTHFR (C677T; A1298C) and PAI-1/SERPINE1 (4G/5G).

Results: Out of 60 Patients with infertility, thrombophilia screening detects no one patient negative for MTHFR gene (C677T; A1298C) mutations. 56.67% (n = 60) was homozygote or compound heterozygote, 10% (n = 60) was homozygote for MTHFR gene (C677T) mutation, 23.33% (n = 60) was homozygote for MTHFR gene (A1298C) mutation. 30% was homozygote for PAI-1/SERPINE1 (4G/4G) polymorphism, 53.33% was heterozygote for PAI-1/SERPINE1 (4G/5G) polymorphism. However, mutations of factor V (G1691A; H1299R) and II (G20210A) were less common, 6.67%, 6.67% and 3.33% respectively.

Almost all patients received 2 month antithrombotic prophylactic therapy before fertilization. It’s noteworthy that in 4 patients previous several IVF attempts were negative without prophylactic antithrombotic treatment.

Conclusion: Our study showed an increased frequency of MTHFR (C677T; A1298C) mutations and PAI-1/SERPINE1 (4G/5G) polymorphism in women with unexplained infertility, who underwent IVF or are preparing for IVF, However, mutations of factor V and II were less common.

Conflict of Interest: None declared.

EP01.032 Characterization of Epilepsy and Neurodevelopmental disorders in familiar and sporadic cases of Poirier-Bienvenu Neurodevelopmental Syndrome

Fabrizia Stregapede 1, Allessandra Terracciano1, Alessia Micalizzi1, Marina Trivisano1, Angela De Dominicis1, Marialisa Dentici1, Chiara Quintavalle1, Simona Cappelletti1, Nicola Specchio1, Antonio Novelli1

1IRCCS Bambino Gesù Children’s Hospital, Rome, Italy

CSNK2B gene encodes a regulatory subunit of casein kinase II, highly expressed in the brain. Heterozygous mutations in CSNK2B are associated to Poirier-Bienvenu Neurodevelopmental Syndrome (POBINDS) (OMIM #618732), characterized by facial dysmorphisms, seizures, intellectual disability and behavioural abnormalities, described for the first time in 2017. In this study, we report eight new cases of POBINDS associated with novel heterozygous variants of CSNK2B gene. In three of these patients, a pathogenic variant was inherited from an affected parent. We describe the molecular and clinical features of our patients, focusing in the epileptic and neurodevelopmental phenotype, and comparing them with the previously reported cases. Moreover, while so far all previously reported patients had a de novo CSNK2B mutation, here we report for the first time two familial cases of POBINDS, confirming the high variable expressivity of the disease, and underlying the importance of a thorough family history collection before performing genetic testing in patients with epilepsy and neurodevelopmental disorders.

Conflict of Interest: None declared.

EP01.033 Telomere length in spermatogonia and spermatocytes I as a new predictor of ICSI with testicular sperm efficiency

Arina Golubeva 1, Yanina Sagurova2, Mikhail Krapivin2, Evgeniia Komarova2, Irina Mekina2, Andrei Tikhonov2, Ekaterina Trusova1, Dmitrii Staroverov1, Olga Efimova2, Anna Pendina1;2

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

Background/Objectives: We have checked whether telomere length (TL) in spermatogonia and spermatocytes I from azoospermic patients whose testicular sperm was used for ICSI can be a predictor of the embryo survival in vitro and of a pregnancy chance.

Methods: Testicular samples were obtained by open biopsy from 25 azoospermic patients. TL was measured by quantitative fluorescence in situ hybridization (Q-FISH) on cytogenetic preparations of mitotic spermatogonia (n = 359) and meiotic spermatocytes I (n = 295). The embryo survival was estimated as a ratio of blastocysts at 5th day (n = 52) to embryos at 2nd day (n = 132). A total of 38 cycles were analyzed including 29 with embryo transfer and 9 with registered pregnancy.

Results: TL in spermatogonia and spermatocytes I was linked to the embryo survival up to the blastocyst stage and to the pregnancy rate. Telomeres were longer in spermatogonia and spermatocytes I in patients: (1) from couples with high (>50%) embryo survival compared to the couples with low (<50%) embryo survival, including developmental arrest at cleavage stages (р<0.0001 and р<0.0001, respectively, the Mann-Whitney U-test); (2) from couples with a pregnancy after embryo transfer compared to the couples in which pregnancy was not registered (р = 0.01, the Mann-Whitney U-test).

Conclusion: The survival of embryos obtained by ICSI with testicular sperm as well as chance of pregnancy are associated with TL in spermatogonia and spermatocytes I. In ICSI cycles with testicular sperm, TL in spermatogonia and spermatocytes I could predict successful embryonic development in vitro and a chance of pregnancy.

Grants: RSF №18-75-10046.

Conflict of Interest: Arina Golubeva: None declared, Yanina Sagurova full-time, RSF №18-75-10046, Mikhail Krapivin full-time, RSF №18-75-10046, Evgeniia Komarova full-time, RSF №18-75-10046, Irina Mekina: None declared, Andrei Tikhonov full-time, RSF №18-75-10046, Ekaterina Trusova: None declared, Dmitrii Staroverov: None declared, Olga Efimova full-time, RSF №18-75-10046 (principal investigator), Anna Pendina full-time, RSF №18-75-10046.

EP01.034 High incidence of CPLANE1 related Joubert syndrome in early pregnancy loss

Gjorgji Bozhinovski 1, Marija Terzikj1, Katerina Kubelka-Sabit2, Dijana Plaseska-Karanfilska1

1Research Center for Genetic Engineering and Biotechnology “Georgi D. Efremov”, Macedonian Academy of Sciences and Arts, Skopje, Macedonia; 2Private Hospital “Acibadem-Sistina”, Skopje, Macedonia

Until now, monogenic causes of early pregnancy losses (EPLs) remain largely unexplored, and only a small number of studies using whole exome sequencing (WES) have been published. Recently, we have reported CPLANE1 double heterozigosity (c.1819delT;7817T > A and c.5820+3_5820+6del) in two EPLs from a couple of Albanian ethnic origin (ESHG 2022; P01.026B). Pathogenic variants in CPLANE1 (C5orf42) are known to cause Joubert syndrome (JS), a primary ciliopathy with multiple system defects. The CPLANE1 c.1819delT;7817T > A allele has been observed with an allele frequency (AF) of 0,64% (7/1090) among our cohort of 545 patients studied by WES. In this study we used allele specific PCR to screen for the presence of CPLANE1 c.1819delT;7817T > A allele among 246 euploid EPLs (<12 gestational age) from families with Macedonian (n = 155), Albanian (n = 80) and other ethnic origin (n = 11). Three c.1819delT;7817T > A homozygous and two heterozygous fetuses were detected, all of Albanian ethnic origin except one heterozygote with Macedonian origin. The subsequent WES analysis showed no second pathogenic mutation in the heterozygous fetuses. Thus, we have detected a high incidence of JS in the total studied group of EPLs (2%; 5/248), and even higher incidence among Albanian families (6.1%; 5/82). In conclusion, to the best of our knowledge this is the highest incidence of monogenic disease reported as a cause of EPLs. Targeted screening of euploid EPLs for the CPLANE1 c.1819delT;7817T > A allele with subsequent NGS analysis in heterozygotes is justified, especially in couples of Albanian ethnic origin since it would detect 1 JS in ~16 EPLs.

Conflict of Interest: None declared.

EP01.036 Detection of genetic causes of miscarriage in the products of conception: Analysis of fetal tissues from a miscarriage using QF-PCR and Array CGH

Natalie Friedova 1;2, Martina Langova1, Jana Tajtlova1, Vladimir Gregor1, Antonin Sipek Jr1;2, Renata Kejkulova1, Stepanka Maskova1

1Thomayer University Hospital, Department of Medical Genetics, Prague, Czech Republic; 2Institute of Biology and Medical Genetics, First Faculty of Medicine, Charles University and General University Hospital, Prague, Czech Republic

Background: From a mathematical point of view is human reproduction relatively inefficient. Studies have shown that only 30% of children from all fertilized eggs are born alive. Repeated spontaneous abortions affect approximately 1-2% (some sources state up to 5%) of women. Generally, early reproductive losses have various causes, as far as genetic causes are concerned, chromosomal aberrations, are clearly the most common cause of early reproductive losses Chromosome aberrations may cause around half of early reproductive losses, in different studies chromosome abnormalities have been detected in 20-70% of cases. Chromosome analysis using microarray methods detected supposed causal CNV (copy number variation) in ~2% of miscarriages and CNVs of unknown significance (mainly of parental origin) in up to 40% of abortions.

Methods: Retrospective analysis of the causes of miscarriage in products of conception (POC) routinely examined at the Department of Medical Genetics TUH using QF-PCR and Array CGH.

Results: Between 2020 and 2022 were 72 POC examined in TUH. Numerous chromosomal aberrations were found in 8 cases. Array CGH revealed the complex structural aberrations as a cause of miscarriage in another 7 cases (examination of the parents showed that the cause of the abortion was mostly „de novo“).

Conclusion: The cause of a large number of miscarriages is still unknown. Whole-exome sequencing has revolutionized the postnatal diagnosis of genetic diseases but is still rarely used to study reproductive disorders. Consequently, we would like to incorporate WES into the routine diagnosis of unexplained miscarriages in our department.

Conflict of Interest: Natalie Friedova Thomayer University Hospital.

Charles University in Prague

Pronatal, Martina Langova Thomayer University Hospital

Charles University in Prague, Jana Tajtlova Thomayer University Hospital, Vladimir Gregor Thomayer University Hospital

Charles University in Prague

Pronatal, Antonin Sipek Jr Thomayer University Hospital

General University Hospital in Prague

Charles University in Prague, Renata Kejkulova Thomayer University Hospital, Stepanka Maskova Thomayer University Hospital

EP01.037 Potential role of FOXL2 gene missense variations in women with history of recurrent miscarriages

Sri Hari Chandan Appikonda 1, Divya Krishnan1, Sudhanshu Kumar1, Reshma Vasudevan1, Neetha John1, Lova Matsa1

1Igenomix FZ LLC, Genomic precision diagnostic dept, Dubai, United Arab Emirates

Haploinsufficient mutations in FOXL2, a forkhead transcription factor gene, are known to cause blepharophimosis-ptosis-epicanthus inversus syndrome (BPES) types I and II, a rare genetic disorder associated with premature ovarian failure. While isolated cases of premature ovarian failure have been reported in woman with missense variations in FOXL2 gene.

Here, we report two couples with early recurrent miscarriages, with a normal chromosomal complement confirmed by karyotyping. Whole-exome sequencing based screening analysis revealed two missense variants of uncertain significance in FOXL2 gene (p.Pro337Ser, p.Gly187Asp) in the female partners, respectively. Based on the available scientific evidence, no other significant variants were detected in these couples. The p.Pro337Ser variant is absent from general population (gnomAD), while p.Gly187Asp variant was reported in only 2 males in the gnomAD population (0.001%; 02/151714 alleles; 0 in females). Both these variations are in the non-forkhead domain of the FOXL2 gene.

The p.Gly187Asp variant has previously been implicated in a woman with non-syndromic premature ovarian insufficiency but with elevated levels of follicle stimulating hormone (FSH), luteinizing hormone (LH) and Low estradiol (E2) levels. Recent studies have shown that the FOXL2 is strongly expressed in the uterine tissue and controls the expression profile of the endometrial genes. Mutation spectrum analysis of the FOXL2 gene revealed that the variations in the forkhead domain cause BPES I and II phenotypes. Thus, we hypothesise that a gain-of-function mutation in the non-forkhead domain of the FOXL2 gene may have significant implication in recurrent miscarriages alongside with their assumed role in the primary ovarian insufficiency.

Conflict of Interest: None declared.

EP01.038 The need of couple carrier genetic screening in absence of an index child: retrospective analysis

Lova Matsa 1, Sarah Elewisy1, Sri Hari Chandan Appikonda1, Neetha John1, Sudhanshu Kumar1, Reshma Vasudevan1, Divya Krishnan1

1Igenomix FZ LLC, Genomic precision diagnostic dept, Dubai, United Arab Emirates

An Egyptian couple had two children who died at an early stage of life (2 and 8 moths respectively) with brain abnormalities and respiratory failure. Clinical features include: clenched hands, club foot, septal defects, oesophageal atresia, seizure, abnormal facies, spasticity, brain atrophy. No molecular testing was performed on the affected children. The couple also have 2 healthy children.

In absence of the index children samples, the couple had come forward for the genetic screening testing. Whole-exome sequencing (WES) based screening was performed. Based on the previous children clinical phenotypes, the couple was found to carry a heterozygous variant of uncertain significance (NM_005787.5:c.1034C>T, p.Ser345Phe) in the ALG3 gene. Pathogenic ALG3 variants are associated to a rare autosomal recessive congenital disorder of glycosylation (CDG) type Id. Clinical symptoms typically develop during infancy and affect many body systems throughout life.

During the next pregnancy of the couple, targeted prenatal testing was performed for the ALG3 variant and the fetus was found to be homozygous. However, due to advanced gestational age, and in view of the variant classification, the pregnancy was continued. The baby was born and developed clinical features consistent with ALG3-CDG. Later, on index-based WES and retrospective TRIO-WES couldn’t find any other significant variant than ALG3 variant. Healthy children were found to be heterozygous for this ALG3 variant.

This case emphasises the importance of carrier genetic testing in the couple in absence of the index child and retrospective approach to identify the disease-causing variant in the families suffering with severe genetic conditions.

Conflict of Interest: None declared.

EP01.039 A combined RNA expression analysis and whole genome sequencing approach for the study of lncRNAsrole in teratozoospermia

Maria-Anna Kyrgiafini 1, Themistoklis Giannoulis2, Alexia Chatziparasidou3, Nikolaos Christoforidis3, Zissis Mamuris1

1University of Thessaly, Department of Biochemistry and Biotechnology, Larissa, Greece; 2University of Thessaly, Department of Animal Sciences, Larissa, Greece; 3Embryolab IVF Unit, Thessaloniki, Greece

Background/Objectives: Male infertility is a major health problem as more than 20 million men are affected worldwide. Genetic background plays an important role in many types of male infertility, such as teratozoospermia which is associated with defects in sperm morphology. However, the genetic causes of male infertility remain still at large unexplored.

Methods: In this study, whole-genome sequencing was used in combination with RNA expression analysis to identify differentially expressed (DE) lncRNAs in teratozoospermia and mutations on these DE lncRNAs that are found only on teratozoospermic men. Several bioinformatics tools were used to explore the effect of the variants on lncRNAs’ structure and function and lncRNAs-miRNAs interactions.

Results: 3582 mutations found only in teratozoospermic men were identified on DE lncRNAs between normozoospermic and teratozoospermic men. Of these, 64 variants on 24 lncRNAs have a potential regulatory role according to 3DSNP and RegulomeDB scores. Furthermore, 14 variants affect the structure of 9 lncRNAs according to lncRNASNP v3 and 65 variants on 27 lncRNAs cause loss or gain of miRNA targets. Pathway enrichment and Gene ontology analyses of the genes targeted by these lncRNAs revealed pathways that are deregulated in teratozoospermia.

Conclusions: The present study confirms the contribution of lncRNAs studied in the past to male infertility and sheds light on new molecular mechanisms by providing a list of variants and candidate lncRNAs associated for the first time with teratozoospermia paving the road for future studies aiming to the improvement of diagnosis and therapy.

Grant references: Spermogene (grant number Τ1ΕΔK-02787).

Conflict of Interest: None declared.

EP01.040 Can we rescue the developmental competence of 3PN zygotes ?

Brian Sperelakis Beedham1, Anne Mayeur2, Nadine Gigarel3, Laura Brosseau4, Jean-Michel Dupont4, Nelly Achour2, Julie Steffann 1

1Université Paris Cité-institut Imagine, Mitochondrial genetics, Paris, France; 2Antoine Béclère Hospital, Reproductive Biology Unit CECOS, Clamart, France; 3Université Paris Cité-Necker Hospital, Genetics, Paris, France; 4Université Paris Cité-Hopital Cochin, Cytogenetics, Paris, France

Because human embryo research is ethically and numerically restricted, alternatives are needed, such as abnormal 3 pronuclei (3PN) zygotes. Correction of triploidy is performed by removing the extrapronucleus at the zygote stage. We aimed to investigate the in vitro development and the genetic component of these corrected zygotes.

A total of 43 3PN zygotes was submitted to extrapronucleus resection, and their in vitro development subsequently monitored. Parental genetic origin was assessed in 29/43 zygotes by segregation analysis of 10 microsatellite markers located on 8 different chromosomes. In 4 blastocyst embryos screening for aneuploidies was performed on trophectoderm biopsies by low pass WGS.

In total, 6/48 manipulated 3PN zygotes reached the blastocyst stage, suggesting that extrapronuclear removal did not modify in vitro development (p = 0,32). Parental origin was assessed for 29/48 embryos. Among them, 10/29 had a biparental contribution, 4/29 a uniparental contribution, and 9/29 had at least one trisomy and 1/29 one tetrasomy. In 5 embryos, multiple trisomies with the same parental origin suggested that embryos were still triploid, despite PN resection. Remarkably, none of the embryos with uniparental contribution reached the blastocyst stage. In 3 blastocysts, comprehensive preimplantation genetic testing showed only one euploid embryo.

In conclusion, our study illustrates the limitations of using 3PN zygotes as a research model. Despite numerous cumbersome micromanipulations, the failure to restore diploidy considerably limits the number of developing embryos available for research purpose.

Grant AFM 20304

Conflict of Interest: None declared.

EP01.041 Low mitochondrial content marks high health status of human euploid embryos

Konstantin Popadin 1;2;3;3, Maxim Ri1;4, Natalia Ree1;4, Maria Tofilo5;6, Irina Zvereva7, Anastasia Kirillova7;8, Ilya Mazunin7;8

1Center for Mitochondrial Functional Genomics, Institute of Living Systems, Immanuel Kant Baltic Federal University, Kaliningrad, Russian Federation; 2School of Life Sciences, École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland; 3Swiss Institute of Bioinformatics, Lausanne, Switzerland; 4AcademGene LLC, Novosibirsk, Russian Federation; 5Center for Molecular and Cellular Biology, Skolkovo Institute of Science and Technology (Skoltech), Skolkovo, Skolkovo, Russian Federation; 6Medical Genomics LLC, Moscow, Russian Federation; 7Fomin Clinic, Moscow, Russian Federation; 8Center for Molecular and Cellular Biology, Skolkovo Institute of Science and Technology (Skoltech), Skolkovo, Russian Federation

Recently we have shown that aneuploid human embryos are characterised by an increased ploidy of their mitochondrial DNA (mtDNA) (https://doi.org/10.1101/2022.10.14.512116). The potential mechanism behind this finding is based on a cellular attempt to compensate for wide spectrum of energetical problems by hyper-replication of mtDNA. If this compensatory mechanism is universal, i.e. it can be activated by a broad range of deleterious and slightly-deleterious factors, we can expect an increased mtDNA content to be a marker of a poor health status even among euploid embryos. Here, using the dataset of 15’000 annotated human embryos with available low-coverage whole-genome sequences, we tested an association of mtDNA content with several phenotypes of euploid embryos such as morphology, expansion grade and outcome (pregnancy and childbirth). In a subset of 7,000 euploid embryos in the dataset, we showed that embryos with the healthiest morphology states and expansion grades had decreased mtDNA content. In a subset of 500 euploid embryos with known outcomes, we observed a gradual decrease in mtDNA content from the cohort of all transferred embryos to those resulting in pregnancy and finally to those leading to childbirth. These results are in line with the hypothesis that increased mtDNA content may indicate poor health status under all else equal. Further research is needed to elucidate the underlying molecular and biochemical compensatory mechanisms of mtDNA hyper-replication. More data are needed to the research consortium INITIATOR (IN vItro ferTIlizATion fOr Research) to uncover new aspects of human embryogenesis.

Conflict of Interest: None declared.

EP02 Prenatal Genetics

EP02.003 Importance and application of whole exome sequencing in prenatal genetic diagnostics

Renata Szalai 1, Agnes Till1, Attila Gyenesei2, Judit Bene1, Kinga Hadzsiev1

1University of Pécs Medical School, Department of Medical Genetics, Pécs, Hungary; 2János Szentágothai Research Centre of the University of Pécs, Pécs, Hungary

Background/Objective: Fetal anomalies are responsible for 20% of perinatal deaths. Prenatal whole exome sequencing (WES) approaches can provide genetic diagnosis when conventional tests are negative. A major challenge in fetal diagnostics is that many diseases may not have a known prenatal phenotype, moreover a prenatal feature may be atypical compared to the postnatally described phenotype. Microcephaly in a pregnancy is a structural abnormality may lead postnatal neurodevelopmental consequences (intellectual disability, autism spectrum disorders, epilepsy) are associated with abnormal brain growth causing morbidity and mortality in infancy or early childhood. Ultrasound examination allows the detection of microcephaly in a fetus. ASPM gene mutations are estimated to account for 10-40% of autosomal recessive congenital microcephaly.

Methods: Based on positive family history and ultrasonography findings suggesting primary microcephaly, prenatal WES analysis was indicated. Prenatal WES was performed using Agilent SureSelectXT library kit and Illumina sequencing technology. Sanger sequencing confirmed the presence of the pathogenic variants.

Results: WES analysis revealed a c.8506_8507delCA (p.Gln2836Glufs*35) and a novel c.3134_3135delTC (p.Leu1045Glnfs*17) pathogenic ASPM mutations in the fetus in compound heterozygous state. The c.3134_3135delTC has never been reported in the literature.

Conclusion: WES proved to be a valuable method in diagnostics, in case of carefully chosen group of patients with appropriate indication. It would be important to make it more widely available in prenatal clinical practice. This method can provide clinically relevant information to manage a pregnancy. The correct diagnosis offers an opportunity for early intervention and effective treatment in prenatal or in postpartum period.

Conflict of Interest: None declared.

EP02.004 Focal dermal hypoplasia presenting with split hand/foot malformation, lack of skin findings, broad thumb, renal agenesis, and coloboma: a fetal case report

Molly Jackson 1, Ian Suchet2, Rati Chadha3, Kristopher Langdon4, Mary Ann Thomas5;6

1Cumming School of Medicine, University of Calgary, Department of Medical Genetics, Calgary, Alberta, Canada; 2Calgary Maternal Fetal Medicine Center, EFW Radiology, Calgary, Alberta, Canada; 3Cumming School of Medicine, University of Calgary, Division of Maternal-Fetal Medicine, Department of Obstetrics & Gynecology, Calgary, Alberta, Canada; 4Cumming School of Medicine, University of Calgary, Department of Pathology and Laboratory Medicine, Calgary, Alberta, Canada; 5Cumming School of Medicine, University of Calgary, Department of Medical Genetics, Department of Pediatrics, Calgary, Alberta, Canada; 6Alberta Children’s Hospital Research Institute, Calgary, Alberta, Canada

Background: Focal dermal hypoplasia (FDH) is a rare X-linked dominant multisystem disorder characterized by cutaneous (skin aplasia/hypoplasia, hypo-/hyper-pigmentation, nodular fat herniation) and limb malformations (syndactyly, oligodactyly, camptodactyly, split hand/foot malformation [SHFM], and long bone shortening), and developmental abnormalities of the eyes, face, and teeth. The prenatal phenotype is emerging. We describe a case with SHFM, lack of skin findings, broad thumb, renal agenesis, and coloboma.

Methods and Results: A 32-year-old G3P1 female presented following anatomy scan at 19 + 2 weeks’ gestation that identified a female fetus with short long bones; syndactyly and camptodactyly on the hands bilaterally; left hand absent middle ray fingers; right foot syndactyly and absent toes, consistent with SHFM. Left microphthalmia and right renal agenesis were identified. Amniocentesis was performed with rapid aneuploidy detection (RAD), chromosomal microarray (CMA), and trio whole exome sequencing (WES). The pregnancy was interrupted at 21 + 4 weeks’ gestation. CMA and RAD were normal. WES identified a de novo likely pathogenic variant in the PORCN gene, c.1016T>G, p.(Leu339Arg), which is associated with FDH.

Autopsy additionally identified a left thumb with enlarged distal phalanx and left choroid/sclera coloboma. There were no cutaneous findings.

Conclusion: FDH presents with variable phenotype even among family members, likely due to skewed X-inactivation. Prenatal cases have been described that are clarifying the prenatal presentation and include major congenital anomalies visible on prenatal ultrasound. This case contributes to the prenatal phenotype with evidence of rare associated features. The lack of cutaneous findings may provide insight into the presentation at this gestational age.

Conflict of Interest: Molly Jackson full, Ian Suchet full, Rati Chadha full, Kristopher Langdon full, Mary Ann Thomas full

EP02.005 The number of non-pathogenic CNVs impacts the risk of preterm birth

Michal Macarov 1, Tal Bamberger2, naama elefant2, Hanah Margalit2, Ayala Frumkin1, Shamir Zenvirt1, Vardiella Meiner1, Shiri Shkedi Rafid1, Hagit Hochner3

1Hadassah Medical Center and Faculty of Medicine, Hebrew University of Jerusalem, Department of Genetics, Jerusalem, Israel; 2Institute for Medical Research Israel-Canada, Faculty of Medicine, The Hebrew University of Jerusalem, Department of Microbiology and Molecular Genetics, Jerusalem, Israel; 3Braun School of Public Health and Community Medicine, Hebrew University of Jerusalem, Jerusalem, Israel

Background: Preterm birth (PTB) is the main condition related to perinatal morbimortality. Environmental and genetic factors have been shown to contribute to PTB risk. Here, we aimed to study the potential impact of benign/non-pathogenic copy number variants (CNVs) on PTB, focusing either on individual CNV-harboring regions (CNVRs) or on the total burden of CNVs across the genome.

Methods: Genomic data from prenatal CMA tests performed at Hadassah Medical Center during 2013-2018 were used to define recurrent CNVRs. Additionally, for each CMA sample the length and number of CNVs that represent loss\gain events were determined (referred hereafter as loss score\gain score). Genomic data were linked with pregnancy outcomes in women who subsequently gave birth at Hadassah. Multivariable logistic regressions were used to examine the associations of PTB with each CNVR and with loss\gain scores, adjusted for covariates.

Results: During the study period 10,650 prenatal CMA test were performed. A total of 48,070 autosomal CNVs (size 50Kb-1Mb) were found and used to define 411 CNVRs. We further analyzed pregnancy outcomes of 3,340 singleton live births with non-pathogenic CMA test results; of these 5.9% were PTB. A positive association was found between loss score and PTB (OR = 1.17, p = 0.035), adjusted to loss length and abnormal ultrasonographic findings. No significant associations were observed between any of the CNVRs and PTB.

Conclusions: Our findings suggest that non-pathogenic loss events have a cumulative effect on PTB risk, that is independent of the length of losses and irrespective of their specific genomic position.

Funding: Israel NIHP grant 2015/82.

Conflict of Interest: None declared.

EP02.006 Bi-allelic variations in CRB2, encoding the Crumbs Cell Polarity Complex Component 2, lead to non-communicating hydrocephalus due to atresia of the Aqueduct of Sylvius and central canal of the medulla

Aude Tessier 1;2;3, Nathalie Roux1, Lucile Boutaud1;2, Elodie Lunel1, Leila Hakkakian1, Mélanie Parisot4, Meriem Garfa Traore5, Amale Ichkou1, Nadia Elkhartoufi1, christine bole4, Patrick Nitschke6, Jeanne Amiel1;2, Jelena Martinovic7, Ferechte Razavi1, Tania Attie-Bitach1;2, Sophie Thomas2

1Hôpital Universitaire Necker-Enfants Malades, Service de Médecine Génomique des Maladies Rares, Paris, France; 2Imagine, INSERM U1163, Paris, France; 3Institute Pathology And Genetic, Charleroi, Belgium; 4Institute Imagine, Genomics Core Facility, Paris, France; 5INSERM-US24-CNRS UMS 3633, Cell Imaging Platform, Paris, France; 6Institute Imagine, Bioinformatics Platform, Paris, France; 7Antoine-Béclère Hospital, Clamart, France

Congenital hydrocephalus is a common condition caused by the accumulation of cerebrospinal fluid in the ventricular system. We report 3 cases from 2 families with congenital hydrocephalus due to bi-allelic variations in CRB2, a gene previously reported to cause nephrotic syndrome, variably associated with hydrocephalus. Neurohistopathological analysis allowed us to demonstrate that the pathological mechanisms underlying hydrocephalus are due to atresia of Sylvius Aqueduct and central medullar canal. While CRB2 has been largely shown crucial for apico-basal polarity, immunolabelling experiments in our fetal cases showed normal localization and level of PAR complex components as well as of tight and adherens junction molecules indicating a priori normal apicobasal polarity and cell-cell adhesion of the ventricular epithelium suggesting another pathological mechanism. Interestingly, atresia of Sylvius aqueduct was also described in cases with variations in MPDZ and CCDC88C encoding proteins previously linked functionally to the Crumbs (CRB) polarity complex, and all 3 being more recently involved in apical constriction, a process crucial for the formation of the central medullar canal. Overall, our findings argue for a common mechanism of CRB2, MPDZ and CCDC88C variations that might lead to abnormal apical constriction of the ventricular cells of the neural tube that will form the ependymal cells lining the definitive central canal of the medulla. Our study thus highlights that hydrocephalus related to CRB2, MPDZ and CCDC88C constitutes a separate pathogenic group of congenital non-communicating hydrocephalus with atresia of both Sylvius aqueduct and central canal of the medulla.

Grant reference : ANR-17-CE16-0003-01

Conflict of Interest: None declared.

EP02.007 Uncertainty in prenatal molecular diagnosis : dual diagnosis in two fetuses as new challenges for further debate on reporting policies

Maud Favier 1;2, Julian Delanne3, Guillaume Gorincour4, Yannis Duffourd1, Antonio Vitobello1, Aurore Garde5, Laurence Faivre1;5, Christophe Philippe1, Thierry Rousseau5, Aude Tessier2;6, Georges Tarris7, Camelia Oualiken7, Christel Thauvin-Robinet1;3;5, Frederic Tran Mau Them1;3

1Inserm UMR1231 GAD - Unité fonctionnelle innovation en diagnostic génomique des maladies rares, CHU Dijon Bourgogne, Dijon, France; 2SoFFoet - Société Française de Fœtopathologie, Paris, France; 3Centre de Référence Maladies Rares Anomalies du Développement et Syndromes malformatifs, Centre de Génétique, FHU-TRANSLAD, CHU Dijon Bourgogne, Dijon, France; 4Institut Méditerranéen d’Imagerie Médicale Appliquée à la Gynécologie, la Grossesse et l’Enfance (IMAGE 2), Marseille, France; 5Centre Pluridisciplinaire de Diagnostic Prénatal, CHU Dijon Bourgogne, Dijon, France; 6Institut de Pathologie et de Génétique, Namur, Région wallonne, Belgium; 7UMR1098 - Service de Pathologie, CHU Dijon Bourgogne, Dijon, France

Background: Prenatal Exome Sequencing (pES) offers parents the opportunity to guide choices regarding their pregnancy. It also allows detection of (likely) pathogenic variants involved in severe disorders but without phenotype-genotype correlation due to an early term of pregnancy or prenatally undetectable phenotype.

Methods: Two pregnancies were referred for trio-pES after ultrasound findings. At 10 + 1 weeks of gestation (WG), fetus 1 presented with persistent hygroma colli. At 24 WG, fetus 2 presented decreased movements, hypoplasic external genitalia, retrognathia, prefrontal edema, dilated tortuous aorta.

Results: In fetus 1, pES identified two homozygous variants, i.e. a ASCC1 nonsense involved in spinal muscular atrophy with congenital bone fractures and a CSPP1 nonsense involved in Joubert syndrome. External fetal examination revealed arthrogryposis but no autopsy was performed. In fetus 2, pES also identified two homozygous variants, i.e. a EFEMP2 missense involved in cutis laxa and a RAG1 nonsense involved in postnatal severe combined immunodeficiencies. Postnatal examination confirmed redundant skin.

Conclusion: pES analysis could be subject to partial or undetected phenotype during pregnancy. Current practice is to return only primary findings, i.e. (probably) pathogenic variants having clear phenotype/genotype correlation. Prenatal reporting of variants of uncertain significance (VUS) remains debated but ultrasound monitoring may reveal new signs to reclassify the variant. Should we use the terms “incidental findings” or VUS when phenotypes are prenatally undetectable? Is it ethical and not harmful to exclude variants whose impact on unborn children is certain and to avoid genetic counselling with consequences for future pregnancies?

Conflict of Interest: None declared.

EP02.008 Fetal genetic factors associated with sonographic abnormalities and pregnancy loss

Andrea Hadjipanteli 1, athina theodosiou1, ioannis papaevripidou1, paola evangelidou1, angelos alexandrou1, nicole salameh1, Violetta Anastasiadou2, Ioannis Kallikas3, Regina Pekri4, Andreas Stavroulis5, Niki Agathokleous6, Maria Agathokleous7, LUDMILA KOUSOULIDOU1, carolina sismani1

1The Cyprus Institute on Neurology and Genetics, Cytogenetics and Genomics, Nicosia, Cyprus; 2Archibishop Makarios III hospital, Nicosia, Cyprus; 3AAK Ultrasound and Fetal Medicine Centre, Nicosia, Cyprus; 4Novita Fetal Medicine Centre, Nicosia, Cyprus; 5American Medical Centre, Nicosia, Cyprus; 6Limassol Medclinic, Limassol, Cyprus; 7The fetal Medicine Centre Ltd, Limassol, Cyprus

Spontaneous pregnancy loss (SPL) is common during the first trimester of pregnancy and can be caused by various factors including large-scale chromosomal abnormalities and submicroscopic aberrations. However, in most SPLs that occur after the first trimester the aetiology remains undetermined. This study aims to resolve SPL cases of unknown aetiology by investigating the fetal genome and its effect on pregnancy outcome.

Twenty-nine samples were collected from fetuses that were spontaneously aborted, terminated or died neonatally. All fetuses had abnormal ultrasounds and no findings after karyotype and array-CGH. Trio-based whole-exome sequencing (WES) was performed to identify causative fetal variants.

Out of eighteen tested trios, causative/potentially causative variants were uncovered in six cases. A known de novo heterozygous missense variant within SCN2A was found in a fetus presenting Developmental and Epileptic Encephalopathy 11 phenotypes. Two inherited novel missense variants in SCN4A were found in a compound heterozygous fetus resulting in severe SCN4A-related congenital myopathy. A known homozygous nonsense variant in KLHL40 was found in a fetus with Nemaline Myopathy 8. Potentially causative heterozygous variants were identified in three cases, in genes USP18, CC2D2A and CPLANE1 with autosomal recessive inheritance.

We identified causative variants in 3/18 cases as well the possible involvement of heterozygous variants in genes USP18, CC2D2A and CPLANE1 in fetal development. Further investigation is required to assess the clinical significance of the latter findings. Accurate identification of variants in such genes creates new genotype-in utero phenotype associations, leading to the prospect of new additions in preconception and prenatal diagnostic panels.

Conflict of Interest: None declared.

EP02.009 Neonatal death due to F10 gene mutation

Lucie Slamova 1;2, Lucie Šrámková1, Martina Sekowska2, Monika Koudová2

12nd Medical School, Charles University, Department of Pediatric Hematology and Oncology, Prague, Czech Republic; 2GENNET - Center of Medical genetics and reproductive medicine, Center of Medical genetics and reproductive medicine, Prague, Czech Republic

Non-consanguineous couples was referred to genetic counseling for neonatal death. First pregnancy was extrauterine, next pregnancy was terminated due to Turner syndrom. After two years female proband got pregnant spontaneously. Prenatal screening was negative and pregnancy was physiological and full term with vaginal delivery. Five hours after delivery new born presented with gastrointestinal bleeding, cyanosis, development of petechiae, dyspnoea, life-threatening bleeding and decease in 18 hours of life due to hemorrhage. Undelying genetic disease was unknown. Clinical exome sequencing was performed with KAPA HyperExome solution (Roche) on NextSeq (Illumina) as trio exome analysis postmortem. Analysis uncover missense variant c.166G>A (p.Glu56Lys) (maternal origin) and terminal variant c.837C>A (p.Tyr279Ter) (paternal origin) in F10 gene. None of the variants have been found yet. Neonate was compound heterozygous in F10 gene´s variants. Factor X is a vitamin K dependent, liver produced serine protease which forms the prothrombine complex convering prothrombin to thrombin. Inherited factor X deficiency is rare autosomal recessive bleeding disorder with prevalence 1: 1000000 individuals, reccurence risk for our couple does 25%. Severity of clinical presentation was unexpectedly grave. In time of second genetic counselling female proband was pregnant after spontaneous conception. Invasive prenatal diagnosis was performed, fetus was as well compound heterozygous for both variants in F10 gene, pregnancy was terminated due to miscarrige. Preimplantation genetic testing (PGT) was proposed, couple went through in vitro fertilization with PGT. Six embryos was retreived but only one was eligible for transfer. Female proband gets pregnant after the embryo transfer, waiting for prenatal screening.

Conflict of Interest: None declared.

EP02.010 Plasma miRNA Profile in High Risk of Preterm Birth during Early and Mid-Pregnancy

Anastasia Maltseva 1, Roman Illarionov1, Olga Pachuliia1, Elena Vashukova1, Alexander Tkachenko2, Tatyana Postnikova1, Andrey Glotov1

1D.O. Ott Research Institute for Obstetrics, Gynecology, and Reproduction, Department of Genomic Medicine, Saint Petersburg, Russian Federation; 2ITMO University, Institute of Applied Computer Sciences, Saint Petersburg, Russian Federation

Background/Objectives: In recent years, evidence has been accumulated showing that miRNAs can act as potential biomarkers or targets for therapy of preterm birth (PTB), one of the most important problems in modern obstetrics. We have performed a prospective study of the miRNA profile in the plasma during the first and second trimesters in pregnant women with high risk of preterm birth.

Methods: Total of 48 blood plasma samples were taken from 24 women in the first and second trimesters. Small RNA isolation and library preparation for sequencing was performed using miRNeasy Serum/Plasma Kit (Qiagen) and QIAseq miRNA Library Kit (Qiagen). Libraries were sequenced on a HiSeq 2500 (Illumina). Bioinformatic data analysis was performed using the GeneGlobe Data Analysis Center and DESeq2 R Package.

Results: We detected differences in the levels of 15 miRNAs (3 upregulated—hsa-miR-122-5p, hsa-miR-34a-5p, hsa-miR-34c-5p; 12 downregulated—hsa-miR-487b-3p, hsa-miR-493-3p, hsa-miR-432-5p, hsa-miR-323b-3p, hsa-miR-369-3p, hsa-miR-134-5p, hsa-miR-431-5p, hsa-miR-485-5p, hsa-miR-382-5p, hsa-miR-369-5p, hsa-miR-485-3p, hsa-miR-127-3p) (log2(FC) ≥ 1.5; FDR ≤ 0.05) during the first trimester compared with the control (non-high-risk of preterm birth pregnant women). All downregulated miRNAs in the first trimester from the placenta-specific C14MC cluster. During the second trimester no differentially expressed miRNAs were found.

Conclusion: Our results suggest that the miRNA profile in plasma during early pregnancy may predict a high risk of preterm birth, which is important in preventing gestational problems as early as possible. Identified miRNAs can be used as PTB biomarkers.

Grant References: This study was financially supported by a Russian Science Foundation grant 19-75-20033.

Conflict of Interest: None declared.

EP02.011 A rare prenatal case of osteogenesis imperfecta

FLORINA MIHAELA NEDELEA1, Diana Prepelita 2;3, Andreea Tutulan-Cunita2, Anca Pavel2, Georgeta Cardos4, Mariana Predescu4, Brandusa Cimpoca4, Simona Duta4, Ana Maria Vayna4, Alina Veduta4, Gheorghe Peltecu3;4, Anca Panaitescu3;4

1Filantropia Clinical Hospital, Genetics, București, Romania; 2Cytogenomic Medical Laboratory, București, Romania; 3Carol Davila University of Medicine and Pharmacy, București, Romania; 4Filantropia Clinical Hospital, București, Romania

Background/Objectives: We present a case referred to our Clinic in the second trimester of pregnancy for “second opinion” because the fetal ultrasound identified bilateral, curved, short femur (below 3rd percentile). Also general shortening of the long bones was noted. Subsequent ultrasound at 29 weeks revealed long bones under 1st percentile, femur bent in a “phone handle”. Rib cage was subjectively smaller than gestational age. Some of the possible diagnoses in this case included skeletal dysplasias, such as thanatophoric dysplasia, campomelic dysplasia osteogenesis imperfecta, chondroectodermal dysplasia, Jeune dystrophy. Due to high risk of genetic syndrome, prenatal diagnosis was advised.

Methods: Amniocentesis was performed after the first evaluation in our Hospital. Apart from fetal karyotype with normal result, a gene panel (10 genes) for skeletal dysplasias was performed.

Results: Gene panel analysis identified a missense heterozygous pathogenic variant, c.2596G>A (p.Gly866Ser) in exon 37 out of 51 of COL1A1 gene, responsible for osteogenesis imperfecta, more commonly associated with types II and III. It should be mentioned that type II has an increased risk of perinatal lethality, and in the case of type III, bone deformities are associated and progressive.

Conclusion: In conclusion, the multidisciplinary approach, involving feto-maternal specialists and geneticist is important for such complex cases. Prenatal testing confirmed the diagnosis, being able to predict the outcome and the familial reccurence risk.

Conflict of Interest: None declared.

EP02.012 Non-invasive prenatal testing (NIPT) in Bizkaia: a 10-year experience

IRATXE HUERTA1;2, Mertxe Télez1, javier muga1, jaime bordas3, Yaima TORRES4, javier suela4, Maria Garcia-Barcina 1

1IMQ Análisis Clínicos, Genetics, Bilbao, Spain; 2University of Deusto, Bilbao, Spain; 3IMQ Análisis Clínicos, Quality Department, Bilbao, Spain; 4NIMGenetics Laboratory, Non invasive Department, Madrid, Spain

Objectives: Prenatal diagnostic testing involves all the analysis done before birth (prenatally) to determine whether the fetus has certain abnormalities, as hereditary or spontaneous genetic disorders. Currently, there are different detection strategies that include serological and genetic markers. Among them, the emerging non-invasive prenatal test (NIPT) is changing the practice of prenatal diagnosis worldwide due to its benefits and its demand. The objective of our study is to evaluate the diagnostic capacity of the NIPT in screening for common aneuploidies.

Methods: This descriptive and retrospective study includes the analysis of cell free fetal DNA (cffDNA) in maternal blood using the non-invasive prenatal test TrisoNIM® (all its modalities) of 4.717 pregnant women in Bizkaia between January 2013 and December 2022.

Results: 1.21% of the total analyses have been classified at high-risk for common chromosomopathies. Trisomy 21 and sexual aneuploidies account for the majority of these cases, specifically 80%. Of the total number of cases confirmed by invasive techniques (chorionic biopsy or amniocentesis), the concordance has been almost complete for trisomies 13 and 21, while it is practically reduced by half for trisomy 18 and sexual aneuploidies.

Conclusión: Our results confirm the high sensitivity and specificity of the NIPT, and support its use as an incomparable prenatal screening test for fetal chromosomal abnormalities.

Grant References: European Society of Human Genetics (2015). Non-invasive prenatal testing for aneuploidy and beyond: challenges of responsible innovation in prenatal screening. Summary andrecommendations.

Conflict of Interest: None declared.

EP02.013 Impact of prenatal ultrasound findings and parental SNP microarray testing in pregnancy management

Irina Ioana Iordanescu 1;2, Anca Teodora NEACSU2, Mariela Sanda Militaru2;3, Andreea Catana2;3, Zina Barabas Cuzmici2, Diana Elena Voicu2, Cristina Dragomir2, Emilia Severin1

1University of Medicine and Pharmacy “Carol Davila”, Bucharest, Romania; 2CMU Regina Maria, Bucharest, Romania; 3University of Medicine and Pharmacy “Iuliu Hatieganu”, Cluj Napoca, Romania

Background: We present two pregnancies of a phenotypically normal couple with the same inherited maternal 1q21.1 microdeletion. The 1q21.1 isolated microdeletion is associated with a variable phenotype from the absence of symptoms to microcephaly, mild to moderate global developmental delay, facial dysmorphism, seizures, heart malformations, ADHD. Also, this microdeletion in trans with a mutation in the RBM8A gene is associated with TAR (Thrombocytopenia-absent radius) syndrome.

Methods: For both pregnancies prenatal diagnosis was performed using QF-PCR and SNP microarray.

Results: In the first pregnancy in the 22nd week of gestation the ultrasound revealed the bilateral absence of the radius. Amniocenteses and SNP microarray analysis were performed and a 453Kb microdeletion was identified in the 1q21.1 region. TAR syndrome was suspected and this pregnancy was terminated.

In the second pregnancy, a NIPT test was performed with a Turner syndrome high risk result. CVS, QF-PCR and SNP microarray were performed. The QF-PCR result was negative. The SNP microarray analysis identified again a 576kb microdeletion in the 1q21.1 region. The ultrasound revealed no malformations. Following this result, the couple was tested using SNP microarray and the mother was found to be a carrier for the 1q21.1 microdeletion. Genetic counseling was performed and considering that the mother is phenotypically normal and the fetal ultrasound was normal, the couple decided to continue the pregnancy.

Conclusion: In this couple’s decision to continue or interrupt the pregnancies, both the fetal ultrasound and the parental SNP microarray testing were decisive.

Conflict of Interest: None declared.

EP02.014 Prenatal diagnosis of mosaic trisomy 15 and maternal uniparental disomy 15

Veronica Barbieri1, Chiara Cattani 1, Simonetta Rosato1, Roberta Zuntini1, Stefano Caraffi1, Giuseppina Comitini2, Mariangela Pati2, Maria Marinelli1, Livia Garavelli1

1Azienda USL-IRCCS of Reggio Emilia, Medical Genetics Unit, Reggio Emilia, Italy; 2Azienda USL-IRCCS of Reggio Emilia, Obstetrics and Gynecology Unit, Reggio Emilia, Italy

We describe a case of mosaic trisomy 15 and maternal uniparental disomy 15 [UPD(15)mat] in a fetus with unilateral hydronephrosis.

Cytogenetic analysis on amniotic fluid revealed a mosaic karyotype of 47,XX, +15/46,XX. Array-CGH analysis detected a whole chromosome 15 duplication of approximately 20%. Microsatellite and MLPA analyses confirmed the same mosaicism percentage of chromosome 15 defining its maternal origin. Furthermore, MLPA showed the presence of hypermethylation of the SNRPN locus at 15q11, indicating the presence of UPD(15)mat in approximately 40% of disomic cells.

Given the extreme rarity of the mosaic trisomy 15 condition (fewer than 20 cases liveborns described) and the variability of the clinical features reported in the literature, predicting the expected phenotype is challenging. In our case, the percentage of trisomic cells would seem comparable with cases with normal outcome, however the presence of UPD(15)mat represents a further reason of uncertainty.

In general, UPD following trisomy rescue mechanism can be found associated with placental or fetal mosaicism. In particular, UPD(15)mat associated with mosaic trisomy seems to be characterized by a distinct phenotype from the classic Prader-Willi Syndrome one, with a higher incidence of congenital heart disease and worse cognitive and behavioral outcome.

The study of the fetal morphology showed a left unilateral hydronephrosis, accentuated echogenicity of the right kidney and absence of heart defects or other abnormalities. A clinical evaluation and genetic investigations are planned at birth to assess the extent of mosaicism and UPD.

Conflict of Interest: None declared.

EP02.015 Maternal translocation t(7;9) resulting in a not previously described unbalanced fetal karyotype with trisomy 9p and partial trisomy 7q

Sabine Naumann 1, Dominique Schwank1, Christine Sattler1, Franziska Klee1, Udo Koehler2, Kai Sendelbach2, Lucas Wilhelm3, Maria Korte1

1IMD Humangenetik Wiesbaden, Wiesbaden, Germany; 2MGZ - Medizinisch Genetisches Zentrum, Munich, Germany; 3Westend Ultraschall, Frankfurt, Germany

Background/Objectives: Cytogenetic analysis of the amniotic fluid of a fetus with conspicuous ultrasound results and suspected trisomy 18 revealed a different cause for fetal symptoms

Methods: Prenatal ultrasound examination; rapid prenatal testing by fluorescence in situ hybridisation (FISH); prenatal chromosome analysis combined with additional FISH analysis; prenatal microarray-based comparative genomic hybridization (array CGH); chromosome analysis of both parents

Results: Ultrasound examination of a fetus at 31 + 4 weeks of gestation showed IUGR with polyhydramnia, ventriculomegaly, complex vitium (AVSD, DORV), rocker bottom feet, flat profile with a receeding chin, and a clenched right hand. Trisomy 18 was suspected based on the clinical picture.

Rapid prenatal testing by FISH showed a regular result; no trisomy 18 was detected.

Prenatal chromosome analysis, additional FISH analysis, fetal array CGH analysis, and chromosome analysis of the parents revealed the following chromosome complements:

Fetal: 47,XY,+der(9)t(7;9)(q31;q21)dmat

Maternal: 46,XX,t(7;9)(q31.2;q21.1)

Paternal: 46,XY

Conclusion: By using complementary cytogenetic analyses, the cause for the abnormal prenatal ultrasound result was found and was of great importance in genetic counselling regarding prenatal clinical prognosis. However, in cases with conspicuous ultrasound results, performance of array CGH analysis as the method of choice would save time. Unfortunately, the German health system complicates the conduct of array CGH analysis, especially for time-sensitive prenatal cases. The fetal karyotype with an additional der(9)t(7;9) resulted from the maternal balanced t(7;9) by quadrivalent forming during meiosis I, followed by 3:1 segregation with tertiary trisomy. A 3:1 segregation is rare in comparison with the more common 2:2 segregation.

Conflict of Interest: None declared.

EP02.017 Three genetic polymorphisms of MTRR, MTR and AGT in association with fetal growth restriction susceptibility

dema alset 1, Elena Butenko1, Tatiana Shkurat1, Ekaterina Zabanova2, Natalia Kuznetsova2

1Academy of biology and biotechnology, Southern Federal University, Department of Genetics, Rostov-on-Don, Russian Federation; 2Rostov State Medical University, Rostov-on-Don, Russian Federation

Background/objectives: Fetal Growth Restriction (FGR) is a pregnancy-associated condition with a frequency of more than 10% of pregnancies worldwide. It is known to cause multiple adverse outcomes on fetal, postnatal and adult quality-of-life. Therefore, prenatal prognosis and diagnosis is of high importance. It does not exist yet, but scientists are focusing on finding new genetic candidates as FGR biomarkers. The purpose of the presented research is to study the association of maternal genetic variants: Methionine synthase reductase-MTRR(A66G), Methionine synthase-MTR(A2756G) and Angiotensinogen-AGT(T704C) with FGR susceptibility.

Methods: For FGR-diagnosed (n = 46) and healthy (n = 57) pregnant women, leukocytes DNA was extracted using NK-sorbent «Base»; (Lytech. Co. Ltd. Russia) kit. Allele-specific PCR was used for genotyping and MDR was used to analyze SNP-SNP interactions.

Results: Our data showed that MTRR(66GG) genotype is significantly associated with higher FGR risk (OR = 3.18, 95%CI:1.31-7.72, P = 0.025). On the other hand, AGT(704C) allele has a protective effect and is associated with lower FGR susceptibility (OR = 0.58, 95%CI:0.32-1, P = 0.049). These findings were supported by the antagonistic interaction of MTRR(A66G) with AGT(T704C) shown in MDR-analysis. MDR also figured that MTRR(A66G) has the highest predictive potential among the three studied polymorphisms.

Conclusion: This study suggests MTRR(A66G) and AGT(T704C) as candidate markers of FGR risk. Results also showed that these polymorphisms have opposite roles in FGR pathophysiology with MTRR(A66G) as a risk factor and AGT(T704C) as protective one. Future trials are recommended to confirm findings.

This study was funded by the Ministry of Science and Higher Education of the Russian Federation #0852-2020-0028.

Conflict of Interest: None declared.

EP02.018 Multiple prenatally detected huge duplications. How to deal with it?

Cornelie Müller-Hofstede 1, Yvonne Stratis1, Malvina Kuschmann1, Renate Rosenberg2, Axel Bohring1, Albrecht Röpke1

1Institut für Humangenetik, Universitätsklinikum, Münster, Germany; 2Praxis für Pränatalmedizin am Bült, Münster, Germany

Background/Objectives: We report on a 27-year-old woman presented at 11th gestational week because of increased nuchal translucency and generalized hydrops in the fetus.

Methods: Cytogenetic analysis and SNP-array were performed.

Results: Chromosome analysis on chorionic villus sampling was apparently normal after short-term culture, however revealed a conspicuous karyotype with a derivative chromosome 17 (46,XY,dup(17)(q2?3q2?5)) after long term culture.

For further characterization, an SNP-array analysis was performed and confirmed a gain of 12Mb in 17q24.2-17q25.3, encompassing 198 genes, many of them disease-associated. Moreover, three additional gains, 6.2Mb in 2q31, 8.9Mb in 4q12-4q13.1, and 7.3Mb in 8q23.1-8q23.3 were detected, all four de novo.

To prove the possibility, that these CNVs might be restricted to the placenta only, an amniocentesis was performed. Contrary to the expectations, all four abnormalities were recovered in the amniotic fluid cells too.

Follow-up-sonography at 16th gestational week then showed shortening of bones, complex heart defect, brain anomalies, hepatomegaly and polyhydramnios.

The boy was born at 35th week of gestation with weight 2490 g and length 46 cm after labor induction with planned palliative care. Initially the boy was “stable” and discharged home on day two. Because of health deterioration parents then requested for maximum care and heart surgery, but the child died at the age of two weeks.

Conclusion: This case illustrates that multiple large duplications encompassing many disease-associated genes, unexpectedly can be present in the fetus itself (and need not be restricted to the placenta) and that these do not per se preclude a (limited) viability.

Conflict of Interest: None declared.

EP02.019 Reduction of screen positive rate of 10qter deletions in noninvasive prenatal testing by paired-end sequencing

Elmira Amiri 1, Victoria Corey2, Franchesca Liao2, Theresa Boomer2, Susan Hancock2, sung kim2, Eileen de Feo2

1Illumina, Inc., Cambridge, United Kingdom; 2Illumina Inc, San Diego, United States

Objectives: Noninvasive prenatal testing (NIPT) sequences both maternal and fetal cell-free DNA (cfDNA) isolated from the plasma of pregnant women. NIPT results, therefore, may be confounded by maternal factors. One such example involves 10qter deletions where maternal mosaicism has been implicated in false positives. cfDNA size has been shown to be correlated to its origin where maternal cfDNA fragments are on average larger than fetal cfDNA. This study will evaluate NIPT screen positive rates (SPR) of 10qter when cfDNA size information is taken into consideration.

Methods: Retrospective data from a low pass paired-end whole genome sequencing NIPT assay of 112,250 clinical samples were used. The samples were not screened for partial chromosomal deletions/duplication at the time of testing. Exploratory analysis of the sequence data collected under IRB was performed to assess the impact of leveraging cfDNA size information on the SPR of 10qter deletions should patients have opted for a genome-wide screen.

Results: 16 samples were initially found to have a deletion spanning 10q23 to 10q26.3. When cfDNA size information was considered, 15 of 16 deletions would screen negative for 10qter potentially resulting in a SPR of less than 1:110,000.

Conclusions: Methods that can differentiate maternal vs fetal signal are important to improve the accuracy of NIPT. This study aims on improving NIPT by taking into consideration known size differences between maternal and fetal cfDNA. Although further clinical validation is needed, these results suggest cfDNA size information may assist in reducing overall screen positive rates potentially incurred by maternal factors.

Conflict of Interest: None declared.

EP02.020 Costello syndrome in our population: report of three cases

Jorge Rodríguez-Afonso1, Nayra Pérez-Delgado 1, Carol Prieto-Morín1, Ruth López-Travieso1, Francisco Martínez Bugallo1, Inmaculada García-Cobaleda1, Alba Rodríguez-González2

1Hospital Universitario Nuestra Señora de la Candelaria, Clinical Analysis Service, Genetic Unit, Santa Cruz de Tenerife, Spain; 2Hospital Universitario Nuestra Señora de la Candelaria, Gynaecology and Obstetrics Service, Santa Cruz de Tenerife, Spain

Background: Costello syndrome (CS) is a RASopathy caused by heterozygous gain-of-function germline variants in HRAS. The majority of cases are described postnatally, however, a series of suggestive prenatal abnormalities such as hypertrophic cardiomyopathy, fetal hydrops or increased nuchal translucency (NT) may occur. Its exact prevalence is unknown, with a range in the literature from 1/1,250,000 to 1/300,000.

Methods: We report 3 cases of CS confirmed by clinical exome sequencing (CES), due to pathogenic or probably pathogenic variants in the HRAS gene (ACMG 2015 criteria).

Results: Case 1. A 25-week gestational premature girl with clinical suspicion of RASopathy. CES identified pathogenic heterozygous variant NM_001130442: c.34G>T(p.Gly12Cys) in HRAS gene. The patient finally died at 15 days of life due to cardiogenic shock secondary to hypertrophic cardiomyopathy.

Case 2. 33-year-old woman, 22 weeks pregnant. The ultrasound examination revealed a fetus with few movements and hands in permanent hyperflexion. CES identified pathogenic heterozygous variant NM_001130442: c.35G>A (p.Gly12Asp) in HRAS gene. The pregnancy was interrupted.

Case 3: 29-year-old woman, 11 weeks pregnant. Ultrasound evidenced cystic hygroma (TN = 15.7 mm). CES identified likely pathogenic heterozygous variant NM_001130442: c.38G>T (p.Gly13Val) in HRAS gene. The pregnancy was interrupted.

Conclusion: Three cases of CS were diagnosed in our center within a period of 3 years, despite a yearly delivery rate of 2,700. These findings suggest a higher prevalence of the disease in our population than that reported in the literature. This fact may be justified because the recent incorporation of CES into prenatal diagnosis reveals a prior underdiagnosis.

Conflict of Interest: None declared.

EP02.021 Prenatal exome sequencing, a powerful tool to describe unreported prenatal features of monogenic disorders

Aurore Garde 1;2, Frédéric Tran Mau-Them2;3, Yannis Duffourd2, Antonio Vitobello2;3, Christophe Philippe2;3, Christel Thauvin-Robinet1;2;3, Laurence Faivre1;2, Nicolas Bourgon2;4

1CHU Dijon Bourgogne, Centre de Référence Maladies Rares “Anomalies du développement et syndromes malformatifs”, Centre de Génétique, FHU TRANSLAD et Institut GIMI, Dijon, France; 2INSERM UMR 1231, Dijon, France; 3CHU Dijon Bourgogne, Unité Fonctionnelle Innovation en Diagnostic Génomique des maladies rares, Dijon, France; 4Necker University Hospital, Obstetrics and Fetal Medicine, Paris, France

Background/Objectives: Prenatal exome interpretation may be difficult because of unknown or restricted fetal features available and limited prenatal data in databases. In this study, we report the prenatal ultrasound phenotype of five monogenic disorders.

Methods: We retrospectively analyzed the 56/150 cases harboring a causal diagnosis in the “AnDDI-Prenatome study”. For each causal monogenic disorder, we investigated if a prenatal onset was previously reported in a public phenotype-genotype databases or in the literature.

Results: 5/56 fetuses presented a prenatal ultrasound phenotype unreported. Causal variants were identified in two gene previously involved in syndromic or not ID/DD (LINS1, GNB2), and three genes associated with a monogenic disorder with a pediatric onset (ZNF148, ASXL1, PGM1).

In three cases (ZNF148, ASXL1, PGM1 variants), causal variants were identified in genes involved in disorders with highly variable postnatal features. The identification of causal variant was possible because of a partial overlap between prenatal and postnatal phenotypes, in silico prediction tools and suspected inheritance. A causal heterozygous de novo GNB2 variant was identified in a fetus with hydrops and IUFD. Although prenatal features have also been reported in one family, we reported an extreme phenotype leading to IUFD. Finally, a bi-allelic truncating LINS1 variant was identified in a male fetus with increased nuchal translucency, IUGR and asymmetric cerebral ventricles. To date, bi-allelic LINS1 variants are involved in non-syndromic ID/DD.

Conclusion: pES allows to accurately describe prenatal phenotype of monogenic disorders. It’s now time to collect all prenatal onset of monogenic disorder in database to help pES analysis.

Conflict of Interest: None declared.

EP02.022 Rare case of androgenetic-biparental mosacism causing placental mesenchymal dysplasia

Ivana Joksic 1, Mina Toljic1, Zagorka Milovanovic2;3, Marko Dzuverovic4, AndjelaS Stankovic1, Zeljko Mikovic2;3

1Gynecology and obstetrics clinic “Narodni front’, Genetic laboratory department, Belgrade, Serbia; 2School of Medicine University of Belgrade, Belgrade, Serbia; 3Gynecology and obstetrics clinic “Narodni front’, Belgrade, Serbia; 4Gynecology and obstetrics clinic “Narodni front’, Pathology laboratory department, Belgrade, Serbia

Background/objectives: Placental mesenchymal dysplasia (PMD) is rare placental vascular anomaly characterized by placentomegaly and grapelike vessels without throphoblast proliferation. It can be associated with adverse fetal outcomes such as intrauterine demise, growth restriction or Beckwith Wiedemann syndrome, but its cause mostly remains unknown.

Case report: A 30-year old gravida was referred to our center at 13 gestation weeks due to enlarged placenta with multiple anechoic cyst detected by ultrasound and normal appearing fetus.

Results: Chorionic villi sampling was performed. QF PCR for common aneuplodies (Aneufast kit) markers showed tetraploidy with extra set being of paternal origin (comparison of fetal short tandem repeat (STR) markers with parental STR markers was done), thus implying presence of triandric teraplody or androgenic biparental mosaicism (ABM). Karyotype analysis showed normal female karyotype, and thus confirming ABM in placental tissue. Subsequent QF PCR and karyotype analysis from amniotic fluid showed normal female karyotype of biparental origin. After pregnancy termination, histology examination and immunohistochemisty showed nuclear expression of p57 in cytothrophoblast cells with stem villous edema and loss of nuclear expression in stromal villous cells, pattern characteristic of ABM.

Conclusion: PDM is usually associated with diploid karyotype. Our case confirms ABM as a rare cause of PMD. Diagnosis of PMD is challenging due to its ultrasound resemblance to more common placental pathologic conditions such as molar pregnancies. Genetic studies as well as immunohistochemisty are need in order to establish appropriate diagnosis, thus enabling adequate pregnancy management.



Conflict of Interest: None declared.

EP02.024 Genetic prenatal diagnosis of Lethal congenital contracture syndrome 11

Miriam Potrony 1;2;3, Antoni Borrell4, Narcis Masoller4, Alfons Nadal5, Leonardo Rodriguez-Carunchio5, Karmele Saez de Gordoa Elizalde5, Juan Francisco Quesada-Espinosa6, José Villanueva-Cañas7, Montse Pauta4, Meritxell Jodar1;2, Irene Madrigal1;2;3, Celia Badenas1;2;3, Maria Isabel Alvarez1;2;3, Laia Rodriguez-Revenga1;2;3

1Hospital Clinic of Barcelona, Biochemistry and Molecular Genetics Department, Barcelona, Spain; 2FRCB-IDIBAPS, Barcelona, Spain; 3CIBER of Rare Diseases (CIBERER), Barcelona, Spain; 4Hospital Clinic of Barcelona, Universitat de Barcelona, BCNatal, Barcelona Center for Maternal-Fetal and Neonatal Medicine, Institut Clínic de Ginecologia, Obstetricia i Neonatologia Fetal i + D Fetal Medicine Research Center, Barcelona, Spain; 5Hospital Clinic of Barcelona, Pathology Department, Barcelona, Spain; 612 de Octubre University Hospital, Genetics Department, UDISGEN (Unidad de Dismorfología y Genética), Madrid, Spain; 7Hospital Clinic of Barcelona, Molecular Biology CORE (CDB), Barcelona, Spain

Background/Objectives: Arthrogryposis is characterized by congenital joint contractures in two or more body areas resulting from reduced or absent fetal movements. Prenatal ultrasound imaging is crucial in early diagnosis and genetic diagnosis is important for counseling. Our aim was to perform the genetic diagnosis of a fetus with ultrasound alterations exhibiting hydrops, short long bones, fixed limb joints, absent fetal movements, and polyhydramnios at 28 weeks of gestation.

Methods: DNA from uncultured amniocytes was analyzed using the qChipPrenatal microarray (qGenomics). Whole exome sequencing (WES) was further performed using DNA Prep with Exome Enrichment on a NextSeq 500 (Illumina). Variant classification was performed according to the ACMG recommendations.

Results: The microarray results revealed a normal female profile, arr(X, 1 − 22) × 2. WES evidenced a compound heterozygous for the GLDN (NM_181789) gene: c.62C>A p.(Ala21Glu) and c.1494G>T p.(Leu498Phe). Lethal congenital contracture syndrome 11 (OMIM # 617194) is an autosomal recessive syndrome caused by GLDN pathogenic variants. The literature was reviewed and 28 additional cases were collected. A distinguishing clinical feature described in the majority of patients was pulmonary hypoplasia and six patients survived beyond the neonatal period.

Conclusion: The present reported case and the literature review confirms the association of biallelic GLDN variants with arthrogryposis and other phenotypic spectra such as pulmonary hypoplasia, reaffirming it should be better classified as fetal akinesia deformation sequence (FADS). Prenatal diagnosis of this condition is challenging since and WES should be recommended when a FADS is suspected.

Grant References: Fundación Mutua Madrileña (Grant/Award Number: AP171442019).

Conflict of Interest: None declared.

EP02.025 Non-immune hydrops fetalis is associated with variants in the MYB Binding Protein 1a (MYBBP1A) gene

Elena Mansilla1;2;3, Jair Tenorio1;2;3, Julián Nevado 1;2;3, Eugenia Antolin4, Roberto Rodriguez-González4, Rita María Regojo Zapata5, Fe Amalia García Santiago1;2;3, Pedro Arias1;2;3, Natalia Gallego1;2;3, alejandro parra1;2;3, Patricia Pascual Vinagre1;2;3, Mario Cazalla1;2;3, Pablo LAPUNZINA1;2;3

1Hospital Universitario la Paz, INGEMM, Madrid, Spain; 2CIBERER, Madrid, Spain; 3ITHACA, Brussels, Belgium; 4Hospital Universitario la Paz, Fetal Physiopathology, Madrid, Spain; 5Hospital Universitario la Paz, Pathology, Madrid, Spain

Background: Non-immune hydrops fetalis (NIHF) is an extremely infrequent entity usually characterized by an excessive accumulation of fetal fluid within the fetal extravascular compartments and body cavities. Etiology of NIFH is highly variable with a proportion of idiopathic unknown cases, or associated with syndromic disorders.

Here we present a fetus death at 27 + 3 weeks with a NIHF presenting with oligohydramnios, cystic hygroma, pleural effusion, echography with generalized hydrops with predominance of subcutaneous edema. The fetus also presented with ascites, severe and precocious IUGR and some skeletal anomalies.

Methods and Results: Whole exome sequencing in a trio way was applied in order to screen for a possible genetic pathogenic variant. Variant prioritization according to a custom in-house algorithm allowed to identify two variants in MYBBP1A, one nonsense (NM_001105538.2:c.238G>T:NP_001099008.1:p.Gly80Ter) and one canonical splice-site variant (NM_001105538.2:c.3196-2A > G:NP_848696.1:p.Leu177Argfs*20), each inherited from a healthy parent. A previous report (PMID:28425981) described another case with similar phenotype with a compound heterozygous variant in MYBBP1A (one identical to our patient). The two variants are predicted to be damaging by the in silico tools applied. The protein encoded by MYBBP1A play a role in many cellular processes including response to nucleolar stress, tumor suppression and synthesis of ribosomal DNA.

Discussion: Therefore, we suspect that MYBBP1A can be a strong candidate gene associated with the development of hydrops fetalis. It is necessary to collect more cases and further studies to understand the role of this gene and the mechanism associated with the development of the prenatal malformation.

Grant: FIS020/01053 & 018/01433

Conflict of Interest: None declared.

EP02.026 NIPT high-risk of trisomy 16 as a predictor of adverse pregnancy outcome

Madina Kaplanova 1, Aleksandra Galaktionova1, Alexander Potapov1, Elena Baranova1;2, Olesya Sagaidak1, Maxim Belenikin1, Ekaterina Kuznetsova1, Sergei Martirosyan3

1LLC Evogen, Moscow, Russian Federation; 2Federal 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; 3Moscow State Budgetary Institution of Healthcare “L.A.Vorohobov City Clinical Hospital No.67 MHD”, Moscow, Russian Federation

Background/Objectives: Trisomy 16 (T16) is a chromosomal abnormality (CA) with extra chromosome 16 in every cell (full trisomy) or in some cells (mosaic trisomy), that may also predict adverse pregnancy outcomes in 68% of cases. The non-invasive prenatal test (NIPT) has become widely used in prenatal screening for CAs. NIPT allows to determine the risks of rare CAs, including T16. Here we present 4 clinical cases with T16 and adverse pregnancy outcome.

Methods: Whole genome NIPT was performed for 4 pregnant women with low PAPP-A levels (<0.3 MoM): 3 from the first trimester prenatal screening high-risk group for CA and 1 from the low-risk group for CA. Patients with high-risk NIPT results were recommended to undergo invasive prenatal diagnostics with karyotyping or/and chromosomal microarray analysis (CMA).

Results: Amniotic fluid karyotyping results indicated that all fetuses had normal karyotypes. For 2 cases amniotic fluid CMA was performed – in 1 case no CA was detected, in the other case mosaic T16 (27%) was detected. In all cases T16 was detected in placenta (CMA or karyotyping was performed). 3 women continued their pregnancies (including mosaic T16 – the newborn had Tetralogy of Fallot), all newborns had growth retardation. 1 woman had late-term loss.

Conclusion: The high-risk T16 detected by NIPT may predict an adverse pregnancy outcome despite the normal fetal karyotype.

The work was supported by the Moscow Department of Health grant.

Conflict of Interest: None declared.

EP02.027 Non-invasive prenatal testing positive results for 22q11.2 deletion syndrome in two consecutive pregnancies of a woman harboring an atypical 22q11.2 deletion

Irena Bradinova 1, Radoslava Vazharova2, Stoyan Bichev3, Radostina Raynova4, Alexey Savov4

1Medical University Sofia, National Genetic Laboratory, Sofia, Bulgaria; 2SU “St. Kl. Ohridski”, Faculty of Medicine, Department of Biology, Medical genetics and Microbiology, Sofia, Bulgaria; 1Medical University Sofia, National Genetic Laboratory, Sofia, Bulgaria; 1Medical University Sofia, National Genetic Laboratory, Sofia, Bulgaria

Background: The 22q11.2 deletion syndrome, also known as DiGeorge or velocardiofacial syndrome, results from submicroscopic deletion of a defined part of chromosomal band 22q11.2. It is the most common microdeletion syndrome (1 in 3000–6000 live births) and has a heterogeneous clinical presentation that can affect multiple organ systems with widely varying degrees of severity. Advances in non-invasive prenatal testing (NIPT) technology have allowed coverage of a set of microdeletions with high penetrance and severe phenotype, including 22q11.2 deletion syndrome.

Methods: A 38-year-old healthy woman with no significant history of genetic disorders was referred for genetic counseling because of a positive NIPT result for 22q11.2 deletion (estimated at 0.65 Mb, likely of maternal origin) of her second pregnancy. NIPT at her first pregnancy was also positive for 22q11.2 deletion and the subsequently performed prenatal diagnosis using chorion villus sampling with MLPA for microdeletion syndromes showed a normal result. Spontaneous pregnancy loss occurred later.

Results: MLPA (P245, P064) for 22q11 deletion of DNA extracted from patient’s blood detected a deletion of 3 close located gene probes SNAP29, MED15, ZNF74 (0,5Mb) and normal amplification of 8 other probes at 22q11.21. After genetic counseling concerning the presence of a small atypical 22q11.2 deletion and its clinical and reproductive implications the couple decided not to undertake an invasive prenatal diagnosis of this pregnancy.

Conclusion: The presence of a submicroscopic chromosomal aberration in the maternal genome causes positive NIPT results precluding the fetus state assessment and creates difficulties in clinical correlation and pregnancy management decisions.

Conflict of Interest: None declared.

EP02.028 Could increased nuchal translucency be an early marker for type 0 spinal muscular atrophy?

Giulia Gori1, Sara Bargiacchi1, Maria Rosaria D’Apice2, Roberto Biagiotti3, Paolo Poggi3, Viviana Palazzo1, Rosangela Artuso1, Angelica Pagliazzi4, Angela Peron1;5, Giuseppe Novelli2;6;7, Elena Andreucci 1

1Meyer Chidren’s Hospital IRCCS, Medical Genetics Unit, Florence, Italy; 2Tor Vergata University of Rome, Medical Genetics Unit, Department of Biomedicine and Prevention, Rome, Italy; 3Meyer Chidren’s Hospital IRCCS, Prenatal DIagnosis Unit, Florence, Italy; 4Leuven University, Nephrology and Renal Transplantation Research Group, Leuven, Belgium; 5University of Florence, Florence, Italy; 6IRCCS Neuromed, Pozzilli, Italy; 7School of Medicine, University of Nevada, Department of Pharmacology, Reno, United States

We describe a case of de novo type 0 spinal muscular atrophy (SMA) diagnosed after the evidence of familial high risk of SMA and prenatal echographic aspecific findings.

After the first trimester ultrasound, which showed increased fetal nuchal translucency (NT), early CVS was performed. Karyotype, chromosomal microarray and NGS analysis of a RASopathy gene panel resulted negative. The couple decided to terminate the pregnancy.

Meanwhile, SMA was diagnosed in a relative of the father and carrier testing was performed on the couple: the father resulted a classic carrier, the mother was found to have two copies of SMN1.

Considering that some authors correlate cardiac defects and high NT values with SMA, we decided to analyze also the fetus’ DNA, which showed no copies of SMN1 and one copy of SMN2.

On their next pregnancy, the couple decided to perform CVS and molecular testing for SMA in the fetus, who had only one copy of SMN1, maternally inherited, and no copies on the paternal allele. This evidence brought doubt on the mother’s hypothesized genotype so, to better understand the familial genotype and define a correct recurrence risk for the couple, we performed the test on their first daughter, who resulted to have two SMN1 copies, one on the maternal and one on the paternal allele, excluding the possibility of a 2 + 0 genotype in the mother.

We discuss potential new scenarios for testing SMN1 in pregnancies with atypical ultrasound findings to identify affected fetuses as early as possible.

Conflict of Interest: None declared.

EP02.029 Shifts and tendencies in Non-Invasive Prenatal Testing (NIPT) - 10 years of experience of the main Polish test provider

Monika Jurkowska 1, Anna Wąsowska1, Ewa Matczyńska1, Przemysław Łyszkiewicz1, Magdalena Kania1, Elżbieta Gregorczyk1, Paweł Ogrodnik1, Maria Jędrzejowska1, Marek Zagulski1, Anna Boguszewska-Chachulska1

1Genomed SA, Warsaw, Poland

In 2013 Non-Invasive Prenatal Testing started to be routinely offered in Poland as an out-of-pocket screening test. It is characterized by no miscarriage risk, accuracy (decreased number of confirmatory testing) and early timing (10w of pregnancy). Over the years, local recommendations defined it as a second-tier test, only recently (2022) acknowledging the fact that its accuracy has been confirmed in both high and low risk groups.

Altogether 40 003 reports, issued 2014-2022, were analysed (15850 based on BGI NIFTY and 24153 on Illumina VeriSeq V1,V2). Data have been extracted from a test-dedicated database. Demographic, technical and medical parameters were compared between two tests and over time.

In line with recommendations, the main reason for testing was constant - abnormal results of the combined screen (35.2%). However, a general shift towards younger maternal age (<35y: 41.0% in 2016 vs 60.5% in 2022) as well as an earlier test take-up (<12w: 16.0% vs 27.6%, respectively) has been noticed over the years. The switch from the BGI to the Illumina test allowed to reduce turn-around time by half (5.5 vs 2.5 days) and to decrease the percentage of both false negative (from 0.044 to 0.020%) and no-call (0.43% to 0.10%) cases. The introduction of genome-wide NIPT (VeriSeqV2 in 2020) decreased the choice of the basic range (T21, T18, T13, sex chromosomes) from 70% in 2020 to 12.6% in 2022. Test price was reduced by 14%.

Improvements in the NIPT performance, availability and range have been allowing gradual shifting it towards the first-tier screening.

Conflict of Interest: None declared.

EP02.030 Prenatally diagnosed arthrogryposis multiplex congenita is associated with variants in the Cardiac muscle alpha-actin (ACTC1) gene

Fe Amalia García Santiago 1;2;3;4, Elena Mansilla1;2;3;4, Luis Fernández García-Moya1;2, Miguel Ruiz de Azua5, Yolanda Nieto5, Eugenia Antolín Alvarado4;6, Beatriz Herrero Ruiz4;6, Isabel Vallcorba1, Carmen Rodriguez Jiménez7, Rita María Regojo Zapata8, Teresa López Jiménez1, Sonia Rodriguez Novoa7, Pablo LAPUNZINA1;2;3

1La Paz University Hospital, Instituto de Genética Médica y Molecular, Madrid, Spain; 2Carlos III Health Institute, CIBERER, Madrid, Spain; 3ITHACA, European Reference Network on Rare Congenital Malformations and Rare Intellectual Disability, Brussels, Belgium; 4La Paz University Hospital, Skeletal Dysplasia Multidisciplinary Unit (UMDE) and ERN-BOND, Madrid, Spain; 5Puerta de Hierro Majadahonda University Hospital, Obstetrics and Gynecology, Majadahonda, Spain; 6La Paz University Hospital, Fetal Medicine Unit. Department of Obstetrics and Gynecology, Universidad Autónoma, Idipaz, Madrid, Spain; 7La Paz University Hospital, Department of Genetics, Idipaz, Madrid, Spain; 8La Paz University Hospital, Anatomic Pathology, Idipaz, Madrid, Spain

Arthrogryposis, also termed arthrogryposis multiplex congenita, is a descriptive term for conditions with multiple congenital contractures (MCC). Prenatal diagnosis is usually based on the detection of diminished fetal movements and joint contractures on ultrasound. The etiology is extremely heterogeneous and is associated with syndromic disorders.

Here we present a fetus with MCC presenting prenatally with polyhydramnios, non-immune hydrops with predominance of subcutaneous edema and ductus venosus agenesis. The fetus also had abnormal limb position with hyperextended legs and flexed arms and placentomegaly.

Trio-based whole exome sequencing was performed to screen for possible pathogenic variants. Ultra-rare, previously unreported variant ACTC1 (NM_005159.4) c.1121G>A; p.(Arg374His) was identified de novo in the fetus. This variant predicts a conservative substitution of a highly conserved residue near the C-terminal end of the protein.

Cardiac muscle alpha-actin is the major component of the sarcomeric thin filament and plays a key role in endomyocardial development and cardiac contraction. Loss of function of this protein has been associated with the development of cardiomyopathy and atrial septal defects. Exceptionally, alteration of some residues as the one reported in this case may result in skeletal muscle disorders, where this actin has a minor expression.

Conflict of Interest: None declared.

EP02.031 Prospective reanalysis of unsolved prenatal exome sequencing for structural defects: feasibility and diagnostic yield

Nicolas Bourgon 1;2, Aurore Garde1;3, Frederic Tran Mau Them1;4, Yannis Duffourd1, Antonio Vitobello1, Christophe Philippe1;4, Laurence Faivre1;3, Christel Thauvin-Robinet1;3;4

1INSERM UMR 1231, Dijon, France; 2Necker University Hospital - APHP, Obstetrics and Fetal Medicine, Paris, France; 3CHU Dijon Bourgogne, Centre de Référence Maladies Rares “Anomalies du développement et syndromes malformatifs”, Centre de Génétique, FHU TRANSLAD et Institut GIMI, Dijon, France; 4CHU Dijon Bourgogne, Unité Fonctionnelle Innovation en Diagnostic Génomique des maladies rares, Dijon, France

Background/Objectives: In case of normal karyotype/CMA, prenatal exome sequencing (pES) provides an additional diagnosis in around 30% of structural defects. Regardless of the pregnancy outcome, reanalysis of unsolved cases is offered using additional postnatal/postmortem data. In this study, we investigate the additive value of postnatal/postmortem features to reanalyze negative pES.

Methods: We conducted a prospective reanalysis of unsolved cases included in the “AnDDI-Prenatome cohort”, a French multicenter study offering pES for major structural defects. For 84/140 cases with unconclusive pES, reanalysis was systematically offered in case of parental request. Referent clinicians were requested to collect all relevant clinical, biological, and imaging data after birth (postnatal or postmortem). Phenotypical data were collected as Human Phenotype Ontology (HPO). Two independent biologists analyze raw data from pES thanks to an update bioinformatic in-house pipeline for annotation and databases.

Results: From 84 unsolved cases, reanalysis was required for 51 and declined for 8 couples. 8 couples are under consideration, 11 cases were lost of follow-up, and 6 not being approached by the referring clinicians. At least one year of life, data were available for 24 cases, 12 cases were lost of follow-up and 38 are under consideration. At the time of writing, reanalysis is ongoing.

Conclusion: We report the first cohort of fetuses investigated during pregnancy by trio pES for structural defect. We investigated the additive value of postnatal/postmortem data for systematic reanalysis after negative pES. Our data may provide significant informatic and influence clinical practice in case of negative pES.

Conflict of Interest: None declared.

EP02.032 Cell free fetal DNA testing in twin pregnancies

Irene Madrigal 1;2, Marta Gracia1, Laura Muñoz1, Celia Badenas1;2

1Hospital Clinic of Barcelona, Biochemistry and Molecular Genetics, Barcelona, Spain; 2IDIBAPS, Barcelona, Spain

Background/Objectives: cell free fetal DNA (cffDNA) study in Catalonia is offered as a screening test for pregnant women with single or multiple pregnancies with high (1/10-1/250) or intermediate risk (1/251-1/1100) in the combined screening. In twin pregnancies, cffDNA is more complex than in singleton pregnancies because: 1) twins could be either monozygotic or dizygotic (DZ), in which only 1 twin is likely to have aneuploidy when present, and 2) in DZ pregnancies each twin can contribute different amounts of cffDNA into the maternal circulation. These two factors may contribute to discordant cffDNA results.

Methods: We have performed cffDNA test in 118 pregnancies, including 89 dichorionic diamniotic (DCDA) twins, 25 monochorionic diamniotic and 4 monochorionic monoamniotic. Four DCDA pregnancies presented a vanishing twin.

Results: cffDNA results detected 3 gestations with a high risk of T21, 1 with a high risk of T18 and 2 with a high risk of T13. Invasive testing was performed in 4 cases and aneuploidies were confirmed in each pregnancy (in one of the twins) except in one case that presented a vanishing twin. In the two gestations that refused an invasive test, 1 twin of each pregnancy was born with T21. In addition, 1 false negative was detected in a pregnancy with a vanishing twin.

Conclusion: Our results reinforce the idea that cffDNA testing in twins is feasible, although special caution is needed in pregnancies with a vanishing twin as they may lead to discordant results for up to 6–8 weeks after fetal demise.

Conflict of Interest: None declared.

EP02.035 The use of whole-genome sequencing in the study of the etiology of complex birth defects in 85 fetuses

Agnieszka Stembalska 1, Malgorzata Rydzanicz2, Rafał Płoski2, Robert Smigiel3

1Medical University of Wrocław, Department of Genetics, Wroclaw, Poland; 2Medical University of Warsaw, Department of Medical Genetics, Warsaw, Poland; 3Medical University of Wroclaw, Department of Paediatrics and Rare Disorders, Wroclaw, Poland

Whole-exome sequencing has become a widely used clinical genetic diagnostic test as a second-tier test after chromosomal microarray analysis in the prenatal evaluation of fetuses with multiple congenital anomalies. The study included 85 fetuses with congenital anomalies. The inclusion criteria were the presence of at least two congenital defects of different organs or numerous defects of one system. It primarily concerned defects resulting in severe organ dysfunction and/or congenital defects with poor prognosis, which may lead to fetus death or baby death after birth. The fetal anomalies were diagnosed in ultrasound scans in fetuses of II and III trimesters of pregnancy. The research materials were amniocytes, fragments of the umbilical cord or other fetal tissues. These were collected appropriately from live fetuses during invasive prenatal diagnosis, after miscarriages or stillbirths. WES was performed in fetuses with normal karyotype and chromosomal microarray analysis results.

Molecular findings were found in 47% of studying fetuses. In 35 % of these, de novo variants were found. Variants in genes with autosomal recessive (17) and autosomal dominant (17) were detected in most cases.

Pathogenic/likely pathogenic variants explained the congenital anomalies in most of studying fetuses. In a few cases, the variants did not correspond with the observed symptoms, in three cases were found germline variants in the genes, which have not been associated with known human diseases.

WES analysis is essential for improving prenatal diagnosis of fetuses with congenital abnormalities. Although in some cases genetic variants potentially relevant for the specific phenotype require functional testing.

Conflict of Interest: None declared.

EP02.036 Investigation of Genes Responsible for Diaphragmatic Developmental Defects with Next Generation Sequencing Technologies

Somayyeh Heidargholizadeh 1, Cagri Gulec2, Gulnihal Bulut3, gözde tutku turgut2, Seher Basaran4, Umut Altunoğlu5, Birsen Karaman6

1Istanbul University Institute of Health Sciences, Genetics, İstanbul, Turkey; 2Istanbul Faculty of Medicine Internal Medicine, Genetics, İstanbul, Turkey; 1Istanbul University Institute of Health Sciences, Genetics, İstanbul, Turkey; 2Istanbul Faculty of Medicine Internal Medicine, Genetics, İstanbul, Turkey; 5Koç University, School of Medicine, Genetics, İstanbul, Turkey; 6Istanbul University, Institute of Child Health, Pediatric Basic Sciences, İstanbul, Turkey

Background/Objectives: Congenital Diaphragmatic Hernia (CDH) that occurs as a result of defects in the development of the diaphragm is identified as a severe anomaly with high mortality. CDH is caused by chromosomal anomalies, copy number variations, or sequence variations in increasing number of genes. Besides the retinoic acid signaling pathway which is known to play crucial role in diaphragmatic development, the genes modulating other processes like cell migration, cytoskeleton organization, and myogenesis are also involved in CDH etiology. Therefore, next generation sequencing (NGS) technology based whole-exome sequencing (WES) method is expected to be effective to identify new canditate genes for the genetic etiology of CDH.

Methods: In order to identify new candidate genes, trio-WES analysis was performed in eight fetuses and their parents, and solo-WES analysis was performed in ten fetuses with excluded CNVs by chromosome analysis and a-CGH study.

Results: Pathogenic variants have been identified in the genes (NR2F2, ZFPM2, ARID1A, CREBBP, PLAT, POGZ, RARB) with known CDH association, and in candidate genes (CDKL4, STAB2, NEIL2, SETD5, STAB2, TAF4, ZBTB38, ZNF423, COL11A1, PCSK5, RBM8A) with function-, database- and literatüre-based association.

Conclusion: The results of this study, support the notion that a single gene or variant is not responsible for the majority of CDH cases. The findings of our study will contribute to the literature on the genes that play a role in the etiology of CDH.

Grant References: This work was supported by the Research Fund of Istanbul University.No.30101

Conflict of Interest: Somayyeh Heidargholizadeh BAP. PROJECT NO: 30101, Cagri Gulec full-time, consultant- project no: 30101, Gulnihal Bulut BAPSİS, project no:30101, gözde tutku turgut full-time, BAPSİS, project no:30101, Seher Basaran full-time, BAPSİS, project no:30101, Umut Altunoğlu full-time, BAPSİS, project no:30101, Birsen Karaman full-time, principal investigator, BAPSİS, project no:30101.

EP02.037 Prenatal WES diagnostic yield in fetuses with congenital defects detected by antenatal ultrasound

Neus Baena 1, Núria Capdevila1, Juan Pablo Trujillo Quintero1, Carmen Manso1, silvia pina2, cristina lesmes2, Montserrat Comas2, Victor Martinez-Glez1

1Corporació Sanitaria Parc Tauli, Centre de Medicina Genòmica, Sabadell, Spain; 2Corporació Sanitaria Parc Tauli, Obstetrics and Gynecology, Sabadell, Spain

Background/Objectives: Whole exome sequencing (WES) is a diagnostic tool in postnatal settings for individuals with a suspected genetic condition. In prenatal settings, WES diagnostic yield ranges from 15% to 35%. The aim is to evaluate the usefulness of WES in identifying the genetic etiology of congenital defects.

Methods: WES was performed in 25 fetal samples with ultrasound anomalies and previous prenatal CGH-array with a normal result. Kit Nextera Flex for enrichment, oligos Illumina Exome using NextSeq (Illumina). Variant and segregation studies were confirmed by Sanger sequencing.

Results: WES detected five pathogenic variants (EFTUD2, TBX5, OFD1, SEC23B, FANCA), one likely pathogenic (PTPN11), and three variants of uncertain significance (VUS). The diagnostic yield for pathogenic and likely pathogenic variants was 24%. In 2 fetuses, the diagnosis followed an autosomal recessive pattern, one homozygous variant (SEC23B) in a nonconsanguineous gypsy couple with recurrent hydrops in two pregnancies and one diagnosed with Fanconi anemia. In addition, two pathogenic variants inherited from a healthy parent were identified in GNB1 and GLI2 genes previous to an accurate compilation of family history that led to a reassessing of the clinical pathogenicity of the variants.

Conclusion: Despite the small sample size, the results show the clinical utility of prenatal WES for detecting pathogenic/likely pathogenic variants in fetuses with congenital defects. As in postnatal WES, detailed phenotyping is necessary to filter and prioritize variants to obtain a molecular diagnosis.

Conflict of Interest: Neus Baena full, Núria Capdevila full, Juan Pablo Trujillo Quintero full, Carmen Manso full, silvia pina full, cristina lesmes full, Montserrat Comas full, Victor Martinez-Glez full.

EP03 Sensory Disorders (Eye, Ear, Pain)

EP03.001 Recurrent benign paroxysmal positional vertigo in DFNB16 patients with biallelic STRC gene deletions

Sophie ACHARD 1, Margaux Campion1, Marine Parodi1, melissa macaskill1, Baptiste Hochet2, François Simon1, Isabelle Rouillon1, Laurence Jonard1, Margaux Serey Gaut1, Françoise Denoyelle1, Natalie Loundon1, Sandrine MARLIN1

1Hôpital Necker, Paris, France; 2Hôpital Foch, Suresnes, France

Objective: Deletions of STRC gene (DFNB16) account for 12% of isolated congenital mild to moderate hearing loss (HL). In mice, the stereocilin protein, encoded by STRC, is present in the vestibular kinocilium embedded in the otoconial membrane of the utricular macula. Despite this, effects on vestibular function have not been widely investigated. The aim of this study was to investigate the prevalence of Benign Paroxysmal Positional Vertigo (BPPV) in a cohort of DFNB16 patients.

Study Design: Observational descriptive epidemiological study.

Setting: Single-centre study, in a tertiary referral center.

Patients: Over 5 years of age, with a genetic diagnosis of HL related to biallelic STRC gene deletions, diagnosed between 2015 and 2021

Intervention: Patients or their parents were interviewed to determine whether they had experienced vertigo or episodes of BPPV.

Main outcome measure: Criteria were at least five acute episodes of rotatory vertigo, each lasting less than one minute, episodes triggered by changes in specific head position, and an absence of neurological symptoms.

Results: 64 patients were included, having mild (33%) to moderate (66%) HL. Median age was 15 years, (range: 6-48). Prevalence of BPPV was 39% (25/64). Median age of first onset was 13 years, (range: 3-18).

Conclusions: This study showed recurrent BPPV and early age of onset in patients with biallelic STRC gene deletions. BPPV may be associated with the HL phenotype in patients with STRC gene deletions. It is important to inform patients and families of this potential risk such that appropriate management can be proposed.

Conflict of Interest: None declared.

EP03.002 The identification of biallelic LAMA1 pathogenic variant in a consanguineous Saudi family with presumed stickler syndrome

Basamat AlMoallem 1

1College of Medicine, King Saud University, Department of Ophthalmology, Riyadh, Saudi Arabia

Purpose: Poretti– Boltshauser syndrome (OMIM # 615960) is a rare autosomal recessive non-progressive cerebellar dysplasia disorder with wide range of ophthalmic manifestations such as high myopia, strabismus, and retinal dystrophy. We aim to study a consanguineous Saudi family with two affected siblings (female 30 yrs.) and (male 28 yrs.) who were primarily diagnosed with stickler syndrome with a positive history of ataxia, delayed speech and a bilateral prophylactic laser that was done 20 years ago for high myopia.

Methods: Comprehensive clinical and molecular approaches were applied including detailed ophthalmological examination followed by whole-exome sequencing (WES).

Results: Detailed ophthalmological examination revealed visual acuity of 20/200 bilaterally associated with pendular nystagmus and esotropia. Anterior segment examination showed a bilateral posterior subcapsular cataract with no sign of lens subluxation. A myopic fundus was observed with a pale optic disc, flat retina, and prominent laser marks. The additional neurological assessment confirmed the presence of mildly delayed gross motor skills that resolved with age with ongoing MRI requests for the full characterization. Genetically, WES revealed novel homozygous laminin alpha-1 (LAMA1) nonsense mutation; NM_005559.4: c.5801C>G(p.Ser1934*), class 4 according to ACMG. This variant was segregated well within available family members confirming the diagnosis of PTBHS.

Conclusions: Our identified novel nonsense LAMA1 pathogenic variant supports the pathogenicity of the LAMA1 gene and expands the phenotypic spectrum of PTBHS. Our study highlights the importance of genetics testing in refining the clinical diagnosis for such heterogeneous cases where ocular features are overlapping with other vitreoretinopathies such as stickler syndrome.

Conflict of Interest: None declared.

EP03.003 Digenic cause for optic atrophy due to heterozygous variants in SPG7 and AFG3L2 genes?

Florina Stoica 1, Andreea Ciubotaru2, Maria Puiu3, Adela Chirita-Emandi4

1Emergency Clinical Municipal Hospital, part of ERN EYE, Timisoara, Romania; 2INFOSAN Ophtalmology Clinic, Bucuresti, Romania; 3Genetics Discipline, Center of Genomic Medicine “Victor Babeș” University of Medicine and Pharmacy, Regional Center of Medical Genetics, “Louis Țurcanu” Clinical Emergency Hospital for Children, part of ERN ITHACA, Timisoara, Romania; 4Genetics Discipline, Center of Genomic Medicine “Victor Babeș” University of Medicine and Pharmacy, Regional Center of Medical Genetics, “Louis Țurcanu” Clinical Emergency Hospital for Children, part of ERN ITHACA, Timisoara, Romania

Background/Objectives: Recently, a case series showed that heterozygous variants in SPG7 and AFG3L2 genes are involved in the regulation of mitochondrial protein homeostasis and maturation by m-AAA proteases to maintain optic nerve physiology (PMID:32548275). Here we present a patient were optic atrophy could be explained by digenic mechanism.

Methods: Clinical evaluation, thorough ophthalmological assessments and extensive genetic testing were performed for the patient and parents.

Results: A 6 years old male presents with decreased visual acuity, stating age 4 years. He had normal neurological development and healthy non-consanguineous parents. Bilateral optic disc pallor, decreased thickness of retinal fiber layer, macular ganglion cell loss (on OCT) and diminished functionality of the rods (on ERG) were documented. WGS solo (including mitochondrial genome) and sanger sequencing in parents showed: AFG3L2 NM_006796.1:c.1286_1288dup,p.(Asn429dup),heterozygous,VUS, paternal and SPG7 NM_003119.4:c.1045G>A,p.(Gly349Ser),heterozygous, pathogenic, maternal. An article (PMID:30252181) reports an m-AAA mitochondrial damage-associated phenotype characterized by early-onset optic atrophy with spastic ataxia and L-dopa-responsive parkinsonism. The affected individual had a heterozygous de novo AFG3L2 mutation (p.R468C) together with a heterozygous maternally inherited intragenic deletion of SPG7. Analysis of the patient’s fibroblasts showed an abnormal pattern of processing of OPA1, a protein essential for mitochondrial fusion and responsible for hereditary optic atrophy. A similar mechanism could explain the optic atrophy in our patient; however, functional studies were not performed.

Conclusion: Possibly, the patient’s optic atrophy is explained by both heterozygous variants in the SPG7 (maternal) and AFG3L2 (paternal), being involved in the same mitochondrial cellular metabolic pathway (mitochondrial m-AAA protease involved in OPA1 processing).

Conflict of Interest: None declared.

EP03.004 Spectrum of congenital and inherited ocular disorders seen in a genetic clinic: experience of a developing ocular genetic service

Anupriya Kaur 1, Animesh Sahu1, Savleen Kaur2, Jaspreet Sukhija2, Priyanka Srivastava1

1Post Graduate Institute of Medical Education and Research, Pediatrics, Chandigarh, India; 2Post Graduate Institute of Medical Education and Research, Advanced Eye Center, Chandigarh, India

Background: Hereditary causes are an important etiological category of childhood blindness. This study reports real world experience of a developing ocular genetic service.

Material and Methods: The study was carried out from Jan 2020 to Dec 2021 jointly by the departments of Pediatrics and Ophthalmology of a tertiary care hospital in North-West India. Children presenting to the genetics clinic with congenital or late onset ocular disorder(s) and any individual (irrespective of age) suffering from an ophthalmic disorder and referred by an ophthalmologist for genetic counseling for himself/herself and/or his/her family member(s) were included. Genetic testing (exome sequencing /panel based sequencing /chromosomal microarray) was outsourced to third party laboratories with the cost of the test been borne by the patient.

Results: 8.6% of the registered patients in the genetics clinic had ocular disorders. Maximum number of patients belonged to category of Anterior Segment Dysgenesis followed by Microphthalmia Anophthalmia Coloboma spectrum, Lens disorders, Inherited retinal disorders in decreasing numbers. The ratio of syndromic ocular to isolated ocular disorders seen was 1.8/1. Genetic testing was accepted by 55.5% of families. The genetic testing was clinically useful for ~ 35% of the tested cohort with the opportunity for prenatal diagnosis being the most useful application of genetic testing.

Conclusion: Syndromic ocular disorders are seen at a higher frequency as compared to isolated ocular disorder in a genetic clinic. Opportunity for prenatal diagnosis is the most useful application of genetic testing in ocular disorders.

Conflict of Interest: Anupriya Kaur Full time, Animesh Sahu Full time, Savleen Kaur Full time, Jaspreet Sukhija Full time, Priyanka Srivastava Full time

EP03.005 3 different iPSC-derived cell models for the study of Stargardt Disease and Retinitis Pigmentosa

Arnau Navinés-Ferrer 1;2, Pilar Mendez1;2, Laura Siles1;2, Paula Gaudó1;2, Esther Pomares1;2

1Fundació de Recerca de l’Institut de Microcirurgia Ocular, Genetics, Barcelona, Spain; 2IMO Grupo Miranza Barcelona, Genetics, Barcelona, Spain

Purpose: Stargardt disease (STGD) and Retinitis Pigmentosa (RP) are two of the most prevalent inherited dystrophies of the retina. The first one is caused by recessive mutations in the ABCA4 gene, expressed in both RPE cells and photoreceptors. RP is the most common form of rod-cone dystrophy. RHO - the gene encoding for the specific opsin of rods – is one of the most prevalent mutated genes in the dominant forms.

Methods: We generated 3 different iPSC-derived cell models: RPE, photoreceptor precursors (PhRPs) and Retinal Organoids (RO) from 3 patients affected by STGD disease or RP (with a dominant RHO mutation). Using qPCR, Western Blot, and immunochemistry, along with other cellular biology methods, we analyzed their phenotype.

Results: The effect of patient-specific mutations induces aberrant splicing, reduced mRNA expression and reduced protein expression of ABCA4 while showing early signs of autophagy and reduced function on RPE, PhRPs and RO. The RP line shows accumulation of the rhodopsin protein in rod photoreceptors, and early signs of endoplasmic reticulum (ER) stress and autophagy.

Conclusions: iPSC are a wonderful tool for the investigation of retinal diseases, through differentiation to different cell types. While some models with a faster differentiation time can be useful for detection of mRNA or protein expression, more complicated models such as RO are necessary for a deeper insight into the pathophysiology of each disease.

Conflict of Interest: None declared.

EP03.006 Whole-exome sequencing allows the identification of new causal mutations and candidate genes in unsolved inherited retinal dystrophy patients

Marta Martín-Sánchez 1;2, Elena Fernández-Suárez1;2, Nereida Bravo-Gil1;2, Cristina Méndez-Vidal1;2, María González-del Pozo1;2, Marcela Mena1;2, José Manuel Mejías-Carrasco1, Alejandro García-Nuñez1, Enrique Rodríguez-de la Rúa3;4, Maria Jose Morillo Sanchez3, Salud Borrego1;2, Guillermo Antiñolo1;2

1Department of Maternofetal Medicine, Genetics and Reproduction, Institute of Biomedicine of Seville, IBiS/Virgen del Rocío University Hospital/CSIC/University of Seville, Seville, Spain; 2Center for Biomedical Network Research on Rare Diseases (CIBERER), Seville, Spain; 3Department of Ophthalmology, University Hospital Virgen Macarena, Seville, Spain; 4Retics Patologia Ocular, OFTARED, Carlos III Health Institute, Madrid, Spain

Background/Objectives: Inherited retinal dystrophies (IRD) are a group of rare diseases leading to visual loss. After genetic analysis of prevalent IRD genes, ~40% of patients remained unsolved. Here, we applied whole-exome sequencing (WES) to address unsolved IRD cases.

Methods: A total of 47 families (55 IRD patients and 88 unaffected relatives), initially screened for variants in 99 known IRD genes, were analysed using SOPHiA Whole Exome Solution and a personalized bioinformatic pipeline, initially designed to analyse whole-genome sequencing (WGS) data, that uses a virtual panel containing 297 IRD-genes as the first prioritization step. Additionally, all genes were evaluated in seven families so far. Segregation studies were conducted by Sanger sequencing.

Results: This approach allowed the identification of likely causal variants in 12 families, and variants of uncertain significance in other three families. These variants were identified in IRD-related genes not previously included in the panel, located in challenging regions (RPGR-Orf15) or not detected due to variant calling errors (NR2E3). Also, a new genotype-phenotype correlation was proposed related to SLC4A7. Additionally, we detected variants in new candidate genes in another family, for which additional studies are currently being completed.

Conclusions: These results have increased our diagnostic yield in IRD patients and have improved the efficiency of our population-specific IRD panel. Also, we have proposed a new genotype-phenotype correlation and new IRD-candidate genes. Since our algorithm has been adapted from a WGS-pipeline, we suggest the use of a single pipeline to analyse NGS-derived data.

Grant References: ISCIII-ERDF/ESF(PI21-00244), Andalusian Government(PEER_0501_2019;RH-0049-2021), F.Isabel Gemio/F.Cajasol(2019-01), ISCIII-IMPaCT(IMP/0009).

Conflict of Interest: None declared.

EP03.007 Towards a better understanding of genotype-phenotype spectra in hereditary pain loss disorders

Katja Eggermann 1, Annette Lischka1, Miriam Elbracht1, Florian Kraft1, Matthias Begemann1, Daniela Dey1, Christopher Record2, Mary M Reilly2, Petra Lassuthova3, Arman Cakar4, Yesim Parman4, Jonathan Baets5;6, Vincent Timmermann7, Jan Senderek8, Angelika Lampert9, David Bennet10, James Cox11, Maike Dohrn12;13, Stephan Züchner13, Thorsten Hornemann14, Michaela Auer-Grumbach15, Christian Hübner16, Geoffrey Woods17, Enrico Leipold18, Jonathan de Winter5;6, Danique Beijer13, Atchayaram Nalini19, Wilson Marques Junior20;21, Ingo Kurth1

1Institute for Human Genetics and Genomic Medicine, University Hospital RWTH Aachen, Aachen, Germany; 2Centre for Neuromuscular Diseases, Department of Neuromuscular Diseases, London, United Kingdom; 3Department of Paediatric Neurology, 2nd Faculty of Medicine, Charles University in Prague and Motol University Hospital, Prague, Czech Republic; 4Neuromuscular Unit, Department of Neurology, Istanbul Faculty of Medicine, Istanbul University, Istanbul, Turkey; 5Translational Neurosciences and Institute Born Bunge, Faculty of Medicine and Health Sciences, Antwerp, Belgium; 6Neuromuscular Reference Centre, Department of Neurology, Antwerp University Hospital, Antwerp, Belgium; 7Peripheral Neuropathy Research Group, Department of Biomedical Sciences, Institute Born Bunge, University of Antwerp, Antwerp, Belgium; 8Friedrich-Baur-Institute, Department of Neurology, Ludwig-Maximilians-University, Munich, Germany; 9Institute of Physiology, Medical Faculty, Uniklinik RWTH Aachen University, Aachen, Germany; 10Nuffield Department of Clinical Neuroscience, Oxford University, Oxford, United Kingdom; 11Molecular Nociception Group, Wolfson Institute for Biomedical Research, University College London, London, United Kingdom; 12Department of Neurology, Medical Faculty of the RWTH Aachen University, Aachen, Germany; 13Dr. John T. Macdonald Foundation, Department of Human Genetics and John P. Hussman Institute for Human Genomics, University of Miami, Miller School of Medicine, Miami, FL, United States; 14Department of Clinical Chemistry, University Hospital Zurich, University of Zurich, Zurich, Switzerland; 15Department of Orthopedics and Trauma Surgery, Medical University of Vienna, Vienna, Austria; 16Institute of Human Genetics, University Hospital Jena, Jena, Germany; 17Cambridge Institute for Medical Research, Keith Peters Building, Cambridge Biomedical Campus, Cambridge, United Kingdom; 18Department of Anesthesiology and Intensive Care, Center of Brain, Behaviour and Metabolism, University of Lübeck, Lübeck, Germany; 19Department of Neurology, National Institute of Mental Health and Neurosciences, Bengaluru, Karnataka, India; 20Division of Neuromuscular Disorders, Department of Neurology, Ribeirão Preto School of Medicine, University of São Paulo, São Paulo, Brazil; 21Department of Neurosciences and Behavioral Sciences, National Insitute for Science and Technology for Translational Medicine, INCT, São Paulo, Brazil

Background/Objectives: Genetic pain loss disorders include congenital insensitivity to pain (CIP) and hereditary sensory (and autonomic) neuropathy (HSN/HSAN). Patients have no or reduced response to pain stimuli, leading to trauma and mutilation with potentially fatal complications. In addition to congenital cases, milder forms with adult onset exist. Here we report on a multicenter study that aims at improving the knowledge of the clinical features with regard to the genetic basis. The collected data are compiled in a registry to enable genotype-phenotype correlations and natural history studies.

Methods: We conducted whole exome or whole genome sequencing and clinical work-up of a large retrospective cohort of CIP/HSAN patients in a global network of neuromuscular centers. When appropriate, we included segregation analyses, sphingolipid profiles from patient plasma, or long-range sequencing techniques to further characterize variants.

Results: We describe 70 patients with previously unpublished disease-associated variants in 11 genes. The most frequently affected genes are NTRK1 and SCN9A in children and SPTLC1 in adult patients. We report mutations in previously rarely described entities such as ATL3-, FLVCR1- and NGF-associated pain loss and combine the molecular results with clinical information.

Conclusion: Our results broaden the knowledge of HSAN and thereby improve patient care. The register established here is the starting point for natural history studies and the identification of biomarkers. In view of the already existing first therapy approaches in clinical trials and expected further therapies, the cohort will allow subtype-specific classifications in the future.

Grant Reference: EJP RD COFUND-EJP N° 825575, NIH (R01NS105755)

Conflict of Interest: Katja Eggermann: None declared, Annette Lischka: None declared, Miriam Elbracht: None declared, Florian Kraft: None declared, Matthias Begemann: None declared, Daniela Dey: None declared, Christopher Record: None declared, Mary M Reilly: None declared, Petra Lassuthova: None declared, Arman Cakar: None declared, Yesim Parman: None declared, Jonathan Baets: None declared, Vincent Timmermann: None declared, Jan Senderek: None declared, Angelika Lampert: None declared, David Bennet: None declared, James Cox: None declared, Maike Dohrn NIH (R01NS105755), Stephan Züchner: None declared, Thorsten Hornemann: None declared, Michaela Auer-Grumbach: None declared, Christian Hübner: None declared, Geoffrey Woods: None declared, Enrico Leipold: None declared, Jonathan de Winter: None declared, Danique Beijer: None declared, Atchayaram Nalini: None declared, Wilson Marques Junior: None declared, Ingo Kurth EJP RD COFUND-EJP N° 825575.

EP03.008 PP1 criteria pitfall assessing autosomal dominant TECTA-variant in non-syndromic hearing loss

Anneli Bolund 1;2, Thomas Blauenfeldt1;3, Allan Thomas Højland1;2;4

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

Introduction: Autosomal dominant (AD) inherited non-syndromic hearing loss (NSHL) is characterized by genetic heterogeneity. More than 50 genes are associated to AD NSHL, including TECTA. NSHL specific ACMG variant interpretation guidelines have been published. However, caution is advised regarding the PP1 criterion.

Material and methods: We describe the finding of NM_005422.44(TECTA):c.5990T>C using gene panel screening of 165 NSHL genes and the subsequent segregation analysis in a Danish NSHL-family. The variant was previously reported to segregate with NSHL in a large Japanese family. The variant was classified as C4 with the following ACMG criteria: PM2_sup, PP3, PM1 and PP1_strong. The latter criterion was decisive and given based on 11 affected segregations in the Japanese family. However, no additional screening of other NSHL genes was reported for the Japanese family. We initially omitted the PP1 criterion and classified the variant as C3 until further segregation analysis had been performed by Sanger sequencing.

Results: The variant was confirmed in the Danish family in two additional affected family members and not found in one unaffected. Based on a total of 13 affected segregations in two families of different ethnicity we included the criterion PP1_strong and reclassified the TECTA variant as C4.

Conclusion: Caution is advised when using the PP1 criterion, as variants could be in linkage disequilibrium with other causative variants. Therefore, PP1 could falsely elevate a C3 variant to C4 with subsequent clinical significance for entire families. In this case NM_005422.44(TECTA):c.5990T>C co-segregated with AD NSHL in both families, supporting a true causative effect.

Conflict of Interest: None declared.

EP03.009 Molecular genetic investigation of CHM in the Czech patient population reveals seven novel pathogenic/likely pathogenic variants including 3 large deletions and one duplication

Petra Liskova 1;2, Monika Pankievic1, Gabriela Storkanova1, Hana Vlaskova1, Marie Vajter1;2, Andrea Vergaro1;2, Arpad Boday3, Lubica Dudakova1

1First Faculty of Medicine and General University Hospital, Department of Paediatrics and Inherited Metabolic Disorders, Prague, Czech Republic; 2First Faculty of Medicine and General University Hospital, Department of Ophthalmology, Prague, Czech Republic; 3Agel Laboratories, Department of Medical Genetics, Novy Jicin, Czech Republic

Objectives: The aim of the study was to define the spectrum of pathogenic variants in 18 Czech families with choroideremia (CHM).

Methods: All probands underwent complex ocular examination including fundus autofluorescence. Initial molecular genetic investigation comprised direct sequencing of the CHM coding region or next-generation sequencing (ocular gene pane or exome), followed by MLPA analysis and genome sequencing to map the exact positions of breakpoints. Segregation analysis was done by Sanger sequencing.

Results: 13 male probands were suspected to suffer from CHM, while 5 (2 males and 3 females) were referred with retinitis pigmentosa and one female with Stargardt disease. Pathogenic or likely pathogenic variants in CHM were identified in 17 probands including four novel mutations predicted to introduce a premature stop codon. In addition, four previously unreported copy number variations were found; specifically 2.8 kb deletion involving exon 3, 692 kb deletion comprising exons 3-15 plus MIR1321 and part of POF1, and a deletion of the entire CHM together with DACH2 and KLHL4. The only duplication identified was 110 kb involving CHM exons 2-8. In one proband the pathogenic c.525_526del variant occurred de novo which is a rare observation in CHM. In one proband with typical CHM phenotype the c.1511-10_1511-6del variant was evaluated as of unknown significance.

Conclusion: Identification of CHM pathogenic/likely pathogenic variants in patients referred with other diagnoses than CHM highlights the utility of molecular genetic testing. Disease-causing structural variants in CHM may be more prevalent than currently anticipated.

Grant references: NU20-07-00182, Solve-RET 8F20004 No. 825575

Conflict of Interest: Petra Liskova Charles University, Prague, Ministry of Health of the Czech Republic NU20-07-00182, Novartis, Monika Pankievic: None declared, Gabriela Storkanova General University Hospital, Hana Vlaskova General University Hospital, Marie Vajter General University Hospital, Andrea Vergaro General University Hospital, Arpad Boday Agel Laboratories, Lubica Dudakova Charles University.

EP03.010 Autosomal Recessive Leber’s Hereditary Optic Neuropathy Caused by a Homozygous Variant in DNAJC30 Gene in Two Unrelated Patients

Laura Mauring 1;2;3;4, Sanna Puusepp1;3, Mall Parik2, Rita Teek1, Tiia Reimand1;3, Sander Pajusalu1;3, Kuldar Kaljurand2;4, Katrin Ounap1;3

1Tartu University Hospital, Genetics and Personalized Medicine Clinic, Tartu, Estonia; 2Tartu University Hospital, Eye Clinic, Tartu, Estonia; 3University of Tartu, Department of Clinical Genetics, Institute of Clinical Medicine, Tartu, Estonia; 4University of Tartu, Department of Eye Clinic, Tartu, Estonia

Recently, Stenton et al., described a new, autosomal recessive inheritance pattern of Leber’s hereditary optic neuropathy (LHON) caused by missense variants in the DNAJC30 gene. The DNAJC30 c.152A>G, p.(Tyr51Cys) variant was by far the most common variant reported in patients originating from Eastern Europe, thus it is believed to be a founder variant in these populations. We report the first two cases of DNAJC30-related autosomal recessive LHON in a young male and a female originating from Estonia. The patients presented severe loss of central vision and clinical features indistinguishable from mitochondrial LHON. The whole exome sequencing carried out in the male patient, and the next-generation sequencing panel in the young female patient identified the same homozygous missense variant in the DNAJC30 gene. Our cases further reinforce the pathogenicity of c.152A>G, p.(Tyr51Cys) DNAJC30 variant causing autosomal recessive LHON. We support the parallel sequencing of the mitochondrial DNA and the DNAJC30 gene in patients with LHON.

Funding information: Estonian Research Council; Grant/Award Number: PRG471, PSG774.

Conflict of Interest: None declared.

EP03.011 A novel HPS5 acceptor splice-site mutation causing Hermansky Pudlak Syndrome type 5

Tomer Poleg 1, ofek freund1, Liba Gradstein1, Diya Tacruri1, ohad wormser1, Marjan Huizing2, Erez Tsumi1, Ohad Shmuel Birk1

1Genetics Institute and Department of Ophthalmology at Soroka Medical Center and the Morris Kahn Laboratory of Human Genetics, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer sheva, Israel; 2Medical Genetics Branch, NHGRI, NIH, Bethesda, Maryland, United States

Objectives: Three affected siblings of two branches of a Bedouin consanguineous kindred presented with nystagmus, mild cutaneous and ocular hypopigmentation, and a history of easy bruising, recurrent epistaxis, and severe bleeding after uneventful surgery, commensurate with a diagnosis of Hermansky Pudlak Syndrome (HPS). We aimed to identify the molecular basis of HPS in the studied kindred.

Methods: Affected individuals were studied following informed consent and approval of Soroka Medical Center IRB. Senior ophthalmologist and geneticist determined the phenotype. Linkage analysis, testing 13 family members, was done using SNP arrays. Segregation analysis of the putative pathogenic variant was done using Sanger sequencing.

Results: Linkage analysis delineated a 17 Mbp disease-associated homozygosity locus on chromosome 11 (LOD score 2.4), containing HPS5. HPS5 sequencing identified a novel homozygous splice-site mutation, c.285-2, T > C, near the end of intron 4. The mutation is in a highly evolutionary conserved residue predicted by SpliceAI to cause acceptor loss (score 0.78). Sanger sequencing of patients’ cDNA blood samples revealed that the mutation led to a frameshift and premature stop codon due to a deletion of five nucleotides.

Conclusions: The novel HPS5 mutation causing HPS in the Bedouin kindred is in a highly conserved splice acceptor site, resulting in an aberrant transcript. The HPS5 protein is part of the Biogenesis of Lysosome-related Organelle Complex 2 (BLOC-2) containing also proteins HPS3 and HPS6. BLOC-2 is involved in the formation and migration of lysosomal-related endosomal compartments and is crucial in melanosome formation and maturation.

Grant: Morris Kahn Family Foundation

Conflict of Interest: None declared.

EP03.012 Audiological phenotyping evaluation in KBG syndrome: Description of a multicenter review

Margaux Serey Gaut1;2, laetitia rhamati3, Anne-Marie Guerrot4, Alice Goldenberg4, Marlene Rio5, Genevieve Baujat5, Stanislas Lyonnet6;7, Elisa Rubinato1;8, Laurence Jonard1;2, Sophie Rondeau6, JACQUEMONT Marie-Line9, Delphine dupin-deguine10, Sebastien Moutton11, Bertrand Isidor12, Alban Ziegler13, Valérie CORMIER-DAIRE6, Marie Vincent12, Sandrine MARLIN 1;2;7

1Hôpital Necker-Enfants Malades, Centre de Référence Surdités Génétiques, UF Développement et Morphogénèse, Service de Médecine génomique des Maladies rares, Paris, France; 2Hôpital Necker-Enfants Malades, Centre de recherche en audiologie, Paris, France; 3Hospital Center University De Rouen, Service d’ORL et Chirurgie Cervicofaciale et Audiophonologie, Rouen, France; 4Chu, Département de Génétique, Centre de Référence des anomalies du Développement, Rouen, France; 5Necker Hospital, UF Neurodeveloppement-Neurologie Mitochondries-Métabolisme, Service de Médecine génomique des Maladies rares, Paris, France; 6Necker Hospital, UF Développement et Morphogénèse, Service de Médecine génomique des Maladies rares, Paris, France; 7Institute Imagine, UMR-1163 INSERM, Paris, France; 8Institute for Maternal and Child Health –IRCCS “Burlo Garofolo”, Medical Genetics, Trieste, Italy; 9St-Pierre hospital, Génétique Médicale Pôle femme-mère-enfant, Saint-Pierre, Reunion; 10Hospital Center University De Toulouse, Service ORL, Otoneurologie et ORL pédiatrique, Toulouse, France; 11Nursing Home Protestant De Bordeaux Bagatelle, Centre Pluridisciplinaire de Diagnostic PréNatal, Talence, France; 12Centre hospitalier universitaire de Nantes, Service de Génétique Médicale, Nantes, France; 13Angers University Hospital Center, Service de Génétique Médicale, Angers, France

KBG syndrome is a rare genetic disorder due to monoallelic pathogenic variations of ANKRD11.The typical phenotype includes facial dysmorphism, costal and spinal malformation and developmental delay. Hearing loss in KBG patients has been reported for many years, but no study has evaluated audiological phenotyping from a clinical and an anatomical point of view.

Our objective was to reinforce clinical knowledge of hearing impairment in KBG syndrome.

This French multicenter study included 32 KBG patients with retrospective collection of data on audiological features, ear imaging and genetic investigations.

We identified a typical audiological profil in KBG syndrome: conductive (70%), bilateral (78%), mild to moderate (84%) and stable (70%) hearing loss, with some audiological heterogeneity.

Among patients with an abnormality on CT imaging (55%), ossicular chain impairment (67%), fixation of the stapes footplate (33%) and inner-ear malformations (33%) were the most common abnormalities.

In this innovative study, we have shown that hearing impairment may be the first clinical sign of KBG syndrome and we describe the specific characteristics of hearing loss, underlining the variability of clinical, anatomical and radiological features.

We recommend a complete audiological and radiological evaluation and an ENT-follow up in all patients presenting with KBG. Imaging evaluation is necessary to determine the nature of lesions in the middle and inner ear.

Conflict of Interest: None declared.

EP03.013 PRPH2-related retinal dystrophies: mutational spectrum in Fundación Jiménez Díaz University Hospital cohort

Lidia Fernández-Caballero 1;2, Inmaculada Martin Merida1;2, Fiona Blanco-Kelly1;2, María José Trujillo Tiebas1;2, Almudena Avila Fernández1;2, Marta del Pozo Valero1;2, Irene Perea-Romero1;2, Saoud Tahsin-Swafiri1;2, Fermina Lopez-Grondona1;2, Ana Isabel Sánchez Barbero1;2, Olga Zurita1;2, Gonzalo Nuñez Moreno1;2;3, Pablo Mínguez1;2;3, Blanca García Sandoval4, Marta Corton1;2, Carmen Ayuso1;2

1Instituto de Investigación Sanitaria-Fundación Jiménez Díaz University Hospital, Universidad Autónoma de Madrid (IIS-FJD, UAM), Department of Genetics and Genomics, Madrid, Spain; 2Centre for Biomedical Network Research on Rare Diseases (CIBERER), Instituto de Salud Carlos III, Madrid, Spain; 3Bioinformatics Unit, Health Research Institute-Fundación Jiménez Díaz University Hospital, Universidad Autónoma de Madrid (IIS-FJD, UAM), Madrid, Spain; 4Fundación Jiménez Díaz University Hospital (FJD), Department of Ophthalmology, Madrid, Spain

Background/Objectives: Inherited Retinal Dystrophies (IRD) are a group of rare diseases with a prevalence of 1:3000-4000 people. PRPH2 is one of the most frequent non-syndromic IRD (NS-IRD) causative genes (3,4-4,6%). It is linked to a wide variety of retinal phenotypes (retinitis pigmentosa, cone-rod dystrophy and different macular dystrophies). Here, we describe the PRPH2 mutational spectrum in the FJD-University Hospital cohort.

Methods: After genetic screening through classic genotyping or targeted next-generation sequencing (NGS) methods, 108 unrelated IRD families from our database (4800 families) were characterized with PRPH2 variants. The variants were classified according to their typology and localization in the protein domain. Patient’s clinical data were obtained from self-reported ophthalmological history. NS-IRDs were classified according to the reported referral phenotype into cone- (NON-RP) or rod-predominant diseases (RP).

Results: In the FJD-cohort, 4% of solved NS-IRDs were genetically characterized with PRPH2 variants. Most patients carried missense (33%) or frameshift (12%) variants. Regarding the location in the protein, the majority of mutations are found in the D2-loop domain (75%). Approximately 4/5 of these patients have NON-RP phenotypes with an age onset ranging from adolescence to the 5th-6th decade. Our findings show high phenotypic variability within patients even belonging to the same family.

Conclusion: Given the PRPH2-associated phenotypic variability, further studies in the phenotype implication of some of the variants are needed. This could help to improve clinical management and genetic counseling and thus, hopefully, help to bring new therapies to patients.

Grant References: CIBERER, ISCIII (PI19/00321-PI22/00321), IIS-FJD_Biobank, University Chair UAM-IIS-FJD Genomic Medicine, ONCE.

Conflict of Interest: None declared.

EP03.014 Identification of common and rare genetic variants reveals a possible role in early-onset maculopathy

Martin Georgiev 1;2, Kunka Kamenarova1;2, Nevyana Veleva3, Kalina Mihova1;2, Yoanna Kaneva3, Elena Mermeklieva4, Silvia Cherninkova5, Alexander Oscar3, Radka Kaneva1;2

1Medical University - Sofia, Molecular Medicine Center, Department of Medical Chemistry and Biochemistry, Sofia, Bulgaria; 2Medical University - Sofia, Laboratory of genomic diagnostics, Department of Medical Chemistry and Biochemistry, Sofia, Bulgaria; 3Medical University - Sofia, Department of Ophthalmology, University Hospital Alexandrovska, Sofia, Bulgaria; 4Sofia University “St. Kliment Ohridski”, Department of Ophthalmology, University Hospital Lozenets, Faculty of Medicine, Sofia, Bulgaria; 5Medical University - Sofia, Department of Neurology, University Hospital Alexandrovska, Sofia, Bulgaria

Background/Objectives: Age‐related macular degeneration (AMD) is one of the leading causes of severe visual impairment among elderly worldwide. Although AMD mainly affects the elderly (≥65y), some individuals develop AMD characteristics, including drusen at a much younger age. Early-onset maculopathy (EOM, ≤65y) can lead to advanced macular degeneration later in life. The aim of this study was to determine the contribution of common AMD‐associated variants in patients with AMD and EOM, and to identify potential disease‐causing rare variants in patients with EOM.

Methods: We performed whole exome sequencing (WES) in 10 ADM and 8 EOM patients. Common genetic variants were analysed in 124 AMD‐associated genes. We searched for rare variants causing inherited retinal degenerations (IRD) that resemble AMD to identify potential deleterious variants that might contribute to EOM development.

Results: Eight common genetic risk alleles in 7 AMD‐associated genes (C2, C3, CFH, CFB, TLR3, ARMS2, CX3CR1) were present in the two phenotype groups. Four SNPs (CFH-rs1061170, ARMS2-rs10490924, C3-rs1047286, TLR3-rs3775291) showed prevalence in AMD probands. Multiple-filtering pipeline identified 14 rare variants in 11 AMD-genes (CFHR4, CFB, ABCG1, HMCN1, APOB, CFI, CSMD2, CD36, SLC16A8, ABCA1, VLDR) in EOM probands. Evaluation of IRD genes revealed 7 rare variants (3 of which in PROM1, SEMA4, and COL2A1), were predicted to be potentially deleterious based on variant type and in silico tools.

Conclusion: Common AMD-associated variants contribute to AMD and EOM. The early-onset nature of EOM and our results suggest a possible role for genetic rather than environmental factors.

References: de Breuk et al. 2022


Conflict of Interest: None declared.

EP03.015 Analysis of VEGFA SNPs towards susceptibility of Diabetic Retinopathy in North West Indian population

Manroop Singh Buttar 1, Vasudha Sambyal1, Kamlesh Guleria1

1Guru Nanak Dev University, Department of Human Genetics, Amritsar, India

Background/Objectives: Diabetic retinopathy (DR) is one of the major microvascular complications associated with Type 2 Diabetes (T2D) and is one of the leading causes of blindness worldwide. Vascular endothelial growth factor (VEGF) is a multifunctional cytokine that promotes angiogenesis and vascular permeability. VEGF overexpression promotes vessel endothelial cell proliferation, migration, tube formation, and sprouting, thereby subserving as a contributing factor for DR. VEGFA-2549 insertion/deletion (I/D) and VEGFA -7 C/T have been implicated in a number of diseases with angiogenic basis, and hence are polymorphisms of particular interest. The present study is an attempt to evaluate the association of selected VEGFA polymorphisms with Diabetic Retinopathy.

Methods: In this case-control study 414 DR patients, 422 T2D patients (internal controls) and 401 healthy controls (CN) were analyzed. DNA samples were screened for VEGFA -2549I/D and VEGFA -7C/T polymorphisms using PCR (polymerase chain reaction) based methods.

Results: The genotypic and allelic frequencies of VEGFA -2549I/D polymorphism showed no significant difference between the study groups. The analysis of VEGFA -7C/T polymorphism showed significant association in T2D + DR vs. CN (p = 0.0339) and DR vs. T2D (p = 0.002).

Conclusion: Our results indicated that VEGFA -7 T allele showed 2.12-fold risk for T2D over CN and showed protection for DR over T2D.

Grant References: Indian Council of Medical Research- Senior Research Fellowship (ICMR-SRF)

Conflict of Interest: Manroop Singh Buttar ICMR-SRF, Vasudha Sambyal Professor, Kamlesh Guleria Associate Professor.

EP03.016 The Genetic Basis and the Diagnostic Yield of Nonsyndromic Hearing Loss in Qatar

Shaza Malik1;2, Karen El Akouri2;3, Nader Aldewik2, Houssein Khodjet Elkhil1, Sarah Okasha1, Tawfeg Bin Omran2;3, Mashael Al-Shafai 1

1Department of Biomedical Sciences, College of Health Sciences, QU Health, Qatar University., doha, Qatar; 2Department of Adult and Pediatric Medical Genetics, Hamad Medical Corporation, Doha, Qatar., doha, Qatar; 3Division of Genetic and Genomic Medicine, Sidra Medicine, Doha, Qatar., doha, Qatar

Background/Objectives: Hearing loss (HL) is the most common sensory defect worldwide, and 50-60% of cases are hereditary. The prevalence of Hereditary Hearing Loss is in Qatar is ~5.2%, and several tests are available including GJB2 sequencing, gene panel, whole exome sequencing and mitochondrial testing. We aim to investigate the genetic spectrum of Nonsyndromic Hearing Loss (NSHL) in Qatar and to assess the diagnostic yield of these test.

Methods: We performed a retrospective chart review for 127 pediatric patients with NSHL who attended the Department of Adult and Pediatric Medical Genetics at Hamad Medical Corporation (between 2014 and 2019).

Results: 39 of cases were genetically solved resulting in a diagnostic yield of 30.7%. The pathogenesis was attributed to 19 single gene variants detected among 11 genes, and two copy number variants. GJB2 variants were the most common cause for NSHL (36.8% of solved cases), and the c.35delG variant was the most common (9 cases). We also associated for the first time the variant c.283C>T in FGF3 with NSHL. 7 case with variants of uncertain significance were solved based on family segregation analysis. The diagnostic yield was significantly associated with the use of GJB2 sequencing and HL gene panel.

Conclusion: Our work provided new insights into the genetic basis of NSHL in Qatar. In the clinical setting, we recommend performing GJB2 gene sequencing as a first-tier genetic test for NSHL and HL gene panel as a second-tier genetic test for NSHL, and familial segregation for cases with uncertain variants.

Grant references: NA

Conflict of Interest: None declared.

EP03.017 Clinical and genetic spectrum of coloboma: a proposal for a comprehensive approach to newly diagnosed pediatric patients with coloboma

Antonio Martinez-Monseny 1, david ferri-rufete2, didac casas-alba1, laia baleta riera2, jaume catala mora3, ester casas3, Ana Llorca-Cardeñosa3, jesus diaz carcajosa3, carlos fresno3, francesc palau martinez4

1Sant Joan de Deu Hospital, Clinical Genetics, Barcelona, Spain; 2Sant Joan de Deu Hospital, Pediatrics, Barcelona, Spain; 3Sant Joan de Deu Hospital, Ophthalmology, Barcelona, Spain; 4Sant Joan de Deu Hospital, Genetic and Molecular Medicine, Barcelona, Spain

Background/Objectives: Coloboma is a defect in the closure of the optic fissure that can affect any part of the eyeball. It has been described in isolation or together with ophthalmological or systemic alterations, forming part of genetic syndromes. We aim to (1) establish the variables with the highest probability of a genetic diagnosis and worse visual outcome (2) develop a comprehensive diagnostic and follow-up protocol.

Methods: Descriptive, retrospective and single-center study. Patients under 18 years of age diagnosed with coloboma under follow-up by a Pediatric Ophthalmology Unit have been selected from January 2012 to December 2020.

Results: We included 214 patients (54% female), 50.9% presented with bilateral coloboma and 62.1% with other ophthalmological alterations (28.5% with microphthalmia). Systemic involvement was observed in 28%: neurological dysfunction (24.8%) and dysmorphic features (18.2%). A molecular diagnosis was reached in 26.2% (CHARGE syndrome was the most prevalent), being the clinical exome the most profitable test (22.2%). The variables associated with the highest probability of a diagnosis were: (1) secondary diagnosis for coloboma (p < 0.001), (2) nystagmus (p < 0.05), (3) systemic affection (p < 0.05), (4) performance of hearing, cardiology, and neuroimaging studies (p < 0.05), and (5) Clinical Genetics assessment (p < 0.001). Bilaterality, macular chorio-retinal coloboma and neurological, cardiovascular, and growth abnormalities were associated with a worse visual prognosis (p < 0.05). A diagnosis and follow-up strategy has been elaborated.

Conclusions: It’s important to identify diagnosis and prognosis indicators in patients with coloboma in order to offer genetic counseling, prevent possible complications and improve the quality of life of patients and their families.

Conflict of Interest: None declared.

EP03.018 Asymptomatic retinal dysfunction in alpha-methylacyl-CoA racemase deficiency

Abrar Alsalamah 1, Arif Khan2;3

1Vitreoretinal and Uveitis Divisions, King Khaled Eye Specialist Hospital, Riyadh, Saudi Arabia; 2Eye Institute, Cleveland Clinic Abu Dhabi, Abu Dhabi, United Arab Emirates; 3Department of Ophthalmology, Cleveland Clinic Lerner College of Medicine of Case Western Reserve University, Cleveland, United States

Background/Objectives: Alpha-methylacyl-CoA racemase (AMACR) deficiency is a peroxisomal disorder due to biallelic mutations in AMACR. At least 13 genetically confirmed patients have been reported to date. Seven had obvious pigmentary retinopathy; however, for the other six, no retinal phenotype was mentioned. Most of the previously reported patients with clinically obvious pigmentary retinopathy did not have electrophysiology studies, and for the few who did the details were limited. The purpose of this report is to document subtle retinal findings in an additional affected family.

Methods: Retrospective case series (three affected siblings and their unaffected parents).

Results: Three Arab siblings (16, 19, and 22 years old) with prior juvenile cholelithiasis had been diagnosed with AMACR deficiency based on biochemical analysis, whole exome sequencing, and confirmatory segregation analysis (AMACR NM_001167595.1: c.877T>C; p.C293R). For all three, there were no visual complaints, but retinal multimodal imaging and electroretinography suggested subtle retinal dysfunction.

Conclusion: Retinal dysfunction is a parameter that should be measured in patients with known or suspected AMACR deficiency even in the absence of visual symptoms. This may be helpful with clinical diagnosis and monitoring response to dietary interventions.

Conflict of Interest: None declared.

EP03.019 Long-read sequencing for improving the characterization of rare inherited eye diseases

Cristina Rodilla 1, Alejandra Damián1, Gonzalo Nuñez Moreno1, Irene Perea-Romero1, Marta del Pozo-Valero1, Lidia Fernández-Caballero1, Marta Rodriguez de Alba1, Inmaculada Martin Merida1, Almudena Avila Fernández1, Gema García-García2, Belén García-Bohórquez2, Pilar Barberán-Martínez2, Marc Delépine3, Claire Jubin3, Cédric Fund3, Aurélie Leduc3, Jean-François Deleuze3, Pablo Mínguez1, José María Millán2, Marta Corton1, Carmen Ayuso1

1Instituto de Investigación Sanitaria Hospital Universitario Fundación Jiménez Díaz, Universidad Autónoma de Madrid (IIS-FJD, UAM) - Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Genetics Department, Madrid, Spain; 2Institut d’Investigació Sanitària La Fe de València (IIS-La Fe)-Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Molecular Biomedicine, Cellular and Genomics Research Department, València, Spain; 3Centre National de Recherche en Génomique Humaine, Évry-Courcouronnes, France

Background/Objectives: Emerging long-read sequencing (LRS) technologies have great potential to improve the detection of structural variants (SVs) in rare diseases. This work presents our experience with nanopore-based sequencing in studying SVs in a cohort of patients with ophthalmogenetic diseases, mainly inherited retinal dystrophies (IRD).

Methods: Long-read genome sequencing was conducted in 35 patients with inherited eye diseases following short-read sequencing approaches. Libraries were prepared using high-weight molecular DNA following Oxford Nanopore Technologies protocols and sequenced at 30x on a PromethION platform. SVs were validated using Sanger sequencing, CGH, MLPA and/or FISH analysis.

Results: To date, LRS revealed causal SVs in 6 patients, including 3 copy number variants (CNVs) in 3 IRD patients and 3 copy-neutral rearrangements, 2 chromosomal inversions and one translocation, in 3 patients with developmental eye diseases. LRS allowed the detection and breakpoint refinement of microdeletions affecting one or multiple exons of three IRD-related genes (EYS, KCNV2 and MYO7A). In the latter patient, LRS also revealed a previously overlooked likely pathogenic single nucleotide variant (SNV), thus allowing a complete genetic characterization. Additionally, we identified other variants of unknown significance, such as a chromosome X duplication rearrangement and chromosome 1 retrotransposon insertion.

Conclusion: Our study underscores the utility of this technology for unveiling and characterize SVs involved in ophthalmogenetic diseases and, thus showcasing advantages for the detection and characterization of complex SVs in repetitive regions and other variants in non-coding regions.

Grant References: H2020 EasiGenomics program (PID10291), ISCII (PI19/00321, PI20/00851 and PI22/00321), University Chair UAM-IIS-FJD Genomic Medicine and ONCE.

Conflict of Interest: None declared.

EP03.020 Genetic diagnostic of inherited retinal dystrophies through clinical exome sequencing

Pilar Barberán-Martínez 1, Belén García-Bohórquez1;2, Cinta Navarro-Moreno1;2, Teresa Jaijo1;2;3, Elena Aller1;2;3, Gema García-García1;2, José M. Millán1;2

1Health Research Institute La Fe, Molecular, Cellular and Genomics Biomedicine Research Group, Valencia, Spain; 2Biomedical Research Network for Rare Diseases, CIBERER, Madrid, Spain; 3La Fe Polytechnic and University Hospital, Genetics Unit, Valencia, Spain

Background/Objectives: Inherited retinal dystrophies (IRD) are a group of diseases that cause visual impairment. There are currently 281 genes related to IDR. However, many patients still remain without a genetic diagnosis. The objective of this study is the genetic diagnosis of IRD patients through clinical exome (CE) sequencing.

Methods: We sequenced the clinical exome “Custom Constitutional Panel 17MB” of 61 IRD patients. The libraries were prepared with the protocol SureSelect XT HS (Agilent) and sequenced in the Illumina platform. The bioinformatic analysis was carried out with the Alissa resource (Agilent) filtering IRD genes. The potential pathogenicity of the variants was evaluated with in silico predictors, databases, and functional assays. Furthermore, we validated the (likely) pathogenic mutations by Sanger sequencing and/or MLPA.

Results: Causative variants were detected in 39/61 IRD patients (64%). The patients were solved with mutations in 25 different genes, being ABCA4 and USH2A the most frequent. Furthermore, one of the patients was solved with variants in RPE65, the first IRD gene with a gene therapy approved.

Overall, we identified 93 variants that were classified as (likely) pathogenic using the standards of the American College of Medical Genetics and Genomics. It is worth to mention the novel c.1299A>G;p.(Glu433=) variant in ABCA4, with a pathogenic effect confirmed by minigene assay.

Conclusions: CE sequencing is an effective strategy for the genetic diagnosis of genetically heterogeneous diseases, such as IDR. Moreover, the minigenes are a good tool to demonstrate the splicing effect of mutations.

Grant References: PI19/00303, ACIF/2019/252, PI22/00213, FPU20/04736, CP22/00028, ONCE.

Conflict of Interest: Pilar Barberán-Martínez Full, FPU20/04736, Belén García-Bohórquez full, Cinta Navarro-Moreno full, Teresa Jaijo full, Elena Aller full, Gema García-García full, José M. Millán full, IP.

EP03.021 When WGS is key to sort out clinical traits: a case of syndromic hearing loss

Margaux Serey-Gaut 1;2, Pierre Blanc3, Laurence Jonard3;4, Ralyath Balogoun3;4, Isabelle Mosnier5, Sandrine MARLIN2;6

1Hôpital Necker, AP-HP.CUP, Centre de Recherche en Audiologie, PARIS, France; 2Hôpital Necker, AP-HP.CUP, CRMR Surdités Génétiques, Service Médecine Génomique des maladies rares, UF Développement et Morphogenèse, Fédération de Génétique et de médecine génomique, PARIS, France; 3SeqOIA, Laboratoire SeqOIA (PFMG2025), Paris, France; 4Hôpital Necker, AP-HP.CUP, Laboratoire de Génétique Moléculaire, Service Médecine Génomique des maladies rares, UF Développement et Morphogenèse, Fédération de Génétique et de médecine génomique, PARIS, France; 5Hôpital de la Pitié-Salpêtrière, APHP, Service d’ORL, PARIS, France; 6Institut Imagine, Unité INSERM UMR1163, PARIS, France

Background: In patients presenting with profound bilateral sensorineural hearing loss, some degree of dysarthria is regularly observed, especially if cochlear implantation is conducted with delay. Vestibular areflexia is regularly associated to hearing loss and can induce delayed walk acquisition and some degree of imbalance.

Case report: A male patient has been investigated in the national reference centre for genetic hearing loss for more than 25 years. Initially, he presented with profound bilateral sensorineural hearing loss, implanted at age 5 years, with persisting dysarthria despite good cochlear implant performance. Fifteen years later, he started complaining of imbalance, leading to the discovery of vestibular areflexia. The association of profound bilateral sensorineural deafness and vestibular areflexia mandated the conduction of several rounds of retinal testing, all showing subnormal results. Lastly, he presents with persisting imbalance with subnormal clinical testing. Imaging revealed mild cerebellar hypoplasia and electrophysiological testing only showed proprioceptive deficit. For years, clinicians taking care of him had trouble delineating which symptoms were consequences of his cochleo-vestibular disorder and which could be associated symptoms.

At 27 years old, whole genome sequencing revealed a probably pathogenic hemizygous de novo c.212G>C p.(Arg71Pro) BCAP31 variation. It matches with a mild form of Deafness, dystonia, and cerebral hypomyelination (DDCH) syndrome, due to missense variations in BCAP31, as described by Whalen et al., (2021).

Concusion: In this case, reaching a diagnosis through WGS and with the recent published work by Whalen et al., was the only way to understand the patient’s clinical presentation.

Conflict of Interest: None declared.

EP03.022 Whole exome sequencing of 20 Spanish families: candidate genes for non-syndromic paediatric cataracts

patricia rodríguez-solana1, carmen gonzález-atienza1, eloísa sánchez-cazorla1, natalia arruti2;3, maría nieves-moreno2;3, Rocío Mena1;4, marta guerrero-carretero2, Ángela del Pozo4;5, victoria e.f.montaño1;4, lucia de dios-blázquez5, Celia Fernandez Alcalde2, María de los Ángeles Gómez Cano6, Luna Delgado de Mora6, Susana Noval2;3, Elena Vallespín 1;3;4

1Molecular Ophthalmology Section, Medical and Molecular Genetics Institute (INGEMM) IdiPaz, Hospital Universitario La Paz, 28046, Madrid, Spain; 2Department of Paediatric Ophthalmology, IdiPaz, Hospital Universitario La Paz, 28046, Madrid, Spain; 3European Reference Network on eye diseases (ERN-EYE), Hospital Universitario La Paz, 28046, Madrid, Spain; 4Biomedical Research Center in the Rare Diseases Network (CIBERER), Carlos II Health Institute (ISCIII), 28029, Madrid, Spain; 5Clinical Bioinformatics Section, Medical and Molecular Genetics Institute (INGEMM) IdiPaz, CIBERER, Hospital Universitario La Paz, 28046, Madrid, Spain; 6Clinical Genetics Section, Medical and Molecular Genetics Institute (INGEMM) IdiPaz, CIBERER, Hospital Universitario La Paz, 28046, Madrid, Spain

Background/Objective: Non-syndromic paediatric cataracts are defined as opacification of the crystalline lens that occurs during the first years of life without affecting other organs. This disease is the most frequent cause of reversible blindness in childhood, so the main objective of this study is to propose new candidate genes responsible for paediatric cataracts that allow a better genetic approach to this pathology.

Methods: We present a whole exome sequencing (WES) study of 20 Spanish families with non-syndromic paediatric cataracts who have obtained a previous inconclusive result in a customised cataract Next Generation Sequencing (NGS) panel. After ophthalmological evaluation and collection of peripheral blood samples of these families, WES was carried out to find genes which could cause the phenotype of the patients.

Results: On the one hand, we found at least one alteration that could cause non-syndromic paediatric cataracts in 45% of the families studied (9/20). Of the 11 variants found in the 9 families, 18.2% (2/11) are classified as pathogenic (P), 72.7% (8/11) as variants of uncertain significance-likely pathogenic (VUS-LP) and 9.1% (1/11) as variants of uncertain significance (VUS). On the other hand, we did not find conclusive results in 55% of the families studied, which suggests that further studies are needed.

Conclusion: The results of this WES study allow us to propose LONP1, ACACA, TRPM1, CLIC5, HSPE1, ODF1, PIKFYVE, CHMP4A, AQP5 and loci 2q37 as possible candidate genes for non-syndromic paediatric cataracts.

Grant References: Instituto de Salud Carlos III (ISCIII) project 507 “18/1234” and ONCE grant number 2020/0197782.

Conflict of Interest: patricia rodríguez-solana: None declared, carmen gonzález-atienza: None declared, eloísa sánchez-cazorla: None declared, natalia arruti: None declared, maría nieves-moreno: None declared, Rocío Mena full, principal investigator, marta guerrero-carretero: None declared, Ángela del Pozo: None declared, victoria e.f.montaño: None declared, lucia de dios-blázquez: None declared, Celia Fernandez Alcalde: None declared, María de los Ángeles Gómez Cano: None declared, Luna Delgado de Mora: None declared, Susana Noval: None declared, Elena Vallespín: None declared.

EP03.023 Aiming at the development of the optimal targeted approach to genetic diagnostics of retinal dystrophies

Ewa Matczyńska 1;2, Marta Beć-Gajowniczek1, Larysa Sivitskaya1, Elżbieta Gregorczyk1, Ewa Suchecka1, Przemysław Łyszkiewicz1, Robert Szymańczak1, Maria Jędrzejowska1, Sławomir Teper2, Maciej Krawczyński3, Anna Boguszewska-Chachulska1

1Genomed S.A., Warsaw, Poland, Warsaw, Poland; 2Medical University of Silesia in Katowice, Chair and Clinical Division of Ophtalmology, Katowice, Poland; 3Center of Medical Genetics Genesis, Poznań, Poland

Previous experiences concerning the genetic diagnosis of inherited retinal dystrophies (IRD), based on whole exome data and the first targeted panel approach, indicated several aspects worth consideration in the development process. Here we present our insights based on the results for the second group of patients.

NGS analysis based on a Twist Bioscience custom panel targeting coding regions of 343 IRD genes, along with selected deep intronic regions and mtDNA sequence, was applied to identify genetic causes of IRD in a group of Polish patients (N = 272). Bioinformatics involved the GATK(v4) best practice pipeline. The CNV analysis was conducted using CoNVaDING and GATK GermlineCNVCaller. The variant classification was performed according to ACMG/AMP guidelines with the help of the in-house interpretation tool BroVar(v3).

Positive results were provided for 176 patients (64.71%). In several cases, this was possible due to the analysis of deep intronic regions. An increase in mean coverage levels (200-250x) and uniformity (F80 1.2-1.4) enabled noise reduction during the CNV analysis (10 patients with the contribution of CNVs in the diagnosis, in two cases diagnosis was based solely on CNV variants). Coverage in the difficult RPGR ORF15 region was improved as a result of additional probe tiling.

Presented changes in the panel construction, as well as the improvement in the QC parameters and the CNV analysis, resulted in a 5% increase in the diagnostic yield compared to previous approaches. Four of the patients could be potentially treated with gene therapy as pathogenic variants were uncovered in the RPE65 gene.

Conflict of Interest: Ewa Matczyńska Employed in Genomed S.A., Marta Beć-Gajowniczek Employed in Genomed S.A., Larysa Sivitskaya Employed in Genomed S.A., Elżbieta Gregorczyk Employed in Genomed S.A., Ewa Suchecka Employed in Genomed S.A., Przemysław Łyszkiewicz Employed in Genomed S.A., Robert Szymańczak Employed in Genomed S.A., Maria Jędrzejowska Employed in Genomed S.A., Sławomir Teper: None declared, Maciej Krawczyński Employed in Center of Medical Genetics Genesis, Anna Boguszewska-Chachulska Employed in Genomed S.A., Member of Genomed S.A. advisory board.

EP03.024 Analysis of 40 index patients with non-syndromic and syndromic hearing impairment using whole exome sequencing (WES) discovers variants in rare or recently identified causative genes

Nanna Dahl Rendtorff1, Lone Sandbjerg Hindbaek1, Lisbeth Tranebjærg1, Mette Bertelsen 1

1The Kennedy Centre/Rigshospitalet, Department of Clinical Genetics, Copenhagen, Denmark

Background: Hearing impairment is a common congenital sensory disorder, which is genetically heterogeneous. Identification of the causative variants underlying hearing impairment is challenging, since >100 different genes for non-syndromic hearing impairment and >450 different genes for syndromic hearing impairment have been reported so far.

Methods: In this study, 40 index patients with hearing impairment and variable additional clinical features underwent whole exome Next-Generation Sequencing (WES). Patients were prescreened for DFNB1 and SLC26A4-related hearing impairment by Sanger sequencing and for most patients also by analyzing 90-150 genes in a genepanel for non-syndromic and syndromic hearing impairment using an Illumina platform.

Results: Of the 40 patients sequenced, we were able to detect causative variants in eleven cases. The study emphasizes the genetic heterogeneity of hearing impairment since the variants were found in different rare and relatively recently identified causative hearing loss genes such as: CDK5RAP2, CEP78, CEP250, COL11A1, COL9A3, FDXR, and TFAP2A. In total, thirteen different likely pathogenic or pathogenic variants were detected, and nine variants were novel (absent from the literature).

Conclusion: Whole exome sequencing, after negative gene panel analysis, allowed us to detect causative variants in eleven of 40 tested cases. The causative variants were found in rare and/or recently identified causative hearing impairment genes. Regarding the non-solved cases, selected families will be analyzed by whole genome sequencing.

Conflict of Interest: None declared.

EP03.025 Bilateral optic nerve coloboma in a floppy infant as a first presentation of Bosch-Boonstra-Schaaf syndrome

Karolina Gadzalska 1, Paulina Jakiel1, Agata Pastorczak1, Ewa Juścińska1, Monika Gorządek1, Tomasz Płoszaj1, Maciej Borowiec1, Agnieszka Zmysłowska1

1Medical University of Lodz, Lodz, Poland

Background: Bosch-Boonstra-Schaaf optic atrophy syndrome - BBSOAS (OMIM: 615722, ORPHA 401777) is a rare, autosomal dominantly inherited syndrome characterized by moderate developmental delay, dysmorphic features and significant visual impairment resulted from optic nerve atrophy. Patients with BBSOAS carry heterozygous mutations in NR2F1 gene, encoding a nuclear hormone receptor and transcriptional regulator.

Methods: The material for the study was DNA isolated from the peripheral blood leukocytes of a symptomatic patient using automated Maxwell system (Promega). The whole exome sequencing (WES) was performed using Twist Human Core Exome kit (Twist Bioscience). The result was confirmed by Sanger sequencing.

Results: We report a 7-month-old infant who was tested due to severe neurodevelopmental delay, generalized hypotonia and bilateral optic nerve coloboma. The first symptoms were observed at the age of 2 weeks when anisocoria occurred. WES revealed that the patient carries a de novo, heterozygotes, pathogenic variant located within DNA-binding domain of NR2F1 gene (NM_005654.6: p.Met151Thr) which causes the phenotype of BBSOAS.

Bioinformatics analysis identified the NR2F1 variant as pathogenic according to the ACMG recommendation. The variant was not found in database collecting polymorphic gene variants (Gnomad, dbSNP), while it is described as probably pathogenic in the ClinVar database.

Conclusions: In conclusion, we found pathogenic variant of NR2F1 in patient with severe neurodevelopmental delay and coloboma. The presented data confirms the whole exome sequencing is a highly useful tool to identify genetic background of severe optic nerve abnormality in children with a no family history of visual impairment.

Conflict of Interest: None declared.

EP03.026 Genetic etiology of non-syndromic hearing loss in Hungarian patients with cochlear implant

Margit Pál 1;2, Dóra Nagy1;3, Alexandra Neller1, Katalin Farkas1, Dóra Leprán-Török1, Nikoletta Nagy1, Dalma Füstös1, Roland Nagy4, László Rovó4, József Géza Kiss4, Marta Szell1;2

1Albert Szent-Györgyi Medical School, University of Szeged, Department of Medical Genetics, Szeged, Hungary; 2Eötvös Loránd Research Network, ELKH-SZTE Functional Clinical Genetics Research Group, Szeged, Hungary; 3Kepler University Hospital Med Campus IV, Johannes Kepler University Linz, Institute of Medical Genetics, Linz, Austria; 4Albert Szent-Györgyi Medical School, University of Szeged, Department of Oto-Rhino-Laryngology and Head-Neck Surgery, Szeged, Hungary

Introduction: Hearing loss is the most prevalent sensory disorder, affecting millions of people worldwide with an incidence of 0.1-0.2% in the newborn population. Most congenital nonsyndromic hearing loss (NSHL) cases are caused by hereditary factors. Previously, the majority of studies focused on Connexin 26 (encoded by the GJB2 gene), however with the improvement of new-generation sequencing (NGS) methods, the identification of novel variants has shown a great improvement in NSHL.

The purpose of this study was to elaborate the most effective screening process in a Central-European population of NSHL.

Methods: 139 non-related NSHL patients were enrolled in our study. All patients underwent cochlear implantation or were candidates for it. A stepwise comprehensive genetic approach, including bidirectional capillary sequencing, MLPA and an NGS panel of 108 HL genes was used in the study.

Results: Genetic diagnosis could be established in 99 patients, resulting in an overall 71% diagnostic yield. Targeted sequencing and MLPA could identify the genetic background of 50% of the cases and the NGS panel added another 21% to this diagnostic yield. The vast majority of the diagnosed cases showed autosomal recessive inheritance (86%). The distribution of GJB2 and non-GJB2-cases was 68% vs. 32%, respectively.

Conclusion: The implementation of this step-wise analysis markedly increased our diagnostic yield and proved to be cost-effective as well. To further improve the diagnostic yield, the implementation of whole exome sequencing (WES) may be taken into consideration.



Conflict of Interest: None declared.

EP03.027 Exploring the Role of CERKL and RIMS1 in Inherited Retinal Disorders: A Case report

Federico Rondot 1, Alessandra Pelle2, Amedeo Primerano2, Matteo Scaramuzzi3, Patrizia Dentelli1, Michele Reibaldi3;4, Barbara Pasini1;2

1University of Turin, Department of Medical Sciences, Turin, Italy; 2AOU Città della Salute e della Scienza di Torino, Medical Genetics Unit, Turin, Italy; 3AOU Città della Salute e della Scienza di Torino, Oculistica U, Turin, Italy; 4University of Turin, Dipartimento di Scienze Chirurgiche, Turin, Italy

Background: We present the case of a 16-year-old man from Pakistan who experienced a progressive and severe visual night vision impairment and maculopathy. The patient presented with bilateral scotoma and alopecia areata; a family history of early-onset blindness was referred in a first-degree cousin. Given the personal and familial history of visual impairment a clinical exome was performed.

Method: SureSelect-Agilent Custom-Constitutional-Panel-17Mb encompassing 5219 genes. Virtual panel “Inherited retinal diseases” (235 genes).

Results: The analysis led to the identification of a homozygous likely pathogenic variant in the CERKL gene (NM_201548.5: c.1073+3_1073+6del, rs1553513437) and a nonsense variant in RIMS1 gene [NM_014989.7: c.3391C>T - p.(Arg1131Ter)]. The former is predicted to disrupt the canonical donor site of CERKL exon 7 with the retention within the transcript of the 79 nucleotides from intron 7. The latter, never reported previously, should introduce a premature termination codon within exon 22 of a gene for which the role in inherited retinal disorders is disputed.

Conclusion: RIMS1 has been considered a candidate gene for dominant cone-rod dystrophy 7 (MIM #603649), but recent evidence challenges the causative role in the disease. The identification of a homozygous pathogenic/likely-pathogenic variant in the CERKL gene seems to explain the patient’s phenotype and challenges the previously reported causative role of RMS1 in inherited retinal disorders. However, we cannot exclude a possible contributory role of the nonsense RIMS1 variant in the clinical expression of our patient. Additional data and segregation analyses are needed to better understand the role of RIMS1 in retinal disorders.

Conflict of Interest: None declared.

EP03.028 Hypoacusia and GJB2 gene: a short report of the c.-22-2A>C variant

Giulia Vinci1;2, Ilaria Fiscarelli1;2, Alessandra Pelle 2, Marco Andrea Barberis2, Luca Sbaiz2, Patrizia Marcella Consolino3, Diego Di Lisi3, Enrico Grosso2, Barbara Pasini1;2

1Università degli studi di Torino, Scuola di Specializzazione in Genetica Medica, Torino, Italy; 2AOU Città della salute e della scienza di Torino, SC Genetica Medica U, Torino, Italy; 3Ospedale Martini, SS Audiologia, Otologia ed impianti Cocleari, Torino, Italy

Biallelic pathogenic variants of GJB2 gene (NM_004004.6) are responsible for autosomal recessive nonsyndromic hypoacusia.

The c.-22-2A > C likely pathogenic variant is a hypomorphic allele, affecting the splicing-acceptor-site of exon2, supposed to trigger a secondary upstream acceptor-site with a longer 5’UTR and a decreased expression of CX26-protein. Expected phenotype is milder.

We report two cases with c.-22-2A > C substitution.

PatientA. 26-year-old female, mild bilateral sensorineural hypoacusia on higher frequencies, with late childhood onset and slow age-related worsening. GJB2 compound heterozygote for c.246C>G and c.-22-2A > C variants: the first inherited from the 64-year-old father with right-ear hypoacusia and tinnitus of unknown origin; the latter variant inherited from the 59-year-old mother, with normal-hearing.

PatientB. 40-year-old female, presently pregnant. Severe bilateral sensorineural deafness with sudden onset at 22 years (autoimmunity and vascular causes were excluded, no benefit with corticosteroid therapy). NGS analysis of 101 deafness-related genes revealed only the heterozygous GJB2 c.-22-2A > C variant. Since B patient’s partner is also a healthy carrier of the c.35del GJB2 pathogenic variant, prenatal diagnosis was offered: both variants were found in the fetus.

Our data support the hypothesis that heterozygote carriers of the c.-22-2A > C variant are usually not hearing impaired, while compound heterozygotes develop non-congenital mild to moderate hypoacusia. However, we cannot rule out a possible contributory role of this variant in hearing loss if associated with unknown other genetic or non-genetic causes. Regarding patientB’s pregnancy, we can’t predict the audiological phenotype of the unborn child but, according to the available data, hypoacusia is expected to occur during their life.

Conflict of Interest: None declared.

EP03.029 Minigenes and long read sequencing for the analysis of splicing variants located in acceptor sites of the PAX6 gene

Carolina Ruiz-Sanchez 1, Alejandra Tamayo1;2, Gonzalo Nuñez Moreno1;2;3, Cristina Rodilla1;2, Carmen Ayuso1;2, Pablo Mínguez1;2;3, Marta Corton1;2

1Health Research Institute - Fundación Jiménez Díaz (IIS-FJD), Genetics Department, Madrid, Spain; 2Center for Biomedical Network Research on Rare Diseases (CIBERER), ISCIII, Madrid, Spain; 3Health Research Institute - Fundación Jiménez Díaz (IIS-FJD), Bioinformatics Unit, Madrid, Spain

Background/Objectives: PAX6 is a transcription factor involved in ocular development whose alterations are associated with congenital aniridia. Approximately 15% of PAX6 mutations affect canonical splicing sites. Generally, spliceogenic variants result in exon skipping but also might enhance the use of unexpected cryptic sites. Our work aimed to study the spliceogenic effects of different variants in the canonical acceptors of PAX6.

Methods: From our aniridia cohort or mutational databases, we selected variants located in the nucleotides -1 and -9, close to the canonical acceptor of any PAX6 exon. After in silico analysis, potential spliceogenic variants were in vitro assessed through exon-trapping minigenes. Three multiexonic minigenes were generated with the genomic sequences of PAX6 exons 5-7, 8-11 and 10-13. Variants were introduced by directed mutagenesis and then, mutated and wild-type minigenes were transfected into HEK293 cells. Subsequently, total RNA was extracted, and splicing patterns were analysed by RT-PCR using long-read sequencing in an Oxford Nanopore MiNION device.

Results: Finally, seven variants on acceptors showing potential splicing effects were analysed in vitro. Of these, half produced exon skipping while the remaining half also participated in the activation of exonic or intronic cryptic acceptors causing partial exon skipping.

Conclusion: Splicing variants located in canonical regions of different exons can affect splicing differently despite being in the same gene. Therefore, in vitro assays with minigenes are a good approach to test their involvement in the splicing process and their pathogenicity in aniridia.

Grants: Spanish Health Institute Carlos III (PI17_01164 and PI20_00851) and ONCE.

Conflict of Interest: None declared.

EP03.030 NGS genetic testing of a Romanian patient and his family identified a novel OPA1 gene mutation associated with hereditary optic atrophy

Cristina Gug1;2, Miruna Gug 3, Ioana Mozos4, Eusebiu Vlad Gorduza5, Maria Puiu6

1”Victor Babes” University of Medicine and Pharmacy, Microscopic Morphology, Timisoara, Romania; 2Medical office and Genetic Laboratory Dr Gug, Genetic Laboratory, Timișoara, Romania; 3”Victor Babes” University of Medicine and Pharmacy, Student at the Faculty of General Medicine, Timisoara, Romania; 4”Victor Babes” University of Medicine and Pharmacy, Functional Sciences-Pathophysiology, Center for Translational Research and Systems, Medicine, Timisoara, Romania; 5”Grigore T. Popa” University of Medicine and Pharmacy, Iasi, Mother and child Medicine, Iasi, Romania; 6”Victor Babes” University of Medicine and Pharmacy, Microscopic Morphology, Timisoara, Romania

Introduction: Hereditary optic neuropathy refers to a group of inherited diseases that cause optic nerve atrophy, a major cause of visual impairment.

Materials and Methods: A 15-year-old male patient was recently diagnosed with optic atrophy. Sequence analysis and deletion/duplication testing of the 772 genes associated with hereditary ophthalmic disease were performed by Next Generation Sequencing (NGS) was used.

Results: A novel variant, likely pathogenic, in the OPA1 gene was identified in this case. Mutations in this gene have been associated with optic atrophy type 1, an autosomal dominant inherited condition. Six more autosomal recessive genes have been identified in heterozygosity. The pathogen variant of the OPA1 gene (3q28-q29, OMIM:165500) that causes the patient’s disease is present in both his mother and the probate’s sister. Maternal grandparents do not have the mutation in the OPA1 gene, therefore the mutation occurred de novo in the patient’s mother she passed it on to both children.

Sequencing analysis of OPA1 gene revealed a splicing mutation (c.1589+1dup), in intron 16, in heterozygous state, that is expected to disrupt RNA splicing. This variant is not present in population databases and has not been reported in the literature in individuals affected with OPA1-related conditions.

Conclusions: The genetic cause of hereditary optic atrophy in the proband has been identified. Extensive family testing has allowed the diagnosis of two other relatives with the same disease, one of them pre-symptomatic. All this information is especially useful in the post-test genetic counseling and for a prenatal diagnosis in the future.

Conflict of Interest: None declared.

EP03.031 Early diagnosis of syndromic deafness by whole exome sequencing

Nerea Gestoso-Uzal 1;2;3, Juan Manuel Ruiz-Robles1;2;3, Carlos Gutiérrez-Cerrajero1;2;3, Juan José Tellería4;5, Luis Antonio Corchete2;5, Hortensia Sanchez-Gomez2;6, José Ignacio Benito-Orejas7, Fernando Benito-González2;6, Ana-Belén Herrero1;2;3, Rogelio González-Sarmiento1;2;3

1Molecular Medicine Unit, Department of Medicine, University of Salamanca, Salamanca, Spain; 2Biomedical Research Institute of Salamanca (IBSAL), University Hospital of Salamanca-USAL- CSIC, Salamanca, Spain; 3Institute of Molecular and Cellular Biology of Cancer (IBMCC), University of Salamanca-CSIC, Salamanca, Spain; 4Department of Cell Biology, Histology and Pharmacology, University of Valladolid, Valladolid, Spain; 5Institute of Molecular Biology and Genetics (IBGM), University of Valladolid-CSIC, Valladolid, Spain; 6Department of Otorhinolaryngology, University Hospital of Salamanca-IBSAL, Salamanca, Salamanca, Spain; 7Otorhinolaryngology Department, University Hospital of Valladolid, Valladolid, Spain

Background: Hearing loss is the most common sensory disorder worldwide. Up to 30% of hereditary hearing impairments are syndromic and affect other organs including the kidneys, eyes and heart. Currently, genetic diagnosis of congenital deafness is limited to high penetrance non-syndromic gene mutational status, like GJB2, GJB5 and OTOF. However, it is a very heterogeneous entity, and these genes only explain around 25% of new congenital deafness cases. The aim of this study was to identify rare mutations associated with congenital deafness using whole exome sequencing.

Methodology: Genomic DNA was extracted from peripheral blood leukocytes in a cohort of 12 patients. WES was performed to identify germline mutations. Selected mutations were confirmed by Sanger sequencing and studied in samples from patients’ relatives.

Results: We selected two patients with hearing loss as their only clinical manifestation, where we identified mutations in syndromic congenital deafness genes. One patient carried the c.1772G>T;p.Arg555Leu KCNQ1 heterozygous pathogenic mutation, associated with long QT syndrome. Manifestations of this syndrome include prolongation of the QT interval, ventricular arrhythmias and high risk of sudden death. The other carried the c.23G>A;pArg8Gln BSND heterozygous pathogenic mutation, related to Bartter syndrome, characterized by congenital deafness and renal affection. This early genetic diagnosis allows prevention, a better follow-up and the improvement of the treatment of these patients.

Conclusion: WES technique is a useful tool to identify mutations in genes with low penetrance, achieving an early diagnosis of syndromic deafness before severe manifestations appear.

This study was funded by GRS1864/A/18.

Conflict of Interest: None declared.

EP03.032 Revisiting PRDM12 polyalanine tract expansion in chronic mutilating itch of MiTES

Yunisa Pamela 1;2, Nivedita Sarveswaran1, Anuradha Bishnoi3, Arun C. Inamadar4, Bayanne Olabi5, Fiona Browne6, Vinay Keshava Murthy3, Manoj Pawar7, Sahana Srinivas8, Samantha Ibbs9, Natarajan Sivakumar10, Vijay Zawar7, Vykuntaraju K Gowda8, Celia Moss9, Geoffrey Woods1

1University of Cambridge, Cambridge Institute for Medical Research, Cambridge, United Kingdom; 2Universitas Padjadjaran, Department of Biomedical Sciences, Bandung, Indonesia; 3Post Graduate Institute of Medical Education and Research, Department of Dermatology, Chandigarh, India; 4Shri BM Patil Medical College & Hospital, BLDE (Deemed to be University), Department of Dermatology, Bijapur, India; 5Edinburgh Royal Infirmary, Department of Dermatology, Edinburgh, United Kingdom; 6Our Lady’s Children’s Hospital, Department of Dermatology, Dublin, Ireland; 7Dr. Vasantrao Pawar Medical College and Research Center, Nashik, India; 8Indira Gandhi Institute of Child Health, Department of Pediatric Neurology, Bangalore, India; 9Birmingham’s Children Hospital, Department of Dermatology, Birmingham, United Kingdom; 10The Newcastle Upon Tyne Hospitals NHS Foundation Trust, Department of Dermatology, Newcastle upon Tyne, United Kingdom

Background: Expansion of PRDM12 alanine repeat can cause two opposite phenotypes: midfacial toddler excoriation syndrome (MiTES) associated with homozygous 18 alanines and congenital insensitivity to pain (CIP) that might be linked to 19 alanines. MiTES is characterised by restricted congenital itch with normal pain sensing whilst in CIP pain sensation is absent but itch behaviour is normal.

Methods: We assessed 4 new cases of MiTES and 3 possible MiTES with atypical features and followed up 16 previous MiTES cases. Using HEK293 expressing normal, MiTES, or CIP alanine repeats (12, 18 and 19 alanines respectively), we examined aggregation and subcellular localisation by confocal microscopy.

Results: Phenotype study shows that chronic mutilating itch in MiTES is specific to the early years of life and entirely separate from PRDM12-CIP phenotype. The genotype-phenotype study shows that 7 – 15 alanines in PRDM12 results in normal phenotype, expansion to 17 – 18 alanines is associated with MiTES, while 19 alanines leads to CIP. The atypical MiTES having unusual clinical features have normal genotype, confirming that they are not MiTES. Both 18- and 19-alanine cell lines demonstrate aggregates that are predominantly nuclear, but no clear distinction between the 18 and 19-alanine aggregation phenotype.

Conclusion: This study validates the diagnostic criteria of MiTES as a congenital itch condition caused by PRDM12 polyalanine tract expansion. The MiTES and CIP exhibit distinct phenotypes despite their genotypes varying by a single amino acid. MiTES could be a protein aggregation disease.

Grant Reference: Indonesia Endowment Fund for Education/LPDP No. 201908222915477.

Conflict of Interest: None declared.

EP03.033 Functional validation of a novel variant in the MYO6 gene identified in a family with postlingual non-syndromic hearing loss from Argentina

Paula Buonfiglio 1, Carlos David Bruque2, Vanesa Lotersztein3, Lucia Salatino4, Paola Plazas4, Ana Belén Elgoyhen4;5, Viviana Dalamón5

1Instituto de Investigaciones en Ingeniería Genética y Biología Molecular “Dr. Héctor Torres”, INGEBI/CONICET, Laboratory of Physiology and Genetics of Hearing, CABA, Argentina; 2Hospital de Alta Complejidad SAMIC - El Calafate, Unidad de Conocimiento Traslacional Hospitalaria Patagónica, Calafate, Argentina; 3Hospital Militar Central “Dr. Cosme Argerich”, Servicio de Genética, CABA, Argentina; 4Facultad de Medicina, Universidad de Buenos Aires, Instituto de Farmacología, CABA, Argentina; 1Instituto de Investigaciones en Ingeniería Genética y Biología Molecular “Dr. Héctor Torres”, INGEBI/CONICET, Laboratory of Physiology and Genetics of Hearing, CABA, Argentina

Hereditary hearing loss (HL) is the most common sensory disorder affecting 1 in 500 newborn children. There are more than 100 hundred genes related to non-syndromic hearing impairment, therefore, the Whole Exome Sequencing (WES) technique becomes the best option to study all the genes at once. However, analysis of novel variants, in particular missense changes which can lead to a spectrum of phenotypes, is not always straightforward. In this work, we aimed to functionally validate a novel variant detected in a family case with HL.

After performing WES followed by the variant priorization process and segregation analysis, the novel variant c.2782C>A p.Arg928Ser in MYO6 gene was identified in a family with post-lingual non-syndromic hearing loss. To further analyse the functional implication of this variant, protein modeling with the AlphaFold2 software was performed, revealing a change in the electrostatic charge on the surface of the single alpha helix domain. Then, in order to functionally validate the novel variant, we carried out a knockdown phenotype rescue assay in zebrafish by inhibiting the expression of the orthologous myo6b gene. Injection with wild type MYO6 mRNA rescued the phenotype whereas the mutant MYO6 (c.2782C>A) mRNA had no effect, demonstrating the deleterious effect of this variant on the auditory system. These results highlight the importance of a combined strategy in order to identify candidate variants, as well as the in silico and in vivo studies we performed to infer their pathogenicity and better understand the mechanisms underlying the physiopathology.

Conflict of Interest: None declared.

EP04 Internal Organs & Endocrinology (Lung, Kidney, Liver, Gastrointestinal)

EP04.001 Simultaneously monitoring liver response and hepatitis B virus infections during pregnancy through plasma cell-free RNA sequencing

Jinghua Sun1, Wen-Jing Wang1, Xuanyou Zhou2, Tingyu Yang 1;3, Songchang Chen4;5, Chenming Xu4;5

1BGI-Shenzhen, Shenzhen 518083, China, Shenzhen, China; 2International Peace Maternity and Child Health Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200030, P. R. China, Shanghai, China; 3College of Life Sciences, University of Chinese Academy of Sciences, Beijing 100049, China, Beijing, China; 4International Peace Maternity and Child Health Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200030, P. R. China, Shanghai, China; 5Obstetrics and Gynecology Hospital, Institute of Reproduction and Development, Fudan University, Shanghai, China, Shanghai, China

Background: Hepatitis B virus (HBV) can be transmitted vertically to the fetus, and pregnancies with HBV infection should be closely monitored for HBV load and maternal liver function. Plasma cell-free RNA (cfRNA) encompasses a broad spectrum of RNA species that can be derived from both human cells and microbes, therefore providing a non-invasive means to simultaneously monitor liver response and HBV RNA level during pregnancy.

Methods: We profiled cfRNA in 211 plasma samples from pregnant women with HBV infection (n = 40) and healthy donors (n = 171). We then analyzed the changes of both the human transcriptome and the HBV RNA of plasma of HBV patients.

Results: We demonstrated that cfRNA can be used to identify HBV infections. The expression level of HBV in the patients with HBeAg positive was higher than in patients with HBeAg negative, and show a positive correlation with clinical liver functional indicators. The comparative analysis identified a group of genes that altered their expression level in the HBV patient, with function annotations consistent with apoptosis, biosynthesis and metabolism, transmembrane transport, and complement and coagulation cascades. In addition, cfRNA signatures from the liver increased in the HBV patients and correlated with the HBV RNA level.

Conclusions: Plasma cfRNA demonstrates the potential to simultaneously monitor liver response and HBV infections during pregnancy, which will be a benefit in HBV prevention.

Grants: none

Conflict of Interest: None declared.

EP04.002 Gene expression profiling through whole transcriptome sequencing predicts immunological mechanisms in Hirschsprung associated enterocolitis

Francesca Rosamilia 1, FRANCESCA CALCATERRA2;3, Francesco Caroli4, Marta Rusmini5, alice grossi5, Paolo Uva6, Alessio Pini Prato7, domenico mavilio2;3, Isabella Ceccherini5, francesca lantieri1

1University of Genoa, Biostatistic Unit, Health Science Department (DISSAL), Genoa, Italy; 2University of Milan, Department of Medical Biotechnologies and Translational Medicine, Milan, Italy; 3IRCCS Humanitas Research Hospital, Laboratory of Clinical and Experimental Immunology, Milan, Italy; 4IRCCS Giannina Gaslini, UOC Genetica Medica, Genoa, Italy; 5IRCCS Giannina Gaslini, UOSD, Laboratory of Genetics and Genomics of Rare Diseases, Genoa, Italy; 6IRCCS Giannina Gaslini, Clinical Bioinformatics Unit, Genoa, Italy; 7The Children Hospital, Azienda Ospedaliera SS Antonio e Biagio e Cesare Arrigo, Pediatric Surgery Unit, Alessandria, Italy

Hirschsprung’s disease (HSCR) is a congenital gut malformation caused by a lack of innervation. One of the most serious is HSCR associated enterocolitis (HAEC), a potentially lethal condition with 30% of incidence. However, the causes of HAEC are still unknown and the onset is difficult to predict.

We have carried out a transcriptome analysis on Intraepithelial lymphocytes (IEL) derived from gut biopsies and on Peripheral Blood Monocyte Cells (PBMCs) from HAEC, HSCR-only and pediatric patients affected by neither Hirschsprung, nor inflammatory related diseases.

The analysis on the IELs showed a clear clustering between the three groups of patients. Several transcripts were nominally significantly over- and under-expressed in HAEC vs HSCR-only patients, among which several transcripts also involved in Inflammatory Bowel Disease (IBDs) pathogenesis. Accordingly, we found an enrichment in immune and inflammatory pathways in the HAEC group of the Intraepithelial lymphocytes. The transcriptome analysis on the gut PBMCs from a subsample of the same patients is still on progress and is aimed to define if PBMCs might be a reliable biomarker. Preliminary results show a good concordance between IELs and PBMCs.

The finding of a shared genetic background with other inflammatory disorders affecting the gut, and the evaluation of gene networks and pathways involved in inflammation provides further knowledge on the mechanisms of gut inflammation. Eventually confirming the concordance between IELs and PBMCs would allow the much more feasible use of blood instead of gut biopsies, making the replication of the present findings in other and bigger samples more reliable.

Conflict of Interest: None declared.

EP04.003 Genetics of rare kidney disease focal segmental glomerulosclerosis (FSGS) in adult central European population

Dana Thomasova 1, Michaela Zelinová2, Malgorzata Libik2, Pavel Votypka2, Jan Geryk2, Silvie Rajnochova Bloudickova3, Karel Krejčí4, Jana Machová5, Jana Reiterova6, Eva Jancova6, Martina Klollarova7, Ivan Rychlik7, Martin Havrda7, Marek Kollar8, Milan Macek1

1University Hospital Motol and 2nd Faculty of Medicine, Charles University, Institute of Biology and Medical Genetics, Prague, Czech Republic; 2University Hospital Motol, Institute of Biology and Medical Genetics, Prague, Czech Republic; 3Institute of Clinical and Experimental Medicine, Nephrology, Prague, Czech Republic; 4University Hospital Olomouc, Nephrology, Olomouc, Czech Republic; 5University Hospital Pilsen, Nephrology, Prague, Czech Republic; 6General University Hospital, Nephrology, Prague, Czech Republic; 7University Hospital Vinohrady, Nephrology, Prague, Czech Republic; 8Institute of Clinical and Experimental Medicine, Pathology, Prague, Czech Republic

Introduction: Focal segmental glomerularsclerosis (FSGS) is a clinically and genetically heterogeneous entity that affects glomerular function of the kidney and manifests itself by proteinuria. It often leads to loss of kidney function, accounting for about 15 % of patients with renal failure. A portion of the FSGS cases is of a monogenic etiology.

Methods: Multicentric study, cohort: adult patients with FSGS confirmed by kidney biopsy, FSGS family history or a sporadic primary FSGS resistant to steroids. Methods: whole exome sequencing, whole genome sequencing, bioinformatics.

Results: The current cohort consists of 239 patients of average age 48 years. The DNA analysis revealed pathogenic variants in genes associated with FSGS at 13,4 % unrelated patients. Genetic testing yielded diagnosis in 40% of FSGS families and in 10% of sporadic cases. The most frequent mutations were variants in COL4A genes (50%), compound heterozygous mutations in NPHS2 gene (17%) and in FSGS families INF2 gene variants (10%). 36% of variants are novel. By the statistical enrichment analysis we identified two Czech-specific founder mutations within NHPS2 (c.G868A) and COL4A4 (c.G1598A). The latter variant is specific for patients with Roma ancestry. The most significantly enriched gene with rare mutations within our cohort is SMARCAL1.

Conclusion: This is the first large-scale adult-onset genetic FSGS study in Czech population. It brings new insights into genetic and clinical features of the FSGS in adults and the results will improve personalized medical care.

Support: by grant NV19-06-00443, Czech Health Research Council, Ministry of Health of the Czech Republic

Conflict of Interest: Dana Thomasova grant NV19-06-00443 from AZV ČR (Czech Health Research Council), Ministry of Health of the Czech Republic, Michaela Zelinová: None declared, Malgorzata Libik: None declared, Pavel Votypka: None declared, Jan Geryk: None declared, Silvie Rajnochova Bloudickova: None declared, Karel Krejčí: None declared, Jana Machová: None declared, Jana Reiterova: None declared, Eva Jancova: None declared, Martina Klollarova: None declared, Ivan Rychlik: None declared, Martin Havrda: None declared, Marek Kollar: None declared, Milan Macek: None declared.

EP04.004 Impact of bread diet on intestinal dysbiosis and irritable bowel syndrome assessed by high-throughput sequencing of the 16S rRNA gene: A pilot study

Aleix Lluansí 1, Marc Llirós2, Robert Carreras-Torres1, Anna Bahí1, Montserrat Capdevila1, Anna Feliu1, Laura Vilà-Quintana1, Núria Elias-Masiques3, Emilio Cuevas3, Laia Peries4, Leyanira Torrealba4, Josep Oriol Miquel Cusachs4, Míriam Sàbat5, David Busquets Casals4, Carmen López Núñez4, Sílvia Delgado-Aros6, Jesús L Garcia-Gil7, Isidre Elias3, Xavier Aldeguer1

1Institut d’Investigació Biomèdica de Girona Dr Josep Trueta (IDIBGI), Digestive Diseases and Microbiota Group, Girona, Spain; 2Universitat de Vic–Universitat Central de Catalunya, Vic, Spain, Bioinformatics and Bioimaging (BI-SQUARED) research group, Biosciences Department, Faculty of Sciences, Technology and Engineerings, Vic, Spain; 3Elias-Boulanger S.L., Vilassar de Mar, Spain; 4Hospital Universitari de Girona Dr. Josep Trueta, Department of Gastroenterology, Girona, Spain; 5Hospital de Santa Caterina, Department of Gastroenterology, Girona, Spain; 6Gastroenterology Scientific advisor to Elias-Boulanger S.L, Vilassar de Mar, Spain; 7Universitat de Girona, Deparment of Biology, Girona, Spain

Background/Objectives: Gut microbiota may be involved in the presence of Irritable Bowel Syndrome (IBS)-like symptomatology in remission Ulcerative Colitis (UC) patients. High-throughput sequencing of the 16S rRNA gene allows an agnostic characterisation of the gut microbiota. Bread is an important source of dietary fibre, and a potential microbiota modulator. We aimed to assess the effect of a traditionally elaborated and baked bread, in comparison to a modern elaboration bread, in changing the gut microbiota and relieving IBS-like symptoms in patients with quiescent UC.

Methods: Thirty-one UC patients in remission with IBS-like symptoms were randomly assigned to a dietary intervention with 200 g/d of either traditional or modern bread for 8 weeks. Changes in faecal microbiota composition were assessed by high-throughput sequencing of the 16S rRNA gene. Clinical symptomatology was tested using questionnaires and inflammatory parameters.

Results: A decrease in IBS-like symptomatology was observed after both the treatment (p-value = 0.003) and control bread (p-value < 0.001) interventions. The treatment bread suggestively reduced the Firmicutes/Bacteroidetes ratio (p-value = 0.058). In addition, the Firmicutes/Bacteroidetes ratio seemed to be associated with abdominal pain mitigation (p-value = 0.059).

Conclusions: The intake of a bread baked using traditional elaboration decreased the Firmicutes/Bacteroidetes ratio, which seemed to be associated with improving IBS-like symptoms in quiescent UC patients. These findings suggest that the traditional bread elaboration has a potential prebiotic effect in improving gut health.

Grant reference: Spanish Ministry of Economy, RETOS program (RTC-2017-6467-2).

Conflict of Interest: None declared.

EP04.005 Linking genetic testing and histopathologic evaluation of the kidney as a way tosuccessfully diagnose Alport syndrome

Maria MALARSKA 1, Hanna Moczulska1, Paulina Pachniak1;2, Paulina Jakiel1, Karolina Gadzalska1, Ewa Jachacz2, Filip Jabłoński3, Andrzej Kałużyński3, Hanna Romanowicz3, Agnieszka Zmysłowska1, Maciej Borowiec1

1Department of Clinical Genetics, Medical University of Lodz, Lodz, Poland, Lodz, Poland; 2Department of Internal Medicine and Nephrodiabetology, Medical University of Lodz, USK im. WAM – CSW, Lodz, Poland, Lodz, Poland; 3Department of Pathomorphology, Institute of the Polish Mother’s Memorial Hospital, Lodz, Poland, Lodz, Poland

Background: Alport syndrome (AS) is a genetic collagen IV disease that causes kidney, hearing and vision damage. In the past, it was diagnosed mainly by kidney biopsy, but now more often by genetic testing. This study aims to combine these two techniques for the AS diagnosis and to test the relationship between the presence of pathogenic variants and renal biopsy lesions.

Methods: The study group included 57 patients (F:M; 31:26), whose average age at the time of genetic diagnosis was 24 years. Genetic studies were performed by NGS technique and confirmed by Sanger sequencing. Kidney biopsy material was analyzed by optical and electron microscopy and immunomorphological studies.

Results: In a group of 57 patients with genetically confirmed AS, 13 underwent renal biopsy prior to genetic diagnosis. Among the biopsies performed, seven led to the suspicion of AS. The most common changes observed were thickening and thinning of the basement membrane and inflammatory infiltrates. Among seven patients in whom the correct suspicion was established after renal biopsy, the most common finding was a pathogenic variant in the COL4A3 gene (42.8%). Two patients with the most severe clinical course of AS showed inflammatory infiltrates on histopathological examination without AS-specific lesions.

Conclusion: In the results presented here, there was no clear relation between the pathogenic variant found by genetic testing in patients with Alport syndrome and the microscopic image of the kidney. Despite the increasing progress of histopathological testing, it seems that it cannot be an alternative to genetic testing in this disease.

Conflict of Interest: None declared.

EP04.006 Clinical exome sequencing as a tool for genetic diagnosis of Alport syndrome and thin basement membrane disease

Giulia Margherita Brach del Prever 1;2, Valeria Bracciamà1;2, chiara vizzuso2, Silvia Kalantari1;2, Angelo Corso Faini1;2, maria luca1;2, carmelo maria romeo1;2, fiorenza mioli1;2, Francesca Arruga1, Diana Carli1;2, elisa longhitano3;4, Licia Peruzzi5, Roberta Fenoglio6;7, domenico santoro3;4, Dario Roccatello6;7, Antonio Amoroso1;2, Tiziana Vaisitti1;2, Silvia Deaglio1;2

1Immunogenetics and Transplant Biology Service, Città della Salute e della Scienza University Hospital, Turin, Italy; 2University of Turin, Department of Medical Sciences, Turin, Italy; 3Unit of Nephrology and Dialysis, Martino Policlinic, Messina, Italy; 4University of Messina, Department of Clinical and Experimental Medicine, Messina, Italy; 5Nephrology Dialysis and Transplantation, Regina Margherita Children’s Hospital, Turin, Turin, Italy; 6SCU Nephrology and Dialysis (ERKnet member)-CMID, Center of Research of Immunopathology and Rare Diseases, San Giovanni Hospital, Turin, Italy; 7University of Turin, Department of Clinical and Biological Sciences, Orbassano, Italy

Background/Objectives: Pathogenic variants in COL4A3, COL4A4 and COL4A5 are associated with Alport syndrome (AS) and thin basement membrane disease (TBMD). Patients with AS show glomerular nephropathy and end-stage renal disease, frequently associated with sensorineural deafness/ocular anomalies, while in TBMD most patients are asymptomatic and have normal renal function. We describe a cohort of patients with AS/TBMD.

Method: Clinical exome sequencing (CES) was performed on a cohort of 95 patients with clinical features suggestive of AS referred to our center from 2019 to 2022.

Results: We identified causative (C4/C5) variants in 43 (45.3%) patients. We confirmed genetic diagnosis of TBMD in 16 (37.2%) patients, of whom 12 (75.0%) with monoallelic variants in COL4A4 and 4 (25.0%) in COL4A3; 19 (44.2%) patients with AS had variants in COL4A3 (n = 5, 26.3%), COL4A4 (n = 1, 5.3%) and COL4A5 (6 females, 7 males, 68.4%). In 2 (4.7%) cases we found variants in COL4A1 and MYH9, associated with other renal diseases. Finally, we found digenic AS in 6 (13.9%) patients. Of note, 3 (50.0%) of them with coexisting variants in COL4A5/COL4A4 and one (16.7%) in COL4A3/COL4A4 had early renal failure. In contrast, 2 male patients (33.3%), with COL4A5/COL4A3 missense variants showed a mild phenotype. The remaining 16 (16.8%) patients had variants of uncertain significance (C3) and 36 (37.9%) tested negative.

Conclusion: CES is a powerful tool to define the AS/TBMD molecular diagnosis, to identify digenic AS and to find variants in other renal genes, improving clinical and therapeutic management.

Grants: Ministero dell’Istruzione, Progetto di Eccellenza Dipartimentale #D15D18000410001

Conflict of Interest: None declared.

EP04.007 Update on Alport syndrome in our healthcare area

Maria del Mar Del Aguila Garcia 1, María Luz Bellido1, Ana Isabel Morales Garcia2, Susana García-Linares2, Rafael Jose Esteban de la Rosa1, Antonio Miguel Poyatos-Andújar1

1Hospital Universitario Virgen de las Nieves, Granada, Spain; 2Hospital Universitario Clínico San Cecilio, Granada, Spain

BACKGROUND/OBJETIVES: Alport syndrome (AS) is considered a rare disease and is underdiagnosed partly due to its clinical heterogeneity and variable expressivity. It can be transmitted X-linked (80%), autosomal recessive (15%) or dominant (5%).

METHODS: Descriptive observational study of 386 individuals who underwent genetic study using a panel of 44 genes related to hereditary renal diseases (SOPHiA Genetics).

Sequencing of the libraries was performed on a MiSeq (Illumina Inc), bioinformatics analysis of the data and variant annotation was performed using SOPHiA DDM software, and variant review by querying major databases (ClinVar, Exac, HGMD, NCBI, PKD Foundation, LOVD).

RESULTS: 132 informative results were obtained, 20% corresponding to Alport syndrome, of which: 35% developed deafness, whereas only 7% had vision loss. One third required renal replacement therapy, in which 70% had variants in COL4A3 or COL4A4.

The correlation between clinical and genetic diagnosis was only 11%, the reasons for the study being mainly unfiliated chronic kidney disease, segmental and focal glomerulosclerosis and even polycystic kidney disease.

65% of the variants found were in heterozygosis and dominant inheritance, with COL4A3 being the most frequently implicated gene.

CONCLUSION: We observed that in AS there is a low degree of concordance between clinical and genetic diagnosis, making the genetic study a basic tool for its correct diagnosis.

In our health care setting, in contrast to the scientific evidence published to date, autosomal dominant inheritance is the majority.

We found few cases of recessive transmission, which could be associated with the variable expressivity of the syndrome.

Conflict of Interest: None declared.

EP04.008 A novel TRPM6 gene variant (c.531delA) causing hypomagnesemia with secondary hypocalcemia

Sorina Schipor 1, Andrei Muresan1, Catalina Poalelungi1, Susana Vladoiu1, ELENA EMANUELA BRAHA1, Aura Madalina Boboc2;3, Iuliana Gherlan2;3

1”C. I. Parhon” National Institute of Endocrinology, Research Department, Bucharest, Romania; 2“C. I. Parhon” National Institute of Endocrinology, Pediatric Department, Bucharest, Romania; 3“Carol Davila” University of Medicine and Pharmacy, Bucharest, Romania

Background: Primary hypomagnesemia with secondary hypocalcemia (HSH) is a rare autosomal recessive disorder caused by mutations in TRPM6 gene encoding TRPM6 (transient receptor potential melastatin 6). This is a channel responsible for epithelial magnesium absorbtion in colon and renal distal convoluted tube.

Objective: To identify and characterize the genetic cause of a marked hypomagnesemia associated with hypocalcemia in a 4 months old Romanian girl.

Methods: The genomic DNA of the proband was extracted for Trusight One Sequencing Panel (Illumina) clinical exome sequencing and analysis of candidate causal variants.

Results: We identified a novel homozygous frameshift variant in TRPM6 gene, c.531delA (p.Gly178fs) which pre-maturely terminates protein and results into a truncated product of 178 amino acid residues instead of 2022. This variant is subject to degradation by nonsense-mediated decay (NMD Prediction Tool (shinyapps.io)). In silico analysis using MutationTaster predicted this variant as disease‐causing and was classified as likely pathogenic according to ACMG guidelines. The variant has not yet been described in the medical literature and has not been identified in population databases (dbSNP, gnomAD, ExAC, 1000 Genomes).

Conclusion: Our results shows that a novel frameshift variant in TRPM6 could be responsible for HSH in this patient. The finding has expanded the mutation spectrum of TRPM6 gene.

Conflict of Interest: None declared.

EP04.009 Novel TCF7L2 familial linkage and association with Type 2 diabetes, depression, and their comorbidity

Laura del Bosque-Plata 1, Mutaz Amin2, Rongling Wu3;4, Teodor Postolache5;6;7, Claudia Gragnoli4;8;9

1National Institute of Genomic Medicine, Nutrigenetics, and Nutrigenomic Laboratory, Mexico City, Mexico; 2Al-Neelain University, Faculty of Medicine, Biochemistry and Molecular Biology, Khartoum, Sudan; 3Penn State College of Medicine, Department of Statistics, Hershey, United States; 4Penn State College of Medicine, Department of Public Health Sciences, Hershey, United States; 5University of Maryland School of Medicine, Mood and Anxiety Program, Department of Psychiatry, Baltimore, United States; 6Military and Veteran Microbiome: Consortium for Research and Education (MVM-CoRE), Rocky Mountain Mental Illness Research Education and Clinical Center (MIRECC), Veterans Integrated Service Network (VISN) 19, Denver, United States; 7Veterans Integrated Service Network (VISN) 5, VA Capitol Health Care Network, Mental Illness Research Education and Clinical Center (MIRECC), Baltimore, United States; 8Department of Medicine, Creighton University School of Medicine, Division of Endocrinology, Omaha, United States; 9Bios Biotech Multi-Diagnostic Health Center, Molecular Biology Laboratory, Rome, Italy

Objectives: Alterations in the activity of transcription factor 7-like 2 (TCF7L2) generate defects previously associated with neuropsychiatric disorders. We investigated the role of the TCF7L2 gene with major depressive disorder (MDD), type 2 diabetes (T2D), and MDD-T2D comorbidity. We tested whether TCF7L2 is linked to and/or in linkage disequilibrium (LD, namely linkage plus association) with MDD, T2D, and MDD-T2D comorbidity.

Patients and Methods: In 212 families with T2D and MDD and from the Italian population, we analyzed 80 microarray-based SNPs using Pseudomarker software for linkage to and LD with T2D and MDD under the recessive model with complete penetrance (R1). In a secondary analysis, we tested the variants under the models dominant with complete penetrance (D1), recessive with incomplete penetrance (R2), and recessive with incomplete penetrance (R2).

Results: We found several novel linkage signals and genetic LD-based associations. In addition, we detected two new transcription-factor (TF) binding sites created by two found risk variants: the MDD-risk variant rs12255179 creates a new TF-binding site for the CCAAT/enhancer-binding protein α (C/EBPα), and the T2D-risk variant rs61872794 creates a new TF-binding site for the organic cation-uptake transporter (OCT1). Both new binding sites are related to insulin metabolism.

Conclusions: These results highlight the cross-interactivity between T2D and MDD. Further replication is needed in diverse ethnic groups.

Conflict of Interest: None declared.

EP04.010 Case report: Tuberous sclerosis/polycystic kidney disease contiguous gene syndrome case originated by atypical non-contiguous PDK1/TSC2 deletions

Marc Ventayol-Guirado 1, Laura Torres-Juan2;3, Victor Asensio-Landa1;4, Rosa Martorell4, Angeles Perez Granero4, Maria Isabel Madrid4, Jessica Hernández1, Javier Lumbreras5, Jordi Roldan6, Monserrat Pons5, Iciar Martínez1;4, DAMIAN HEINE SUÑER3;7, Fernando Santos1;4

1Fundación Instituto de Investigación Sanitaria Islas Baleares (IdISBa), Health Genomics, Palma, Spain; 2Health Research Institute of the Balearic Islands (IdISBa), Genomics of Health research group, Palma de Mallorca, Spain; 3Hospital Universitari Son Espases, Unit of Molecular Diagnostics and Clinical Genetics, Palma, Spain; 3Hospital Universitari Son Espases, Unit of Molecular Diagnostics and Clinical Genetics, Palma, Spain; 5Hospital Universitari Son Espases, Pediatrics Department, Palma, Spain; 6Hospital Universitari Son Espases, Radiology Department, Palma, Spain; 7Health Research Institute of the Balearic Islands (IdISBa), Genomics of Health research group, Palma, Spain

Background: Mutations in PKD1 or PKD2 cause autosomal dominant polycystic kidney disease (ADPKD) while mutations in TSC1 or TSC2 cause the tuberous sclerosis complex (TSC). PKD1 lies immediately adjacent to TSC2 and large deletions can result in the PKD1/TSC2 contiguous gene deletion syndrome (CGS) with a distinctive phenotype of early onset PKD plus various manifestations of TSC.

Case presentation and Methods: A 16 month-old girl, first child of healthy non-consanguineous parents, was initially evaluated in the neonatal period due to cystic kidney disease. Her course was complicated with several TSC associated manifestations (West syndrome, Wolf-Parkinson-White syndrome, subependymal nodules and cortical tubers on brain MRI) and a likely diagnosis of PKD1/TSC2 CGS was considered. Molecular investigations to confirm the diagnosis were undertaken.

Results: An initial 60K array-CGH was normal. Whole exome sequencing (WES) detected 2 heterozygous variants of unknown significance (VOUS), a maternally inherited variant in PKD1(NM_001009944.2): c.5245G>A; p.V1749I and a de novo variant in TSC2 (NM_000548.4:): c.4166C>G p.P1389R, not allowing full confirmation of the clinical diagnosis. Subsequent reanalysis of WES data allowed the detection of 2 de novo non-contiguous partial gene deletions of both PKD1 (exons 1 to 10) and TSC2 (exons 27 to 32), confirmed by MLPA.

Conclusions: We present a case with clinical manifestations of PKD1/TSC2 CGS caused by 2 non-contiguous partial gene PKD1 and TSC2 deletions, probably mediated by a complex genomic rearrangement. Further results and discussion on the possible mechanism will be presented.

Conflict of Interest: None declared.

EP04.011 Case study: the key role of whole-genome sequencing (WGS) in the diagnostics of kidney disorders

Slavyana Staykova 1, Maya Atanasoska1;2, Lubomir Balabanski1, Irena Bradinova1, Maria Gaydarova3, Draga Toncheva1, Radoslava Vazharova1;4

1Cellgenetics laboratory, Sofia, Bulgaria; 2Department of Genetics, Faculty of Biology, Sofia University “St. Kliment Ohridski”, Sofia, Bulgaria; 3Pediatric Hospital „Prof. Dr. Ivan Mitev“, Sofia, Bulgaria; 4Department of Biology, Medical Genetics and Microbiology, Faculty of Medicine, Sofia University “St. Kliment Ohridski”, Sofia, Bulgaria

We present eleven patients referred for genetic testing based on symptoms of polycystic kidney disease, nephrocalcinosis, glomerulopathies and tubulopathies. For the purpose of the study, whole-genome sequencing and targeted analysis of 906 genes associated with kidney disorders was performed.

Five patients received genetic diagnosis. A three-year old boy was compound heterozygous carrier of one pathogenic and one likely pathogenic variant (c.1397G>A and c.3545G>C) in the PKHD1 gene, associated with polycystic kidney disease 4. A likely pathogenic variant in PKD1:c.11390A>G was found in a 45-year-old male with family history of polycystic kidney disease type 1. Another proband harbored one likely pathogenic variant in COL4A5:c.4538del, associated with X-linked Alport syndrome 1. Two unrelated children carried compound heterozygous pathogenic mutations in CYP24A1 (c.428_430del and c.443T>C) causing infantile hypercalcemia 1.

Two variants of unknown significance (VUS) that could be related to two of our patients’ phenotypes were detected: HOGA1:c.410C>T, combined with a known pathogenic mutation (HOGA1:c.700+5G > T) causing primary hyperoxaluria 3 and LRP5:c.3403C>T, associated with polycystic liver disease 4 with or without kidney cysts.

One incidental finding was detected in a 15-year-old girl, referred for genetic analysis based on clinical evidence of proteinuria, intellectual disability and dysmorphic features with unknown etiology. The patient carried one pathogenic homozygous missense variant in the TAF6 gene (c.212T>C) and was diagnosed with the ultrarare Alazami-Yuan syndrome.

Clarification of our patients’ genetic diagnosis provided precise genetic counseling for their families, allowed doctors to take appropriate measures for treatment and gave options for disease prevention in the future offspring.

Conflict of Interest: None declared.

EP04.012 The role of oxidative stress in chronic pancreatitis and pancreatic cancer risk

Laura Vilà-Quintana 1, Esther Fort1;2, Laura Pardo1;2, Maria T. Albiol-Quer3, Maria Rosa Ortiz-Duran4, Montserrat Capdevila1, Anna Feliu1, Anna Bahí1, Marc Llirós5, Adelaida García-Velasco6, Mireia M Ginestà7, Berta Laquente8, Débora Pozas1, Aleix Lluansí1, Ville N Pimenoff9, Victor Moreno10, Jesús L Garcia-Gil11, Eric J Duell12, Robert Carreras-Torres1, Xavier Aldeguer1

1Institut d’Investigació Biomèdica de Girona Dr Josep Trueta (IDIBGI), Digestive Diseases and Microbiota Group, Girona, Spain; 2Hospital Universitari de Girona Dr Josep Trueta, Department of Gastroenterology, Girona, Spain; 3Hospital Universitari de Girona Dr Josep Trueta, Department of Surgery, Hepato-Pancreato-Biliary unit, Girona, Spain; 4Hospital Universitari de Girona Dr Josep Trueta, Department of Pathology, Girona, Spain; 5Bioinformatics and Bioimaging (BI-SQUARED) research group, Biosciences Department, Faculty of Sciences, Technology and Engineerings, Universitat de Vic–Universitat Central de Catalunya, Vic, Spain; 6Hospital Universitari de Girona Dr Josep Trueta- Institut Català d’Oncologia (ICO), Girona, Spain; 7Institut d’Investigació Biomèdica de Bellvitge - Institut Català d’Oncologia (ICO), Hereditary Cancer Program, Oncobell Program, CIBERONC, Barcelona, Spain; 8Institut d’Investigació Biomèdica de Bellvitge - Institut Català d’Oncologia (ICO), Medical Oncology Department, Barcelona, Spain; 9Karolinska Institutet, Stockholm, Sweden; 10Institut Català d’Oncologia (ICO), Oncology Data Analytics Program (ODAP), Barcelona, Spain; 11Universitat de Girona, Department of Biology, Girona, Spain; 12Institut d’Investigació Biomèdica de Bellvitge - Institut Català d’Oncologia (ICO), Unit of Nutrition and Cancer, Cancer Epidemiology Research Program, Barcelona, Spain

Background/Objectives: Oxidative stress plays an important role in the pathogenesis of pancreatic diseases such as pancreatic cancer (PC) and chronic pancreatitis (CP). This study aims to assess the role of antioxidant enzymes and oxidative stress markers in PC and CP diseases.

Methods: Enzyme activities of superoxide dismutase (SOD) and glutathione peroxidase (GPx), plus concentrations of plasma total glutathione (GSH) and lipid peroxidation markers such as malondialdehyde (MDA), were assessed in 6 PC patients, 20 CP patients and 20 healthy controls using enzyme-linked immunosorbent assays (ELISA). In addition, genetically predicted gene expression association analyses (TWAS) were performed for genes involved in oxidative stress homeostasis, combining genetic prediction models and genome-wide association results for PC risk from the PanScan and PanC consortia (7,110 cases and 7,264 controls), accessed through dbGaP.

Results: Controls showed higher activity of SOD (20.44 ± 6.76 u/ml) compared to PC patients (11.80 ± 7.42 u/ml, p = 0.02), and higher activity of GPx (52.37 ± 27.35 nmol/min*ml) compared to patients with CP (23.56 ± 23.54 nmol/min*ml, p = 0.002). In addition, MDA levels were lower in controls (63.60 ± 32.20 nmol/ml) compared to PC (142.00 ± 51.97 nmol/ml, p = 0.01) and CP patients (152.52 ± 71.70 nmol/ml, p = 4.1 × 10−5). No statistically significant differences were found in total GSH concentrations among groups. Finally, higher levels of predicted expression of GPX1 were inversely associated with PC risk (Zscore = -2.98, p = 0.003).

Conclusions: Reduced antioxidant capacity and increased levels of oxidative stress markers were found associated to pancreatic diseases.

Grant references: Fundació La Marató 201912-31.

Conflict of Interest: None declared.

EP04.013 Molecular characterization of RCCX module in 21-hydroxylase argentine patients

Melisa Taboas1, Cecilia Fernandez2, Aldana Claps1, Carlos David Bruque3, Lucia Espeche1, Marisol Delea1, Liliana Dain 1;4

1Centro Nacional de Genética Médica, Buenos Aires, Argentina; 2Laboratorio Novagen, Buenos Aires, Argentina; 3Unidad de Conocimiento Traslacional Hospitalaria Patagónica, Hospital de Alta Complejidad SAMIC, El Calafate, Provincia de Santa Cruz, Argentina; 4Departamente de Fisiología, Biología Molecular y Celular, FCEN-UBA, Buenos Aires, Argentina

Background: Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency, accounts for 90–95% of CAH cases. The gene encoding 21-hydroxylase, CYP21A2 mapped to at 6p21.3, in the RCCX (RP-C4-CYP21-TNX) module with another duplicated copy composed of the pseudogenes RP2, CYP21A1P, TNXA. CYP21A1P shares 98% sequence identity with CYP21A2, therefore, unequal crossover may generate structural rearrangements and copy number changes in the RCCX module. Here we present our results on the molecular characterization of the RCCX module in affected patients from Argentina.

Methods: We analyzed a total of 314 patients: 226 (55 classical and 171 nonclassical, NC) consecutively samples by Southern blot (SB, 34 of them also analyzed by Multiplex-ligation probe amplification, MLPA), and 88 selected patients by MLPA (18 classical, 70 NC) representing 611 non-related alleles.

Results: We found 11 different haplotypes. The most frequent haplotypes were the bimodular standard RCCX module (50.8 and 43.3%, for patients studied by SB and MLPA, respectively), and a RCCX module with a duplicated CYP21A1P (34.6 and 44.6%, for patients studied by SB and MLPA, respectively). Chimeric genes account for 4.6 and 6.4 % respectively, more frequent (16.3%) for classical patients than for NC (1.5%). Haplotypes bearing a duplicated CYP21A2 gene represent 1.2 and 2.3% of the analyzed alleles by SB and MLPA, respectively.

Conclusions: The data provided add to the knowledge of the complexity of the RCCX region and for the characterization of a large cohort of patients from our region.

Grant References: PICT2006-00208; UBACYT 20020110100005BA; FOCANLIS: 2010, 2013 and 2015

Conflict of Interest: None declared.

EP04.015 Influence of MUC5B common sequence variant on the development and progression of fibrotic interstitial lung disease

Ariadna Padró Miquel 1, Guadalupe Bermudo Peloche2, Celia Montaño Montaño2, Maria Sanz Felisi1, Anna Esteve Garcia1, Cinthia Aguilera Roman1, Nuria Llecha1, Lurdes Planas Cerezales3, Maria Molina Molina2

1Bellvitge University Hospital, Clinical and Molecular Genetics Unit, Clinical Laboratory, L’Hospitalet de Llobregat, Spain; 2Bellvitge University Hospital, Pneumology Department, L’Hospitalet de Llobregat, Spain; 3Hospital de Viladecans, Pneumology Department, Viladecans, Spain

Background/Objectives: A genetic variant in the promoter region of MUC5B gene (NG_031880.1:g.1927G > T,rs35705950) has been associated controversially not only with an increased risk of developing IPF (idiopathic pulmonary fibrosis), the paradigm of fibrotic interstitial lung disease (ILD), but also with improved survival. The aim of this work is to assess the influence of MUC5B on the development and progression of ILD.

Methods: This is an observational, retrospective study. MUC5B variant was tested to 213 patients: 74 patients diagnosed with ILD and family history (cases), and another group of 139 asymptomatic subjects (controls). Fibrotic progression was defined as a forced vital capacity (FVC) loss ≥5% and/or carbon monoxide diffusing capacity (DLCO) loss ≥10% over 1 year. The influence of MUC5B on disease development and progression was tested by chi-square test (χ2).

Results: There were statistically significant differences (p-value = 2.478e-9) in the prevalence of the MUC5B variant between the control group (28%) and the cases group (71,6%).

Main diagnosis were IPF (31%), followed by hypersensitivity pneumonitis (24%). MUC5B variant was also more prevalent within the IPF group than no-IPF (p-value = 0,023). Although only a minority of cases shared consistent usual interstitial pneumonia pattern, functional progression was observed in 45,9% (41% of which were IPF). No statistically significant relationship of disease progression and MUC5B genotype was found.

Conclusions: The presence of the MUC5B variant is a risk factor for developing fibrotic ILD. However, no relationship with disease progression could be identified, raising doubts about the previously reported protective role of MUC5B.

Conflict of Interest: None declared.

EP04.016 Double trouble: an interesting case report with a double diagnosis

Özlem Baysal 1, Jolijn Verseput1, Rolph Pfundt1, Bert B.A. de Vries1

1Radboud University Medical Center, Human Genetics, Nijmegen, Netherlands

A five week old boy who was admitted at the pediatric ward of the Radboudumc hospital because of a viral respiratory tract infection causing central apnea’s.

His medical history showed a premature birth at a gestational age of 35 weeks and 6 days and a weight of 3780 grams (97th percentile). Next to the macrosomia, a hypospadias, a congenital heart defect and hypercalcemia were noted.

In his family a mitral valve insufficiency in his father and type 1 diabetes and a hypothyroidism in his mother were present

During physical examination some mild facial dysmorphisms were noticed such as bitemporal narrowing, short and upslanting palpebral fissures, upturned nasal tip and a relatively small mouth.

By using rapid trio whole exome sequencing, a double diagnosis was established within 2 weeks. First, a de novo likely pathogenic variant in the CASR gene (NM_000388.4:c.1626C>G p.(Cys542Trp)) was found, likely explaining his hypocalciuric hypercalcemia. Furthermore, a paternally inherited central 22q11.21 deletion was found, explaining his congenital heart defect.

In conclusion, this case report shows the added value of combined SNV/CNV analysis by rapid whole exome sequencing in neonates with multiple medical problems, not only by limiting the diagnostic journey but also by providing clarity for parents and healthcare providers.

Grant References: Not applicable.

Conflict of Interest: Özlem Baysal Radboud university medical center, Jolijn Verseput Radboud university medical center, Rolph Pfundt Radboud university medical center, Bert B.A. de Vries Radboud university medical center

EP04.017 Mutation screening using whole exome sequencing and extended gene panels for individuals who first present with advanced kidney disease

Olga Beltcheva 1, Radosveta Bozhilova1;2, Kunka Kamenarova1;2, Kalina Mihova1;2, Greta Mihneva-Kirilova3;4, Simona Kerezieva5;6, Boriana Deliyska5;6, Maria Gaydarova3;4, Radka Kaneva1;2

1Medical University of Sofia, Molecular Medicine Center, Department of Medical Chemistry and Biochemistry, Sofia, Bulgaria; 2Medical University of Sofia, Genome Diagnostics Laboratory, Department of Medical Chemistry and Biochemistry, Sofia, Bulgaria; 3Medical University of Sofia, Deptartment of Pediatrics, Sofia, Bulgaria; 4SBAL Children’s Hospital „Prof. Dr. Ivan Mitev“, Sofia, Bulgaria; 5Medical University of Sofia, Department of Nephrology, Sofia, Bulgaria; 6UMHAT “Tsarica Yoanna-ISUL”, Nephrology Clinic, Sofia, Bulgaria

Background/Objectives: For patients who first present with advanced kidney disease determining the nature of the underlying pathology may be difficult. In cases such as these, genetic diagnosis may be used to guide the treatment, prognosis and counselling.

Methods: After obtaining informed consent, we collected samples from 10 patients with chronic kidney disease (CKD) with uncertain etiology, aged between 30 and 70. Whole exome sequencing on NovaSeq 6 000 (Illumina) was performed and a panel of 773 gene, previously associated with kidney pathologies, was analysed for the presence of genetic variants. Relevant variants were confirmed using Sanger sequencing. When needed, segregation analysis was carried out using the same method. The pathogenicity of new variants was evaluated using ACMG criteria.

Results: Pathogenic or potentially pathogenic variants were found in several genes, known to be involved in kidney development and/or function. Among those were PKHD1, FOXC2 and SEC61A1. The genetic findings led to establishing the correct diagnosis for several individuals, allowing the clinical team to make a treatment plan and offer adequate genetic counselling. Notably, a woman was first diagnosed with autosomal recessive polycystic kidney disease at the age of 41.

Conclusion: We tested the applicability of targeted exome sequencing for diagnosis in Bulgarian CKD patients. For individuals with advanced kidney disease, where renal function tests and biopsy may not give conclusive results, mutation screening using extended panel of genes may be of great help in establishing formal diagnosis.

Grant references: MU180/14.06.2022; MES: D01-395/18.12.2020, D01-278-14.12.2022, D01-302/17.12.2021 and D01-165/28.07.2022

Conflict of Interest: None declared.

EP04.018 De novo mutation in NR5A1 (SF1) in a child with gonadal dysgenesis

Silvia Andonova 1, Maria Sredkova-Ruskova2, Todor Ruskov2, Tsvetina Veleva2, Trayan Delchev2, Alexey Savov1, Daniela Avdjieva-Tzavella2

1National genetic laboratory, UHOG “Maichin dom”, Sofia, Bulgaria; 2Department of Clinical Genetics, University Pediatrics Hospital, Medical University, Sofia, Bulgaria

Background/Objectives: Steroidogenic factor-1 is a nuclear receptor associated with 46,XY DSD (disorders of sex development). A spectrum of phenotypes has been reported including gonadal dysgenesis. We report a child with gonadal dysgenesis and mutation in NR5A1 (SF1).

Methods: Six-months-old child (assigned as female) presented due to phallus enlargement with size of 2 cm. The investigations revealed 46,XY karyotype, normal adrenal function with normal levels of 17-OH-progesterone and elevated testosterone of 6.34 nmol/l [0.07-0.17]. Blind-ended vagina with length of 29 mm was found - micturition takes place from the urethra below the clitoris. MRI scan identified 2 gonads and laparoscopy was performed subsequently. No uterus was found, the gonads in abdomen were determined histologically as testes with preserved architecture after biopsy. DNA from K2EDTA blood was extracted from the proband and the parents. Sanger sequencing of coding regions and intron-exon boundaries of NR5A1 (SF1) gene was performed after PCR amplification.

Results: A heterozygous genetic variant NR5A1:c.985C>T (p.Gln329Ter) with de novo origin was identified in the patient.

Conclusion: The diagnosis and management of 46,XY infant with genital ambiguity is always a challenge as precise genetic and clinical diagnosis has implication for gender assignment and further treatment. NR5A1 mutations in 46,XY DSD patients are often associated with intact adrenal steroid biosynthesis. Weather our patient will develop adrenal failure remains to be pursued. Genotype-phenotype correlation needs to be determined as it will allow a better follow-up of the DSD patients and proper genetic counselling of their families.

Conflict of Interest: None declared.

EP04.019 Exome sequencing identifies oligogenic inheritance in familial Primary Biliary Cholangitis

Giulia Solda1;2, Alessio Gerussi3, Elvezia Maria Paraboschi1;2, Giulia Cardamone1, Benedetta Terziroli4, Pietro Invernizzi3, Rosanna Asselta1;2

1Humanitas University, Biomedical Sciences, Pieve Emanuele, Milan, Italy; 2IRCCS Humanitas Research Hospital, Rozzano, Milan, Italy; 3University of Milano-Bicocca, Division of Gastroenterology and Center for Autoimmune Liver Diseases, Department of Medicine and Surgery, Monza, Italy; 4Epatocentro Ticino, Lugano, Switzerland

Background/Objectives: Primary biliary cholangitis (PBC) is a rare autoimmune disorder with multifactorial etiology leading to a progressive damage of small bile ducts, eventually causing liver failure. Most of the efforts performed till now to explain PBC heritability were based on genome-wide association studies and meta-analyses, which however miss rare and ultra-rare deleterious variants with high impact. Here, we report the detailed genetic characterization of a Spanish family with the aim to identify additional risk loci for PBC.

Methods: A family with multiple affected individuals was subjected to genome-wide linkage analysis and exome sequencing. Genotyping was performed on 9 individuals from the family using the Illumina Infinium HumanCore-24 v1.1 BeadChip whereas exome sequencing was performed on 3 affected subjects using the Agilent Sure Select exome v7 Library prep kit and an Illumina NovaSeq6000 sequencer.

Results: Parametric multi-point data analysis did not evidence any significant linkage signal with LOD score ≥3. Rare variant analysis by exome sequencing identified potentially pathogenic variants in three genes (SLC30A10, SHROOM2, and KRT18). SLC30A10 is highly expressed in the liver, and rare variants in this gene are associated with elevated levels of liver transaminases and cirrhosis. SHROOM2 is a X-linked gene that was found differentially methylated in PBC. KRT18 pathogenic variants are known risk factors for developing liver disease, and knock-out mice develop antimitochondrial autoantibody – a hallmark of PBC.

Conclusion: Our data suggest an oligogenic inheritance for PBC, with multiple rare variants each likely explaining different phenotypic features of the patients.

Grant References: -

Conflict of Interest: None declared.

EP04.020 Application of Clinical Exome in genetic nephropathies: example of Nephronophthisis

Ourayna Batta 1;2, imane jaouad cherkaoui2, ilham ratbi1;2, abdelali zrhidri2, Yasmina Rahmuni1;2, Nada Amllal1;2, kenza soulami3, abdelaziz sefiani1;2, lyahyai jaber1;2

1Faculty Of Medicine And Pharmacy Of Rabat, research team in genomics and molecular epidemiology of genetic diseases, genomics center of human pathologies, Rabat, Morocco; 2National Institute of Hygiene, Rabat, Morocco; 3Dr Kenza SOULAMI, Dar-el-Beida, Morocco

Background: Nephronophthisis is a chronic tubulointerstitial nephropathy with an autosomal recessive inheritance, characterized by renal cystic disease evolving to end-stage renal disease (ESRD). It is the most frequent genetic nephropathy in children (10% of ESRD cases), with an incidence of 1/50000 to 1/900000 births. Three forms of nephronophthisis are described: infantile, juvenile and adult. These forms are distinguished by clinical and radiological signs and the age of development of ESRD. Nephronophthisis can be isolated or associated with extra-renal signs, defining a variety of other more complex syndromes. Several genes have been identified in nephronophthisis. Given this clinical and genetic heterogeneity, molecular diagnosis using Next-Generation Sequencing (NGS) represents the most efficient and rapid means of diagnosis for this group of diseases.

Methods: Three patients were referred for genetic investigation suffering from clinical signs of nephronophthisis with chronic ESRD, and we report here clinical features and molecular findings. Considering the clinical heterogeneity and the diagnosis difficulties, we performed clinical exome screening in the patients. A virtual panel of 12 genes involved in nephronophthisis was applied to filter variants.

Results: Three novel mutations have been detected in three different genes: NPHP1, INVS and NPHP4. These results were confirmed by Sanger sequencing and qPCR.

Conclusion: The present case report illustrates the important role of Clinical Exome Sequencing in the accurate molecular diagnosis of genetic kidney disease. This approach allows for an appropriate management of patients and adequate genetic counseling. Moreover, this molecular analysis would be more cost-benefit for patients and their families.

Conflict of Interest: None declared.

EP04.021 Identification of NR6A1 as a new gene involved in congenital urogenital anomalies

Adeline Jacquinet 1, Lydie Flasse2, Hélène Pendeville3, Erwan Plougonven4, Angélique Léonard4, Daniel Guerrier5, Bernard Peers2, Vincent Bours1

1ULiège, GIGA-Human Genetics Lab, Liège, Belgium; 2ULiège, GIGA-Laboratory of Zebrafish Development & Disease Models, Liège, Belgium; 3ULiège, GIGA-Zebrafish Facility, Liège, Belgium; 4ULiège, Department of Chemical Engineering, Liège, Belgium; 5University of Rennes, CNRS, IGDR (Institut de Génétique et de Développement de Rennes)-UMR6290, Rennes, France

Background: Retinoic acid and posterior Hox genes are both required for renal and uterine development in mammals, their alteration resulting in renal and uterine malformations. The nuclear receptor NR6A1 is implicated in anteroposterior patterning, possibly by regulating Hox gene expression and/or by modulating retinoic acid gradient.

Results: By WES, we identified a heterozygous missense variant in NR6A1 in one family with recurrence of renal and uterine malformations. Sequencing of the gene in a cohort of individuals with Mayer-Rokitansky-Küster-Hauser syndrome identified one additional predicted deleterious variant. Generation of Nr6a1 mutant models was performed by knocking out the zebrafish orthologs nr6a1a and nr6a1b thanks to crispr/cas9 technology. Developmental kidney defects were detected using transgenic reporter line and characterized by in situ hybridization. nr6a1a-/- mutants show impaired development of the kidney with anomalies in pronephros segmentation, which translate in hypoplasia of kidney tissues in adults. As nr6a1a-/- females are infertile, analysis of the genitalia was performed. MicroCT scan showed precocious degeneration of oocytes while the oviduct seems to be formed. In addition, we detected various defects in the mutant skeleton such as abnormal number of vertebrae and absence of the anal fin.

Conclusion: Renal anomalies in our zebrafish model provide the first evidence for causality of NR6A1 mutations in kidney malformations. The skeletal defects observed in our mutant confirm a role for Nr6a1 in anteroposterior patterning of the paraxial mesoderm. Moreover, we show that Nr6a1 is also involved in anteroposterior patterning of the intermediate mesoderm as revealed by abnormal segmentation of the pronephros.

Conflict of Interest: None declared.

EP04.022 Mutational spectrum of MEN1 and MEN2 syndrome in Croatian patients

Ana Merkler Sorgic 1, Hana Ljubic1, Ana Acman Barisic1, Domagoj Caban1, Sanja Kusacic-Kuna2, Nevena Krnic3, Darko Kastelan4, Ivana Rako1

1University Hospital Centre Zagreb, Department of Laboratory Diagnostics, Zagreb, Croatia; 2University Hospital Centre Zagreb, Department of Nuclear Medicine and Radiation Protection, Zagreb, Croatia; 3University Hospital Centre Zagreb, Department of Pediatrics, Zagreb, Croatia; 4University Hospital Centre Zagreb, Department of Internal Medicine, Zagreb, Croatia

Introduction: Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant syndrome caused by mutations in MEN1 gene. One of its main hallmarks is endocrine and non-endocrine tumor growth (parathyroid tumors, pituitary tumors, gastrinomas, insulinomas, cutaneous tumors, etc.).

Mutations in the RET proto-oncogene are the primary cause of the hereditary syndrome called multiple endocrine neoplasia type 2 (MEN2). It is divided into three autosomal dominant subtypes (MEN2A, MEN2B, and FMTC) with a high risk of developing medullary thyroid cancer.

Materials and Methods: For MEN1 and MEN2, respectively, genomic DNA samples from 176 and 115 individuals were examined. Sequencing of MEN1 included all 10 exons with flanking intronic regions, whereas, for RET, only hotspot exons (exon 10, 11, 13, 14, 15 and 16) were analyzed. Applied Biosystems 3130xl Genetic analyzer and BigDye® Terminator v3.1 Cycle Sequencing Kit were used to identify pathogenic variants in the coding region of these genes.

Results: Heterozygous pathogenic variants were detected in 32 individuals with MEN1. The most prevalent variants were p.Arg516Profs*15 (8 patients) and p.Glu191Glyfs*5 (6 patients). There were 16 individuals with MEN2 heterozygous pathogenic variant. The most frequent variant was p.Cys620Arg (4 patients), followed by p.Cys634Tyr and p.Leu790Phe (found in 3 patients each).

Conclusions: The genotype-phenotype correlations that can be used to determine the likelihood of aggressive medullary thyroid cancer make molecular diagnostics of MEN2 significant. Contrarily, despite the fact that MEN1 syndrome does not exhibit genotype-phenotype correlations, molecular diagnostics of MEN1 is important for identifying at-risk family members.

Conflict of Interest: None declared.

EP04.025 A pathogenic variant in THRA as the cause of Congenital non-goitrous Hypothyroidism in a pair of monozygotic twins

Violetta Anastasiadou 1, Andri Miltiadous1, Petroula Gerasimou1, rafaella metaxa1, Nicos Skordis2, Paul Costeas1

1Karaiskakio Foundation, nicosia, Cyprus; 2Paedi Center for Specialized Pediatrics, Nicosia, Cyprus., Division of Pediatric Endocrinology, nicosia, Cyprus

Introduction: Congenital non-goitrous Hypothyroidism, 6 (CHNG6) is an autosomal dominant condition characterised by growth and developmental retardation, low serum T4 and high serum T3 levels and delayed bone development. THRA is one of several nuclear receptors for thyroid hormone that when mutated confers a resistance to thyroid hormone.

Methods: Twin sisters presenting with ataxic gait, poor fine motor coordination, dysarthria, truncal obesity, dysmorphic features and mild intellectual disability. Personal history was significant for severe generalized hypotonia, feeding difficulties, global developmental delay and learning difficulties. They are treated for hypothyroism since early childhood. Previous investigations including karyotype, metabolic screening and molecular karyotype were non- conclusive. Family history was non-contributory. Exome sequencing performed with Agilent’s Exome V8 NGS panel and data analysed using Franklin. Proband, parents and the rest of the family were additionally tested by Sanger sequencing.

Results: The THRA c.1187dupT p.(Phe397LeufsTer10) truncating pathogenic variant, was found in heterozygous state in the two probands, and was absent from their parents. The variant was the first reported mutation in THRA and since then has been reported in literature in at least three affected individuals.

Conclusion: To our knowledge about 10 individuals from 8 families were found to harbour a THRA pathogenic variant and this study can be incorporated to the limited reported cases published. In addition, this study highlights the importance of the pathogenicity of truncating mutations even if these are located at the C-terminal of a protein.

Conflict of Interest: None declared.

EP04.026 Nonsense mutation in the novel PERCC1 gene as a genetic cause of Congenital Diarrhea and Enteropathy

Dina Yagel1;2;3, emily stenke4, Ben Pode-Shakked 1;2;5, cara dunne4;6, Ellen Crushell7, anthea bryce-smith4, Michael McDermott8, maureen j o’sullivan8, alvit veber1, Mansa Krishnamurthy9;10, james m. wells5;9;10, Yair Anikster1;2;11, Billy Bourke4;12

1Sheba Medical Center, Metabolic Disease Unit, Edmond and Lily Safra Children’s Hospital, Ramat Gan, Israel; 2Tel-Aviv University, Sackler Faculty of Medicine, Tel-Aviv, Israel; 3Clalit Research Institute, Ramat Gan, Israel; 4Children’s Health Ireland-Crumlin, National Centre for Paediatric Gastroenterology, Dublin, Ireland; 5Cincinnati Children’s Hospital Medical Center, Division of Developmental Biology, Cincinnati, United States; 6St James’ Hospital, Department of Gastroenterology, Dublin, Ireland; 7Children’s Health Ireland-Temple Street, National Centre for Inherited Metabolic Disorders, Dublin, Ireland; 8Children’s Health Ireland-Crumlin, Department of Histopathology, Dublin, Ireland; 9Cincinnati Children’s Hospital Medical Center, Center for Stem Cell and Organoid Medicine (CuSTOM), Cincinnati, United States; 10Cincinnati Children’s Hospital Medical Center, Division of Endocrinology, Cincinnati, United States; 11Sheba Medical Center, The Wohl Institute for Translational Medicine, Ramat Gan, Israel; 12University College Dublin, School of Medicine, Dublin, Ireland

Background/Objectives: Congenital diarrheas and enteropathies (CODEs) constitute a heterogeneous group of individually rare disorders manifesting with infantile-onset chronic diarrhea. Genomic deletions in chromosome 16, encompassing a sequence termed the ’intestine-critical region (ICR)’, were recently identified as the cause of an autosomal recessive congenital enteropathy. The regulatory sequence within the ICR is flanked by an unannotated open reading frame termed PERCC1, which plays a role in enteroendocrine cell (EEC) function. We investigated 2 unrelated children with idiopathic congenital diarrhea requiring home parenteral nutrition attending the Irish Intestinal Failure Programme.

Methods: Currently 12- and 19-years old, these Irish male patients presented with watery diarrhea and hypernatremic dehydration in infancy. Probands were phenotyped by comprehensive clinical investigations, including endoscopic biopsies and serum gastrin level measurements. Following negative exome sequencing, PCR and Sanger sequencing of the entire coding region and intron boundaries of PERCC1 were performed for each proband and their parents.

Results: In both patients, serum gastrin levels were low and failed to increase following a meal challenge. While no deletions involving the ICR were detected, targeted sequencing of the PERCC1 gene revealed a shared homozygous c.390C>G stop gain variant.

Conclusion: We report clinical and molecular findings in two unrelated patients harboring a shared homozygous variant in PERCC1, comprising the first description of a point mutation in this gene in association with CODE. That the 2 parenteral nutrition dependent children with unexplained diarrhea at our institution harbored a PERCC1 mutation underscores the importance of its inclusion in exome sequencing interpretation.

Conflict of Interest: None declared.

EP04.027 First proved case of autosomal dominant ATP6V1B1-related renal tubular acidosis: experience with whole genome sequencing data

Faisal Alkandari 1, Ahmad Almulla1, Lova Matsa2, Haya AL-Balool3, Divya Krishnan2, Ahmad Alaqeel1

1Kuwait Oil Company (KOC); Medical Group, Paediatric Department, Ahmadi City, Kuwait; 2Igenomix FZ LLC, Bioinformatics department, Dubai Healthcare City, United Arab Emirates; 3Kuwait Medical Genetic Centre (KMGC), Molecular Genetics and Genomics, Sulaibikhat, Kuwait

Background/Objectives: In the genomic era, it is not uncommon to have unusual mode of inheritance (MOI) for various genes. Here we present unusual dominant-form of ATP6V1B1-related renal tubular acidosis. To our knowledge, this is the first case proved to have dominant inheritance by covering all parts of ATP6V1B1 gene using whole genome sequencing (WGS).

Case: Five-year-young girl presented in early infancy with H/O poor weight gain and failure to thrive. She was admitted at 6-months of age to the ICU with H/O lethargy and severe dehydration. Initial blood investigation showed hyperchloraemic - normal anion gap - metabolic acidosis with hypokalaemia causing ECG changes. Urine PH was consistently >5.5 in spite of metabolic acidosis. Renal ultrasound showed bilateral medullary nephrocalcinosis. She was diagnosed with Distal renal tubular acidosis.

Result: whole exome sequencing (WES) revealed a heterozygous c.1181G>A (p.Arg394gln) variant in ATP6V1B1 gene. None of parents carry the variant. To rule-out possible variant in the 2nd allele; whole genome sequencing (WGS) was done to virtually cover all parts of the gene including promoter, deep intronic variants and 3`-end; which revealed the same variant in heterozygous state.

Conclusion: This study shows it is more common than originally thought to have different MOI even within the same gene. This phenomenon of having multiple MOI’s is still relatively new and therefore it is the duty to report unusual MOI of a gene in order to have better understanding of function of a gene and thus better understanding of disease mechanism.

No Grants

Conflict of Interest: None declared.

EP04.028 Genetic analysis in patients with androgen insensitivity syndrome – detection of a novel AR gene variant

Agnieszka Gach 1, Iwona Pinkier1, Kinga Sałacińska1, Urszula Wysocka1, Tadeusz Kałużewski1

1Polish Mother’s Memorial Hospital Research Institute, Department of Genetics, Lodz, Poland

Background: Androgen insensitivity syndrome (AIS) is a rare condition with an X-linked pattern of inheritance, in which patients with 46,XY karyotype demonstrate complete (CAIS) or partial (PAIS) impairment of pre- and postnatal virilisation. The differential diagnosis in CAIS is limited, whereas in PAIS, numerous other causes of disorders of sex development (DSD) can also produce the typical phenotype of micropenis, severe hypospadias and bifid scrotum. The majority of the cases is determined by the presence of variants in the androgen receptor (AR). More than 1100 mutations in the AR gene have been reported. We aimed to study the genetic basis of androgen insensitivity syndrome in patients with clinical diagnosis of the condition.

Material and Methods: Eight patients were recruited from the cohort of 92 individuals with DSD referred to the Department of Genetics, Polish Mother’s Memorial Hospital RI. The diagnosis of CAIS or PAIS was made based on their karyotype (46,XY), phenotype and corresponding hormone alternations. Custom DSD NGS panel was used.

Results: A novel pathogenic AR variant c.1344_1345insTA was found in a patient with clinical diagnosis of CAIS. In 5 other cases known AR missense variants were identified.

Conclusions: Despite the fact that AR gene has been thoroughly studied in several DSD cohorts we identified new, yet undescribed variant within its sequence. We believe, that continually increasing access to genomic sequencing technology worldwide will allow for further characterization of genomic variants associated with AIS translating into a better personalised healthcare service.

Conflict of Interest: None declared.

EP04.029 Uniparental Disomy of chromosome 16 as a cause of Primary Ciliary Dyskinesia

Lidon Carretero-Vilarroig 1, Alba Berzal-Serrano1, Ana Reula2, Rosana Blanco-Mañez3, Noelia Muñoz-Fernandez4, Gema García-García1;5, Elena Aller1;5;6, José M. Millán1;5, Miguel Armengot-Carceller1;4;7, Teresa Jaijo1;5;6

1Health Research Institute Hospital La Fe (IIS La Fe), Molecular, Celular and Genomic Biomedicine Group, Valencia, Spain; 2San Pablo CEU University, Castellón, Spain; 3La Fe Polytechnic and University Hospital, Pathology Department, Valencia, Spain; 4La Fe Polytechnic and University Hospital, ENT Department, Valencia, Spain; 5Biomedical Research Network for Rare Diseases, CIBERER, Madrid, Spain; 6La Fe Polytechnic and University Hospital, Genetics, Valencia, Spain; 7Valencia University, Valencia, Spain

Background/Objectives: Primary Ciliary Dyskinesia (PCD) is a rare genetic condition affecting cilia structure and function. Patients manifest chronic respiratory infections beginning in early childhood, with or without abnormally positioned internal organs and infertility. About 50 genes have been described, almost all with an autosomal recessive inheritance. We present a patient with neonatal respiratory distress, situs inversus, cough and mucus production.

Methods: Ciliary beat pattern and ultrastructure were studied on samples of nasal curettage using a high-speed camera accoupled to an inverted microscope and electronic microscopy (EM) respectively. Clinical Exome (CE) was sequenced from blood samples and segregation analysis of the candidate variant was assessed with Sanger sequencing. Finally, CytoScan HD trio array (Affymetrix) was carried out to seek copy number variants and runs of homology (ROH).

Results: Cilia were immobile and EM showed outer dynein arm absence. CE revealed the p.Gly343Profs*4 pathogenic variant in the autosomal recessive DNAAF1 gene in homozygous state. Although paternity was genetically confirmed, segregation analysis failed at detecting this variant in the father. Finally, the SNP-array revealed two ROH of 38,73Mb and 6,68Mb at 16p11.2-16q22.1 and 16q23.3-16qter respectively. The DNAAF1 gene is located at 16q24.1, thus explaining the homozygous state of the detected variant as a result of a segmental maternal uniparental isodisomy (UPiD(16)).

Conclusion: PCD is a genetically heterogeneous disorder related to motile cilia. Here we describe the first PCD patient due to a UPD of chromosome 16. This information is crucial to genetic counselling.

Grant References: Instituto de Salud Carlos III PI19/00949 and PI22/01010.

Conflict of Interest: Lidon Carretero-Vilarroig Full, Alba Berzal-Serrano Full, Ana Reula Part-time, Rosana Blanco-Mañez Full, Noelia Muñoz-Fernandez Full, Gema García-García Full, Elena Aller Full, José M. Millán Full, Miguel Armengot-Carceller Full, Instituto de Salud Carlos III PI22/01010, Teresa Jaijo Full, IP Instituto de Salud Carlos III PI22/01010.

EP04.030 Bartter syndrome vs Gitelman syndrome: a case report

Carol Prieto-Morín 1, Nayra Pérez-Delgado1, Francisco Martínez Bugallo1, Ruth López-Travieso1, Inmaculada García-Cobaleda1

1Hospital Nuestra Sra. de Candelaria, Clinical Analysis Service, Genetic Unit, Santa Cruz de Tenerife, Spain

Introduction: Bartter syndrome and Gitelman syndrome are autosomal recessive renal disorders characterized by fluid, electrolyte, urinary and hormonal abnormalities, including renal potassium, sodium, chloride, and hydrogen wasting; hypokalemia; hyperreninemia and hyperaldosteronism without hypertension; and metabolic alkalosis. Clinical findings include electrolyte, growth, and sometimes neuromuscular abnormalities.

Case report: we performed clinical exome sequencing (CES) in a 32 year-old female patient presenting chronic hypokalemia, hypomagnesemia and intense asthenia.

Results: Data analysis revealed the c.1783C>T (p.Arg595Ter) pathogenic variant. Copy number variant analysis from CES data identified a heterozygous complete deletion of CLCNKB gene.

The c.1783C>T mutation was previously reported in homozygous state and as compound heterozygous in families with Bartter syndrome. A functional study determined that this variant abolished chloride conductance in vitro (PMID:28381550). This variant is present in population databases (gnomAD allele frequency 0.0003633). The patient presented a heterozygous complete deletion of the CLCNKB gene: (hg19)NC_00001.10(NM_000085.5):g.(?_16370987)_(16383412_?)del. This copy number variant has been reported in homozygous and compound heterozygous state in Bartter syndrome patients.

Conclusion: This patient was refered with clinical diagnosis of Gitelman syndrome but the CES-based genetic study stablished the diagnosis of Bartter syndrome. To our knowledge, this is the first patient described in the literature with this two variants. Gitelman syndrome and Bartter syndrome are two highly overlapping renal conditions. In this case, the genetic study allows a better diagnosis and follow up of patients.

Conflict of Interest: None declared.

EP04.031 Advantages of the MS-MLPA in the diagnosis of Russell-Silver syndrome

Iryna Lastivka 1, Vita Antsupova2, Ludmila Khlunovska3, Larysa Sheiko3, Ljudmila Brisevac3, Anastasiya Babintseva1, Iryna Malieieva2, Iana Ushko2

1Bukovinian State Medical University, Chernivtsi, Ukraine; 2Bohomolets National Medical University, Kyiv, Ukraine; 3Shupyk National Healthcare University of Ukraine, Kyiv, Ukraine

Russell-Silver Syndrome (RSS) is a genetically heterogeneous disease manifested by growth retardation. Most often, the genetic defect is associated with impaired methylation of the H19 and IGF2 genes located in 11p15, or with maternal uniparental disomy of chromosome 7. The use of methylation-dependent multiplex legase probe amplification (MS-MLPA) for genetic disorders in RSS is important in the search. Case from practice. Boy, 6 years old, height 106 cm (-3DS), weight 12.5 kg (-3DS), BMI 11.1 (-3DS), protruding forehead, triangular face, narrow chin, clinodactyly 5 fingers. Anamnesis: signs of intrauterine growth retardation of the III degree were detected at the 33rd week of gestation during ultrasound screening of the fetus. The boy was born at 37-38 weeks of pregnancy. Birth weight 1700 g (-3DS), body length 41 cm (-3DS), head circumference 29 cm (-1DS). At the age of 1 year 3 months, there was a delay in physical development, a deficiency in body weight of the III degree. A diagnosis of RSS has been proposed. However, when studying the IGF2 gene by real-time PCR and the study of the 7q33-34 locus by microsatellite analysis did not reveal any pathology. Karyotype 46,XY.

We proposed to conduct an additional examination using the MS-MLPA method to determine the methylation status and the presence of microdeletions/microduplications in the NSD1, H19, CDKN1C, KCNQ1OT1 (11p15) genes. Hypomethylation of the H19DMR/IC1 domain was found. The use of MS-MLPA made it possible to verify the diagnosis in a 6-year-old boy: Q87.1. Russell-Silver syndrome, abnormal methylation of the H19 gene.

Conflict of Interest: None declared.

EP04.032 Further evidence of a novel PPP1R12A frameshift variant involved in anomalies of sexual development

Ina Ofelia Focsa 1;2, Andreea Tutulan-Cunita2, Anca Pavel2, Diana Prepelita2, Cristian Strugaru1, ELENA EMANUELA BRAHA3, Laurentiu Bohiltea1, Danai Stambouli2

1University of Medicine and Pharmacy Carol Davila, Medical Genetics, Bucharest, Romania; 2Cytogenomic Medical Laboratory, Bucharest, Romania; 3National Institute of Endocrinology C.I. Parhon, Bucharest, Romania

Abnormalities of sexual development are caused by genetic defects, ranging from chromosomal aberrations to single nucleotide changes. They may be limited to the genitourinary system or can be associated with impairment of different organs.

Here we present a rare case of sexual development anomaly determined by a pathogenic variant in PPP1R12A. The boy, aged 3 years 6 months, was referred to our clinic for genetic consultation and molecular diagnosis.

He is the first child of a nonconsanguineous couple, born at term, after a pregnancy complicated with polyhydramnios in the third trimester. Duodenal atresia, prenatally suspicioned, was confirmed at birth, when bilateral cryptorchidism was noted, as well. Later, an intraabdominal testis, a rudimentary fallopian tube and uterus were detected. The child has a mild facial dysmorphism and low levels of estrogen, testosterone, AMH and DHEA. Classical karyotype, SNP array and WES were normal, except for a paternal-inherited inversion on chromosome 9.

WGS was performed by Illumina technology and data was analyzed using Congenica platform.

A novel, heterozygous frameshift variant c.2182_2185delGAAA in PPP1R12A, disrupting exon 16 of 25 was detected, which leads consequently to a truncated protein. The variant was omitted by WES due to low coverage. PPP1R12A is a key regulator involved in cell cycle, cell adhesion and migration during embryogenesis and in morphogenesis. It has been recently (2020) linked with genitourinary syndrome with or without cerebral malformations by Hughes et al., who reported 12 patients with truncating variants. Our study brings new evidence for a better characterization of PPP1R12A-related anomalies.

Conflict of Interest: None declared.

EP04.033 Altered microRNA expression in asymptomatic and mild COVID-19 cases

Angélica Domínguez-de-Barros 1, Malena Gajate-Arenas1, Omar García-Pérez1, Javier Chao-Pellicer1;2, Roberto Dorta Guerra1;3, Jacob Lorenzo-Morales1;2;4, Elizabeth Córdoba-Lanús1;2

1Instituto Universitario de Enfermedades Tropicales y Salud Pública de Canarias (IUETSPC). Universidad de La Laguna, La Laguna, Spain; 2Consorcio Centro de Investigación Biomédica (CIBER) de Enfermedades Infecciosas (CIBERINFEC), Instituto de Salud Carlos III, Madrid, Spain; 3Departamento de Matemáticas, Estadística e Investigación Operativa. Facultad de Ciencias. Universidad de La Laguna, La Laguna, Spain; 4Departamento de Obstetricia y Ginecología, Pediatría, Medicina Preventiva y Salud Pública, Toxicología, Medicina Legal y Forense y Parasitología. Facultad de Ciencias de la Salud. Universidad de La Laguna, La Laguna, Spain

Background/Objectives: Host-encoded microRNA (miRNA) response to SARS-CoV-2 infection has been reported to be altered mostly in relation to severe COVID-19 cases. However, there is scarce information in relation to host miRNAs profiles in asymptomatic or mild symptomatic COVID-19 cases, that might also provide important insights into both viral pathogenesis and patient management.

Methods: Circulating miRNAs from serum and nasopharyngeal samples belonging to eight asymptomatic or mild COVID-19 patients and eight age and gender matched healthy controls were determined by NGS. RNA was isolated and converted into miRNA NGS libraries to subsequent sequence on a NextSeq (Illumina Inc.) instrument.

Results: We observed 18 miRNAs dysregulated in serum from asymptomatic or mild symptomatic COVID-19 cases when compared to healthy controls, being miR-485-3p and miR-143 the most up-regulated (Log2FC = 3.77 and 3.23, FDR = 2.2e-7 and 0.0018, respectively) while miR-12136 and miR-1275 were the most down-regulated ones (Log2FC = -4.70 and -3.05, FDR = 5.2e-8 and 0.000073, respectively). The nasopharyngeal samples from the same individuals presented the miR-3195 as the strongly upregulated miRNA (Log2FC = 5.24, FDR = 0.00027). The principal component analysis (PCA) revealed that the miRNA profile based on 500 dysregulated miRNAs could independently classified serum from nasopharyngeal samples of COVID-19 cases.

Conclusion: This study demonstrates that SARS-CoV-2 infection induces a robust host miRNA response also in asymptomatic or mild COVID-19 cases. miR-485-3p is a promising useful inflammatory biomarker while miR-143 may efficiently combat SARS-CoV-2 by reducing host cell apoptosis via mTOR. Validation of the present findings in a large patient cohort are warranted.

Grant References: CB21/13/00100 (CIBERINFEC), ISCIII, Spain.

Conflict of Interest: None declared.

EP04.034 Genetic modifiers of Cystic Fibrosis-Related Diabetes in Georgian CF Patients with 1677DelTA mutation

Mariam Ghughunishvili 1;2, Tinatin Tkemaladze1;2, Sandro Surmava1, Maia Gagua3, Elene Abzianidze1, Ludmila Livshits4, Eka Maisuradze3, Eka Kvaratskhelia1;3

1Tbilisi State Medical University, Department of Molecular and Medical Genetics, Tbilisi, Georgia; 2Tbilisi State Medical University, G.Zhvania Pediatric Academic Clinic, Tbilisi, Georgia; 3Tbilisi State Medical University, V. Bakhutashvili Institute of Medical Boitechnology, Tbilisi, Georgia; 4Institute of Molecular Biology and Genetics, Laboratory of Human Genomics, Kyiv, Ukraine

Introduction: CF Genome Wide Association Studies (CF GWAS) identify cystic fibrosis-related diabetes (CFRD) loci. Genome-wide significance was obtained for variants at PTMA, TCF7L2 and SLC26A9 loci. PTMA and SLC26A9 variants are CF-specific; TCF7L2 variants also associated with T2D. The objective of our study was to assess CFRD genetic risk factors in Georgian CF patients.

Materials and Methods: The study was approved by the ethics committee of the Tbilisi State Medical University. Informed contents will be obtained from all patients or their representatives. 18 CF patients (age: 5-15) with 1677DelTA mutations were enrolled in this study. We genotyped patients regarding the TCF7L2 rs7903146, PTMA rs838440 and SLC26A9 rs4077468 using a TaqMan assay (Applied Biosystems, USA).

Results: The allele distributions of rs7903146, rs838440 and rs4077468 are summarized in Table 1. We observed 2 CF patients double homozygotes regarding the risk alleles of both, TCF7L2 and SLC26A9 genes which may suggest an increased susceptibility to the CFRD.

Table 1. Allele distributions of CFRD gene variants




Major allele/Minor allele



P Value






















MAF, Minor allele frequency

Conclusions: Individuals with cystic fibrosis with the additional diagnosis of CFRD have a higher risk of early death than those without CFRD. Early diagnosis and treatment correlate with slower rates of pulmonary decline and improved growth.

This work was supported by the Shota Rustaveli National Science Foundation of Georgia, Fundamental Research Grant #FR-22-2601

Conflict of Interest: None declared.

EP05 Skeletal, Connective Tissue, Ectodermal and Skin Disorders

EP05.001 A novel heterozygote LORICRIN variant in a father and daughter with palmoplantar keratoderma

Sofie Fredberg 1, Stine Bjoern Gram1;2, Ulrikke Lei3, Anette Bygum1;2, Klaus Brusgaard1;2, Lilian Ousager1;2

1Department of Clinical Genetics, Odense University Hospital, Odense, Denmark; 2Department of Clinical Research, University of Southern Denmark, Odense, Denmark; 3Department of Dermatology and Allergy, Herlev and Gentofte Hospital, Gentofte, Denmark


Introduction: Loricrin Keratoderma (LK) is a rare early-onset autosomal dominant skin disorder, characterized by honeycomb-like palmoplantar keratoderma (PPK) and diffuse ichthyosiform dermatosis. There might also be symptoms such as pseudoainhum/autoamputations, knuckle pads, secondary infections and hyperhidrosis. Affected children can be born as collodion babies. To the best of our knowledge 10 different disease-causing variants in LORICRIN have been published, of these nine have been related to LK.

Material & Methods: A father and daughter presented with PPK with a honey-comb like pattern and fine-scaling ichthyosis since adolescence and childhood respectively. The daughter had associated hyperhidrosis, discrete knuckle pads and constriction bands. Diagnostic analysis was performed by next-generation sequencing, using an exome-based in-silico panel targeting 95 genes related to palmoplantar keratoderma. A novel variant in LORICRIN was identified in both father and daughter.

Results: A heterozygote frameshift variant was identified in LORICRIN (c.792dupC,p.(Ile265Hisfs*71)). The variant was absent in gnomAD and ClinVar. The variant is predicted to result in a delayed stop codon and elongation of the protein to 336 amino acids as opposed to the native length of 312 amino acids. Nine of 10 disease-causing variants reported in the literature were also frameshift variants and six of these variants lead to an elongation of the protein to 336 amino acids, supporting our interpretation of the variant as likely pathogenic.

Conclusion: We identified a novel heterozygote variant in LORICRIN in a father and daughter with PPK. We classified the variant as likely pathogenic.

Conflict of Interest: None declared.

EP05.003 Novel mutations and atypical presentation of Papillon Lefevre Syndrome

Maha Abouzaid 1, Nermeen Ahmed1, Mohamad Abdelhamid2, Mostafa Mostafa1

1National Research Centre, Orodental Genetics, Cairo, Egypt; 2National Research Centre, Medical Molecular Genetics, Cairo, Egypt

Background Papillon Lefevre syndrome (PLS) is an autosomal recessive condition that is caused by mutations in cathepsin C (CTSC) enzyme. Typically, it presents with severe periodontitis that leads to teeth loss in early youth and palmoplantar keratosis.

Aim We present two PLS cases with novel mutations and unusual clinical presentations.

Results We present two PLS cases with confirmed novel CTSC mutations; the first case had the frameshift mutation c.285_286delGT (p.L96Efs*2) and clinically had all teeth existing with minimal attachment/ bone loss along with severe enamel structural defect and open root apices, while the second case had the homozygous frameshift variant c.1331delinsAAAAA (p.G444Efs*4) and presented with extensive buphthalmos secondary to congenital glaucoma and loss of vision in her right eye.

Conclusion The spectrum of PLS causing gene have additional two novel mutations with atypical clinical presentations.

Funded by Science, Technology and Innovation Funding Authority (STDF), Egypt.

Conflict of Interest: Maha Abouzaid National Research Centre, Science,Technology and Innovation funding Authority (STDF), Nermeen Ahmed National Research Centre, Science, Technology & Innovation Funding Authority (STDF)., Mohamad Abdelhamid National Research Centre, Science, Technology & Innovation Funding Authority (STDF), Mostafa Mostafa National Research Centre, Science, Technology & Innovation Funding Authority (STDF).

EP05.004 the potential of mummy substance to stimulate healing in mesenchymal stem cells cultured with human fibroblasts

Sepideh Hassanpour Khodaei 1, shahnaz sabetkam2, Havva Ozgen Eyupoglu3;4, Şükrü Tüzmen3

1Eastern Mediterranean University, Faculty of Dentistry, Famagusta via Mersin 10 Turkey, Cyprus; 2Kyrenia University, Faculty of Medicine, Kyrenia, Cyprus; 1Eastern Mediterranean University, Faculty of Dentistry, Famagusta via Mersin 10 Turkey, Cyprus; 4Marmara University, Medical Biology and Genetics, Istanbul, Turkey

the potential of mummy substance to stimulate healing in mesenchymal stem cells cultured with human fibroblasts

Background: The objective of this research is to determine the effect of mummy substance on the enhancement of cell proliferation and matrix protein synthesis in wound healing.

Methods: The methodology used for this study involves isolating mesenchymal stem cells and human fibroblasts procured at Pastor Institute, Iran. The cells were treated with mummy substance separately and co-cultured between ASCs and WJSCs and fibroblasts. Proliferation was assessed by Ki67 method in monolayer condition. Synthesis of components of extracellular matrix (ECM) such as collagen type I, type III and fibronectin 1 (FN1) were determined by qPCR.

Results: The effective concentration of Mummy substance on fibroblasts, adipose-derived stem cells, and Wharton jelly was determined to be 1000 µm/ml through the MTT assay. The results were analyzed using Graph Pad Prism 6.01 software. The level of Ki67 was found to be higher in adult stem cells compared to neonatal stem cells (p < 0.0001). The study concluded that Mummy material enhanced the expression of collagen I, III, and fibronectin in fibroblast-ASCs co-culture but not in fibroblast-WJ-SCs co-culture.

Conclusion: This research presents a successful in vitro method for promoting healing. Therefore, the potential use of Mummy substance and stem cell-based treatments in wound healing as a new therapeutic strategy is promising.

Conflict of Interest: Sepideh Hassanpour Khodaei full time, shahnaz sabetkam full time, Havva Ozgen Eyupoglu: None declared, Şükrü Tüzmen full time

EP05.005 Hereditary mucoepithelial dysplasia: A report of two patients

Carlos Gutiérrez-Cerrajero 1;2, Nera Gestoso-Uzal1;2, Lucía Rodríguez-González1, Ana-Belén Herrero1;2, Ángela Hernández-Martín3, Rogelio González-Sarmiento1;2

1University of Salamanca, Department of Medicine, Faculty of Medicine, Salamanca, Spain; 2Biomedical Research Institute of Salamanca (IBSAL), IIMD-07, Salamanca, Spain; 3Hospital Niño Jesús, Unit of Dermatology, Madrid, Spain

Background/Objectives: Ichthyoses are disorders of cornification characterized by dry, scaly skin and a disruption of skin barrier function (1). Some severe forms of ichthyosis, called syndromic ichthyoses, show extracutaneous symptoms (1). One such ichthyosis is hereditary mucoepithelial dysplasia (HMD), which has sometimes been called ichthyosis follicularis, atrichia and photophobia syndrome 2 (IFAP2) (1). To date, four SREBF1 mutations in 18 families have been linked to HMD.

Patients/Methods: The patients were admitted to Hospital Infantil Niño Jesús in Madrid. Genomic DNA was extracted from peripheral blood by standard phenol/chloroform protocol, mutations were found using whole-exome sequencing and validated by Sanger sequencing.

Results: Both patients showed diffuse non-scarring scalp alopecia, widespread follicular keratosis, gingival and palatal erythema, and fissured tongue. Whole exome sequencing revealed the patients were heterozygous for mutation c.1669C>T, p.Arg557Cys in SREBF1 (NM_001005291.3) and Sanger sequencing validated it. These results match those found previously by other groups and seem to further point to disease-causing SREBF1 mutations being localized to the same three clustered amino acids (arginine 557, asparagine 558, and leucine 560)

Conclusions: The present study reports two additional patients with the p.Arg557Cys SREBF1 mutation.

Grants: This project was funded by FIS-FEDER PI20/01569

(1) Gutiérrez-Cerrajero, C. et al., Ichthyosis. Nat. Rev. Dis. Prim. 9, 2 (2023).

Conflict of Interest: None declared.

EP05.006 Novel likely pathogenic variant c.588_612del(p.Trp196CysfsTer5) in EBP gene in a Cypriot family with Conradi-Hünermann Syndrome

Emilia Athanasiou 1, irene savvidou1, Sofia Ourani1, Andri Miltiadous2, Petroula Gerasimou2, Paul Costeas2, Violetta Anastasiadou3

1Clinical Genetics,Archbishop Makarios III Hospital, Nicosia, Cyprus, Nicosia, Cyprus; 2Karaiskakio Foundation, Nicosia, Cyprus, Nicosia, Cyprus; 3Karaiskakio Foundation, Nicosia, Cyprus, Genetics Clinic, Nicosia, Cyprus

Background/Objectives: Conradi-Hünermann syndrome is an X-linked disorder caused by mutation in the gene encoding delta(8)-delta(7) sterol isomerase emopamil-binding protein (EBP; 300205) on chromosome Xp11. The clinical findings are highly variable, ranging from fetuses with multiple malformations and severe growth retardation to milder manifestations. Almost all liveborn individuals with CDPX2 are female. Characteristic features are growth deficiency, chondrodysplasia punctata, asymmetric shortening of the limbs, scoliosis, linear or blotchy scaling ichthyosis in the newborn and later follicular atrophoderma, scarring alopecia, and cataracts.

Methods: We report a female newborn referred in our clinic because of antenatally reported short limbs, and postnatal lower limb asymmetry, ichthyosis, and early stippling in the left epiphysis in the hip x-ray. The family history was indicative as the mother showed also short stature, ichthyosis, lower limb asymmetry and severe scoliosis.

Results: Trio exome sequence analysis revealed a novel maternal inherited frameshift likely pathogenic mutation c.588_612del(p.Trp196CysfsTer5) in EBP gene. The mutation has not been reported before, lies at a functional domain and is absent from gnomAD exomes and genomes. Loss-of-function is a known mechanism of disease in EBP gene.

Conclusions: We described a family with X-linked chondrodysplasia punctata caused by a novel mutation in the EBP gene. Our study further extends the mutation spectrum of the EBP gene in CDPX2.

Grant References Chondrodysplasia Punctata 2, X-Linked, genereviews, https://www.ncbi.nlm.nih.gov/books/NBK55062/, Clinical, molecular and biochemical characterization of nine Spanish families with Conradi-Hünermann-Happle syndrome: new insights into X-linked dominant chondrodysplasia punctata with a comprehensive review of the literature,PMID: 22121851, None

Conflict of Interest: Emilia Athanasiou State health services organisation, SHSO Cyprus, irene savvidou Ministry of Health, Cyprus, Sofia Ourani SHSO, CYPRUS, Andri Miltiadous karaiskakio foundation, Petroula Gerasimou karaiskakio foundation, Paul Costeas karaiskakio foundation, Violetta Anastasiadou Karaiskakio Foundation.

EP05.007 Variants in the TB5 domain of FBN1 are responsible for short stature phenotype in Weill-Marchesani syndrome

Pauline Marzin 1;2, Caroline Michot1;2, jean-luc alessandri3, Sandra Mercier4, Oana Moldovan5, Massimiliano Rossi6, Sophie Rondeau1;2, Valérie Cormier-Daire1;2

1Fédération de Génétique et Institut Imagine, AP-HP, Hôpital Necker-Enfants Malades, Paris, France; 2Université de Paris, UMR1163, INSERM, Paris, France; 3Pôle Femme-Mère-Enfants, CHU de la Réunion - Hôpital Félix Guyon, Bellepierre, Saint-Denis, France; 4Service de génétique médicale - Unité de Génétique clinique, CHU de Nantes - Hôtel Dieu, Nantes, France; 5Serviço de Genética Médica, Hospital de Santa Maria, Centro Hospitalar Universitário de Lisboa Norte, Lisbones, Portugal; 6Service de génétique, CHU de Lyon HCL - GH Est-Hôpital Femme Mère Enfant, Bron Cedex, France

- Background: Fibrillin, encoded by FBN1, is a key component of microfibrillar network, responsible for mechanical properties of extracellular matrix and a reservoir for numerous cytokines, including TGFß. FBN1 is associated with several connective disorders, characterized by tall or short phenotype. Among them, acromelic dysplasia which include Weill-Marchesani syndrome (WMS), geleophysic dysplasia (GD) and acromicric dysplasia (AD), is defined by short stature, short extremities, thick skin and joint limitations. GD and AD are associated with severe short stature present all patients, whereas height seems to be more variable in patients with WMS. While pathogenic variants in FBN1 responsible for AD and GD are all located in exons 41-42 encoding TB5 domain, variants responsible for WMS are located throughout the gene. The aim of this work was to establish a correlation between FBN1 variant location and height in WMS patients.

- Methods: Retrospective multicenter study and review of literature (NCBI PubMed database:“Weill-Marchesani + FBN1”). Inclusion criteria: clinical diagnosis of WMS with identified FBN1 mutations.

- Results: 20 WMS patients with FBN1 mutation have been studied, 12 and 8 from literature and cohort, respectively. Half of the patients presented with normal stature. Patients with a variant located in exons 41 or 42 were significantly smaller than other patients (Mann–Whitney test, p = 0.0040).

- Conclusion: We conclude that variants located in TB5 domain are associated with severe short stature in acromelic dysplasias. This observation supports the key role of the TB5 domain in growth.

Conflict of Interest: None declared.

EP05.008 De novo balanced translocations disrupting the FBN1 gene: an uncommon cause of Marfan syndrome

Caroline Racine 1, Patrick Callier2;3, Antonio Vitobello2;3, Nadine Hanna4;5, Pauline Arnaud4;5, Jondeau Guillaume4;5, Christel Thauvin-Robinet1;2;3, Isabelle Creveaux6, Vincent Gatinois7, Renaud Touraine6, Marjolaine Willems8, Laurence Faivre1;2

1Centre de Référence Anomalies du Développement et Syndromes Malformatifs, FHU TRANSLAD, Hôpital d’Enfants, CHU Dijon, DIJON, France; 2UMR1231 GAD, Inserm - Université Bourgogne-Franche Comté, DIJON, France; 3Unité Fonctionnelle Innovation en Diagnostic génomique des maladies rares, FHU-TRANSLAD, CHU Dijon Bourgogne, DIJON, France; 4Laboratory for Vascular Translational Science, INSERM U1148, Centre Hospitalo-Universitaire Xavier Bichat, PARIS, France; 5Centre de référence Syndrome de Marfan et pathologies apparentés, APHP, Hôpital Bichat, Paris, France; 6Laboratoire de Génétique Chromosomique et Moléculaire, CHU-Hôpital Nord, Laboratoire AURAGEN (Plan France Médecine Génomique 2025), SAINT-ETIENNE, France; 7Plateforme ChromoStem, Unité de génétique chromosomique, Département de génétique moléculaire et cytogénomique, CHU de Montpellier, Université de Montpellier, MONTPELLIER, France; 8Département de Génétique Médicale, Maladies Rares et Médecine Personnalisée, CHU Montpellier, MONTPELLIER, France

Background: Marfan syndrome is a well-characterized rare genetic connective tissue disease. Main features involve the skeletal, ocular, and cardiovascular systems, and are mainly caused by FBN1 (MIM #134797) variants.

Patients and Methods: We report two patients, from unrelated families, with sporadic clinically early-diagnosed Marfan syndrome. Among other phenotypic features, both patients presented with marfanoid habitus, ectopia lentis, aortic root dilatation, positive wrist and thumb signs and high-arched palate. Targeted sequencing and multiplex ligation-dependent probe amplification (MLPA) of the FBN1 gene, gene panel of Marfan-related disorders, and an exome-sequencing for the older patient, were performed but did not provide any molecular basis.

Results: Both patients benefited from genome sequencing, retrieving a balanced translocation involving chromosome 15 and disrupting the FBN1 gene. Theses translocations were characterized by specific fluorescence in situ hybridization probes and karyotype. The 25-year-old male carries a de novo t(9;15)(p13.3;q21.1) translocation. The breakpoint disrupts the intron 40 of the FBN1 gene. The 9-year-old female carries a de novo t(15;16)(21.1;13.13) translocation, disrupting the intron 45. The other breakpoints are not clinically relevant in both patients.

Conclusion: In the literature, one patient can be found, leading to three cases of Marfan syndrome caused by de novo balanced translocation. In case of clinical certainty of diagnosis, structural variants should also be screened, along with sequencing of the FBN1 and Marfan-related genes. Our patients illustrate the great contribution of genome sequencing, even in well-known diseases.

Conflict of Interest: None declared.

EP05.009 COL1A2 multiexon deletion in Osteogenesis Imperfecta type 2 – clinical case

Daniela Oliveira 1;2;3, Pedro Almeida1, Rita Cerqueira4, Silvia Modamio-Hoybjor5;6, Sérgio Sousa1;2;3, Karen E. Heath5;6;7, Fabiana Ramos1;8

1Pediatric Hospital, Coimbra Hospital and University Centre, Medical Genetics Unit, Coimbra, Portugal; 2Faculty of Medicine, University of Coimbra, University Clinic of Genetics, Coimbra, Portugal; 3Clinical Academic Center of Coimbra, Coimbra, Portugal; 4Centro de Genética Clínica, Porto, Portugal; 5Institute of Medical and Molecular Genetics (INGEMM), IdiPAZ, Hospital Universitario La Paz, UAM, Madrid, Spain; 6Skeletal Dysplasia Multidisciplinary Unit (UMDE) e ERN-BOND, Hospital Universitario La Paz, Madrid, Spain; 7CIBERER, ISCIII, Madrid, Spain; 8Prenatal Diagnosis Centre, Bissaya Barreto Maternity Hospital, Coimbra Hospital and University Centre, Coimbra, Portugal

Context: Osteogenesis Imperfecta (OI) is characterized by liability to bone fractures mainly due to pathogenic variants in COL1A1 and COL1A2. OI type 2 is at the most severe end of the clinical spectrum of OI usually leading to early death and is typically caused by a dominant-negative effect of glycine substitutions in the triple helical domain.

Case report: We report the first pregnancy of a healthy couple with a high risk in Down syndrome screening and ultrasound anomalies detected at 13 weeks – increased nuchal translucency, subcutaneous edema of lower limbs, shortening of the long bones and bilateral clubfeet. Later, detection of bowing of the long bones, low amniotic fluid, reduction of fetal movements and a small abdominal circumference led the couple to opt for termination of pregnancy. Post-mortem radiological studies confirmed the diagnosis of OI type 2. Molecular studies identified a heterozygous multiexonic COL1A2 deletion (exons 4 to 17). This deletion was also detected in the healthy mother, in mosaic state, who also carries a second multiexonic deletion (exons 12 to 17), in heterozygosity, in the same gene. RNA studies from the mother’s fibroblasts confirmed the presence of both deletions and demonstrated that they were both in-frame.

Conclusion: This case expands the current knowledge of collagen type I variants reinforcing the role of multiexonic deletions in OI type 2. This clinical case also illustrates the challenges in prenatal diagnosis of fetuses with OI and highlights the importance of molecular studies, including familiar studies, to provide accurate genetic counselling.

Conflict of Interest: None declared.

EP05.010 New evidence of the effect of parental age on the likelihood of having children with achondroplasia

Olga Lagutina 1, Svetlana Deryabina1

1Institute of Medical Cell Technology, Molecular Genetics Laboratory, Ekaterinburg, Russian Federation

Background/Objectives: Achondroplasia (ACH; MIM:100800) occurs with an estimated prevalence between 1/16,000 and 1/26,000 live births, representing the most common genetic form of human dwarfism. Affected patients have short limbs with macrocephaly and characteristic facial features such as frontal bossing and midface hypoplasia. Mutations in FGFR3 causing skeletal dysplasia are all inherited in an autosomal dominant pattern, but frequently occur de novo on the paternal allele. It is thought that there are factors that affect DNA replication or repair during spermatogenesis, but not during oogenesis, and may predispose to the G380R mutation in the FGFR3 gene. In European countries there is a trend of increasing the average age of childbearing.

Methods: We analyzed 38 families with a sick child diagnosed with achondroplasia. The control group consisted of 259 families.

Results: The average age of fathers in the control group was 30.3. The average age of the fathers of children with achondroplasia was 36.2 years old. Comparison of the average father’s age in the study and control groups showed significant differences between the groups (U = 2771.5, p = 0.000014). Thus, the age of the father does increase the probability of having a sick child with achondroplasia. Therefore, it is necessary to create a complex of procedures (such as prenatal diagnosis or preimplantation genetic testing) to prevent the birth of a sick child in high-risk couples.

Conflict of Interest: None declared.

EP05.011 Marfan syndrome in case of 14-years old girl with confirmed missense pathogenic variant in FBN1 gene

Magdalena Ivanova 1;2, Slavena Nikolova1;2, Irina Halvadjian2;3, Stanimira Elkina1;2, Zornitsa Kamburova1;2, Katya Kovacheva1;2

1Medical University - Pleven, Pleven, Bulgaria; 2University Hospital “Dr. Georgi Stranski” – Pleven, Pleven, Bulgaria; 1Medical University - Pleven, Pleven, Bulgaria

Marfan syndrome is an autosomal dominant multisystem disorder caused by a mutation in the FBN1 gene with an incidence of 1:5000. Currently, the revised Ghent nosology is used in clinical practice. Molecular analysis of the FBN1 gene has reduced the uncertainty of diagnosis, especially in young patients (before 20 years of age) who do not meet the clinical criteria for Marfan syndrome.

We report a 14-year-old Bulgarian girl who did not meet the Ghent criteria for Marfan syndrome but in whom a pathogenic FBN1 variant was detected.

The girl was referred by a pediatrician for genetic counseling because she was with tall stature, had pectus excavatus, and arachnodactyly. The patient was born after the first uneventful pregnancy in the family (mother 27 years old, father 33 years old). At the age of 14 years, she was with height 175 cm (over 97 percentile) and weight 46 kg (between 25 and 50 percentile). She was not diagnosed with cardiovascular and ocular symptoms. Both parents were with normal stature (mother - 164 cm, father - 170 cm) and there was no other family member with Marfan-like features.

The girl underwent genetic testing by NGS with a target panel comprising the exons of 6699 genes. Analysis revealed a pathogenic variant (missense mutation) c.7532G > A in exon 61 of the FBN1 gene in the heterozygous state.

With the clinical case presented, we confirm the need for NGS technology in cases with suspected Marfan syndrome and not fulfilled clinical criteria, especially in young patients.

Conflict of Interest: None declared.

EP05.012 Two novel mutations in the PLEC gene in a subject with non-lethal epidermolysis bullosa simplex with pyloric atresia

Ivona Sansović 1, Katarina Vulin1, Ljubica Odak1, Leona Morožin Pohovski1, Lara Šamadan2, Slobodna Murat-Sušić3, Suzana Ožanić Bulić2

1Children’s Hospital Zagreb, Scientific Centre of Excellence for Reproductive and Regenerative Medicine (CERRM), University of Zagreb School of Medicine, Department of Medical Genetics and Reproductive Health, Zagreb, Croatia; 2Children’s Hospital Zagreb, University of Zagreb School of Medicine, Department of Medical Genetics and Reproductive Health, Zagreb, Croatia; 3University Hospital Centre Zagreb, University of Zagreb School of Medicine, Department of Dermatology and Venereology, Zagreb, Croatia

Background: Epidermolysis bullosa simplex (EBS) with pyloric atresia (PA) caused by autosomal recessive mutations in PLEC, which encodes plectin, is characterized by severe skin blistering at birth and congenital PA and is usually lethal within the first year of life. Since EBS-PA is most often caused by mutations in ITGB4, which encodes integrin-beta-4, it is likely that the disease-causing mutations abolish the interaction of plectin with integrin-beta-4.

Subject and method: We report a two-year-old boy born to healthy non-consanguineous parents with non-lethal EBS with congenital pyloric atresia and rather mild bullous lesions noted shortly after birth on the trunk and later at the lower limbs. Clinical exome sequencing was performed in the subject using Illumina TruSight One Kit.

Results: In the PLEC gene, two novel heterozygous mutations, c.613G>A (p.Glu205Lys) in exon 3 (actin binding domain, ABD) and c. 11471_11472del (p.Tyr3824Trpfs) in exon 32, were identified. According ACMG classification c. 11471_11472del is a pathogenic variant. Although c.613G>A is a variant of uncertain significance (ACMG), six Meta scores and CADD score predict a damaging effect.

Conclusion: We believe that the newly reported PLEC mutations located in the ABD and C-terminal domain likely contribute to a milder disease course via plectin-integrin-beta-4 interactions already reported for ITGB4-related EBS-PA. This report will further contribute to the mutational and phenotypic spectrum of EBS-PA.

Acknowledgment: 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”.

Conflict of Interest: Ivona Sansović 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”., Katarina Vulin 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”., Ljubica Odak 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”., Leona Morožin Pohovski 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”., Lara Šamadan: None declared, Slobodna Murat-Sušić: None declared, Suzana Ožanić Bulić: None declared.

EP05.013 Familiar Craniofrontonasal syndrome - case reports

Lenka Horáková 1, Monika Koudová2, Věra Kavánová3, Anna Křepelová4

1GENNET - Center of Medical genetics and reproductive medicine, Department of Medical Genetics, Liberec, Czech Republic; 2GENNET - Center of Medical genetics and reproductive medicine, Department of Medical Genetics, Prague, Czech Republic; 3Department of endocrinology, Liberec, Czech Republic; 4Second Faculty of Medicine, Charles University, Department of medical genetics, Prague, Czech Republic

Introduction: Craniofrontonasal syndrome (CFND) is a rare X linked dominant condition caused by a mutation in the EFBN1 gene coding a protein ephrin B1. A deficiency of ephrin B1 blocks the adhesion and communication between cells with clinical picture of various skeletal and soft tissue abnormalities. The reported physical characteristics include curly and frizzy hair, widely spaced eyes, flat and broad nose with a vertical groove on the top, coronal craniosynostosis with brachycephaly, midline defects, scoliosis, facial and body part asymmetry.

Despite mode of inheritance, female patients with CFND exhibit more severe symptoms than men. This genetic paradox can be explained by ephrin-B1 deficit compensation by other ephrin molecules in male patients and mosaic pattern of wild type and mutant ephrin-B1 protein expression in women.

We present the case of a family where 2 sisters and their father were affected. 23 years old female presented with facial asymmetry, growth retardation and typical CFND facial features. Her 3 years younger sister was less affected and the father of both who denied clinical testing was only mildly affected.

Methods: Sanger sequencing analysis of EFBN1 gene was performed.

Results: In our case a heterozygous pathogenic missense mutation c.161C>T, p.(Pro54Leu) in exon 2 of EFNB1 gene was found in both sisters.

Conclusions: Correct diagnosis based on characteristic facial features in combination with targeted genetic testing is essential for confirmation of diagnosis with adequate follow up and complex therapeutic care. Identification of a causative mutation in family allows to offer prenatal or preimplantation genetic testing.

Conflict of Interest: Lenka Horáková part time, Monika Koudová full, Věra Kavánová: None declared, Anna Křepelová full.

EP05.014 Ser40Leu of IFITM5 can manifest as prenatal Caffey disease

Jiin Ying Lim 1, Celeste Jia Ying Yap2, Anju Bhatia3, Gen Nishimura4, Nikki Fong1, Saumya Shekhar Jamuar1

1KK Women’s and Children’s Hospital, Genetics Service, Department of Paediatrics, Singapore, Singapore; 2KK Women’s and Children’s Hospital, Nephrology Service, Department of Paediatrics, Singapore, Singapore; 3KK Women’s and Children’s Hospital, Department of Maternal Fetal Medicine, Singapore, Singapore; 4Musashino-Yowakai Hospital, Department of Radiology, Tokyo, Japan

Background/Objectives: Skeletal dysplasias (SDs) are a heterogeneous group of heritable bone and cartilage disorders. We report a prenatal-onset SD where whole exome sequencing (WES) provided an unexpected result and raised novel implication.

Case presentation: We report a case of prenatal Caffey disease or prenatal cortical hyperostosis (PCH) due to a monoallelic pathogenic variant of IFITM5. The first trimester screening at 12 + 5 weeks of gestation for a 30-year-old mother showed shortening of long bones and increased risk for trisomy 18 and 21. Ultrasonography at 16 weeks showed a clover shaped skull, a narrow chest, and bowing of all long bones. Polyhydramnios was noted at 27 + 1 weeks requiring amnioreductions. A girl was born at 32 + 1 weeks but passed away shortly after birth. Findings of severely bowed long bones with diaphyseal hyperostosis and mildly thick ribs on postnatal X-rays led to the suspicion of PCH. A COL1A1 variant has previously been associated with PCH, thus an osteogenesis imperfecta (OI) panel consisting of COL1A1, COL1A2, CRTAP, P3H1 genes was done, with negative results. Research WES subsequently identified a heterozygous de novo IFITM5 pathogenic variant. The c.119C>T(p.Ser40Leu) variant has been reported in patients with atypical OI, presenting with bowing and broadening of long bones at birth, with multiple fractures developing later. Pathogenesis of PCH is largely unknown, and prenatal manifestation of IFITM5-associated OI may constitute a subset of PCH.

Conclusion: Accurate diagnosis of prenatal SDs is challenging, yet important in the prognostication of these disorders. WES can improve the diagnostic accuracy of prenatal SDs.

Conflict of Interest: Jiin Ying Lim Full time, Celeste Jia Ying Yap Full time, Anju Bhatia Full time, Gen Nishimura Full time, Nikki Fong Full time, Saumya Shekhar Jamuar Full time.

EP05.016 Functional analysis of splicing variants in COL2A1 gene helps in interpretation of clinical picture in patients with skeletal dysplasia

Iuliia Viakhireva 1, Igor Bychkov2, Olga Shatokhina3, Tatiana Markova4, Oksana Ryzhkova3, Mikhail Skoblov1

1Research Centre for Medical Genetics, Laboratory of Functional Genetics, Moscow, Russian Federation; 2Research Centre for Medical Genetics, Hereditary Metabolic Diseases Laboratory, Moscow, Russian Federation; 3Research Centre for Medical Genetics, The Shared Resource Centre “Genome”, Moscow, Russian Federation; 4Research Centre for Medical Genetics, Research and Counseling Department, Moscow, Russian Federation

Background/Objectives: Pathogenic variants in COL2A1 are known to cause a large spectrum of skeletal dysplasia. Splicing variants are one of the most curious variants in interpretation as they can affect mRNA and protein structure by various molecular events.

Methods: SpliceAI tool used for prediction of splicing events. To experimental splicing study we created 8 minigene vectors, containing 47 COL2A1 exons out of 54. Variants were introduced in corresponding vectors by site-directed mutagenesis. Splicing changes were analyzed with Sanger sequencing and fragment analysis.

Results: We chose splicing variants from cohort of Russian patients with different forms of COL2A1-related skeletal dysplasia and previously published variants: 1 variant found in foetus with hypochondrogenesis, 6 variants in patients with Kniest dysplasia, 3 variants in patients spondyloepiphyseal dysplasia congenita (SEDc), 11 variants in patients with Stickler syndrome. It was demonstrated that 10 splicing variants led to in-frame exon skipping, 9 variants to frameshift events, 2 variants had no influence on splicing. We showed that severe form of skeletal dysplasia such as hypochondrogenesis, Kniest dysplasia, SEDc were mainly associated with dominant-negative effect due to in-frame protein shortening caused by exon skipping. Stikler syndrome as most frequent mild phenotype was mainly caused by haploinsuffiency due to splicing events with frameshift.

Conclusion: We successfully analyzed 21 splicing variants in COL21 gene. 19 of them had different effect on splicing: exon skipping leads to in-frame deletion, out-of-frame changes in exon length leads to frameshift with premature stop-codon formation. These data helps to clarify diagnosis and management in affected patients.

Conflict of Interest: None declared.

EP05.017 TGF- β signalopathies between clinics and laboratory: case series with overlapping features of Marfan, Loeys-Dietz, Ehlers Danlos and Osteogenesis Imperfecta syndrome

VASILICA PLAIASU 1, Elena Neagu2, Diana Ozunu3, Gabriela Motei3, Mihaela Ivan1, Alexandra Coltoiu4

1INSMC Alessandrescu-Rusescu, Clinical Genetics, Bucharest, Romania; 2National Clinical Center for Neuropsychomotor Recovery for Children “Dr. Nicolae Robanescu, Genetics, Bucharest, Romania; 3INSMC Alessandrescu-Rusescu, Genetics Laboratory, Bucharest, Romania; 4INSMC Alessandrescu-Rusescu, Dermatology, Bucharest, Romania

Background/Objectives: Heritable connective tissue disorders (HCTDs) are a complex group of disorders involving various organ systems: heart, blood vessels, skin, joints, bone, eyes, lungs, and exhibit a wide range of clinical symptoms showing marked phenotypic variability that can be difficult to diagnose only through clinical examination. Many genes have been identified as a cause of HCTD and they encode for structural proteins, modifying enzymes or components of the TGFβ-signaling pathways. Other genes still await their discovery.

Four notable connective tissue conditions: Marfan syndrome, Loeys–Dietz syndrome, Ehlers-Danlos syndrome, and Osteogenesis imperfecta, show some clinical overlap regarding cardiovascular, skeletal, craniofacial, ocular, and cutaneous features. These diseases are caused by or result in pathological alterations of the complex relationship between the TGF-β family of signalling mediators and the extracellular matrix in connective tissues.

Methods: In this study, we report 7 unrelated cases who showed various degree of phenotypical overlap of skeletal, cardiovascular and cutaneous manifestations and also we highlight the importance of Next Generation Sequencing as a molecular diagnostic tool.

Results: The clinical suspicion of hereditary connective tissue disorders was confirmed by identifying different classes of variants in FBN1, TGFBR2, SMAD3, COL5A1, COL5A2, COL1A1 genes. Two cases presented with additional features were explained by coexisting pathology.

Conclusion: These four typical examples of HCTD are clinically and genetically heterogeneous diseases, sharing some clinical features and the risk of cardiovascular morbidity and mortality. A multispecialty approach for these patients should be encouraged to reach a more personalized management for treatment and prevention. Genetic counselling is mandatory.

Conflict of Interest: None declared.

EP05.018 A homozygous pathogenic variant in DYSF gene caused Miyoshi myopathy in an Iranian family

asiyeh jebelli1, saba baghshomali 1, mohammad yazdchi2, Fahimeh Firyaei3, Leila Emrahi Govar4;5

1Department of Biological Sciences, Faculty of Basic Sciences, Higher Education Institute of Rab-Rashid, tabriz, Iran; 2Neurosciences Research Center, Tabriz University of Medical Sciences, tabriz, Iran; 3Department of Molecular Medicine and Genetics, Research Center for Molecular Medicine, School of Medicine, Hamadan University of Medical Sciences, Hamadan, Iran; 4Department of Medical Genetics, Faculty of Medical Science, Tarbiat Modares University, tehran, Iran; 5Legal Medicine Research Center, Iranian Legal Medicine Organization, tehran, Iran

Background/Objectives: Miyoshi myopathy is a type of muscular dystrophy that is characterized by weakness mainly in the distal parts of the legs. Affected individuals show difficulty in walking, climbing stairs, and tiptoeing standing. This disease is a member of Dysferlinopathies and shows an autosomal recessive inheritance that is caused by pathogenic variants in the DYSF gene. In this study, we reported the molecular analysis of a family affected by muscular myopathy using whole exome sequencing (WES).

Methods: After neurological examination, WES was done on an affected proband, then research was followed by co-segregation analysis on the participating family members.

Results: The proband was a 25-year-old male whose Miyoshi myopathy has been started 5 years ago. The proband has two affected and one non-affected sisters. Their parents have not shown any disease-related symptoms. WES analysis revealed a homozygous c.6001C>T (p.Gln2001Ter) pathogenic nonsense variant in exon 52 of the DYSF. Co-segregation analysis revealed this homozygous variant in affected sisters and a wild variant in a non-affected sister. Also, parents were heterozygous for this variant.

Conclusion: DYSF gene encodes dysferlin protein that is involved in membrane repair and regeneration. c.6001C>T was found as a compound heterozygote with c.706C>T(p.Arg236Ter) in two brothers with dysferlinopathy. Dysferlin was severely reduced in membrane and absent in cytoplasm of their skeletal muscle cells. Also, this variant was reported in a man affected by LGMD2B as a homozygous variant. This variant is predicted to damage normal protein function through nonsense-mediated mRNA decay or protein truncation.

Conflict of Interest: asiyeh jebelli Higher Education Institute of Rab-Rashid, saba baghshomali: None declared, mohammad yazdchi Tabriz University of Medical Sciences, Fahimeh Firyaei Hamadan University of Medical Sciences, Leila Emrahi Govar Iranian Legal Medicine Organization.

EP05.019 Variant c.895_904del (p.Val301SerfsTer8) in a newborn girl with isolated postaxial polydactyly – a case report

Slavena Nikolova 1, Katya Kovacheva1, Zornitsa Kamburova1, Magdalena Ivanova1

1Medical University - Pleven, Medical genetics, Pleven, Bulgaria

Polydactyly is one of the most common inherited limb malformations with an overall incidence of 0.3-3.6/1000 in live births. It is characterized by one or more extra digits on the hands and feet. The most prevalent type of polydactyly is postaxial and is characterized by the presence of extra fingers on the ulnar side of the hand and foot. The most common mode of inheritance of isolated polydactyly is autosomal dominant, but autosomal recessive inheritance is also possible.

We present a case of a female baby with non-syndromic postaxial polydactyly with an established pathogenic variant in ICQE gene associated with autosomal recessive polydactyly type A7. The newborn was a product of a first, uneventful pregnancy of healthy, unrelated parents (mother - 27 years old, father - 29 years old). Isolated polydactyly of all four limbs of the baby without any other dysmorphic features was found at birth. The newborn and both of the parents underwent genetic testing via NGS with a target panel including 6699 gene exons. Analysis revealed a pathogenic frameshift deletion c.895_904del (p.Val301SerfsTer8) in ICQE gene in homozygous state in the baby and heterozygous - in both parents. The identified ICQE mutation is associated with autosomal recessive postaxial polydactyly, type A7. Based on the result of the test, genetic counseling of the family was conducted.

NGS testing contributes to a better understanding on the genetic etiology of isolated polydactyly and provision of genetic counselling of the affected family.

S.Nikolova: None. K.Kovacheva: None. Z.Kamburova: None. M.Ivanova: None. I.Stankov: None.

Conflict of Interest: None declared.

EP05.020 A mosaic PDGFRB variant in a patient with Kosaki overgrowth syndrome

Andri Miltiadous 1, Petroula Gerasimou1, gabriella shianiou1, marina johnson1, yiannos kyprianou1, agathi elpidophorou1, antonia achilleos1, andri mitsidou1, efi georgiou1, christina ioannou1, katerina nicolaou1, Jason Chi1, Paul Costeas1, Violetta Anastasiadou1

1Karaiskakio Foundation, nicosia, Cyprus

Background: PDGFRB mutations are associated with different conditions from overgrowth (Kosaki overgrowth syndrome) and connective tissue abnormalities to myofibromatosis, intellectual disability and cutaneous phenotypes.

Methods: A 5-year old boy with overgrowth, dysmorphic facial features, patchy skin areas with extreme hyperlasticity resembling cutis laxa.Skin biopsy histopathology reported a cutis laxa diagnosis. DNA was extracted from the peripheral blood, buccal swab and skin biopsy from the patient and his parents. Exome sequencing performed with Agilent’s Exome V8 NGS panel and data were analysed using Franklin.

Results: A PDGFRB pathogenic c.1685A>G, p.Tyr562Cys variant was detected at an allelic balance of around 22% in the patient’s skin biopsy and was absent from his peripheral blood and the buccal swab sample, denoting a mosaic de novo origin. The PDGFRB: c.1685A>G is located at a mutational hot spot, has extremely low frequency in gnomAD population databases and computational prediction tools unanimously support a deleterious effect on the gene. This variant has previously been described as mosaic in several patients presenting with intracranial aneurysms or segmental hemihypertrophy of the one arm and hand or one sided aneurysm with an ipsilateral cutaneous phenotype.

Conclusion: The presence of a de novo PDGFRB pathogenic variant in a patient presenting with overgrowth and skin hyperelasticity might comprise to our knowledge the first mosaic PDGFRB-associated Kosaki overgrowth syndrome. This study can be incorporated to the limited reported cases published

Conflict of Interest: None declared.

EP05.021 Novel homozygous missense variant in GTF2E2 causes non-photosensitive trichothiodystrophy type 6

Brian Sperelakis Beedham1, LYSE RUAUD1, Yoann Vial1, Myriam Rachid1, Louise Goujon1, Alain Verloes1, Anne-Claude Tabet1, BOURRAT Emmanuelle2, Jonathan Levy 1

1Genetics Department, AP-HP, Robert-Debré University Hospital, Paris, France, PARIS, France; 2Department of Dermatology, Hôpital Saint Louis, AP-HP, Paris, France., France, PARIS, France

Trichothiodystrophy (TTD) is a rare autosomal recessive neurocutaneous disorder, characterized by brittle sulfur deficient hair and multisystem abnormalities. The main core of the phenotype associates brittle hair with a characteristic tiger tail banding under polarized light microscopy with a variable set of additional features including developmental delay (DD)/intellectual disability (ID), microcephaly, anemia, decreased fertility, and progeroid features. TTD is divided into two forms: photosensitive (PS-TTD) and non-photosensitive (NPS-TTD).

Several genes have been associated with autosomal recessive NPS-TTD, including GTF2E2 in very rare cases. GTF2E2 encodes for TFIIE-β, the beta subunit of the general transcription factor TFIIE. This transcription factor HE (TFIIE) is an essential component for transcription. So far, only two pathogenic variants in GTF2E2 from four unrelated families were reported, both affected the wing helix 2 (WH2) region of TFIIEβ protein.

Here, we report clinical data of a novel homozygous missense variant located in the WH2 domain in two brothers with NPS-TTD. Our two cases and the review of the literature allow us to broaden the genotypic and phenotypic spectrum of the disease, underlining the intra- and interfamilial variability, notably on the neurodevelopmental phenotype.

Conflict of Interest: None declared.

EP05.022 USP7 is a new candidate gene for nonsyndromic polydactyly

Shabir Hussain 1, Abdullah Raza2, Mari Muurinen1, Amir Hayat2, Petra Loid1, Sulman Basit3, Outi Mäkitie1;4;5, Wasim Ahmad2

1University of Helsinki, Faculty of Medicine, Clinical and Molecular Metabolism (CAMM) Research Program, Helsinki, Finland; 2Quaid-i-Azam University, Department of Biochemistry, Islamabad, Pakistan; 3Taibah University, Center for Genetics and Inherited Diseases, Madinah, Saudi Arabia; 4Folkhälsan Research Center, Institute of Genetics, Helsinki, Finland; 5Karolinska Institute, Department of Molecular Medicine and Surgery and Clinical Genetics, Stockholm, Sweden

Background: Polydactyly is a rare developmental disorder characterized by growth of additional digits on the preaxial, postaxial and central axes of hands and feet. Nonsyndromic postaxial polydactyly can be inherited in a recessive or dominant manner. Nine genes (GLI3, GLI2, GLI1, SHH (ZRS), ZNF141, FAM92A, IQCE, KIAA0825, DACH1) have previously been reported for postaxial polydactyly. Genetic analysis of families with polydactyly is very useful to understand human limb development. The purpose of this study was to identify the genetic cause of postaxial polydactyly in two individuals from a Pakistani family.

Methods: Whole exome sequencing was performed to search for the underlying genetic cause and Sanger sequencing was used to check variant segregation in the family. Several bioinformatics tools were used to identify the variant and assess its pathogenicity.

Results: A rare missense variant c.1738G>A, p.(Gly580Arg) was identified in USP7 gene that encodes ubiquitin specific peptidase 7. The variant segregated with the phenotype dominantly with reduced penetrance. In silico analyses revealed damaging effects of the variant.

Conclusion: USP7 is previously known to cause limb defects such as 5th finger clinodactyly, hallux valgus and small hands in Hao-Fountain syndrome. ClinVar search showed two variants (c.383+1G > C, c.333C>G:p.(His111Gln)) for a syndromic form of preaxial polydactyly. Based on these phenotypic observations and our finding, USP7 can be considered as a new candidate gene for nonsyndromic polydactyly. This study expands the phenotypic spectrum of USP7 which will help in clinical diagnosis. Future studies should be performed to define the role of this gene in limb development.

Conflict of Interest: None declared.

EP05.023 Musculocontractural type of Ehler-Danlos syndrome with severe skeletal findings in the novel variant of CHST14 gene

Öznur YILMAZ BAYER 1, nuray ozturk1, sezin yakut uzunuer2, Gokcen Karamik1, banu nur1, ercan mihci1

1akdeniz university, department of pediatrics, division of pediatric genetics, antalya, Turkey; 2akdeniz university, department of medical biology, antalya, Turkey

Background/Objectives: Ehlers-Danlos syndrome (EDS) is a connective tissue disorder characterized by joint hypermobility, hyperextensibility of the skin and generalized connective tissue fragility. Musculocontractural EDS is an autosomal recessive disorder. Here we present this very rare syndrome with severe skeletal and radiological findings.

Methods: We presented a 16 years old girl born at 38 weeks of gestation from 39-year-old mother by spontaneous vaginal delivery. There was parental consanguinity. She walked independently by 3 years. The body weight, height, head circumference were <3th percentile at the time of her most recent assessment. On physical examination; brachycephaly, prominent forehead, micrognathia, facial asymmetry, low-set and rotated ears, prominent ears, hypertelorism, downslanting palpebral fissures, blue sclerae, hypoplastic columella, long philtrum, thin upper lip, small mouth, thorax deformation, severe scoliosis, skin hyperextensibility, atrophic scars, joint hypermobility, skin fragility, scars of operations, multiple contractures, bilateral adducted thumbs, arachnodactyly, distal arthrogryposis, low fat and muscle mass, muscle weakness. Echocardiography showed a mitral valve prolapse.

Results: Whole-exome sequencing analysis displayed a novel homozygous frameshift likely pathogenic variant in the exon 1 of CHST14 gene (NM_130468.4 c.660_667del p.Ser221ProfsTer17).

Conclusion: The major characteristics of the musculocontractural form of EDS include distinctive craniofacial dysmorphism, congenital contractures of thumbs and fingers, clubfeet, severe kyphoscoliosis, muscular hypotonia, hyperextensible thin skin with easy bruisability and atrophic scarring, wrinkled palms, joint hypermobility, and ocular involvement. Since 1997, less than 100 patients have been described. We aimed to contribute to phenotype diversity with a new case with severe skeletal findings.






Conflict of Interest: None declared.

EP05.024 Update on the allelic heterogeneity and phenotypic diversity in CBFB-related cleidocranial dysplasia

Ewa Hordyjewska-Kowalczyk1, Tessi Beyltjens2, Eveline Boudin2, Nicole Revencu3, Nele Boeckx2, Miriam Bertrand4, Leon Schütz4, Tobias Haack4, Axel Weber5, Eleni Biliouri5, Mateja Vinkšel6, Anja Zagožen6, Borut Peterlin6, Shashidhar Pai7, Aida Telegrafi8, Lindsay Henderson8, Courtney Ells9, Lesley Turner9, David Geneviève10, Wim Wuyts2, Wim Van Hul2, Gretl Hendrickx11, Geert Mortier 12

1Laboratory for Skeletal Dysplasia Research, Department of Human Genetics, Leuven, Belgium; 2Antwerp University Hospital and University of Antwerp, Department of Medical Genetics, Edegem, Belgium; 3Cliniques Universitaires Saint-Luc and University of Louvain, Centre for Human Genetics, Brussels, Belgium; 4University of Tuebingen, Institute of Medical Genetics and Applied Genomics, Tuebingen, Germany; 5Justus-Liebig-University, Institute of Human Genetics, Giessen, Germany; 6University Medical Centre Ljubljana, Clinical Institute of Genomic Medicine, Ljubljana, Slovenia; 7Medical University of South Carolina Children’s Health, Division of Genetics, Charleston, United States; 8GeneDx- Inc., Gaithersburg, United States; 9Eastern Health- St. John’s, Provincial Medical Genetics Program, Newfoundland, Canada; 10Montpellier University Hospital, Competence Center for Bone Diseases, Montpellier, France; 1Laboratory for Skeletal Dysplasia Research, Department of Human Genetics, Leuven, Belgium; 1Laboratory for Skeletal Dysplasia Research, Department of Human Genetics, Leuven, Belgium

Background: Cleidocranial dysplasia (CLCD) is a rare skeletal dysplasia with significant clinical variability manifested by delayed closure of fontanels and cranial sutures, dental and clavicular anomalies and short stature. RUNX2 has been the only known disease-causing gene for CLCD until we recently identified pathogenic variants in CBFB in eight individuals from five families with a CLCD-like phenotype (CLCD2). CBFB encodes the core-binding factor β subunit (CBFβ) that interacts with all RUNX proteins to form heterodimeric transcription factors, which may explain phenotypic differences between CBFB- and RUNX2-related CLCD.

Methods: We provide an update on the genotypic and phenotypic data of our current series of individuals with CBFB-related CLCD.

Results: We had previously reported five pathogenic CBFB variants, all located in the RUNX-binding domain of CBFβ. We have now ascertained a sixth family with a sporadic case presenting with clavicula bipartita, pseudo-epiphyses of the 2nd metacarpal, shortening of distal phalanges, delayed carpal ossification and normal stature. The novel heterozygous missense variant (c.314G>A, p.(Gly105Glu)) of CBFB is located in the RUNX-binding domain and in silico programs uniformly predicted its high pathogenicity. Functional studies are currently performed to investigate how these different CBFB variants affect the function of CBFβ-RUNX complexes, and how this may contribute to the development of CLCD2.

Conclusion: We now added a sixth family with CLCD2 to our cohort, herewith expanding the genotypic spectrum of this novel rare bone disease. By including this variant in our ongoing functional studies, we aim to improve our knowledge on genotype-phenotype correlations in CLCD2.

Conflict of Interest: Ewa Hordyjewska-Kowalczyk: None declared, Tessi Beyltjens: None declared, Eveline Boudin: None declared, Nicole Revencu: None declared, Nele Boeckx: None declared, Miriam Bertrand: None declared, Leon Schütz: None declared, Tobias Haack: None declared, Axel Weber: None declared, Eleni Biliouri: None declared, Mateja Vinkšel: None declared, Anja Zagožen: None declared, Borut Peterlin: None declared, Shashidhar Pai: None declared, Aida Telegrafi GeneDx, Inc, Lindsay Henderson GeneDx, Inc, Courtney Ells: None declared, Lesley Turner: None declared, David Geneviève: None declared, Wim Wuyts: None declared, Wim Van Hul: None declared, Gretl Hendrickx: None declared, Geert Mortier: None declared.

EP05.026 Pathogenetic mechanisms in the Darier disease (DD): a gene expression and protein interactions’ study

Erika De Sensi 1, Alessia Croce1, Ilenia Rita Cannizzaro1, Antonietta Taiani1, Miryam Di Capo1, Davide Martorana2, Vera Uliana2, Patrizia Caggiati2, Enrico Ambrosini1, Andrea Gherli1, Anna Montanaro1, Giovanni Roti1;3, Antonio Percesepe1;2, Valeria Barili1

1University of Parma, Department of Medicine and Surgery, Parma, Italy; 2University-Hospital of Parma, Unit of Medical Genetics, Parma, Italy; 3University-Hospital of Parma, Hematology and BMT Unit, Parma, Italy

Background/Objectives: Darier Disease (DD) (OMIM #124200) is a rare autosomal genodermatosis which affects 1-4:100,000 people, which is characterized by loss of cell-to-cell adhesion (acantholysis), premature and abnormal keratinization (dyskeratosis) and rounded keratinocytes. DD is caused by pathogenic variants in the ATP2A2 gene (12q23-24.1), which encodes for the ATPase Sarcoplasmic/Endoplasmic Reticulum Calcium ATPase isoform 2 (SERCA2), a ubiquitously expressed cellular pump responsible for the calcium translocation from cytosol to endoplasmic reticulum. SERCA2 alterations impair intracellular calcium homeostasis leading to ER stress response and cell apoptosis and deregulate the NOTCH1 pathway in several disease models. The project aims include the identification of a DD transcriptional signature associated to SERCA2 defects and of the role of SERCA2 mutations on the NOTCH1 signaling pathway.

Methods: we collected patients’ skin biopsies: i) to perform transcriptomic RNA-sequencing (RNA-seq) analysis on affected and unaffected subjects; ii) to generate keratinocytes and fibroblasts primary cell lines for expression analysis of the NOTCH1-downstream effector proteins (HES1, HEY1, c-MYC) and of the autophagy-related mediators to investigate this lysosome-dependent regulated mechanism of degradation; iii) to investigate on FFPE bioptic samples, NOTCH1 signaling deregulations through immunohistochemistry.

Results: we defined a DD transcriptomic gene profile by RNA-seq analyses which show reduction on the NOTCH1 signaling pathway mediated by ATP2A2 patients’ variants. NOTCH1 protein deregulations have been confirmed in vivo by IHC of the nuclear intracellular domain of NOTCH1 and of c-MYC, a downstream Notch1 target.

Conclusions: we are defining the relationships between SERCA2 and NOTCH1 proteins and the pathogenetic molecular mechanisms underlying the DD.

Conflict of Interest: None declared.

EP05.028 Genotype-phenotype correlation and effects of bisphosphonates in rare forms of osteogenesis imperfecta : a retrospective study

Maelle Charpie 1, Perrine Brunelle2, Genevieve Baujat1, Caroline Michot1, Julien Van Gils3, Bruno Leheup4, Elise Schaefer5, Zagorka Péjin6, Graziella Pinto7, sophie monnot1, Valérie Cormier-Daire1

1Necker Hospital, Department of Genomic Medicine for Rare Diseases, Paris, France; 2Lille University Hospital Center, Lille, France; 3Pellegrin Hospital, CHU Bordeaux, Department of Medical Genetics, Bordeaux, France; 4Nancy Regional Hospital, Department of Medical Genetics, Nancy, France; 5Strasbourg Regional Hospital, Department of Medical Genetics, Strasbourg, France; 6Necker Hospital, Department of Pediatric Orthopedic Surgery, Paris, France; 7Necker Hospital, Department of Pediatric Endocrinology, Paris, France

Osteogenesis Imperfecta (OI) is a clinically and genetically heterogeneous group of diseases characterized by brittle bones. Though genetic mutations in COL1A1 and COL1A2 account for approximately 85-90% of OI cases, there are now more than twenty genes described. Treatment is based on the wide use of bisphosphonates and though it is well established that they increase lumbar spine (LS) bone mineral density (BMD), the clinical impact on fracture reduction is still debated.

In this study, we first investigated the clinical characteristics of 40 patients with variants in non-COL1A1/COL1A2 genes in order to study genotype-phenotype correlations as the natural history of these rare forms is still little known. We then studied the usefulness of bisphosphonate treatment by evaluating the effects on LS BMD, annual non-vertebral fracture rate, bone turnover markers, and height.

This study allowed us to further define two phenotypes. Indeed, patients with CRTAP variants had an antenatal presentation with a short (<3rd p) and curved femur. They can also present with more moderate forms, further extending the phenotypic spectrum of OI forms linked to CRTAP.

In patients with SERPINF1 variants, we consistently observed progressive deformities and loss of mobility.

Regarding treatment by bisphosphonates, all patients showed a significant increase in LS BMD while treated and this increase was dependent on the dose received. The increase in LS BMD also translated in a reduction of fracture rate during treatment. Finally, our study showed that the earlier bisphosphonates are initiated, the greater the fracture rate is reduced.

Conflict of Interest: None declared.

EP05.029 A targeted next-generation sequencing panel outcomes for the molecular diagnosis of Ectodermal Dysplasia in Spanish population

M Martínez-Romero 1;2;3, María Juliana Ballesta Martínez2;3;4, Ana Teresa Serrano-Antón2;3;4, María José Sánchez Soler2;3;4, Lidia Rodríguez-Peña4, Vanesa López González2;4;5, María Barreda-Sánchez2;3;4, María Elena Pérez-Tomás4, Marta Domínguez Jiménez4, Teresa Martínez-Menchón6, pablo carbonell meseguer1;2, Encarna Guillén-Navarro2;4;5

1Molecular Genetics Section, Biochemistry and Clinical Genetics Center, Virgen de la Arrixaca University Clinical Hospital, IMIB-Pascual Parrilla., Murcia, Spain; 2CIBERER-ISCIII., Murcia, Spain; 3Faculty of Medicine and Health Sciences, UCAM Catholic University of Murcia., Murcia, Spain; 4Medical Genetics Section, Pediatrics Department. Virgen de la Arrixaca University Clinical Hospital. IMIB-Pascual Parrilla., Murcia, Spain; 5Faculty of Medicine, University of Murcia (UMU)., Murcia, Spain; 6Dermatology Department.Virgen de la Arrixaca University Clinical Hospital. IMIB-Pascual Parrilla., Murcia, Spain

Background: Ectodermal Dysplasia (ED) includes several clinically and genetically heterogeneous disorders shown isolated or as part of a genetic syndrome. Due to the vast number of genes implicated, establishing a molecular diagnosis can be challenging. We aim to develop a targeted next-generation-sequencing (NGS) panel to know the most prevalent mutated genes.

Methods: We designed a panel of 125 genes involved in ED (SureSelect_XT-HS®_Agilent). If negative result, MLPA or CGH-SNP array were used for CNVs detection. We screened a cohort of 142 unrelated patients (89 males/53 females) referred two or more impaired ectodermal derivatives or only one if were hypohidrosis or Selective-Tooth-Agenesis (STHAG).

Results: The causative mutation was identified in 95 unrelated patients (66.9%), involving likely pathogenic/pathogenic variants in 25 genes. EDA variants associated to Hypohidrotic Ectodermal Dysplasia (XLHED, OMIM#305100) were the majority, 47.4%(45/95) following by variants in WNT10A, 15.8%(15/95) involved in STHAG (OMIM#150400), Odonto-Onycho-Dermal dysplasia (OMIM#2579809) or Schöpf-Schulz-Passarge syndrome (OMIM#224750) and TP63 (OMIM*603273), 6.3%(6/95) cases. Surprisingly, TSPEAR is most frequent ED gene identified for autosomal recessive inheritance (ECTD14, OMIM#618180), 6.3%(6/95) cases above EDAR (OMIM*604095), 4.2%(4/95). Mosaicisms in PORCN (Focal-Dermal Hypoplasia, OMIM#305600) in 2.1%(2/95), mixed variants (CNV/SNV) in GJB6 (Clouston syndrome, OMIM#129500) and new clinically overlapping entities as CLDN10 (Helix syndrome, OMIM#617671) or FOXN1 (OMIM#618806) were also identified.

Conclusions: We have developed a representative NGS panel for molecular diagnosis of a wide variety of ED with high throughput that will help us better understanding of the contribution of usual and uncommon genes in our population.

Grant References: PI14/01259_PI17/00796_PI21/01082. ISCIII, Madrid (Spain).

Conflict of Interest: None declared.

EP05.030 RMRP-related spectrum – clinical and molecular characterization of a patient cohort in the Portuguese population

Catarina Silva Rosas 1, Fabiana Ramos1, Lina Ramos1;2, Alice Mirante3, Sónia Lemos4, Frederico Regateiro5;6;7, Janet Pereira8, Silvia Modamio-Hoybjor9, Karen E. Heath9;10, Sérgio Sousa1;11

1Medical Genetics Unit, Hospital Pediátrico, Centro Hospitalar e Universitário de Coimbra (ERN-BOND), Coimbra, Portugal; 2Faculty of Health Sciences, Universidade da Beira Interior, Covilhã, Portugal; 3Paediatric Endocrinology Unit, Hospital Pediátrico, Centro Hospitalar e Universitário de Coimbra, Coimbra, Portugal; 4Hospital Pediátrico, Centro Hospitalar e Universitário de Coimbra, Coimbra, Portugal; 5Allergy and Clinical Immunology Unit, Centro Hospitalar e Universitário de Coimbra, Coimbra, Portugal; 6Institute of Immunology, Faculty of Medicine, University of Coimbra, Coimbra, Portugal; 7Coimbra Institute for Clinical and Biomedical Research (iCBR), Faculty of Medicine, University of Coimbra, Coimbra, Portugal; 8Molecular Hematology Functional Unit, Hospital Pediátrico, Centro Hospitalar e Universitário de Coimbra, Coimbra, Portugal; 9Institute of Medical and Molecular Genetics (INGEMM), IdiPAZ, Skeletal dysplasia multdisciplinary unit (UMDE-ERN BOND), Hospital Universitário La Paz, UAM, Madrid, Spain; 10CIBERER, ISCIII, Madrid, Spain; 11University Clinic of Genetics, Faculty of Medicine, Universidade de Coimbra, Coimbra, Portugal

Background: Cartilage-Hair Hypoplasia is a RMRP-related disorder comprising a continuum phenotypic spectrum (CHH-spectrum) characterized by disproportionate short stature and other findings such as gastrointestinal dysfunction, immunodeficiency, increased risk for malignancy or anemia.

Methods: Clinical and molecular characterization of 8 CHH-spectrum cases from a Portuguese hospital centre, with performed or ongoing RMRP molecular study, based on retrospective analysis of medical records.

Results: We describe 6 male and 2 female patients, from 6 unrelated families, with a CHH-spectrum diagnosis, currently between 1 and 49 years old. Prior to molecular study, most had clinical or radiological diagnosis suggested (5/8), from birth to 46 years old. Two families had skeletal dysplasia panel performed in prenatal setting. In one, only one variant was detected and in the other the diagnosis was missed (RMRP not included in exome). Most patients had disproportionate short stature (7/8), 3/5 with prenatal onset, normal occipitofrontal circumference (5/5), normal intellect (5/5), impaired lymphocyte proliferation (6/6) and 2/2 with reported recurrent infections. Variable hypotrichosis was identified in 4/7 patients, 4/5 patients had gastrointestinal dysfunction or failure to thrive and one patient had a persistent idiopathic thrombocytopenic purpura. No malignancies were reported. A total of 10 previously described RMRP variants were identified.

Conclusion: Our data are generally in accordance with the literature. In 2/8 cases, one without short stature, the diagnosis was only achieved by reverse phenotyping. We aim to include patients from other Portuguese hospital centres. Detailed description of national cohorts of CHH-spectrum patients contributes to awareness and better-informed counselling and management.

Conflict of Interest: None declared.

EP05.031 A new variant in the LTBP4 gene likely leads to splicing failure

Darya Tsibulskaya 1;2, Natalia Shcherbakova2;3, Anna Zabrodina2, Alla Semyachkina2, Inna Povolotskaya2;3

1Digital Genomics LLC, Belgrade, Serbia; 2Veltischev Research and Clinical Institute for Pediatrics of the Pirogov Russian National Research Medical University, Moscow, Russian Federation; 3Digital Genomics LLC, Belgrade, Serbia

Background/Objectives: The LTBP4 gene encodes for a protein known as latent transforming growth factor beta-binding protein 4 (LTBP4). This protein is a member of the extracellular matrix (ECM) protein family and plays a critical role in the regulation of the transforming growth factor beta (TGF-β) signaling pathway.

LTBP4 is particularly important in the development of several organs and tissues, including the lungs, heart, and skeletal muscle. Pathogenic variants in the LTBP4 gene cause several genetic disorders, including autosomal recessive cutis laxa type 1C (OMIM: 613177). Here we present a patient with cutis laxa and two mutations in the LTB4 gene.

Methods: Bioinformatic analysis of the WES data was performed using a custom-developed bioinformatics pipeline. Polymerase chain reaction (PCR) Sanger sequencing was used for validation of genetic variants detected by exome sequencing. Amplification products of appropriate size were identified using agarose gel electrophoresis.

Results: A 6-year-old male patient underwent whole exome sequencing. As a result of bioinformatics analysis, two variants were identified. The first variant is a known pathogenic genetic variant resulting in the acquisition of a stop codon and premature termination of translation (ENST00000308370.11:c.1453C>T). The second variant is a new variant possibly leading to the destruction of the branch point and disruption of splicing (ENST00000308370.11:c.452-22A > C). We confirmed these variants with Sanger sequencing and showed that they are formed compound heterozygosity.

Conclusion: We classify the new variant as a variant of unknown clinical significance (ACMG classification). A functional analysis will be done to confirm the pathogenicity of the variant.

Conflict of Interest: None declared.

EP05.032 Homozygous variegate porphyria: a novel PPOX variant in two patients from a family

Ezgi Gökpınar İli 1, Mustafa Doğan1

1Başakşehir Çam and Sakura City Hospital, Department of Medical Genetics, İstanbul, Turkey

Background: Variegate porphyria (VP) is one of the acute hepatic porphyrias caused by the deficient activity of protoporphyrinogen oxidase. VP is an autosomal dominant disease, resulting from the pathogenic variants in protoporphyrinogen oxidase (PPOX) gene. It is characterized by adult-onset cutaneous photosensitivity, blistering, fragility of sun-exposed skin, thickening, and hyperpigmentation. Homozygous VP (HVP) with biallelic PPOX variants is very rare, presenting with severe infantile-onset cutaneous and neurological findings. Here, we present two patients from a family with HVP.

Patients and Methods: The proband was a 10-year-old female who was referred for skin lesions suspected as epidermolysis bullosa. She developed blistering skin lesions on her face and hands when she was seven months old. She also had developmental delay and epilepsy. On examination, microcephaly, bilateral brachydactyly and camptodactyly of the hands, and ataxic gait were noted. Erosions, scarring, hyperpigmentation, and thickening were observed on sun-exposed skin. Her 7-year-old first cousin had a similar phenotype, and hyperactivity in addition.

Whole exome sequencing (WES) followed by an analysis focused on rare homozygous variants was performed on the proband. Sanger sequencing was used for confirmation and segregation analysis of the PPOX gene variant.

Results: WES revealed the novel homozygous PPOX(NM_000309.5):c.164A>C(p.Glu55Ala) variant in the proband. The affected cousin was also homozygous. Segregation analysis confirmed autosomal recessive inheritance.

Conclusion: Here, we report a novel PPOX pathogenic variant expanding the genotypic spectrum of HVP. HVP is an extremely rare disease and should be considered in patients with photosensitive skin lesions, microcephaly, brachydactyly/camptodactyly, and neurodevelopmental manifestations.

Conflict of Interest: None declared.

EP05.033 Report of first Endocrine-cerebro-osteodysplasia patient to reach childhood age

Sinem Kocagil 1, Derya Hazal Özbakır1, Uygar Kabaoglu1, Ebru Erzurumluoglu Gokalp1, Oğuz Çilingir1

1Eskisehir Osmangazi University, Eskisehir, Turkey

Endocrine-cerebro-osteodysplasia (OMIM #612651, ECO) is an extremely rare, neonatal lethal, autosomal recessive disorder with multiple anomalies involving the endocrine, cerebral, and skeletal system. To date, seven cases diagnosed with ECO, that all died in utero or in neonatal period, have been described in the literature.

Here we report a 18 month-old female patient who was born at 37 + 6 weeks to consanguineous parents. Physical examination revealed hydrocephalic appearance, short nose, depressed nasal bridge, broad nasal ridge, long philtrum, thin lips, low-set ears, brachydactyly, bilateral postaxial polydactyly of the hands, sandal gap and short micromelia. In her cranial MRI, hypoplasia of the brainstem and intensity changes in peripheral white matter in both cerebral hemispheres that may be due to delayed myelination, and external hydrocephaly due to cortical atrophy was detected. In her abdominal ultrasonography dilated pelvicalyceal structures of left kidney and increased echogenicity in both kidneys were noted.

Whole exome sequencing analysis revealed a homozygous likely pathogenic CILK1(NM_014920.5):c.1664_1665del (p.Tyr555CysfsTer48) variant at patient and segregation analysis showed the healthy parents as carriers. In addition to that the patient had a pathogenic LMNA(NM_170707.4):c.808A>C (p.Lys270Gln) variant that was inherited from healthy mother.

This is the first report of ECO diagnosed at the childhood. There might be several underlying mechanisms that lead to prolonged survival for our patient; such as CILK1 variant being a null variant located at 13th exon which is the penultimate exon therefore leading partial decrease of protein expression and/or it may be caused by the presence of a secondary pathogenic LMNA variant.

Conflict of Interest: None declared.

EP05.035 An indel variant in BHLHA9 causes Mesoaxial Synostotic Syndactyly with Phalangeal Reduction

Safeer Ahmad 1;2, Muhammad Zeeshan Ali1, Muhammad Muzammal1, Mari Muurinen2;3, Shabir Hussain2, Muzammil Ahmad Khan1, Outi Mäkitie2;3;4

1Gomal University, Gomal Centre of Biochemistry and Biotechnology, Dera Ismail Khan, Pakistan; 2University of Helsinki, Research Program for Clinical and Molecular Metabolism, Faculty of Medicine, Helsinki, Finland; 3Folkhälsan Institute of Genetics, Helsinki, Finland; 4University of Helsinki, Children’s Hospital, Helsinki University Hospital, Helsinki, Finland

Background: Mesoaxial synostotic syndactyly with phalangeal reduction (MSSD) presents a characteristic combination of clinical features that includes mesoaxial osseous synostosis at a metacarpal level, absence of one or more phalanges, and hypoplasia of distal phalanges, clinodactyly, and preaxial fusion of toes.

Methods: We evaluated a large consanguineous Pakistani family with autosomal recessive MSSD with polydactyly. Whole Exome sequencing, segregation analysis and in silico predictions were performed.

Results: The family had presented mesoaxial reduction of digits, cutaneous syndactyly, camptodactyly, clinodactyly and postaxial polydactyly. Whole-exome sequencing and Sanger sequencing identified in affected subjects a homozygous frameshift-indel mutation (NM_001164405:exon1:c.251_270del, c.251_254insCGC A, p.Phe85Glnfs*108) in BHLHA9 that results in a truncated BHLHA9 protein. In silico study revealed major changes in 3-D structure of BHLHA9 protein that affect its interaction with other proteins.

Conclusion: This study reports second family with a previously identified frameshift-indel variation in BHLHA9 gene and will facilitate genetic counseling in Pakistani families with a MSSD-related phenotype.

Conflict of Interest: Safeer Ahmad EDUFI Fellowship, Muhammad Zeeshan Ali: None declared, Muhammad Muzammal: None declared, Mari Muurinen full, Shabir Hussain full, Muzammil Ahmad Khan full, Outi Mäkitie full.

EP05.036 genotype and phenotype characteristics of osteogenesis imperfecta in Saudi Arabia

Anwar Alhamad 1;2, Eissa Ali Faqeih1, mohammed alamin1, mohammed saleh1, Ali Alasmari1, aziza mushiba1, Manar Samman1, mohammed bamajboor1

1Children’s Specialized Hospital, King Fahad Medical City, Riyadh, Saudi Arabia; 2Maternal and Children Hospital, Alahsa, Saudi Arabia

Background/Objectives: Osteogenesis imperfecta (OI) is an inherited skeletal disease characterized by variable and recurrent fractures and extra skeletal manifestations. Up to date, there is 20 types of OI, majority are autosomal recessive disorders. We aim to describe clinical, molecular, and radiological features of OI and to evaluate the impact effectiveness of bisphosphonate therapy in variable genetic types of OI.

Methods: We performed a retrospective cohort analysis for 71 patients with variable ages between 2005 and 2022. Clinical and radiological assessment performed and confirmed by genetic study. In-house bisphosphonate therapy protocol is applied to all types regardless the type or severity.

Results: 71 patients with confirmed OI included. Collagenous related OI represent 51.43% with majority being de novo. Non-collagenous OI spectrum shown in 49.30%. The Most common mutation is the heterozygous mutation c.903+1G > A in COL1A1 gene followed by the homozygous mutation c.570_571delTG in P3H1 gene and c.831dupC in FKBP10 gene. Collagenous OI present with typical blue sclera, dentogeneous imperfecta and variable failure to thrive while non-collagenous OI have more hearing loss, physical deformities, short stature secondary to multiple fracture, walking disabilities and respond effectively to earlier bisphosphonate therapy with variable outcome especially, patients with TMEM38B-related OI in whom 50% reach normal bone density.

Conclusion: Almost half of Saudi OI are collagenous and other half belongs to the non-collagenous forms. We observe very dramatic improvement for bisphosphonate therapy even in those a previously lethal OI form.

Conflict of Interest: None declared.

EP05.037 Exome sequencing of nonsyndromic cleft palate trios reveals interesting candidate genes

Nina Ishorst 1, Sarah L. Mehrem1, Dmitriy Drichel2, Sugirthan Sivalingam3;4;5, Carine Carels6, Iris van Rooij7, Andreas Buness3;4;5, Kerstin Ludwig1, Elisabeth Mangold1

1Institute of Human Genetics, University of Bonn, School of Medicine & University Hospital Bonn, Bonn, Germany; 2Cologne Center for Genomics, University of Cologne, Cologne, Germany; 3Core Unit for Bioinformatic Analysis, Medical Faculty, University of Bonn, Bonn, Germany; 4Institute for Genomic Statistics and Bioinformatics, University of Bonn, School of Medicine & University Hospital Bonn, Bonn, Germany; 5Institute of Medical Biometry, Informatics and Epidemiology, University Hospital Bonn, Bonn, Germany; 6Department of Human Genetics, KU Leuven, Leuven, Belgium; 7Department for Health Evidence, Radboud University Medical Center, Nijmegen, Netherlands

Background: Nonsyndromic cleft palate only (nsCPO) is one of the most common forms of orofacial clefting and has a multifactorial etiology with a high heritability of >90%. Genome-wide association studies have identified ten genome-wide significant risk loci for nsCPO. Accordingly, common variants are involved in nsCPO development. However, genetic and epidemiological data suggest that also rare variants with a larger effect size play a strong role. The aim of this project was to perform exome sequencing (ES) in nsCPO trios and multiply affected families to identify rare highly penetrant (de novo) variants localized in potential candidate genes for nsCPO.

Methods: We performed ES in 342 individuals, 124 with nsCPO and 218 healthy relatives (84 trios, 9 quattros and 9 multiplex families ranging from 5 to 8 family members), the majority of European ancestry. Exome capture was performed using Twist Human Core Exome Kit. Libraries were sequenced on Illumina Nova Seq with 2 × 100 bp read length.

Results: A preliminary analysis of 43 trios identified 461 potential de novo variants. After stringent quality control, filtering and validation, the analysis revealed 46 de novo mutations in potential candidate genes for nsCPO, e.g. in KDM6B, a paralog of KDM6A, a gene for Kabuki Syndrome. A protein-protein-interaction analysis using STRING (v.11.5) showed an enrichment of interactions among the candidate genes (p = 0.009).

Conclusion: Our preliminary analysis shows the high potential of our dataset and adds further evidence for a strong contribution of rare variants to nsCPO.

Grant References: DFG grant MA 2546/6-1.

Conflict of Interest: Nina Ishorst: None declared, Sarah L. Mehrem: None declared, Dmitriy Drichel For the sake of completeness, we declare that Dmitriy Drichel provides compensated consulting services outside of academia as an independent consultant. Although past and current clients might include biotech, life science, and pharma companies, the services are not related to the presented work and the author is unaware of any possible conflict of interest., Sugirthan Sivalingam: None declared, Carine Carels: None declared, Iris van Rooij: None declared, Andreas Buness: None declared, Kerstin Ludwig Speaker at trainee workshops by Hans-Riegel Foundation, Co-founder and stake holder LAMPseq Diagnostics Inc., Elisabeth Mangold: None declared.

EP06 Cardiovascular Disorders

EP06.001 The relationship between the MTNR1B polymorphism and myocardial infarction susceptibility

Ivana Škrlec 1, Snježana Džijan1;2, Jasminka Talapko1, Vera Cesar1;3

1Josip Juraj Strossmayer University of Osijek, Faculty of Dental Medicine and Health, Osijek, Croatia; 2Genos Ltd., DNA Laboratory, Zagreb, Croatia; 3Josip Juraj Strossmayer University of Osijek, Department of Biology, Osijek, Croatia

Introduction: Melatonin is a circadian hormone with antioxidant properties and protects against myocardial ischemia-reperfusion injury. Genetic variations of the melatonin receptor gene (MTNR1B) play an important role in the development of type 2 diabetes, a risk factor for cardiovascular disease. Accordingly, MTNR1B polymorphisms are crucial in numerous diseases of the cardiovascular system. Therefore, the aim of the present study was to investigate a possible association between the MTNR1B polymorphism rs10830963 and susceptibility to myocardial infarction.

Methods: The study group consisted of 199 myocardial infarction patients (57% men) and 198 control participants (52% men) without a history of cardiovascular disease. Genotyping for MTNR1B rs10830963 was performed from peripheral blood samples using the TaqMan SNP genotyping assay on the Applied Biosystems QuantStudio 5 real-time PCR system.

Results: In patients with myocardial infarction, the rs10830963 polymorphism was associated with cardiovascular risk factors such as type 2 diabetes (p = 0.04), hypertension (p = 0.04) and shift work (p = 0.05). However, no significant association, estimated by the chi-square test, was found in the distribution of alleles and genotypes between myocardial infarction patients and controls.

Conclusion: In this study, no significant association was found between rs10830963 polymorphism and myocardial infarction susceptibility. The present negative results do not exclude the role of this polymorphism in the development of myocardial infarction, as some previous studies have shown. Rather, they may indicate that rs10830963 is a minor risk factor for myocardial infarction.

Grant no.: IP1-FDMZ-2022

Conflict of Interest: None declared.

EP06.002 Haploinsufficiency in MYH7 as a possible mechanism of pathogenicity in a Portuguese family with Hypertrophic Cardiomyopathy

Diogo Fernandes da Rocha 1, Márcia Baixia2;3, Ana Grangeia1;4, Raquel Silva2;3, Sérgio Castedo2;3;4, Ana Sofia Correia5, pedro louro1;6

1São João Universitary Hospital Center, Serviço de Genética Médica, Porto, Portugal; 2i3S - Instituto de Investigação e Inovação em Saúde da Universidade do Porto, Porto, Portugal; 3IPATIMUP - Instituto de Patologia e Imunologia Molecular da Universidade do Porto, Porto, Portugal; 4Faculdade de Medicina da Universidade do Porto - FMUP, Porto, Portugal; 5Hospital Pedro Hispano, Serviço de Cardiologia, Senhora da Hora, Portugal; 6Universidade da Beira Interior - Faculdade de Ciências da Saúde, Covilhã, Portugal

Background/Objectives: Hypertrophic cardiomyopathy (HCM) is one of the most common inherited cardiac conditions, mostly caused by pathogenic variants in sarcomere-encoding genes. Predicted deleterious missense (pDM) variants in MYH7 have been often described as having a dominant negative effect on sarcomeric function in HCM patients. The ACMG/AMP variant classification framework for MYH7-associated inherited cardiomyopathies also states that loss-of-function (LOF) variants should not be considered as an isolated cause of HCM, although it has been admitted that compound heterozygosity of LOF with missense variants could lead to extremely severe presentations. In addition, a recent paper describes a heterozygous frameshift MYH7 variant segregating in a multiplex family affected by HCM (PMID: 34460321).

Methods: We present a family of six brothers, two of which with previous clinical diagnosis of HCM. One of these brothers’ son died suddenly at 16-years-old, and another brother’ son was diagnosed with HCM in his early 20s.

Results: The NGS panel requested in one of the affected brothers identified a frameshift variant in MYH7 which introduces a premature termination codon. Genetic testing in the remaining relatives identified the same MYH7 variant in a total of six individuals, all with echocardiographic involvement. Based on that, we reclassified this variant as likely pathogenic and offered preimplantation genetic testing to a family carrier.

Conclusion: This case strongly suggests that LOF variants in MYH7 may be associated, albeit rarely, with HCM. It also illustrates the importance of periodically reviewing MYH7-specific pathogenicity criteria.

Grant References: Nothing to declare.

Conflict of Interest: Diogo Fernandes da Rocha Serviço de Genética Médica, Centro Hospitalar Universitário de São João, Márcia Baixia Ipatimup, Ana Grangeia Serviço de Genética Médica, Centro Hospitalar Universitário de São João, EXPL/DTP-EPI/0376/2012, Raquel Silva Ipatimup, Sérgio Castedo Ipatimup, Ana Sofia Correia Serviço de Cardiologia, Unidade Local de Saúde de Matosinhos, Porto, Portugal, pedro louro Serviço de Genética Médica, Centro Hospitalar Universitário de São João.

EP06.003 The MT-ND1 m.3460G>A pathogenic variant as a cause of isolated cardiac disease in a 77 year old man

Sivan Koka 1, Yoav Michowitz2;3, Itshak Amsalem2;3, Moshe Rav Acha2;3, Michael Glikson2;3, ephrat lahad1;3

1Shaare Zedek Medical Center, Medical Genetics Institute, Jerusalem, Israel; 2Shaare Zedek Medical Center, Department of Cardiology, Jesselson Integrated Heart Center, Jerusalem, Israel; 3Hebrew University of Jerusalem, Faculty of Medicine, Jerusalem, Israel

Introduction: Pathogenic variants in MT-ND1, encoding the core protein of mitochondrial respiratory Complex I, cause several disorders, including Leber hereditary optic neuropathy (LHON), Leigh syndrome and MELAS. Cardiomyopathy, particularly hypertrophic (HCM) occurs in ~30% of adults with mitochondrial diseases.

Materials and Methods: We evaluated a 77-year-old man with heart failure secondary to apical HCM. Medical history revealed Wolff-Parkinson-White (WPW) diagnosed at age 14, treated with ablation at age 49 and pacemaker implantation ~10 years ago due to atrioventricular block, and lately upgraded to cardiac resynchronization therapy defibrillator due to ventricular arrhythmia. He has no siblings and family history was limited. No ophthalmological or neurological problems were reported.

Results: Cardiomyopathy multi-gene panel testing revealed a known pathogenic variant in MT-ND1 (m.3460G > A (c.154G>A), p.Ala52Thr) with 29% heteroplasmy. This variant is one of three common mtDNA pathogenic variants underlying LHON.

Conclusions: MT-ND1 m.3460G > A, usually associated with LHON, was found to cause a purely cardiac phenotype. Manifestations included cardiac pre-excitation, found in ~10% of LHON patients, and HCM. The very few reports of left ventricle myocardial thickening in LHON m.3460G > A patients, were all in men with symptomatic optic neuropathy. Optic neuropathy is very unlikely to appear after age 75, so to the best of our knowledge this is the first report of MT-ND1 m.3460G > A presenting as an isolated cardiac disease. The cardiac-limited phenotype may be explained by low heteroplasmy level (29%), compared to >70% leukocyte heteroplasmy in LHON patients with optic pathology. These results underscore the importance of unbiased genetic testing in cardiomyopathy patients.

Conflict of Interest: None declared.

EP06.004 Prevalence of hyperlipidemia causing genetic variants in patients with coronary artery disease

Darius Čereškevičius 1;2, Ali Aldujeli1, Kristina Zubielienė1, Vacis Tatarūnas1

1Lithuanian University of Health Sciences, Institute of Cardiology, Laboratory of Molecular Cardiology, Kaunas, Lithuania; 2Hospital of Lithuanian University of Health Sciences, Department of Genetics and Molecular Medicine, Kaunas, Lithuania

Background/Objectives: Hypercholesterolemia is the main cause of atherosclerosis which is the leading cause of death in the developed world. Recent studies have shown that certain genetic variants related to hypercholesterolemia are much more prevalent than previously thought. Thus, familial hypercholesterolemia (FH) which is an autosomal dominant genetic disorder, might be underdiagnosed and undertreated.

Methods: During this study 93 patients with coronary artery disease (CAD) who have not been previously diagnosed with FH were included in this study. All patients had high low-density Lipoprotein cholesterol levels in blood plasma (>5 mmol/L). Next generation sequencing was used to sequence the coding regions and exon/intron boundaries of the LDLR, PCSK9, APOB and LDLRAP1 genes.

Results: One patient had a heterozygous pathogenic variant in the LDLR gene which has been described previously (c.1013G>A (p.Cys338Tyr)). Another patient was a carrier of a heterozygous pathogenic variant in the APOB gene (c.10580G>A (p.Arg3527Gln)). A third patient was a carrier of a previously undescribed variant in the LDLR gene (c.734A>G (p.Asp245Gly)). Based on the American College of Medical Genetics criteria, this variant was determined to be likely pathogenic. A further 10 patients were carriers of variants of uncertain significance in the LDLR and APOB genes.

Conclusion: This study highlights that patients with FH remain underdiagnosed and undertreated. There is a need to expand molecular testing for patients with CAD. More complex studies are needed to determine the pathogenicity of variants of uncertain significance.

Conflict of Interest: Darius Čereškevičius Employed in Lithuanian University of Health Sciences hospital “Kauno Klinikos” department of genetics and molecular medicine., Research was funded by the Lithuanian University of Health Sciences scientific fund., The Lithuanian University of Health Sciences hospital “Kauno Klinikos” department of genetics and molecular medicine allowed the use of their next generation sequencers., Ali Aldujeli Employed in Lithuanian University of Health Sciences hospital “Kauno Klinikos” cardiology clinic., Kristina Zubielienė Employed in Lithuanian University of Health Sciences hospital “Kauno Klinikos” cardiology clinic., Vacis Tatarūnas Employed in Lithuanian University of Health Sciences Institute of Cardiology Laboratory of Molecular Cardiology.

EP06.005 Cardiogenetic examination of non-ischemic cardiac arrest survivors: concealed arrhythmogenic cardiomyopathy is the most frequent cause, while hypertrophic cardiomyopathy is underrepresented in a representative Czech cohort

Pavel Votypka1, Alice Krebsová2, Petra Peldova1, Terezia Tavacova3, Veronika Zoubková1, Milos Kubanek2, Petr Peichl2, Marketa Segetova2, Jana Haskova2, Hana Wunschova2, Jan Janousek3, Marek Sramko2, Milan Macek 1, Josef Kautzner2

12nd Faculty of Medicine of Charles University and Motol University Hospital, Department of Biology and Medical Genetics, Prague, Czech Republic; 2Institute of Clinical and Experimental Medicine, Department of Cardiology, Prague, Czech Republic; 32nd Faculty of Medicine of Charles University and Motol University Hospital, Children´s Heart Centre, Prague, Czech Republic

Background/Objectives: We aimed to determine the genetics of non-ischemic cardiac arrest (CA) in a representative cohort of CA survivors (CAS) in the Czech Republic.

Methods: A total of 200 CASs (120 M/80 F; mean age at CA: 35.6 ± 16 years), referred from regional centres underwent cardiogenetic counselling and NGS sequencing using a candidate gene panel (150+ genes) utilizing the SOPHiA GENETICS DDM platform. The presence of variants was confirmed by Sanger DNA sequencing and cascade screening. Additional cardiac exams (e.g. TTE, SaECG, exercise ECG) were performed to clinically validate obtained results.

Results: CASs presented mainly as idiopathic ventricular fibrillation (iVF, 124/200). Other diagnoses comprised arrhythmogenic cardiomyopathy (ACM), including forms with involvement of the right ventricle (RVACM, 11/200), left ventricle (LVACM – 13/200), or both ventricles (BiVACM – 13/200), hypertrophic cardiomyopathy (HCM-1/200), hereditary arrhythmic syndromes (LQT – 23/200, BrS-9/200, CPVT-2/200) and arrhythmogenic mitral valve prolapse (MVP -6/200). Molecular genetic testing identified a causative DNA variant (P/LP) in 54/200 (27%) of CASs. The highest detection rate was in RVACM (73%). Genetic testing clarified the diagnosis in 21% of the iVF cohort, mostly as concealed ACM.

Conclusion: Most cases of CA are of unknown aetiology (iVF -62%). Cardiogenetic examination confirmed approximately one third of these cases, primarily concealed ACM (PKP2 gene; 12/200). Other frequent causes of CA were LQT2 (9/200), LQT3 (7/200) and CPVT (5/200). Conversely, HCM was underrepresented in this cohort (1/200 and 2/200 after genetic testing).

Conflict of Interest: None declared.

EP06.006 Genetic screening in patients with suspected heritable thoracic aortic disease in Japan

Takeshi Yagyu 1

1National Cerebral and Cardiovascular Center, Suita, Japan

Background/Objectives: Heritable thoracic aortic diseases (HTAD), such as Marfan syndrome, Loeys–Dietz syndrome, and vascular Ehlers–Danlos syndrome, cause life-threatening aortic events at a young age, and require accurate diagnosis for appropriate treatment. This study aimed to investigate the genetic background of patients with suspected HTAD in Japan.

Methods: The study investigated probands who were suspected of having HTAD and underwent genetic testing in our institute from July 2016 to December 2022. Genetic testing was performed by panel sequencing and Sanger sequencing for the following 11 causative genes: ACTA2, COL3A1, FBN1, MYH11, MYLK, SLC2A10, SMAD3, TGFB2, TGFB3, TGFBR1, and TGFBR2.

Results: A total of 244 probands (male/female 128/116, median age 37 [0–78] years) were included. One hundred forty patients (57.3%) experienced aortic dissection or aortic surgery prior genetic testing, or had aortic root dilatation without a history of aortic events. Sixty-eight patients (27.9%) had a family history of aortic events. Pathogenic/Likely Pathogenic variants of HTAD were detected in 118 probands (48.3%): 87 FBN1, 8 COL3A1, 6 SMAD3, 6 TGFBR2, 4 ACTA2, 3 MYH11, 3 TGFBR1, 1 TGFB3, and none in other genes.

Conclusion: This study presented the positive rate of genetic testing and the composition of causative genes in patients with suspected HTAD in Japan.

Grant reference: JSPS KAKENHI Grant Number 18K15909

Conflict of Interest: None declared.

EP06.007 The role of MRAS in atherosclerosis and relevant cardiovascular risk factors

Pashmina Wiqar Shah 1, Dr. rer. nat. Tobias Reinberger1, Jeanette Erdmann1

1Institute for Cardiogenetics, University of Lübeck, Lübeck, Germany

A study by Erdmann et al., in 2009, revealed a region on 3q22.3, which encompasses the MRAS gene as a risk factor for Coronary Artery Disease (CAD). MRAS encodes muscle Ras, a member of the Ras family of small membrane associated GTPases associated with TNF-α signaling. According to GTEx and other eQTL datasets, MRAS risk variants for CAD increase MRAS mRNA levels primarily in the arterial tissue. Moreover, recently it has been indicated that functional MRAS variants are Macrophage- and SMC-specific. The role of MRAS in atherogenesis is still elusive. Therefore, we aimed to study the function of MRAS in vascular smooth muscle cells (SMCs), one of the key cell types in the etiology of atherosclerosis and in plaque stabilization. To assess the impact of MRAS deficiency, human primary aortic SMCs transfected with MRAS-specific siRNA and murine aortic SMCs derived from Mras/Apoe knockout (ddKO) mice were subjected to functional assays including proliferation and migration. Mras/Apoe ddKO murine SMCs significantly proliferate more and migrate faster as compared to wild type SMCs when stimulated with TNF-α (n = 4, p < 0.001), but not without stimulation. Similar results were obtained from human SMCs after TNF-α stimulation where the knockdown of MRAS increases migration and proliferation (n = 6, p < 0.01). Control experiments with PDGF stimulation showed no impact of MRAS deficiency on cellular behavior, indicating that MRAS is specific to TNF-α signaling. In conclusion, MRAS deficiency modulates SMC biology in response to TNF-α. However, further studies are needed to decipher its exact role in plaque stability. Keywords: MRAS, TNF-α

Conflict of Interest: Pashmina Wiqar Shah Full timw, Full, Student, Dr. rer. nat. Tobias Reinberger Full time, Co-supervisor, Jeanette Erdmann Full time, Prinicpal Investigator, Prof. Jeanette Erdmann is the PI of this project. I am doing my Phd under her kind supervision. I am funded by the DAAD (Deutsches Akademischer Austausch Dienst) for a period of four years to complete my PhD.

EP06.008 Isthmic aortic coarctation, a rare complication of Vascular Ehlers-Danlos syndrome?

ophélie boutfol 1, Alinoe Lavillaureix1, Anne-Sophie Leborgne2, Xavier Jeunemaitre3, Clarisse Billon3, Tristan Mirault4, Sylvie Odent1

1CHU Rennes, Service de Génétique Clinique, Rennes, France; 2Clinique Mutualiste La Sagesse, Cardiologie, Rennes, France; 3Hôpital européen Georges Pompidou, Service de Génétique Clinique, Paris cedex 15, France; 4Hôpital européen Georges Pompidou, Médecine vasculaire, Paris cedex 15, France

Background: Vascular Ehlers-Danlos syndrome (vEDS) (OMIM #130050) is characterized by arterial, intestinal, and uterine fragility, thin and translucent skin, easy bruising, characteristic facial appearance, and an aged appearance to the extremities.

Clinical report: A heart murmur, not present during his previous medical follow-ups, was diagnosed in an 11-year-old child, justifying the performance of a cardiac ultrasound to demonstrate tight isthmic aortic coarctation, an atypical diagnosis given his advanced age. The intraoperative aspect found a friable and very fragile aortic tissue.

Associated symptoms consisting of thin and translucent skin with visible vascularization, easy bruising, postoperative scalp sore, early-onset lumbar scoliosis, and pes planus led to the COL3A1 gene testing. A heterozygous missense variant c.2806G>A (p.(Gly936Ser)) confirmed the diagnosis of vEDS. This variant with a dominant negative effect is inherited from his mother. She presented with two complete perineal tears intrapartum. An aneurysm of the celiac trunk, aneurysms of the right renal artery, an ostial stenosis of the left renal artery with post-stenosis dilation, and ischemic sequelae on both kidneys, had been found with no lesion on the supra-aortic trunks on imaging following a traffic-induced trauma. Maternal grandparents were tested, proving that the COL3A1 variant occurred de novo in the mother.

Conclusion: We report the first case of a child with confirmed vEDS and aortic coarctation, more patients may be affected by this rare complication. vEDS can have dramatic consequences during surgery due to vascular fragility. A diagnosis before surgery could therefore allow additional precautions to be taken to prevent these risks.

Conflict of Interest: None declared.

EP06.009 Prevalence of genetic variants associated with thrombophilia in patients with intracoronary stents

Marina Stratrova 1, Hristo Pejkov2, Zan Zimbakov2, Slavica Josifovska1, Sasho Panov1

1Laboratory for Molecular Biology, Faculty of Natural Sciences and Mathematics, Ss. Cyril and Methodius University in Skopje, Skopje, Macedonia; 2University Clinic of Cardiology, Medical Faculty, Ss. Cyril and Methodius University in Skopje, Skopje, Macedonia

Background/Objectives: Atherosclerosis and intravascular thrombosis play a central role in acute coronary syndromes treated by implantation of an intracoronary stent. In recent years, the role of thrombophilia in the pathogenesis of arterial thrombosis has become increasingly relevant. The aim of this study was to determine the genetic association of coronary artery disease (CAD) with the genotype of variants in the genes coding for prothrombin (F2), coagulation factor V (F5), plasminogen activator inhibitor-1 (PAI-1), methylene-tetrahydrofolate reductase (MTHFR) and coagulation factor XIII (F13A1).

Methods: The genotyping was performed by TaqMan-based RT-PCR on samples from 60 patients with CAD who underwent intracoronary stent implantation and 29 control subjects, followed by statistical analysis.

Results: Multivariate logistic regression was used to constructs a model for predicting the coronary artery disease with investigated variants genotypes. The AA genotype of the F5 1601G > A Leiden mutation (rs6025), the GA genotype of the F2 20210G > A rs1799963 variant, the 4G/5G genotype of the PAI-1 -675 4G/5G rs1799889 polymorphism, and the AC genotype of the MTHFR 1298A > C variant rs1801131 have the effect of increasing the probability of CAD. In contrast, the GT genotype of the F13A1 103G > T (rs5985) variant has an effect of reducing the odds for CAD. The effects of the CC and CT genotypes of the MTHFR 677C > T rs1801133 variant on the model are statistically marginal.

Conclusion: The investigated gene variants have a potential in clinical prediction of the risk for developing coronary artery disease.

Conflict of Interest: Marina Stratrova Genomika Medical, Skopje, Hristo Pejkov University Clinic of Cardiology, Medical Faculty, Ss. Cyril and Methodius University in Skopje, Macedonia, Zan Zimbakov University Clinic of Cardiology, Medical Faculty, Ss. Cyril and Methodius University in Skopje, Macedonia, Slavica Josifovska Laboratory for Molecular Biology, Faculty of Natural Sciences and Mathematics, Ss. Cyril and Methodius University in Skopje, Sasho Panov Laboratory for Molecular Biology, Faculty of Natural Sciences and Mathematics, Ss. Cyril and Methodius University in Skopje.

EP06.010 Rapid exome sequencing for children with acute cardiomyopathy in the PICU – a case series

Tova Hershkovitz 1;2, Clair Habib1, Tamar Paperna1, Rinat Zaid1, Josef Ben Ari2;3, Galit Tal2;4, Asaad Khoury5, Karin Weiss1;2

1The Genetics Institute, Rambam Health Care Campus, Haifa, Israel; 2The Ruth and Bruce Rapport Faculty of Medicine, Technion, Haifa, Israel; 3Pediatric Intensive Care Unit, Rambam Health Care Campus, Haifa, Israel; 4Metabolic Clinic, Rambam Health Care Campus, Haifa, Israel; 5Department of Pediatric Cardiology, Haifa, Israel

Background: Rapid exome sequencing (ES) is increasingly being utilized as an efficient diagnostic tool in the critical care setting with high diagnostic yield. Acutely presenting cardiomyopathy (CM) can often require critical care and has numerous possible etiologies including various genetic disorders, particularly in children.

Here we describe a brief series of pediatric patients hospitalized in the intensive care unit (ICU) with acute cardiomyopathy of unknown cause. Rapid ES provided a timely diagnosis and informed clinical decisions including eligibility for extracorporeal membrane oxygenation ECMO or heart transplant.

Methods: ES was performed for 5 unrelated patients ages 8 days to 10 years with acute cardiomyopathy hospitalized in the ICU. Turnaround time was 5 to 60 days

Results: ES was diagnostic in 3/5 cases including a likely pathogenic variant in ACTC1,c.664G>A, in a 6 year old boy with left ventricular non-compaction and a homozygous pathogenic variant in NRAP,p.Gln1113Hisfs*40, in a 10 year old boy with dilated CM. In both cases, as results confirmed isolated cardiac involvement, the patients were eligible for transplant and received bridging therapies.

A homozygous pathogenic variant in MYBPC3 :c.3491-2A > C was detected in a neonate confirming the diagnosis of fatal neonatal CM. Results were delivered within 5 days precluding ECMO.

In two cases, a variant of unknown significant was identified in genes, related to broader phenotypes These results prompted further investigation and extended metabolic workup.

Discussion and conclusion: Rapid ES for pediatric patients with acute CM had a high diagnostic yield and a significant impact on patient care.

Conflict of Interest: None declared.

EP06.011 A homozygous rare TTR variant of transthyretin amyloidosis

Dovile Zebrauskiene 1, Egle Sadauskiene2, Sigita Aidietiene2, Agnė Šiaudinienė3, Valdas Pečeliūnas3;4, Jurate Barysiene2, Egle Preiksaitiene1

1Department of Human and Medical Genetics, Institute of Biomedical Sciences, Faculty of Medicine, Vilnius University, Vilnius, Lithuania; 2Clinic of Cardiac and Vascular Diseases, Institute of Clinical Medicine, Faculty of Medicine, Vilnius University, Vilnius, Lithuania; 3Center of Haematology, Oncology and Transfusion Medicine, Vilnius University Hospital Santaros klinikos, Vilnius, Lithuania; 4Clinic of Internal Medicine, Family Medicine and Oncology, Institute of Clinical Medicine, Faculty of Medicine, Vilnius University, Vilnius, Lithuania

Background/Objectives: Hereditary transthyretin amyloidosis (ATTRv) is a rare autosomal dominantly inherited disease caused by mutations in the transthyretin (TTR) gene. More than 140 TTR gene variants have been reported in ATTRv. Few cases have been described in which the rare TTR variant c.302C>T, p.(Ala101Val) was mostly associated with cardiac ATTRv.

Methods: We present a 45-years-old patient with diagnosed sensomotor polyneuropathy of the upper and lower extremities and pronounced tetraparesis. Cardiac involvement (amyloidosis) was suspected only 4 years later. Cardiac MRI was performed showing asymmetric LV hypertrophy with a suspicion of hypertrophic cardiomyopathy. Haematological analysis for AL amyloidosis were negative. 99mTc-PYP bone scintigraphy showed no myocardial uptake (Grade 0). Endomyocardial biopsy was performed for differential diagnosis. Amyloid deposits were found, but immunohistochemistry showed a likely non-specific reaction to transthyretin. Mass spectrometry was not available.

Results: Next-generation sequencing revealed a likely pathogenic homozygous variant of the TTR gene NM_000371.3:c.302C>T, NP_000362.1:p.(Ala101Val). As parental testing was not available, real-time PCR analysis was performed and no heterozygous deletion of exon 3 of the TTR gene was detected. This variant in the homozygous state was not described in the literature before. The same c.302C>T TTR variant in the heterozygous state was also found in 3 other unrelated probands (2 females and 1 male) with predominant cardiac involvement at our centre. Their ages at diagnosis were 57, 74 and 77.

Conclusion: The homozygous c.302C>T TTR variant is associated with earlier disease onset and neurological involvement compared to the heterozygote state.

Conflict of Interest: Dovile Zebrauskiene Created a presentation about ATTR cardiomyopathy which was funded by Pfizer., Egle Sadauskiene: None declared, Sigita Aidietiene: None declared, Agnė Šiaudinienė: None declared, Valdas Pečeliūnas: None declared, Jurate Barysiene: None declared, Egle Preiksaitiene: None declared.

EP06.012 Generation of antibodies against α-Klotho protein using canarian camels

VICTOR GARCIA TAGUA 1;2, Yeray Brito-Casillas3, Ernesto Martín-Núñez2, Javier Donate-Correa2, Guido Santos-Rosales1, Emma Carmelo1, Pilar Foronda Rodríguez1, Carmen Mora-Fernández2, Juan Francisco Navarro-González2

1Universidad de La Laguna, San Cristóbal de La Laguna, Spain; 2Unidad de Investigación, Hospital Universitario Nuestra Señora de Candelaria, Santa Cruz de Tenerife, Spain; 3Universidad de Las Palmas de Gran Canaria, Las Palmas de Gran Canaria, Spain

Cardiovascular disease (CVD) is the leading cause of death worldwide. Atherosclerosis is the substrate responsible for the vast majority of cardiovascular events. Reductions in α-Klotho protein levels have been related with the pathophysiology of CVD, particularly in chronic kidney disease patients.

The main objective of our project is to obtain mini- and nanoantibodies (nanobodies) from Canarian camels to be employed in the detection of human α-Klotho protein in different tissues, and serum and urine samples.

Nanobodies are variable domains of heavy chain-only antibodies (HCAbs) that can be isolated from camelids. In spite of their single domain structure, nanobodies display many unique features, such as small size, high stability, and cryptic epitopes accessibility, which make them ideal for sophisticated applications in plants and animals.

We carried out immunizations in camels with the different regions of the protein to detect the different forms in which the protein is presented. We isolated lymphocytes from camel blood in order to clone the DNA regions codifying for antibodies against α-Klotho, that will be further expressed in bacteria. Effectiveness and sensitivity of these new generation antibodies are being tested in clinical samples and cell lines that express α-Klotho through different approaches, e.g. western blot, ELISA and immunohistochemistry assays. These mini- and nanobodies will allow us to use α-Klotho as a biomarker with clinical applicability.

This work is supported by Cabildo de Tenerife, Tenerife 2030, FDCAN, MEDI, in the Agustín de Betancourt programme and PIFIISC21/23 from Fundación Canaria Instituto de Investigación Sanitaria de Canarias (FIISC).

Conflict of Interest: None declared.

EP06.013 Clinical and laboratory characteristics of congenital heart defects, caused by microdeletion of 22q11.2

Nikita Pozhar 1, Vira Galagan1, Valentyna Kurakova1, Andrii Kurkevych2, Yuliia Dudierina3, Maryna Tsyhankova1, Yurii Hryshuk4, Diana Mykhailova4

1Okhmatdyt, The Center of Medical Genetics, Kyiv, Ukraine; 2Government Agency “Scientific Practical Medical Centre of Pediatric Cardiology and Cardiac Surgery”, Kyiv, Ukraine; 3Communal non-commercial enterprise “Kyiv city maternity hospital №5”, Kyiv, Ukraine; 1Okhmatdyt, The Center of Medical Genetics, Kyiv, Ukraine

Introduction: Isolated congenital heart defects (CHD) are the most frequent among all congenital defects (CD). Their frequency among live births of Kyiv is 8-9:1000 (2019-2021). Microdeletion of 22q11.2 and trisomy of chromosome 21 are the most common causes of CHD.

Materials and methods: Prospective genetic counseling and examination of 77 children, aged 1 day to 12 years from different regions of Ukraine for the last 10 years was undergone at the Center of Medical Genetics of Children’s Hospital “OKHMATDYT” (hospital cohort). All cases of CHD were confirmed by pediatric cardiologist. Cytogenetic examination included G-banding karyotyping according to the standard protocol. The FISH using loci-specific microdeletion of 22q11.2 DNA-probes was applied to verify the microdeletion.

Results: Sixty-seven full-term children (87%) were born via physiological delivery, with body weight more than 2,5 kg (81%). Prenatally diagnosed CHD before 22 week of pregnancy were in 16 women (21%), invasive prenatal diagnostics took place in 12% of cases. Phenotype of probands: presence of stigmas was in 51 children (66%), hypo- and aplasia of thymus were in 50 children (65%), other CD - 27%, among them – multiple CD in 8 cases (38%), polydactyly in 4 cases (19%). Four probands (5%) with microdeletion of 22q11.2 did not have any CHD. Conotruncal defects were in 55% of all CHD, critical CHD were in 14%. Microdeletion of 22q11.2 was confirmed in all probands.

Conclusion: With the aim of study the etiology and differential diagnostics of CHD in children, applying of whole range of molecular studies are recommended.

Conflict of Interest: Nikita Pozhar full, Vira Galagan full, Valentyna Kurakova full, Andrii Kurkevych full, Yuliia Dudierina full, Maryna Tsyhankova full, Yurii Hryshuk full, Diana Mykhailova full.

EP06.014 Time to downgrade genetic testing? Review of pathogenic variants in cardiogenetic

Diana Antunes 1;2, Ana Coutinho1, Yuri Chiodo1, Brigida Meireles1, Sofia Pérez3, Maria Carmo-Fonseca4

1GenoMed - Diagnósticos de Medicina Molecular, S.A, Lisboa, Portugal; 2Santa Marta Hospital, Medical Genetics Department, Lisboa, Portugal; 3Dona Estefânia, Medical Genetics Department, Lisboa, Portugal; 4Instituto de Medicina Molecular (IMM), Lisboa, Portugal

The financial and societal impact of hereditary cardiac diseases (HCD) is widely acknowledged. One of the most prevalent of these conditions is hypertrophic cardiomyopathy – it affects 1:200 to 1:500 people – where is reported that in half of cases it is found a pathogenic mutation. In 2022 it was published international “Expert Consensus Statement on the state of genetic testing for cardiac diseases”.

We reviewed the results of genetic testing performed for HCD in our laboratory between January 2019 and January 2023. A total of 675 genetic analysis were carried out for, confirmed or suspected: hypertrophic cardiomyopathy (HCM N = 243), dilated cardiomyopathy (DCM N = 215), arrhythmogenic cardiomyopathy (ACM N = 38), among others.

We found pathogenic or likely pathogenic variants in 130 samples with genetic diagnostic rates for:

  • HCM 22,2% (N = 54) – with pathogenic (P) or likely pathogenic (LP) variants on ACTN2, ALPK3, FHOD3, MYBPC3, MYH7, MYL2, PRKAG2, RBM20, RYR2, SLC25A4, TNNI3, TNNT2, TPM1;

  • DCM 21.4% (N = 46) –DSP, FLNC, GLA, LMNA, MYBPC3, MYH7, PKP2, RBM20, RYR2, SCN5A, TNNT2, TPM1, TTN;

  • ACM 31,6% (N = 12) – DSP, GLA, HAMP, KCNJ2, MYBPC3, PKP2, RYR2.

Currently, the evident benefits of genetic testing in HCD is widely accepted, namely for early disease detection and management. In our cohort it was found P/LP variants on genes with clinical implications for medical management of patients, not included in the 2022 international recommendations. This work aims to contribute to the ongoing discussion of potential benefits of expanded genetic testing in HCD, namely the possibility of reverse phenotyping on cases of clinical uncertainty.

Conflict of Interest: Diana Antunes Genomed Part-time

Chulc/santa marta full time, Ana Coutinho full-time, Yuri Chiodo full-time, Brigida Meireles full-time, Sofia Pérez CHULC/Estefânia Hospital, Maria Carmo-Fonseca IMM full time, Genomed

EP06.015 Prognostic value of microRNA-126-3p for cardiovascular events in a Spaninsh general population

Olga Martinez-Arroyo 1, Ana Flores-Chova1, Juan C Martin-Escudero2, Josep Redon3, Raquel Cortés4, Ana Ortega1

1Biomedical Research Institute Hospital Clinico - INCLIVA, Cardiometabolic and Renal Risk Research Group, Valencia, Spain; 2Rio Hortega Universitary Hospital, Internal Medicine, Valladolid, Spain; 3Hospital Clinico Universitary, Internal Medicine, Valencia, Spain; 4Biomedical Research Institute Hospital Clinico - INCLIVA, Cardiometabolic and Renal Risk Research Group, Valencia, Spain

Background and objectives: Measurement of circulating levels of microRNAs (miRNAs) is emerging as potential biomarkers for cardiovascular disease. Here we estimate the predictive value of a plasma miRNAs signature associated with albuminuria in the incidence of cardiovascular events.

Methods: Plasma miRNAs quantified in hypertensive patients by next generation sequencing were validated in a cohort of patients and controls by real-time quantitative PCR. The microRNAs associated with albuminuria were tested and their prognostic value for cardiovascular event incidence were analyzed on a retrospective, population-based study (Hortega Study) using Cox proportional hazard models.

Results: A plasma microRNA profile was identified in the discovery cohort associated with albuminuria and three microRNAs (miR-126-3p, miR-1260b and miR-374a-5p) were confirmed in the validation cohort. The microRNA signature discriminates urinary albumin excretion at baseline (n = 1025), and predicts the incidence of cardiovascular events and coronary heart disease and stroke in a general population retrospective study within a 14-year follow-up (n = 926). Thus, high miR-126-3p levels were associated with a shorter time free of both cardiovascular events (HR = 1.48, (1.36–1.62), p < 0.0001), as well as coronary artery disease and stroke combined (HR = 2.49, (2.19–2.83), p < 0.0001).

Conclusion: An increased plasma microRNAs profile was identified in hypertensive patients with albuminuria. Increased miR-126-3p emerges as a prognostic marker for cardiovascular events in a long-term general population. Further studies will assess the potential role of miR-126-3p as a guide for the status of endothelial dysfunction.

Grants: Health Institute Carlos III [PI12/02615; PI19/01796; FI20/00096 FI22/00032] IJC2020-045308-I, by MCIN/AEI. European Regional Development Fund (ERDF).

Conflict of Interest: None declared.

EP06.016 Differentially expressed genes in peripheral blood mononuclear cells of coronary artery disease patients

Elena Nosova1, Alexander Dergunov2, Mikhail Popov3, Alexandra Rozhkova1, Margarita Vinogradina 1, Veronika Baserova2, Svetlana A. Limborska1, Liudmila Dergunova1

1National Research Center “Kurchatov Institute”, Department of Molecular Bases of Human Genetics, Moscow, Russian Federation; 2National Medical Research Center for Therapy and Preventive Medicine, Laboratory of Structural Fundamentals of Lipoprotein Metabolism, Moscow, Russian Federation; 3Moscow Regional Research and Clinical Institute MONIKI, Moscow, Russian Federation

Aim: To reveal the differentially expressed genes (DEGs) of HDL-cluster and atherogen-cluster in peripheral blood mononuclear cells (PBMC) of CAD patients.

Methods: 77 male patients with CAD diagnosed by angiography and 65 control patients were enrolled. Two sets of genes related to HDL metabolism (HDL-cluster with 23 genes) and atherosclerosis-prone (atherogen-cluster with 41 genes) were selected by bioinformatic approaches. Transcript levels of 64 genes in RNA isolated from PBMC were measured by real-time RT-PCR and compared by REST software. KEGG pathway and STRING analyses of protein-protein interaction of DEGs were exploited.

Results: Thirty DEGs in CAD patients were identified. Compared with control, 8 genes in HDL-cluster (AMN, APOE, CETP, LDLR, LPL, PLTP, PRKACA, and ZDHHC8) were upregulated and 6 genes (ABCA1, ABCA5, ALB, APOA1, LCAT, and SCARB1) were downregulated in CAD. Analogously, 8 genes in atherogen-cluster (CXCL5, IL1B, ITGB3, NR1H2, NR1H3, TLR5, TLR8, and TNFRSF1A) were upregulated and 8 genes (CD14, F5, IL1R1, ITGAM, PCTP, S100A12, S100A8, and TNFRSF1B) were downregulated.

Conclusion: The expression of genes involved in cholesterol efflux decreased in CAD while the expression of genes involved in (re)generation of nascent HDL-like particles increased. The concomitant increase of APOE and LDLR expression may be responsible for the increased cellular uptake of remnants of TG-rich lipoproteins. Higher expression of ZDHHC8 may evidence the mitochondrial-regulated apoptosis. Expression pattern of atherogen-cluster genes generally relates to inflammatory processes and immune response through MYD88 and TRAF6, leading to NF-kappa-B activation. Opposite changes of IL1B and IL1R1 expression may weaken the net effect.

Conflict of Interest: Elena Nosova full, Alexander Dergunov full, Mikhail Popov full, Alexandra Rozhkova full, Margarita Vinogradina full, Veronika Baserova full, Svetlana A. Limborska full, Liudmila Dergunova full.

EP06.017 Arrhythmiogenic right ventricular cardiomyopathy (ARVC) phenotype investigated by exome sequencing reveals a Naxos disease case

Georgia Christopoulou 1, Stavros Bournazos1, Aikaterini Oikonomaki1, Stavroula Samara1, Antigoni Miliou2, Charalambos Vlachopoulos2, Pantelis Constantoulakis1

1Genotypos Science Labs M.S.A., Genetics and Genomics, Athens, Greece; 2National and Kapodistrian University of Athens, Unit of Inherited Cardiac Conditions and Sports Cardiology, First Department of Cardiology, Athens, Greece

Background: Arrhythmiogenic right ventricular cardiomyopathy (ARVC) is a disorder with increased risk for arrhythmia including sudden cardiac death. ARVC is usually inherited following an autosomal dominant pattern, with variable penetrance and expressivity. In rare cases, mutations in JUP may lead to a rare autosomal recessive condition in the spectrum, namely Naxos disease, presenting additionally with woolly hair and palmoplantar keratoderma. We present a case of Naxos disease diagnosed at infancy.

Methods: A 13.8 m.o. male was referred to our lab for ARVC investigation, following clinical evaluation and no cardiological signs. Woolly hair, patches of rough/dry skin at knees/palms just after the initiation of crawling and both healthy, non-consanguineous parents originating from the island of Naxos, Greece, set the clinical suspicion. Exome sequencing (ES) was applied on DNA extracted from peripheral blood: library preparation was performed with Clinical Exome Solution (Sophia Genetics) and sequenced on Illumina NextSeq-550 (Illumina). Bioinformatics analysis was conducted by SOPHiA DDM® bioinformatics pipelines.

Results: Naxos disease has a prevalence of 1:1000 in the population of the Greek islands and was suspected based on phenotypic traits and origin of the toddler. A virtual panel of 51 genes related to arrhythmiogenic syndromes was analyzed. Tertiary analysis revealed a homozygous pathogenic JUP variant; c.2038_2039del or p.(Trp680Glyfs*11), classic for Naxos disease.

Conclusion: ES permitted early genetic diagnosis of Naxos disease. Although principles of evaluation and treatment are based on ARVC and general heart failure guidelines, early diagnosis is essential for patient management, timely interventions and relative cascade screening for risk stratification.

Conflict of Interest: Georgia Christopoulou Full-time employment at Genotypos M.S.A., Full-time employment at Genotypos M.S.A., Stavros Bournazos Full-time employment at Genotypos M.S.A., Aikaterini Oikonomaki Full-time employment at Genotypos M.S.A., Stavroula Samara Full-time employment at Genotypos M.S.A., Antigoni Miliou: None declared, Charalambos Vlachopoulos: None declared, Pantelis Constantoulakis Full-time employment at Genotypos M.S.A.

EP06.018 Correlations of BHMT (rs3733890) and PAI-I (rs1799889) gene variants with blood parameters in pediatric patients with hereditary thrombophilia

Natalia Gorovenko 1, Liliia Fishchuk1, Kateryna Vilchevska2, Iryna Bakhchyvandzhy2, Ianovska Viktoriia2, Andrii Zakusylo2, Inna Parkhomenko2, Krystyna Yeroshkina3, Larysa Sheiko1, Zoia Rossokha3

1State Institute of Genetic and Regenerative Medicine National Academy of Medical Sciences of Ukraine, Department of Genetic Diagnostic, Kyiv, Ukraine; 2National Specialized Children’s Hospital OKHMATDYT under the Ministry of Health of Ukraine, Kyiv, Ukraine; 3State Institution “Reference-Centre for Molecular Diagnostic of Public Health Ministry of Ukraine”, Kyiv, Ukraine

Background/Objectives: Clinical presentation of hereditary thrombophilia in childhood leads to a deterioration in quality of life, and sometimes to disability, recurrence and death. The aim of the study was to analyze a correlation between genes variants and hemostasis parameters to find new targets in prevention and treatment.

Methods: The study involved 116 children (mean age 9.44 ± 5.01 years) with a clinical presentation of hereditary thrombophilia (thrombosis, stroke, etc.). The investigations of PAI-1 (rs1799889), FV (rs6025), FII (rs1799963), ITGA2 (rs1126643), ITGB3b (rs5918), FGB (rs1800787, rs1800790), MTHFR (rs1801133, rs1801131), MTRR (rs1801394), MTR1 (rs1805087), RFC1 (rs1051266), BHMT (rs3733890) genes variants were performed using PCR and PCR-RFLP. All children patients underwent standard hemostasis examination. Parents of the children gave informed consent. There were used nonparametric Spearman test (SPSS v.27).

Results: Power significant correlations were found between BHMT gene variant and D-dimer level, as well as variant of the PAI-I gene and the activities of plasminogen and plasminogen inhibitor. D-dimer levels were significantly lower in patients with the 742GG genotype of the BHMT gene. Patients with the 4G/4G genotype had the highest levels of plasminogen and plasminogen inhibitor activities.

Conclusion: The identified correlation of BHMT gene variants with D-dimer levels may be the basis for the development of a new strategy for the prevention and treatment of hereditary thrombophilia through betaine consumption. At the same time, analysis of PAI-I gene variants will be important to assess the risk of thrombotic events and compensatory capacity in patients.

References: No

Grants: No

Conflict of Interest: None declared.

EP06.019 Identification of new risk loci for ischemic stroke in genome-wide data with clustering methods

Nikita Sapozhnikov 1, Gennady Khvorykh1, Andrey Khrunin1

1Institute of Molecular Genetics of National Research Center “Kurchatov Institute”, Moscow, Russian Federation

Background/Objectives: Stroke is the second cause of death worldwide. Despite a few dozens of genomic loci found to be associated with the ischemic stroke (IS), the genetic bases of the disease remain underexplored. We applied clustering methods to genome-wide data of 5580 individuals with IS and controls to search for genetic loci (groups of single nucleotide polymorphisms, SNPs) associated with the risk of IS.

Methods: The genotypes of 883908 SNPs were transformed into autosome based linkage disequilibrium matrices and clustered with DBSCAN and HDBSCAN algorithms. Haplotypes were inferred for each cluster and tested for association with IS using Plink, p-values were adjusted by the total number of clusters revealed. The sets of SNPs associated significantly with IS were annotated. Genes obtained were tested for overrepresentation in sets of human genes given in MSigDB.

Results: There were identified 97 and 122 candidate genes by DBSCAN and HDBSCAN, respectively, and 88 of which were common. Sixteen genes of protocadherin gamma gene cluster were found to be overrepresented in cell adhesion.

Conclusion: To date little is known about the role of protocadherin genes in IS but there are several studies showing their involvement into neurodevelopmental disorders. Our research showed for the first time the association of several protocadherin genes of gamma subfamily with IS. This suggests protocadherins can be a common part of base mechanisms underlying pathological processes in nervous system.

Grant: The study was funded by Russian Foundation for Basic Research (grant No 19-29-01151).

Conflict of Interest: None declared.

EP06.020 Establishment of Sidra Cardiac Registry in Qatar and the dissection of genetic causes in patients with congenital heart disease

sara okashah1, Dhanya Vasudeva2, Aya El Jerbi3, Houssein Khodjet Elkhil1, Mashael Al-Shafai1, Asma Jamil2, Mona El Chawli2, Najeeb Syed2, Marios Kambouris4, Sharda Udassi5;6, Luís R. Saraiva2;7;8, Hesham Al-Saloos3, Jai Udassi3;9, Kholoud Al-Shafai 2

1Qatar University, Department of Biomedical Sciences, doha, Qatar; 2Sidra Medicine, Research, doha, Qatar; 3Sidra Medicine, Heart Center Department, doha, Qatar; 4Sidra Medicine, Pathology & Laboratory Medicine Department, doha, Qatar; 5Sidra Medicine, Division of General Academic Pediatrics, doha, Qatar; 6WVU Medicine, Pediatric Hospital Medicine Division, doha, Qatar; 7Hamad Bin Khalifa University, College of Health and Life Sciences, doha, Qatar; 8Monell Chemical Senses Center, Philadelphia, United States; 9WVU School of Medicine, Children’s Heart Center, Virginia, United States

Background: Congenital heart disease (CHD) is one of the most common birth defects worldwide, with a prevalence of 1–1.2% in liveborn infants. Many factors are involved in the causality of CHD including genetic defects like chromosomal abnormalities and single gene mutations.

Methods: In this study, we establish the Sidra Cardiac Registry at Sidra Medicine in Qatar, which includes so far 92 patients with cardiac problems, including CHD, cardiomyopathy and channelopathy, in addition to their relatives. We investigated the genetic causes of 52 registered patients with CHD and reviewed the results of the genetic testing conducted in those patients as part of their clinical evaluation, including chromosomal testing. Furthermore, we performed whole exome sequencing (WES) analysis to identify potential causative variants.

Results: Sixteen CHD patients had chromosomal aberrations, that explained their complex disease phenotype, including six patients with trisomy 21. Moreover, from the WES analysis, we describe 65 potential variants in 56 genes identified in 24 CHD patients. Four out of the 65 variants were pathogenic/likely pathogenic based on the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP) classification; these variants were detected in four patients.

Conclusion: Our study presented potential variants that could contribute to the development of CHD in our patients. Thus, further functional assessments are needed to better understand the role of the identified variants in relation to CHD pathogenesis.

Grant Reference: Sidra Internal Research Fund (SDR200038).

Conflict of Interest: None declared.

EP06.022 Complex congenital heart disease in the 8q21.11 deletion

Rebekah Jobling 1;2;3, Qiliang Ding1, Kelsey Kalbfleisch1;2, Raymond Kim1;2;4, David Chitayat2;5

1The Hospital for Sick Children, Cardiac Genome Clinic, Ted Rogers Centre for Heart Research, Toronto, Canada; 2The Hospital for Sick Children, Division of Clinical and Metabolic Genetics, Toronto, Canada; 3The Hospital for Sick Children, Genome Diagnostics, Department of Paediatric Laboratory Medicine, Toronto, Canada; 4University Health Network, Fred A. Litwin Family Centre in Genetic Medicine, University Health Network, Department of Medicine, Toronto, Canada; 5Mount Sinai Hospital, The Prenatal Diagnosis and Medical Genetics Program, Department of Obstetrics and Gynecology, Toronto, Canada

Congenital heart diseases (CHD) are the most common human birth defects with an incidence of 0.8-1% livebirths and are a major contributor to morbidity and mortality. Most cases are multifactorial; however, known single gene disorders associated with CHD is increasing gradually with the use of next generation sequencing technology. Pathogenic mutations in over 70 genes have been linked to isolated and syndromic CHD, with most mutations causing a loss-of-function. Identification of CHD-causative genes is significant in preimplantation, pre- and postnatal genetic diagnosis, early prophylaxis and personalized treatment of CHD.

A critical region for 8q21.11 deletion was recently defined, including the gene HEY1. Patients with deletions of this critical region frequently have CHD, typically septal defects. However, complex CHD has not been reported. We report a 2-year-old patient with hypoplastic right heart, hyploplastic tricuspid valve, pulmonary atresia, total anomalous pulmonary venous return and ventricular septal defect as well as hypotonia, developmental delay, short palpebral fissures, and low set ears. Microarray analysis revealed 4.34 Mb deletion at GRCh37/hg19 8q21.11q21.13 (chr8:78001401-82340000) overlapping the critical region and encompassing HEY1. Whole genome sequencing revealed no other candidate variants related to the phenotype.

HEY1 is involved in the embryonic development of the heart, central nervous system, and vascular system and is part of the NOTCH1-HEY1 pathway. Hrt1/Hey1null mice show perinatal lethality due to congenital malformations of the aortic arch and its branch arteries. This case expands the cardiac phenotype of 8q21.11 deletion syndrome and the cardiac defects seen in association with haploinsufficiency of HEY1.

Conflict of Interest: None declared.

EP06.023 Reporting a new ACVRL1 gene variant involved in Rendu-Osler-Weber syndrome

JOEL MARTIN PADRON1, Alba María Casado Prieto2, Manuela Moreno Higueras3, Susana García-Linares 1

1San Cecilio University Hospital, Clinical Analysis Unit, Granada, Spain; 2San Cecilio University Hospital, Hematology Unit, Granada, Spain; 3San Cecilio University Hospital, Internal Medicine Unit, Granada, Spain

Background/Objectives: Rendu-Osler-Weber syndrome, also known as Hereditary Hemorrhagic Telangiectasia (HHT), is a rare autosomal dominant disorder characterized by arteriovenous malformations due to defects in angiogenesis. The main clinical manifestation is bleeding that can occur in any location, the most frequent being recurrent and spontaneous epistaxis. We performed a genetic study in a family with several members clinically diagnosed with HHT.

Methods: 13 whole blood samples were obtained from members of the three youngest generations in order to perform a targeting exome of the main five genes associated with HHT (ACVRL1, BMPR2, ENG, GDF2, SMAD4). Variants annotation criteria were: changes with a number of readings >20x and a frequency >30%. Variants were double-checked by Sanger sequencing, compared to the reference sequence (HGMD database), and faced to different databases and in silico prediction tools.

Results: Five members were found to be heterozygous for the variant c.752_772+2dup in the ACVRL1 gene, which is a 23 nucleotide duplication in intron 6 splice donor site. Interestingly, it is the first time this variant has been reported and 3/5 of the tested patients have presented with classical symptoms (mainly epistaxis and cutaneous telangiectasia).

Conclusion: Although the variant c.752_772+2dup in the ACVRL1 gene has not been previously described, its location in the splice donor site and the presence of clinical manifestations in a high number of probands suggest a possible pathogenic effect on ARNm processing. In agreement, a similar intronic variant (c.772+3_772+4dupAA) has been previously reported as pathogenic.

Grant References: not applicable.

Conflict of Interest: None declared.

EP06.024 Sequence analysis unveils a “hot-spot” for microhomology-mediated CNVs in FLNC linked to arrhythmogenic/dilated cardiomyopathy

Laura Cazón 1, Emilia Maneiro1, Luis De la Higuera Romero1, Marlene Perez Barbeito1, Rosalía Peteiro1, Iria Gómez Díaz1, Paula Velez1, Maria Sanchez1, Anahi Sanluis Verdes1, Guillermo Smith Ramos1, Xusto Fernandez1, Diego Cabrera Argaña1, Almudena Amor1, María Valverde1, Soledad García Hernández1;2, Ivonne Cárdenas Reyes1, Martin Ortiz Genga1, Juan Pablo Ochoa1

1Health in Code, A Coruña, Spain; 2Hospital Universitario Clínico San Cecilio, Granada, Spain

Background/Objectives: Loss-of-function variants in the FLNC gene have been associated with a specific overlapping phenotype of arrhythmogenic/dilated cardiomyopathy. To date, only splicing, frameshift, or nonsense variants have been reported. In this study, we sought to identify the exact breakpoint of three different copy number variations (CNVs) detected in FLNC in patients with this phenotype, as well as to review the sequence near the breakpoints to ascertain the putative mechanism underlying their formation.

Methods: Patients’ genomic DNA was genotyped using next-generation sequencing (NGS) with a panel of genes that included FLNC. The CNVs were detected using a read-depth approach, and Sanger sequencing was used to confirm the breakpoints.

Results: In a cohort of 6,750 patients diagnosed with dilated and/or arrhythmogenic cardiomyopathy sequenced by NGS, we identified three different CNVs affecting FLNC. The breakpoints were characterized at the sequence level. The 5’ ends of all three CNVs were found in intron 2 of the gene. According to an analysis of the sequence close to the breakpoints, the CNVs were likely generated by breakpoint microhomology.

Conclusion: Breakpoint sequencing of three different CNVs involving the FLNC gene revealed the presence of microhomology at nearby sites that promote DNA breaks, highlighting the importance of intron 2 of the FLNC gene as a possible hot spot for CNVs.

Conflict of Interest: Laura Cazón Full time, Emilia Maneiro Full time, Luis De la Higuera Romero Full time, Marlene Perez Barbeito Full time, Rosalía Peteiro Full time, Iria Gómez Díaz Full time, Paula Velez Full time, Maria Sanchez Full time, Anahi Sanluis Verdes Full time, Guillermo Smith Ramos Full time, Xusto Fernandez Full time, Diego Cabrera Argaña Full time, Almudena Amor Full time, María Valverde Full time, Soledad García Hernández Full time, Ivonne Cárdenas Reyes Full time, Martin Ortiz Genga Part-time, Juan Pablo Ochoa Full time.

EP06.025 Effect of MYBPC3 c.913_914del variant: a cohort study

Nina Vodnjov 1;2, Janez Toplišek3, Aleš Maver1;4, Nataša Teran1, Borut Peterlin1, Karin Writzl1;4

1University Medical Centre Ljubljana, Clinical Institute of genomic medicine, Ljubljana, Slovenia; 2University of Ljubljana, Biotechnical Faculty, Ljubljana, Slovenia; 3University Medical Centre Ljubljana, Department of Cardiology, Ljubljana, Slovenia; 4University of Ljubljana, Faculty of Medicine, Ljubljana, Slovenia

Objective: Hypertrophic cardiomyopathy, the most common genetic heart disease, is frequently caused by the c.913_914del pathogenic variant in sarcomeric gene MYBPC3. This variant was previously reported as highly penetrant and causative for high rate of adverse cardiac events, including sudden cardiac death (SCD), aborted SCD (aSCD), and heart failure deaths. The aim of present study was to determine the impact of the variant on clinical presentation and prognosis in our cohort.

Methods: Our database of genetic testing results was screened for probands with (likely) pathogenic variants in MYBPC3 and their relatives. Clinical characteristics, rate of adverse events and penetrance were collected and compared between individuals with c.913_914del and those with other (likely) pathogenic variants in MYBPC3.

Results: We identified 30 (19 probands/11 relatives) aged 42 ± 16 years having c.913_914del (NM_000256.3) and 48 (31 probands/17 relatives) aged 33 ± 21 years with other pathogenic variants in MYBPC3. No significant difference in clinical characteristics were observed between probands with c.913_914del and other MYBPC3 variants (left ventricular hypertrophy: 21 ± 11mm vs 18 ± 7mm; p = 0,33). The penetrance for c.913_914del was 73,3% and 72,9% for other MYBPC3 variants. During follow up (9 ± 8 years), none of probands in either group died of SCD or heart failure. One patient in each group had heart transplantation or experienced aSCD (1/19 vs 1/31; p = 0,72). The prevalence of non-sustained ventricular tachycardia was similar in both groups (3/19 vs 5/31; p = 0,97).

Conclusion: MYBPC3 c.913_914del does not lead to significantly increased rate of SCD, different clinical presentation and penetrance in comparison with other pathogenic MYBPC3 variants.

Conflict of Interest: None declared.

EP06.027 Frequency of the most common mutations in the LDLR gene in patients with familial hypercholesterolemia from RN Macedonia

Sanja Kiprijanovska 1, Enes Shehu2, Hristina Dichevska1, Marija Vavlukis2, Sasko Kedev2, Aleksandar Dimovski1;3

1Macedonian Academy of Sciences and Arts, Research Center for Genetic Engineering and Biotechnology, Skopje, Macedonia; 2Faculty of Medicine, University “Ss. Cyril and Methodius” in Skopje, University Clinic for Cardiology, Skopje, Macedonia; 3Faculty of Pharmacy, University SS Cyril and Methodius in Skopje, Center for Biomolecular Pharmaceutical Analyses, Skopje, Macedonia

Background: Familial hypercholesterolemia (FH) is a highly prevalent disorder and a risk factor for early coronary artery disease, caused by inherited changes (mutations) in the LDLR, APOB, and PCSK9 genes, which affect processes of regulation and elimination of cholesterol from the blood. Literature data have implicated the involvement of defects in the LDLR gene as the most common cause of this condition.

Methods: We evaluated the frequency of the most common mutations in the LDLR gene from 110 patients (72 males, 38 females) with clinically defined characteristics of FH. The methodology included TaqMan genotyping analysis for four variants in the LDLR gene (c.81C>G p.(Cys27Trp), c.858C>A p.(Ser286Arg), c.1646G>A p.(Gly549Asp) and c.1285G>A p.(Val429Met).

Results: A total of 25.3 % of the patients had a mutation in the LDLR gene. The most common mutation was c.81C>G detected in 18 (16.3%), followed by c.858C>A in 7 (6.3%) and c.1646G>A in 3 (2.7%) patients. Three patients had two mutations (one homozygote for c.81C>G, and 2 double heterozygotes for c.81C>G c.858C>A and c.81C>G/c.1646G>A, respectively). No significant difference was found in the distribution of these defects between men and women, whereas a higher frequency of defects was detected in patients >50 (73.1%), compared to patients <50 years of age (26.9%) (p < 0.05).

Conclusion: We identified distribution of molecular defects for FH similar to the neighboring countries. The identification of the most common mutations in the LDLR gene will improve the genetic diagnosis of FH in our population.

Conflict of Interest: None declared.

EP06.028 Association between hypertension and coffee drinking based on CYP1A2 rs762551 single nucleotide polymorphism in a Romanian population

Laura Popa 1, Maria Puiu1;2, Simona Farcas1, Cristian Zimbru1, Nicoleta Andreescu1;2

1Victor Babes University of Medicine and Pharmacy, Medical Genetics, Timișoara, Romania; 2Emergency Hospital for Children Louis Turcanu, Clinical Genetics, Timișoara, Romania

Background: Hypertension is of importance mainly as a risk factor for heart, brain and kidney disease. To date, efforts have been made to reduce and treat high blood pressure levels. Cytochrome P450 1A2 (CYP1A2) is known to be the main enzyme, directly responsable for caffeine metabolism. Rs762551, is a SNP encoding the CYP1A2*1F allele of the CYP1A2 gene, and it known mostly for metabolizing caffeine and its protective role against developing high blood pressure. We aimed to determine if habitual coffee intake has a protective role on hypertension onset, related to CYP1A2 polymorphism.

Methods: For this study 358 hypertensive patients have been considered. PCR technique, analyzing rs762551 (assay C_8881221) on LightCycler 480 (Roche) with Gene Scanning software version 1.5.1 (Roche) has been proceeded.

Results: Absence of statistical association could be observed between the following pairs: gender –hypertension, coffee intake – hypertension and genotype – coffee intake. In addition, a lack of a significant association between the genotype and the presence of hypertension has been observed (p = 0.530). A statistically significant difference between the three genotypes regarding the average amount of coffee has been identified (F = 0.38; p = 0.684). Bonferroni test proved that there are differences between the three genotypes, but they are not statistically significant.

Conclusions: Individuals with the CYP1A2 rs762551 CC genotype proved to consume lower quantities of coffee while the AA genotype tend to consume the highest quantities. No association between coffee intake and high blood pressure have been identified, regardless the genotype.

Conflict of Interest: None declared.

EP06.029 Disconcordant genotype-phenotype in a family with arrhythmogenic right ventricular cardiomyopathy

Marius Šukys 1, Eglė Ereminienė2;3, Inga Nasvytienė1, Kristina Aleknavičienė1, Rimvydas Jonikas1, Darius Čereškevičius1, Sandra Žėkienė1, Rasa Traberg1, Rasa Ugenskiene1

1Lithuanian University Of Health Sciences, Department of Genetics And Molecular Medicine, Kaunas, Lithuania; 2Lithuanian University Of Health Sciences, Institute of Cardiology, Kaunas, Lithuania; 3Lithuanian University Of Health Sciences, Department of Cardiology, Kaunas, Lithuania

Background: PKP2 gene pathogenic variants are the most common cause of arrhythmogenic right ventricular cardiomyopathy (ARVC). ARVC has variable expressivity and penetrance which might complicate managing patients, especially in cases not fulfilling all ARVC criteria.

Methods: We report a family case with PKP2 gene variant and ARVC

Results: 17 yo patient was referred to cardiologist with suspicion of cardiomyopathy because of brother’s sudden cardiac death at 20 yo and uncle (mother side) at 19 yo. Episodes of ventricular arrhythmia were noticed, heart MRI showed 4 mm atrial septal defect with shunt to right, dilation of right ventricle. Cardioverter defibrillator was implanted. Hereditary cardiac disease next generation sequencing gene panel was performed – PKP2 gene 2-13 exon deletion was found which was confirmed with MLPA. Variant was interpreted as likely pathogenic as truncating variants are known to cause ARVC and no reports in databases of such variant. The same variant was found for patient’s sister (no cardiac pathology) and other brother (ongoing cardiac investigation), mother (changes in cardiac MRI – hypokinetic right ventricular segments, lower RV ejection fraction and ventricular arrhythmias), mother’s sister (no cardiac pathology). Cousin of the patient from mother side had episodes of chest pain, reduced RV ejection fraction and slight apical dyssynchrony in cardiac MRI, but PKP2 gene variant was not found.

Conclusion: A patient with phenotype suggesting an AVRC and no PKP2 gene familial variant might have additional genetic cause for the disease. This complicates family screening as genotype PKP2-negative family members could develop disease and vice versa.

Conflict of Interest: None declared.

EP06.030 Analysis of the distribution of the ANRIL gene rs1333049 polymorphism in patients with acute coronary syndrome in the Ukrainian population

Polina Kniazkova 1, Viktoriia Harbuzova1, Olha Obukhova1

1Sumy State University, Sumy, Ukraine

Background/Objectives: To analyze the distribution of the ANRIL gene rs1333049-polymorphism (CDKN2B-AS1) in patients with acute coronary syndrome.

Methods: Venous blood of 195 patients with ACS and 234 without heart pathology was used for the study. DNA was isolated from whole venous blood. ANRIL gene polymorphism rs1333049 was studied by Real-time PCR.

Results: The distribution of GG, GC and CC genotypes in patients with coronary artery disease was 23.6%, 46.7%, 29.7%, respectively. In the control group, distribution of genotypes was 29.5%, 50.0%, 20.5%, respectively. Comparison of genotype frequencies did not reveal the existence of a reliable difference in their distribution (P = 0.071; X2 = 5.292). The distribution of C-allele and G-allele in patients with ACS was 46.9% and 53.1%, and in the control group – 54.5% and 45.5%, respectively. The significant difference in alleles distribution between ACS patients and the control group was found (P = 0.027; X2 = 4.871). Genotypic analysis in the recessive model has shown that CC-genotype carriers had more risk of coronary artery disease development compared to patients with GC-genotype and GG-genotype almost twice as high (P = 0.028, OR = 1.641, 95% CI = 1.055–2.551).

Conclusion: There is statistically significant link between ANRIL rs1333049 polymorphism and coronary artery disease development in Ukrainian population. Carriers with CC-genotype have more risk of coronary artery disease development compared to patients with major G-allele (GG and GC-genotypes).

Grant References: The present study is the part of the project “The study of the role of genetic factors in the pathogenesis of multifactorial diseases”, supported by Ministry of Education and Science of Ukraine (no.0120U102166).

Conflict of Interest: None declared.

EP06.031 the power of collaboration: institutional partnership initiatives for data sharing leads to tnxb phenotype expansion

ARPITA NEOGI 1, meghan towne2, daniel j dykas1, arya mani1

1Yale University, New Haven, United States; 2Ambry Genetics, Aliso Viejo, United States

Background/Objectives: Biallelic alterations in TNXB are associated with Ehlers-Danlos, classic type; however, vascular aneurysms and dissections have not previously been associated.

Methods: Clinical exome sequencing at our academic medical center lab identified two TNXB variants in a patient with aortic aneurysm and vertebral artery dissections. A retrospective review of additional research exome sequencing found 5/350 cases with two or more TNXB variants in patients with vascular aneurysms or dissections. To evaluate the strength of this association, replication analysis on a cohort of 1844 individuals who underwent clinical testing for suspicion of vascular aneurysm and dissection syndromes was carried out using data from a diagnostic laboratory.

Results: Only 3/22 diagnostic laboratories identified offered genetic testing for aneurysms and dissections that included TNXB in their analysis due to its association with connective tissue disease. After incorporating data from one laboratory, there was a statistically significant difference (p < 0.01) in the presence of two or more TNXB variants between cases and controls, suggesting an expansion of phenotype associated with TNXB.

Conclusion: This project reinforces the value of transparent data sharing, within the confines of privacy regulations, between researchers, clinicians, and laboratories in the identification of candidate genes. It also illuminates the utility of broad panel testing and the lack of standardization of gene panel content between laboratories. The variability of panel content has implications for test selection, as clinicians sort through seemingly similar test offerings to identify the best one for patients.

Conflict of Interest: ARPITA NEOGI Yale University, meghan towne Ambry Genetics, daniel j dykas Yale, arya mani Yale, NIH grant.

EP06.032 Genetic background of Czech patients with idiopathic ventricular fibrillation

Jana Zídková 1, Iva Synková1, Tereza Kramářová1, Samuel Lietava2, Tomáš Novotný2, Milan Sepši2, Lenka Fajkusova1

1University Hospital Brno, Centre of Molecular Biology and Genetics, Brno, Czech Republic; 2University Hospital Brno, Department of Internal Medicine and Cardiology, Brno, Czech Republic

The sudden cardiac death (SCD) can be a fatal outcome of several cardiac diseases. SCD is generally caused by ventricular arrhythmias, especially ventricular fibrillation (VF). SCD survivors undergo extensive clinical testing to reveal the underlying cause of VF. Nevertheless, in some cases, neither any signs of the structural heart disease are found, nor diagnostic criteria for any electrical disease of the heart are present. The diagnosis of idiopathic ventricular fibrillation (IVF) is made.

We analyzed a group of 26 patients with SCD aborted by cardiopulmonary resuscitation; structural heart disease was excluded and no clinical signs of primary electrical diseases of the heart were present. We use panel sequencing with 130 genes associated with hereditary cardiomyopathies and arrhythmogenic syndromes for variant detection and CNV analysis.

We present genetic findings in 10 patients with idiopathic ventricular fibrillation. Four variants are classified as pathogenic/likely pathogenic (in RYR2, KCNH2, FLNC and TTN), the rest as of uncertain significance (in CACNB2, DSP, PKP2, RYR2, SCN4B). Majority of these genes has been related to arrhythmogenic syndromes.

Although the patients with IVF do not have a clinical presentation of any familial electrical heart disease, they may have rare genetic variants that may predispose them to malignant life-threatening arrhythmias in certain stress situations. Knowledge of presence of these rare variants may contribute to the understanding of the pathogenesis of SCD. The precise mechanism requires further study. Functional analysis of selected variants is undergoing.

Supported by the Ministry of Health of the Czech Republic ̶ grant project NU22-02-00348.

Conflict of Interest: None declared.

EP06.033 Haplotype R157-V378-T681 in TRPV6 possible risk factor for Ventricular Fibrillation in Acute Myocardial Infarction

Ricardo Pan-Lizcano 1, Luis Mariñas Pardo2, Juan Fernandez-Tajes3, Domingo López-Vázquez4, Fernando Rebollal4, Xacobe Flores4;5, Ramón Calvino-Santos4;5, Lucía Núñez Fernández1;6, José Manuel Vázquez Rodríguez4;5, Manuel Hermida-Prieto1

1Instituto de Investigación Biomédica de A Coruña (INIBIC), GRINCAR-Universidade da Coruña (UDC), Cardiology Research Group, A Coruña, Spain; 2Universidad Internacional de Valencia (VIU), Facultad de Ciencias de la Salud, Valencia, Spain; 3Instituto de Investigación Biomédica de A Coruña (INIBIC),), Complexo Hospitalario Universitario de A Coruña (CHUAC), Grupo de Investigación de Reumatología (GIR)., A Coruña, Spain; 4Complexo Hospitalario Universitario de A Coruña (CHUAC), Sergas, Instituto de Investigación Biomédica de A Coruña (INIBIC), Universidade da Coruña(UDC), Department of Cardiology, A Coruña, Spain; 5Instituto de Salud Carlos III, CIBERCV (Centro de Investigación Biomédica en Red Enfermedades Cardiovasculares), Madrid, Spain; 6Universidade da Coruña, Departamento de Ciencias de la Salud. GRINCAR Research Group., A Coruña, Spain

Background/Objectives: Ventricular fibrillation (VF) during acute myocardial infarction (AMI) has been proposed as the main cause of sudden cardiac death. The genetic basis underlying the development of this pathology are still unclear, being many possible related clusters of genes not explored yet. Transient receptor potential (TRP) channels are superfamily of 27 genes coding to cation-permeable ion channels, deeply related to the homeostasis of Ca+. Ion homeostasis is fundamental for the proper function of the cardiovascular system, making this set of genes an interesting target.

Methods: By NGS technologies, the genetic variants present in the 27 TRP genes of 78 patients, 39 with VF during AMI vs 39 without VF during AMI, were analyzed. Depending on their frequency reported in the genomic databases, variants were classified as common or rare. Association of common variants was tested using R package: SNPassoc. Statistical analysis was performed using GraphPad Prism.

Results: A total of 174 common variants were found in the 27 genes analyzed. The haplotype R157-V378-T681 in the TRPV6 gene was identified in 13 VF patients whereas it was not found in any control patients (P.value 0.00007).

Conclusions: The presence of R157-V378-T681 haplotype in the TRPV6 is statistically significant in the VF patients, this correlation has never been reported before. Ca+ homeostasis is essential for the excitation and contraction coupling in cardiomyocytes and the inefficient function of channels could represent a risk factor for the development of VF.

Grants: Instituto de Salud Carlos III (PI18/01737)-FEDER funds and a nonconditional grant from Abbott Vascular.

Conflict of Interest: None declared.

EP06.034 Somatic mosaicism in myocardial samples of patients with severe hypertrophic cardiomyopathy

Maria Balashova 1;2, Mariam Sadekova1;3, Sergey Dzemeshkevich3, Elena Zaklyazminskaya1;3

1PJSC Center of Genetics and Reproductive Medicine “Genetico”, Moscow, Russian Federation; 2I.M. Sechenov First Moscow State Medical University (Sechenov University), Moscow, Russian Federation; 3B.V. Petrovsky Russian Research Centre of Surgery (RRCS), Moscow, Russian Federation

Background/Objectives: Hypertrophic cardiomyopathy (HCM) is a mainly dominant disorder caused by germline mutations in the >40 genes. Apparent phenotypic anticipation is observed in several HCM families but remains unexplained. We hypothesize that myocardial somatic mosaicism might be a factor driving that phenomenon.

Methods: Ten young probands (15-30 y.o.) were selected who underwent septal myectomy and in whom parents had from no to moderate HCM. For 5 of them, we performed WES of DNA extracted from peripheral blood leukocytes (100x coverage) and myocardial samples (200x coverage) taken intra-operatively. Comparison was performed using a domestic bioinformatics pipeline.

Results: On average, we found 185465 variants on the leukocyte WES and 192427 variants on the myocardial WES. Causative variants of IV-V classes of pathogenicity in target genes (MYBPC3, MYH7, ALPK3) were detected in 3 out of 5 probands. All causal variants were confirmed in both type of tissues. In 4 of 5 myocardial samples, LOF variants in the CTBP2 gene were found. In all cases, more variants, as well as a tendency to increase with age, were identified in the myocardium. No new somatic pathogenic variants were found in myocardium that could explain phenotype anticipation in probands.

Conclusion: There is a tendency to an age-dependent increase in the number of somatic mutations. We were unable to detect new pathogenic variants in the genes of sarcomeric proteins. We plan to increase the number of examined patients in the near future.

Grant References: The research was funded by the Russian Science Foundation (project №22-75-00134, https://rscf.ru/project/22-75-00134/)

Conflict of Interest: Maria Balashova Centre of Genetics and Reproductive Medicine GENETICO.

Sechenov University, Principal investigator, Mariam Sadekova B.V. Petrovsky Russian Research Centre of Surgery (RRCS)

Centre of Genetics and Reproductive Medicine “GENETICO”, collaborator, Sergey Dzemeshkevich B.V. Petrovsky Russian Research Centre of Surgery (RRCS), Elena Zaklyazminskaya B.V. Petrovsky Russian Research Centre of Surgery (RRCS)

Centre of Genetics and Reproductive Medicine “GENETICO”

EP06.035 Child with dilated cardiomyopathy and a novel homozygous splice site variant in FLNC gene

Afaf Alsubhi 1, Manar Aldarwish1, Mohammed Almannai1

1King Abdulaziz Medical City, Genetics & Precision Medicine, RIYADH, Saudi Arabia

FLNC gene encodes for Filamin-C (FLNC) protein, a sacromeric protein with important structural and signaling functions in the myocyte. Mutations in FLNC gene were linked initially to myofibrillar myopathy. In 2014, dominant mutations in this gene were linked to familial hypertrophic cardiomyopathy and over time, evidence showed association of this gene with other forms of cardiomyopathy including dilated and restrictive forms.

Recently, a single case report of a child with congenital onset of myopathy with no cardiac involvement associated with a homozygous missense variant in FLNC c.1325C>G (p.Pro442Arg) was reported and the early onset suggest that homozygosity is associated with more severe disease, a phenomena observed with some other dominant disorders.

In this report, we described a boy who presented at the age of 10 years with shortness of breath and found to have dilated cardiomyopathy (DCM) with ejection fraction of less than 20%.

He doesn’t have myopathy and family history is unremarkable apart from consanguinity. Whole exome sequencing showed a novel homozygous splice site variant (c.2122-1G > C) in FLNC. The patient underwent cardiac transplantation recently. Sanger confirmation in parents as well as their cardiac evaluation is pending. To our knowledge, this is the second case in literature with biallelic variants in FLNC. While the previous case presented with isolated myopathy, our case presented with isolated DCM. This case suggested that the phenotype associated with variants in FLNC is very heterogenous and can be inherited in dominant or recessive forms, with later being more severe and of earlier onset.

Conflict of Interest: Afaf Alsubhi Full time, Manar Aldarwish Full time, Mohammed Almannai Full time.

EP06.036 Evaluation of the variant spectrum of patients diagnosed with hereditary cardiomyopathy

Gulay Gulec Ceylan1;2, Büşranur Çavdarlı1, Esra Habiloglu1, Cavidan Nur Semerci Gündüz 1;2

1Ankara Bilkent City Hospital, Medical Genetics, Ankara, Turkey; 2Ankara Yıldırım Beyazıt University, Medical Genetics, Ankara, Turkey

Background/Objectives: Hereditary cardiomyopathies can be defined as structural and functional disorders of heart muscle with a wide range of clinical and genetic heterogeneity. They are classified into 5 main groups as hypertrophic cardiomyopathy(HCM), dilated cardiomyopathy(DCMP), restrictive cardiomyopathy(RCMP), arrhythmogenic right ventricular cardiomyopathy(ARVD) and left ventricular noncompaction cardiomyopathy(NCCMP). The aim of our study is to define the responsible genetic etiology and explain its relationship with patients’ phenotype for non-syndromic cardiomyopathy.

Methods: Patients referred to Medical Genetics Outpatient Clinic with a preliminary diagnosis of cardiomyopathy between 01.03.2019-31.07.2022 were evaluated with anamnesis,physical examination,pedigree analysis and cardiac examination results. A panel covering MYBPC3,TNNT2,TPM1,MYH6,MYH7,TNNI3 and MYL2 genes was applied to 144 patients who were thought to be clinically non-syndromic with next generation sequencing (NGS) method.

Results: Pathogenic/likely pathogenic variants explaining the clinic was detected at 35/144 patients and the diagnosis rate was determined as 26% in our study. It was found to be 25%(23/90) in HCMP, 20%(7/34) in DCMP, 50%(2/4) in RCMP and 18%(3/16) in NCCMP. The most common pathogenic/likely pathogenic variants were detected in MYBPC3 and MYH7 genes. Variants of unknown clinical significance were detected in a total of 17 patients, and segregation analyzes are ongoing for the validation of variants.

Conclusion: Molecular diagnosis of cardiomyopathies is essential for arranging treatment options and genetic counseling. In this study, genetic etiology related to cardiac pathology was shown at 35/144 patients. Detection of molecular etiology in this disease group is very important in terms of treatment strategy for patients, prediction of prognosis and early diagnosis of asymptomatic family members.

Conflict of Interest: None declared.

EP06.037 The prevalence of mutations in ion channels genes in an unselected cohort of young sudden unexplained death cases

Ainur Akilzhanova1, Madina Zhalbinova1, Ayaulym Chamoieva1, Zhanel Mirmanova1, Diana Samatkyzy1, Aidana Gabdulkayum1, Gulbanu Akilzhanova2, Saule Rakhimova 1, Ulykbek Kairov3, Anargul Kuanysheva4, Ulan Kozhamkulov1, Kenes Akilzhanov2;5, Makhabbat Bekbossynova6, Dos Sarbassov1;7

1National Laboratory Astana, Nazarbayev University, Laboratory Of Genomic and Personalized Medicine, Center for Life Sciences, Astana, Kazakhstan; 2Medical University Semey, Pavlodar Branch, Pavlodar, Kazakhstan; 3National Laboratory Astana, Nazarbayev University, Laboratory Of Bioinformatics and Systems Biology, Center for Life Sciences, Astana, Kazakhstan; 4Medical University Semey, Semey, Kazakhstan; 5City Hospital #1, Pavlodar, Kazakhstan; 6National Research Cardiac Surgery Center, Astana, Kazakhstan; 7Nazarbayev University, School of Sciences and Humanities, Astana, Kazakhstan

The purpose of the study was to identify the mutational spectrum in Kazakhstani cohort of young sudden cardiac death (SCD) victims revealed by next-generation sequencing (NGS) of 96 genes associated with cardiac diseases and compare the diagnostic yield of postmortem genetic testing in (1) cases with structural findings of uncertain significance at autopsy (sudden unexplained deaths (SUDs), (2) cases with autopsy findings diagnostic of cardiomyopathy.

Methods: We screened 37 unselected SCD cases (<45 years) using the customized HaloPlex Target Enrichment System (Agilent) and NGS for 96 genes associated with inherited cardiac syndromes and cardiomyopathies. 27 cases had non-diagnostic structural cardiac abnormalities and 10 cases, diagnosed with a cardiomyopathy post-mortem. ACMG/AMP guidelines were applied for variant interpretation of clinical significance.

Results: 31 rare variants were identified in 17 (63%) of the deceased individuals with non-diagnostic structural cardiac abnormalities. Among them pathogenic variants in KCNQ1, KCNJ2, SCN5A, RYR2 genes were identified. The corresponding frequency in deceased individuals with cardiomyopathies was 35%. The most abundant mutations observed in MYBPC3, LAMA2, MYH6 and GAA.

Conclusion: Genetic screening revealed variants with likely functional effects at high rates, in 63% and 35% of the SCD cases with non-diagnostic and diagnostic cardiac abnormalities, respectively. Targeted NGS screening can support the forensic investigation and help the cardiologist’s decision to offer counselling and clinical evaluation to relatives of young SCD victims.

Study was supported by grants from the Ministry of Science and Higher Education of the Republic of Kazakhstan (АР14869903, BR10965164).

Conflict of Interest: Ainur Akilzhanova Principal Investigator, Madina Zhalbinova: None declared, Ayaulym Chamoieva: None declared, Zhanel Mirmanova: None declared, Diana Samatkyzy: None declared, Aidana Gabdulkayum: None declared, Gulbanu Akilzhanova: None declared, Saule Rakhimova: None declared, Ulykbek Kairov: None declared, Anargul Kuanysheva: None declared, Ulan Kozhamkulov: None declared, Kenes Akilzhanov: None declared, Makhabbat Bekbossynova: None declared, Dos Sarbassov: None declared.

EP06.038 Lack of expression of the rare KCNQ1 allele containing two missense variants in the same haplotype

Elena Zaklyazminskaya1, Vera Komoliatova2, Maria Karlova3, Denis Abramochkin3, Olga Sokolova 3

1Petrovsky National Research Centre of Surgery, Medical Genetics Laboratory, Moscow, Russian Federation; 2Centre of Syncope and Cardiac Arrhythmias in Children and Adolescents, Moscow, Russian Federation; 3Lomonosov Moscow State University, Moscow, Russian Federation

Genetic variations in the Kv7.1 channel alter membrane repolarization duration and cause inherited rhythm disorders.

We identified two heterozygous variants c.1011C>G (p.Ile337Met) (Class III, VUS) and c.1025T>C (p.Leu342Pro) (Class IV, Likely Pathogenic) in the KCNQ1 gene in two Russian probands with QTc interval prolongation. Segregation analysis revealed that both variants were presented in same haplotype. Interestingly that lone variant c.1011C>G (p.Ile337Met) was reported by 2 independent groups. We assume that kcnq1-Met337 variant might be raised first and represent more “archaic” allele.

We introduced two variants in three plasmids: kcnq1-Met337, kcnq1-Pro342, and kcnq1-Met337-Pro342, and compared mutant channels with wt. The plasmids were co-transfected into the CHO-K1 cells along with MinK (auxiliary subunit) coding plasmid. The protein expression level was assessed with immunoblot and confocal microscopy. Electrophysiological recordings of integral IKs current were performed using whole-cell patch clamp. The Ile337Met mutation leads to the slowing of K+ current kinetics and right shift of activation curve, which might decrease the extent of IKs current transferred by mutant Kv7.1 channels at the same values of membrane potential in cardiomyocytes.

Cells transfected with plasmids containing either lone p.Leu342Pro variant or both mutations together, possess no current. Immunofluorescent staining demonstrated that mutant channels contating p.Leu342Pro mutation were not exposed to the cell surface. It testifies that Leu342 variant affects normal Kv7.1 expression. We assume that missense mutation Leu342Pro realizes in vivo through haplo-insufficiency with or without rare variant p.Ile337Met.

We suggest that p.Leu342Pro may eliminate the consequences of the traffic changes Kv7.1-Met337 and influence the phenotype.

Conflict of Interest: None declared.

EP06.039 Copy number variants in familial hypercholesterolemia genes using targeted NGS, validated through optical genome mapping

Anna Green 1, Consuelo Alonso2, Jon Jonasson1, Aniruddh Kashyap1, Emma Adolfsson1, Anna Nordenskjöld3

1Örebro University, Department of Laboratory Medicine, Faculty of Medicine and Health, Örebro, Sweden; 2Bionano Genomics, Evry Cedex, France; 3Örebro University, Department of Cardiology, Faculty of Medicine and Health, Örebro, Sweden

Background/Objectives: Familial hypercholesterolemia (FH) is a common genetic disorder which is primarily caused by pathogenic variants in the LDLR, APOB, and PCSK9 genes. Approximately 10% of pathogenic variants in LDLR may be CNVs. Here, we combine NGS, MLPA, and Optical Genome Mapping (OGM) to investigate CNVs in LDLR.

Methods: A NGS panel was designed for whole gene sequencing (8 genes) of 100 FH patients using Twist technology and Illumina platform. CNVs were detected using CNVexpo, and an in-house pipeline for base-resolved normalized coverage. Identified CNVs were validated using MLPA and OGM. Bionano Services Lab performed the OGM procedure. Purified gDNA was labeled using Direct Label and Stain DNA Labeling Kit. Saphyr chip was run aiming for 100X coverage. De novo assembly and Variant Annotation pipelines were executed on Bionano Solve v3.7. Bionano Access v1.7 was used for CNV reporting and visualization.

Results: In five out of 100 samples NGS and MLPA data showed heterozygous deletions in LDLR. Three deletions, affecting different exons, was analyzed and confirmed using OGM. In two samples, OGM better defined the breakpoints as well as the size of the event, which expanded far beyond the gene of interest. In one sample, an additional CNV of SLCO1B1, a pharmaco-gene, important for transport of statins used for FH treatment was identified.

Conclusion: CNVs in FH genes in FH patients could be detected using targeted NGS, which was further confirmed by MLPA and characterized using OGM.

Grant References: The study received grants from Örebro County Research Committee.

Conflict of Interest: Anna Green: None declared, Consuelo Alonso employed at Bionano Genomics, Jon Jonasson: None declared, Aniruddh Kashyap: None declared, Emma Adolfsson: None declared, Anna Nordenskjöld: None declared.

EP06.040 Mutation spectrum of potassium channel genes in Kazakhstani patients with Primary Electrical Diseases

Ayaulym Chamoieva 1, Madina Zhalbinova1, Zhannur Abilova1, Saule Rakhimova1, Tolkin Zhakupova2;3, Tatiana Polyakova2;3, Ulykbek Kairov1, Ainur Akilzhanova1

1Center for Life Sciences, National Laboratory Astana, Nazarbayev University, Astana, Kazakhstan; 2Medical University Astana, Astana, Kazakhstan; 3Institute of Forensic Expertise, Astana, Kazakhstan

Background/Objectives: Primary electrical diseases (PEDs) or channelopathies are group of genetically determined diseases caused by cardiac arrhythmias. It leads to sudden cardiac death (SCD). Potassium channel genes are particularly important in manifestation of primary electrical disorders. Our research aimed to identification of mutations in potassium channel genes by targeted next generation sequencing (NGS) in Kazakhstani patients with PEDs.

Methods: Venous blood samples were recruited from 79 patients (47.5 ± 17.5 years old) with PEDs. All patients were sequenced for coding regions of 96 cardiac risk genes by Haloplex Target Enrichment System (Agilent Technologies). Genetic profiles of patients were analysed using SureCall Design (Agilent Technologies) software in order to identify mutated variants. Genetic variants were classified using ACMG/AMP guideline.

Results: According to genetic analysis, 52 variants were detected in potassium channel genes. Annotated data demonstrated 3.8% pathogenic, 1.9% likely pathogenic, 15.4% variants of uncertain significance (VUS), 19.2% likely benign and 46.2% benign variants according to ACMG classification. Among them KCNH2, KCNQ1 genes showed pathogenic status.

Conclusion: The study evaluated mutation spectrum of potassium channel genes in our group of patients by using targeted NGS. Genetic screening identified clinically significant variants within 96 genes associated with inherited cardiac diseases. Targeted NGS screening can be useful to clinicians for early diagnosis, risk stratification of complications and SCD development.

Grant References: Study was supported by grants from the Ministry Education and Science, Republic of Kazakhstan (АР14869903, BR10965164).

Conflict of Interest: None declared.

EP06.041 TAF1A variants in siblings with arrhythmogenic cardiomyopathy

Mafalda Mucciolo1, Giovanni Parlapiano2, Rosario Ruta1, Fabrizio Drago2, Marianna Cicenia2, Anwar Baban2, Antonio Novelli 1

1Bambino Gesù Children’s Hospital, IRCCS, Laboratory of Medical Genetics, Translational Cytogenomics Research Unit, Rome, Italy; 2Bambino Gesù Children’s Hospital, IRCCS, Pediatric Cardiology and Arrhythmia/Syncope Complex Unit, Rome, Italy

Background/Objectives: Arrhythmogenic cardiomyopathy is a genetic disorder characterized by the risk of life-threatening arrhythmias, myocardial dysfunction and fibrofatty replacement of myocardial tissue. About 35–50% of patients have no identifiable disease-associated variant. We reported here a family with ACM in a sibling pair. The older sister received heart transplant (HT) at 10 years of age. Post explant cardiac examination showed globular right ventricle (RV) with myocardial adipose replacement. Histological analysis confirmed the presence of arrhythmogenic cardiomyopathy (ACM) signs. She died after HT rejection complications. The younger sister started cardiological checkup for family screening. Magnetic Resonance Imaging (MRI) showed mild to moderate dilatation RV and systolic dysfunction, right atrial dilatation and initial LV dilation with systolic dysfunction, extensive epicardial fat at the free wall of the RV. She presented supraventricular tachycardia and paroxysmal atrial fibrillation episodes requiring preventive ICD implantation.

Methods: To identify the genetic basis of ACM in the sibling pair, exome sequencing was performed using a custom panel kit (Twist Bioscience) on NovaSeq6000 platform (Illumina). Exome sequencing data were filtered for rare, deleterious and recessive variants.

Results: Compound heterozygous recessive mutations in TAF1A, encoding an RNA polymerase I complex protein, were identified in the two sister. The identified variants were rare, predicted to be damaging by in silico tools and located in the functional domain of the protein.

Conclusion: Our family confirm the role of TAF1A gene in a wide spectrum of cardiomyopathy. Identification of additional mutations in TAF1A are required to further establish this genotype–phenotype relationship.

Conflict of Interest: None declared.

EP06.042 Association between Exome Sequencing and Metabolomics to investigate coronary heart diseases in a prospective cohort

ANGELO SAVOCA 1, Chiara Catalano1, Carla Debernardi1, Elisabetta Casalone1, Alessandra Allione1, clara viberti1, Alessia Russo1, miriam rosselli1, elton jalis herman1, cecilia di primio1, Marcello Manfredi2;3, Paolo Vineis4, carlotta sacerdote5, Giuseppe Matullo1;6

1University of Turin, Department of Medical Sciences, Turin, Italy; 2University of Piemonte Orientale, Department of Translational Medicine, Novara, Italy; 3University of Piemonte Orientale, Center for Translational Research and Autoimmune and Allergic Diseases, Novara, Italy; 4Imperial College London, Department of Epidemiology and Biostatistics, School of Public Health, London, United Kingdom; 5Città della Salute e della Scienza University-Hospital and Center for Cancer Prevention (CPO), Unit of Cancer Epidemiology, Turin, Italy; 6AOU Città della Salute e della Scienza, Medical Genetics Unit, Turin, Italy

Background/Objectives: Coronary artery diseases (CADs) are linked to metabolic alterations and cause most of the cardiovascular deaths in the adult population in Western countries. The clinical manifestations of CADs (e.g. myocardial infarction and heart failure) are usually acute events that require timely diagnosis and intervention. We aimed at investigating how genetics and metabolomics could improve CADs prevention.

Methods: Whole Exome Sequencing (WES) and untargeted metabolomic data were collected and analyzed for 84 pre-diagnostic individuals, who developed CAD at the follow-up, and 90 healthy controls, selected within the Turin component of the large European Prospective Investigation into Cancer and Nutrition (EPIC) cohort (EPICOR study).

Results: In the WES analysis, we prioritized rare variants (MAX AF < 1%) based on the status according to ACMG annotation and for all individuals we created a score for each gene given by the addition of specific values based on variant severity. The logistic regression model built with anthropometric variables (age, sex and BMI) had an AUC = 0.55, while adding the genetic score to this model the prediction power increased, AUC = 0.68 (p-value = 0.006). From metabolomic analyses, we selected 49 metabolites which, added to the anthropometric variables, showed an AUC = 0.80. Following the integration of covariates, genetic score and metabolites, the model reached an AUC = 0.84.

Conclusion: This integrated approach could be useful to clarify how the genetic background and metabolite profiles could influence CAD risk development.

Grant: This work was supported by Ministero dell’Istruzione, dell’Università e della Ricerca - MIUR project “Dipartimenti di Eccellenza 2018–2022”.

Conflict of Interest: None declared.

EP07 Metabolic and Mitochondrial Disorders

EP07.001 Laboratory challenges by the interpretation of slightly decreased branched chain amino acids concentrations: A case of BCKDK deficiency

Andžela Lazdāne 1, Svetlana Vorslova1, Pārsla Vēvere1, Ieva Mičule2, Zanda Daneberga1, Gita Taurina2, Sabīne Laktiņa2, Madara Auzenbaha2, INGA NARTISA1

1Children’s clinical university hospital, Metabolic genetics laboratory, Riga, Latvia; 2Children’s clinical university hospital, Clinic of Medical Genetic and Prenatal Diagnostics, Riga, Latvia

Background: Branched-chain keto acid dehydrogenase kinase deficiency (BCKDKD) is a rare disease caused by pathogenic variants in the BCKDK gene located on chromosome 16p11.2. Clinically manifests by autism, epilepsy, developmental delay, seizures, microcephaly, and decreased plasma branched chain amino acids (BCAA).

Methods: We report a case of BCKDKD where first amino acid spectra by HPLC-UV showed only slight decrease in two of BCAA – leucine and isoleucine. The result was considered as non-specific secondary changes.

Results: An 18-month-old girl had the first geneticist consultation due to developmental delay and microcephaly. The examination of amino acid spectra showed isoleucine 26 µM/L (reference range 32-92 µM/L), leucine 40 µM/L (53-149 µM/L) and valine 103 µM/L (79-267 µM/L). At the time of second examination reference range changed due to patient’s age, only leucine was identified as slightly decreased 59 µM/L (64-164 µM/L). Diagnosis confirmed by WES analysis identified two heterozygous likely pathogenic variants in the BCKDK gene. Treatment by BCAA mixture dose 4 g 3 times a day was not effective. In next three examinations all BCAA was decreased almost half of reference range lowest value. Only after increasing treatment frequency (dose 2 g 6 times a day) isoleucine was 39 µM/L, leucine 73 µM/L and valine 171 µM/L, which were within the reference range.

Conclusions: Determination of slightly decreased some or all BCAA in plasma amino acid spectra should be evaluated as specific changes indicative for an BCKDK deficiency. The final diagnosis is based on the results of molecular genetic studies.

Conflict of Interest: None declared.

EP07.002 The phenylalanine hydroxylase genotype and the expected responsiveness to sapropterin dihydrochloride in the adult Irish population

Aya Ibrahim 1, Jessica Ivory1, Barbara Gillman1, Darragh Nerney1, Loai Shakerdi1, Karl Kavanagh1, Robert O’Byrne1, Fionnuala Redmond1, Emma Corcoran1, Kevin Ilagan1, Alison Sheerin1, Eileen Treacy1;2, James O’Byrne1;2;3

1The Mater Misericordiae University Hospital, National Centre for Inherited Metabolic Disorders, Dublin, Ireland; 2Trinity College, School of Medicine, Dublin, Ireland; 3University College Dublin, School of Medicine, Dublin, Ireland

Background: Phenylketonuria (PKU) is an autosomal recessive inborn error of metabolism resulting from a deficiency of phenylalanine hydroxylase that catalyzes the hydroxylation of phenylalanine to tyrosine with the help of a cofactor (tetrahydrobiopterin; BH4). Ireland has one of the world’s highest incidences of PKU; currently, ~ 400 patients attend the National Centre for Inherited Metabolic Disorders (NCIMD), Mater Misericordiae University Hospital (MMUH).

Sapropterin dihydrochloride, a synthetic form of BH4, is a genotype-specific treatment that has recently been added to the precision genomic medicine program at NCIMD, MMUH. To service the plan for the roll-out of this treatment, all patients with PKU were genotyped, including prediction around sapropterin dihydrochloride responsiveness.

Methods: A study, analyzing the PAH genotype of patients (>18 years) with PKU attending the NCIMD, was performed along with sapropterin dihydrochloride responsiveness analysis.

Results: Two hundred eighty-three patients had PAH genotyping performed. One hundred and four different genotypes were identified in the population, with the R408W/R408W (13.78%) being the most common. The most frequent allelic variants were R408W (34.63%), F39L (12%), I65T (10.78%), L348V (5.48%) and IVS12 + 1G > A (4.78%). Sapropterin dihydrochloride responsiveness was predicted as likely in 12%, unlikely in 47% and difficult to predict in 41% of patients accurately.

Conclusion: The up-to-date PAH genotype landscape in the adult Irish PKU population is presented, detailing percentages of patients who will likely respond to sapropterin dihydrochloride. A large number will require a trial period to investigate responsiveness; information important for service planning as NCIMD continues to develop a precision genomic medicine program.

Conflict of Interest: None declared.

EP07.003 a homozygous c.1604G>A p.(Arg535His) variant of GBA gene with severe phenotype and a rare complication of splenectomy

Solaf Elsayed 1, Abdullah Abdullah1

1Faculty of Medicine, Ain shams University, Medical Genetics Dept, Cairo, Egypt

Background: The c.1604G>A variant in GBA has been reported in only compound heterozygous form with mild or asymptomatic non-neurological cases. Here we report a 15-year-old patient with a homozygous form with early severe manifestations and a splenectomy complication.

Case presentation: At the age of 7 years, a girl presented with poor weight gain, easy fatigability, and thrombocytopenia. Splenectomy done 2 years later. She was referred at 13 years of age to our clinic. She had below average weight and height, moderate hepatomegaly, and a machinery murmur on left scapular area.

CBC showed mild anemia, normal platelets. Liver enzymes and synthetic functions were normal. Liver biopsy revealed Gaucher cells but glucocerebrosidase was normal while lyso G1 was increased. Therefore molecular testing was done revealing homozygous c.1604G>A p.(Arg535His) variant in GBA gene. Echocardiography and oxygen saturation were normal. CT pulmonary angiography showed left sub-diaphragmatic tortuous convoluted vessels following contrast enhancement of the aorta with no definitive thoracic connection. CT angiography of abdomen and aorta showed splenic arteriovenous fistula. The patient received ERT regularly with satisfactory response and following with the hepatology clinic for portal hypertension.

Conclusions: The homozygous c.1604G>A variant of GBA gene could have an early severe Gaucher disease phenotype with normal enzyme level so a biomarker and molecular testing is necessary. Splenic arteriovenous fistula is a rare complication of splenectomy. Possible causes include mass ligation of splenic artery and vein during splenectomy. Awareness for avoiding splenectomy in Gaucher disease is still needed

Conflict of Interest: None declared.

EP07.004 L-2 Hydroxyglutaric aciduria case in Lithuania

Kristina Aleknavičienė1;2, Inga Poceviciene 1, Rimvydas Jonikas1, Marius Šukys1;2, Rasa Ugenskiene1;2

1Hospital of Lithuanian University of Health Sciences Kauno klinikos, Department of Genetics and Molecular Medicine, Kaunas, Lithuania; 2Lithuanian University of Health Sciences, Department of Genetics and Molecular Medicine, Kaunas, Lithuania

Background/Objectives: L-2 Hydroxyglutaric aciduria (L2HGA) is a rare (about 140 cases have been reported to date), progressive, autosomal recessively inherited metabolic disorder of organic acid metabolism. It is characterized by macrocephaly, developmental delay, seizures, speech difficulties, cerebellum abnormalities, typical brain MRI findings and the presence of L-2 hydroxyglutaric acid in urine samples.

Methods: 19 years old woman was reffered to geneticist because of suspicion of metabolic disease. From 1 years of age she had reccurent febrile seizures till 17 yo. Early psychomotor development was normal but with hypotonia. Reaching school age she had intellectual deficit (especially verbal). At 17 yo sleep electroencephalogram showed no abnormalities. Brain MRI – simetrical white matter intense signals in subcortical, basal ganglia and nucleus dentatatus. She was tested for organic acids in urine sample (liquid chromatography-mass spectrometry method). Whole exome sequencing was also performed to confirm the molecular diagnosis.

Results: Very high (12 time increased) 2-hydroxyglutaric acid concentration was detected after organic acids test. Other organic acids were not indicative of other metabolic disorders. Using this method we could not differentiate between L and D configurations of 2-hydroxyglutaric acid. Two likely pathogenic variants in L2HGDH (NM_024884.3) gene were found after whole exome sequencing: c.853delT (p.Tyr285fs) (PVS1 very strong, PM2 moderate) and c.164G>T (p.Gly55Val) (PP3 strong, PM2 moderate, PM5 supporting). Variants were classified based on the ACMG guidelines.

Conclusion: Increased urinary excretion of 2-hydroxyglutaric acid, typical MRI findings, clinical symptoms (seizures, developmental delay) and sequencing results in this patient established the diagnosis of L2HGA.

Conflict of Interest: None declared.

EP07.005 Uselfulness of biochemical analyses as functional studies to confirm the pathogenicity of VUS in inherited metabolic diseases

Laura Gort 1;2;3, Sonia Pajares1;3, Blai Morales-Romero1;2;3, Joan Anton Puig-Butille4, José Villanueva-Cañas4, Xavier Solé4, Aina Montalban-Casafont4, Esther Titos4, Eva Gonzalez-Roca4, Carles Zaragoza-Bonet1, Cristina Cutillas-Casado1, Cristina Fernandez-Sierra1, Patricia Alcalá-Barrera1, Laura Pacheco1, Sonia Moliner1, Ana Maria Valle-Duran1, Tatiana Collado1, Judit Pérez1, Antonia Ribes1;2;3, Judit Garcia-Villoria1;2;3

1Section of Inborn Errors of Metabolism - IBC. Department of Biochemical and Molecular Genetics. Hospital Clínic de Barcelona., Barcelona, Spain; 2IDIBAPS, Barcelona, Spain; 3CIBER of Rare Diseases, Madrid, Spain; 4Molecular Biology CORE. Department of Biochemical and Molecular Genetics. Hospital Clínic de Barcelona, Barcelona, Spain

Background/Objectives: With the implementation of the Next Generation Sequencing (NGS), the process of diagnosing inherited metabolic diseases (IMD) has undergone a substantial change. This powerful tool helps to reach the diagnosis faster when clear mutations in a candidate gene are found, but in other cases, the high number of variants of unknown significance (VUS) detected, may be a nightmare to elucidate the real diagnosis. In this study we checked how the biochemical studies help to reach the diagnosis of the patients when VUS were detected in homozygosity or in compound heterozygosity in a candidate gene.

Material and methods: We analyzed 413 patients with suspected IMD using Whole Exome sequencing (WES). Data were analyzed using virtual gene panels. Biochemical analyses were performed using routine techniques in IMD.

Results: A total of 413 patients were analyzed and the genetic diagnosis was reached in 44% of the patients (181/413). In 29/181 (16%) of these patients, at least one VUS in a candidate gene was detected, questioning the diagnosis. In all these patients, the corresponding biochemical analyses carried out revealed an alteration indicative of the suspicion of disease, confirming allegedly the pathogenicity of the variants and establishing the definitive diagnosis.

Conclusions: In the diagnosis of IMD, the biochemical analyses are essential and very useful functional studies to confirm the pathogenicity of VUS when found in a candidate gene. These studies increase the diagnosis rate of patients presenting with VUS, confirming their usefulness and the important role of IMD biochemical diagnostic laboratories.

Conflict of Interest: None declared.

EP07.006 Identification of obesity-related genetic variants in a family with an exclusively breastfed obese infant

Hazal Banu Olgun Celebioglu 1;2, Ayse Pinar Ozturk3, Şükran Poyrazoğlu3, Feyza Tuncer2

1Istanbul University Graduate School of Health Sciences, Istanbul, Turkey; 2Istanbul University Aziz Sancar Institute of Experimental Medicine, Genetics, Istanbul, Turkey; 3Istanbul University Faculty of Medicine, Paediatric Endocrinology, Istanbul, Turkey

Background/Objectives: Obesity is a multifactorial disease with genetic and environmental components. Obesity in infancy has been correlated with later childhood and adult obesity, which is prone to increased risk for mortality caused by type 2 diabetes, heart disease, or cancer. We aimed to identify genetic risk factors in a family having an exclusively breastfed obese infant with a history of paternal obesity.

Methods: Four members in a three-generation family were recruited. Physical examination, body mass index (BMI) calculation, biochemical analysis, hormonal evaluations, and hepatic ultrasonography were performed on a 7-month-old boy. Whole exome sequencing (WES) was performed on the case utilizing Illumina NextSeq550, followed by bioinformatic analyses on the Genomize SEQ platform. Variant confirmations and family segregation were performed by Sanger sequencing.

Results: Index had severe obesity (+3.8SD-p > 99.98) on admission with excess weight gain at 2 months, which paralleled paternal history. He showed no sign of syndromic obesity and hepatomegaly was detected at 4 months of age. He reached the normal percentile at 2 years old. His father and grandmother were obese (BMIs: 38 and 31, respectively), while the mother was lean. WES analysis revealed deleterious variants in SH2B1, GHRL, SRA1, ACADVL, PDE11A, and CAPN10 genes, which were previously associated with obesity. Family segregation confirmed paternal inheritance.

Conclusion: Despite reaching a normal percentile, the infant is under periodic follow-up due to shared genetic variants with his obese father that increase his risk for later childhood obesity.

Grant Reference: Supported by the Scientific Research Projects Coordination Unit of Istanbul University (TDP-2020-37103).

Conflict of Interest: Hazal Banu Olgun Celebioglu Istanbul University, Graduate School of Health Sciences, Istanbul, Turkey, Scientific Research Projects Coordination Unit of Istanbul University (TKP-2020-37103), Ayse Pinar Ozturk Istanbul University, Faculty of Medicine, Department of Paediatric Endocrinology, Istanbul, Turkey, Şükran Poyrazoğlu Istanbul University, Faculty of Medicine, Department of Paediatric Endocrinology, Istanbul, Turkey, Feyza Tuncer Istanbul University, Aziz Sancar Institute of Experimental Medicine, Department of Genetics, Istanbul, Turkey, Scientific Research Projects Coordination Unit of Istanbul University (TKP-2020-37103).

EP07.009 CFTR mutation spectrum in Georgian CF patients

Ia Khurtsilava1;2, Malgorzata Libik3, Dodo Agladze 2;4;5, Tsitsino Parulava6, Lali Margvelashvili2;7, Milan Macek8, Oleg Kvlividze1;2

1New Vision University Eco Campus, T’bilisi, Georgia; 2Georgian Foundation for Genetic and Rare Diseases (GeRaD), Tbilisi, Georgia; 3Charles University, Department of Biology and Medical Genetics, Prague, Czech Republic; 4Petre Shotadze Tbilisi Medical Academy, T’bilisi, Georgia; 5KidCo, Children’s Health Center, Department of Medical Genetics and Laboratory Diagnostics, T’bilisi, Georgia; 6Tsitsishvili Children’s Clinic, Tbilisi, Georgia; 7KidCo, Children’s Health Center, Department of Medical Genetics and Laboratory Diagnostics, Tbilisi, Georgia; 3Charles University, Department of Biology and Medical Genetics, Prague, Czech Republic

Background/Objectives: Cystic fibrosis (CF) is a life-threatening autosomal recessive disease caused by mutations in the CFTR gene. F508del is the most frequent mutation in the world. Other mutations are relatively rare and population specific. The aim of the study was to analyze distribution of CFTR mutations in Georgian CF patients.

Materials and Methods: 91 patients with a clinically confirmed CF diagnosis were tested (70.5% of registered CF patients in the country). Patients have been analyzed for CFTR variants by massively parallel sequencing of CFTR coding region and introns combined with the analysis of intra-CFTR rearrangements.

Results: CFTR gene analysis revealed 29 different mutations in Georgian CF patients. The most common mutation is c.1545_1546delTA (1677delTA) (42.7%), the second most common mutation, W1282X (11.2%). All other 27 CFTR mutations have low frequency, including F508del (6.7%). 3 novel mutations were found (c.708dupT; CFTRdele16_17; c.3170C>G) and reported to CFTR2 database.

Conclusions: Distribution of CFTR mutations in the Georgian CF population differs regarding the high frequency of mutation c.1545_1546delTA (1677delTA) and the low frequency of the predominant F508del mutation. Patients with 1677delTA have typical clinical manifestations and complications of the disease. Genotyping the Georgian CF patients was important to register population eligible for CFTR modulator therapy, and to come up with the better care plan for patients who are not eligible for personalized therapy.

Grant References: Supported by grants from Vertex Pharmaceuticals (CG-2015-104643; unrestricted charitable donation).

Conflict of Interest: None declared.

EP07.010 Diagnosis of ethylmalonic encephalopathy in a patient with a preliminary diagnosis of fanconi anemia: a case report

Özge Güngör 1, Gizem KÖK KILIÇ2, Burçak Tatlı Güneş3, emin karaca1, haluk akın1

1Ege University School of Medicine, Medical Genetics, izmir, Turkey; 2Tepecik Training and Research Hospital, Medical Genetics, izmir, Turkey; 3Tepecik Training and Research Hospital, Pediatric Hematology and Oncology, izmir, Turkey

Introduction: Ethylmalonic encephalopathy (EE) is a rare, autosomal recessive metabolic disease caused by dysfunction of the ETHE1 protein. EE is usually an early onset fatal disease with developmental delay, recurrent petechiae, orthostatic acrocyanosis, and chronic diarrhea. Rarely, cases with mild clinical findings have been reported in the literature. Different clinical findings are seen even in patients with the same pathogenic variant. This illustrates the clinical heterogeneity of EE and the difficulty of its diagnosis.

Methods: Clinical exome and Next-Generation Sequencing (NGS) based copy number variation (CNV) analysis was performed on the patient who applied with a preliminary diagnosis of Fanconi anemia.

Case Presentation: The case presented here is a 6-year-old girl with consanguinity between her parents. The patient had short stature, hyperpigmentation, microcephaly with bone marrow failure, and also persistent diarrhea from birth and acrocyanosis. Genetic analysis has revealed a homozygous, 2 kilobase pathogenic deletion including exons 1-3 of the FANCA gene, and a homozygous ETHE1 c.3G>T pathogenic variant. Interestingly, our case had a milder phenotype than other patients with ETHE1 pathogenic variants in the literature.

Conclusion: This case shows that patients with EE can have a milder phenotype and can be affected by another autosomal recessive genetic disease due to the high rate of consanguineous marriages. Therefore, it should be kept in mind that different genetic diseases may manifest together, and clinicians should not always be focused on a single diagnosis. It is also aimed to emphasize the contribution of NGS-based CNV analysis in reverse phenotyping.

Conflict of Interest: None declared.

EP07.011 Undiagnosed monogenic diabetes among young-onset type 2 diabetes – searching beyond current clinical guidelines

Clara Tan 1, Kai Xiang Kee1, Wan Ting Lovynn Chan1, Yuzhen Song1, Rashida Farhad Vasanwala2, Fabian Yap2, Ziliang Lim3, Tavintharan Subramaniam1;4, Chee Fang Sum4, Su Chi Lim1;4;5

1Khoo Teck Puat Hospital, Clinical Research Unit, Singapore, Singapore; 2KK Women’s and Children’s Hospital, Department of Paediatric Medicine, Endocrine Service, Singapore, Singapore; 3National Healthcare Group Polyclinics, Singapore, Singapore; 4Diabetes Centre, Admiralty Medical Centre, Singapore, Singapore; 5Saw Swee Hock School of Public Health, National University of Singapore, Singapore, Singapore

Background: Identification of monogenic diabetes is clinically important for personalized treatment. However, monogenic diabetes remains underdiagnosed and may be overlooked, in the absence of typical monogenic diabetes phenotype (young-onset, non-insulin dependent, non-obese, strong family history). We aim to identify monogenic diabetes among young-onset diabetes patients (onset ≤35 years old) clinically assigned as type 2 diabetes (T2D) by extending genetic testing beyond current guidelines (non-lean and/or absence of family history).

Methodology: 271 patients (median BMI 32.0 kg/m2) were screened for 16 monogenic diabetes-associated genes using massive parallel sequencing method. The m.3243A > G variant (associated with mitochondrial-inherited diabetes) was detected using quantitative PCR. Multiplex ligation-dependent probe amplification was used to detect copy number variation in HNF1A, HNF4A, GCK and HNF1B. All pathogenic variants (classified based on ACMG guidelines) were validated using Sanger sequencing.

Results: We identified 8 (3%) patients with monogenic diabetes, including 3 patients with clinically actionable variants in HNF1A and GCK and one patient with m.3243A > G variant. In comparison with patients without monogenic diabetes, patients with monogenic diabetes had lower median BMI (30.0 kg/m2 vs. 32.2 kg/m2, p = 0.133), lower prevalence of family history of diabetes (37.5% vs. 84.4%, p = 0.004), and lower median MODY PPV based on the Exeter MODY calculator (5.5% vs. 15.1%, p = 0.657).

Conclusion: Current clinical guidelines including the Exeter MODY calculator, may not be adequately efficient in selecting patients for genetic testing in the clinics particularly among young-onset obese T2D patients (BMI ≥30 kg/m2).

This study is supported by AHPL Science-Translational and Applied Research grants STAR18107 and STAR19204.

Conflict of Interest: Clara Tan Yes, Yes, Kai Xiang Kee Yes, Yes, Wan Ting Lovynn Chan Yes, Yes, Yuzhen Song Yes, Yes, Rashida Farhad Vasanwala Yes, Yes, Fabian Yap Yes, Yes, Ziliang Lim Yes, Yes, Tavintharan Subramaniam Yes, Yes, Chee Fang Sum Yes, Yes, Su Chi Lim Full time, Principal investigator, collaborator.

EP07.012 Molecular and biochemical characterization of a novel missense variant in COQ5 causing primary coenzyme Q10 deficiency

Roberta Zuntini1, Marzia Pollazzon 1, daniele frattini2, Stefano Caraffi1, Veronica Bizzarri1, Luca Pagliai1, Antonietta Vezzani1, Emanuele Coccia1;3, Carlotta Spagnoli2, Susanna Rizzi2, Maria Marinelli1, Carlo Fusco2, Livia Garavelli1

1Azienda USL-IRCCS di Reggio Emilia, Medical Genetics Unit, Maternal and Child Health Department, Reggio Emilia, Italy; 2Azienda USL-IRCCS di Reggio Emilia, Child Neurology and Psychiatry Unit, Department of Pediatrics, Reggio Emilia, Italy; 3University of Bologna, Postgraduate School of Medical Genetics, Department of Medical and Surgical Science, Bologna, Italy

Introduction: Coenzyme Q10 Primary Deficiencies (MIM#607426) are caused by mutation in each of the thirteen coenzymes involved in CoQ-Synthome. COQ5 methylase is crucial for maintaining the coenzyme Q10 level in human cells.

Methods: We described a 9-year-old girl born to consanguineous parents who showed ataxia, distal action tremor, ponto-cerebellar atrophy, intellectual disability, speech delay, neonatal jaundice, hypoglycaemia. CGH array, Angelman Syndrome and MECP2- disorders were excluded. Whole Exome Sequencing (WES) was performed and homozygous or compound heterozygous variants were filtered out.

Result: WES revealed the homozygous missense variant in the COQ5 gene c.352G>A causing the aminoacid substitution p.(Gly118Ser). Glycine118 is highly conserved among species mapped in Methyltransferase Motif I. This variant, absent in gnomAD, affects the last nucleotide of exon 2. In silico effector splice tools predict that the substitution will weaken the exon 2 splice donor. RNA was obtained from peripheral blood cells of the proband and her parents. In the patient we demonstrated exon 2 skipping and a residual full-length product in which glycine 118 was replaced by serine. Instead, her parents showed a full-length product with a mixture of glycine and serine (~20%) at same position and low exon 2 skipping level. Moreover, muscle biopsy was processed for histochemical analysis and CoQ10 level.

Conclusion: Our family is the to be second reported with CoQ10 deficiency caused by loss of function of the COQ5 gene. Ataxia and intellectual disabilities are clinical features of impairment of this coenzyme. This result is crucial for target treatment with CoQ10 supplementation.

Conflict of Interest: None declared.

EP07.015 Urine-derived cells: a non-invasive approach to the analysis of mitochondrial functions and mitochondrial diseases diagnosis

matthieu denis1, louisa paris2, naïg gueguen2;3, tobias chapron2, Valérie Desquiret2;3, Magalie Barth1, Marco Spinazzi2;4, guy lenaers2, Dominique Bonneau1;2, Pascal Reynier2;3, Patrizia Bonneau3, franck letournel5;6, Vincent Procaccio1;2, Céline Bris 1;2

1Angers University Hospital Center, Genetics, Angers, France; 2Université d’Angers, MitoLab, UMR MitoVasc, CNRS 6015, Inserm U1083, Angers, France; 3Angers University Hospital Center, Biochemistry, Angers, France; 4Angers University Hospital Center, Neurology, Angers, France; 5Angers University Hospital Center, Neurobiology, Angers, France; 6UMR INSERM 1066 - CNRS 6021, MINT, Angers, France, Angers, France

Purpose: Mitochondrial diseases are common metabolic disease of complex diagnosis, particularly due to the phenomenon of mtDNA heteroplasmy. Historically, molecular diagnosis was always performed on affected tissues (e.g. muscle, liver), but for the past few years the use of urine appeared to be a good alternative to invasive sampling. Coupled with NGS, it has improved the diagnosis of mitochondrial diseases allowing the identification of many variants of uncertain significance (VUS). However, the functional impact of these VUS is often difficult to assess due to the lack of invasive samples enabling the analysis of mitochondrial functions. The aim of this study was to develop mitochondrial functions analysis on urine-derived cells to allow a fully non-invasive diagnosis of mitochondrial diseases.

Methods: Urine-derived cells were obtained from voiding samples for 2 patients carrying one known pathogenic mtDNA variant (m.3243A > G and m.13513G > A) and 6 controls. The activity of the different mitochondrial respiratory chain complexes, COX, SDH and NADH histochemical staining and the OXPHOS analysis through western blot were performed on primary cultures.

Results: In comparison with the controls, complex I defect was observed for the patient carrying the m.13513G > A and a deficit of complexes I and IV for the patient carrying the m.3243A > G. For the latter, histochemistry also revealed the presence of COX-negative urothelial cells.

Conclusion: This study shows that urine-derived cells are a promising alternative to invasive sampling for the analysis of mitochondrial functions and functional validation of VUS, and confirms the value of urine samples for the diagnosis of mitochondrial diseases.

Conflict of Interest: None declared.

EP07.016 Mitochondrial DNA copy number variation in patients with suspected mitochondrial disease

Kristina Grigalionienė 1, Birutė Burnytė1, Algirdas Utkus1, Laima Ambrozaitytė1

1Vilnius University, Department of Human and Medical Genetics, Institute of Biomedical Sciences, Faculty of Medicine, Vilnius, Lithuania

Mitochondrial diseases (MDs) comprise genetically and phenotypically diverse group of disorders, characterized by dysfunctional mitochondria. Point mutations, large deletions and duplications are observed in MDs. However, mitochondrial function can as well be indirectly evaluated by measuring mitochondrial DNA (mtDNA) copy number (CN) that is usually increased in MD patients carrying point mutations compared with the control individuals (Filograna R., et al., 2020).

In this study, RT-PCR was applied to detect mtDNA CN in peripheral blood in a cohort of 71 affected individuals with suspected MD. Sanger sequencing of whole-length mtDNA and long-range PCR were used for comprehensive analysis of mtDNA sequence variants, deletions, including haplogroup profiling. mtDNA CN was also analysed in a control group of 15 healthy individuals.

mtDNA CNs were statistically significantly higher in the pediatric (under 18 years of age) patients (n = 50; median (IQR) CN = 291.5 (95.75) compared to adults (over 18 years of age, n = 21, median CN = 174.6 (55.00), p = 0.00003). MD was confirmed in 10 patients and the diagnosis of non-MD was confirmed in 9 patients of the study group. The difference of mtDNA CN in patients and control groups were not statistically significant (p > 0.05). However, patients with MD had lower mtDNA CN (median CN = 176.25 (55.5)) compared to patients confirmed with non-mitochondrial disease (median CN = 313 (108.0), p < 0.05).

This study indicates the increased mtDNA content in the peripheral blood of non-mitochondrial disease patients manifesting with clinical symptoms relevant to MDs that may be a consequence of compensatory response to secondary mitochondria damage.

Conflict of Interest: None declared.

EP07.018 identification of a novel SLC25A4 mutation causing cardiomyopathy and myopathy in a patient from a consanguineous Saudi family

Mazhor Aldosary 1, Hanan AlQudairy2, Noura Alshalan3, Mohammad Al Muhaizea4, Namik Kaya1

1Neurogenetics Section, Translational Genomics Department, Centre for Genomic Medicine (CGM), King Faisal Specialist Hospital & Research Centre, Riyadh, Saudi Arabia; 2Neurogenetics Section, Translational Genomics Department, Centre for Genomic Medicine (CGM), King Faisal Specialist Hospital & Research Centre, Riyadh, Saudi Arabia, Riyadh, Saudi Arabia; 3College of Science and General Studies, Alfaisal University, Riyadh, Saudi Arabia; 4Neuroscience Centre, King Faisal Specialist Hospital & Research Centre, Riyadh, Saudi Arabia

Background/Objectives: Mitochondrial DNA Depletion Syndrome 12B (MTDPS12B) is an autosomal recessive depletion syndrome caused by various mutations in SLC25A4. Mutations in ANT1 are associated with complex human diseases associated with mitochondrial myopathy and cardiomyopathy [1-3].

Methods: Blood samples were obtained from the family for genetic testing. Skin biopsy was taken from the patient. mtDNA sequencing on the patient DNA sample revealed no pathogenetic variants. We performed a comprehensive muscular dystrophy/myopathy gene panel to identify the genetic cause of the disease. We utilized Sanger sequencing for variant confirmation, and RT-PCR, mtDNA depletion, mitochondrial respiration and glycolysis experiments for further characterization and pathobiological understanding of the variant detected during the gene panel screening.

Results: We identified a novel presumably splicing mutation (c.112-1G > C) in SLC25A4. The variant was fully segregated with the phenotype in the family and absent among large ethnically matching controls. The variant was predicted to be pathogenic by different classifiers. Sequencing of the RT-PCR products showed 6 basepairs deletion in the exon 2/3 junctions. Quantitative PCR results indicated decreased copy numbers of mitochondria in both cell types. Functional analyses on fibroblasts obtained from this patient revealed a decrease in both mitochondrial respiration and glycolytic functions as compared to healthy controls.

Conclusion: This homozygous splice site mutation in SLC25A4 is a strong candidate in causing the disease in our patient and our study expands the genotypic spectrum of SLC25A4 deficiency.


1. Dorner, A., et al., 1999.

2. Jordens, E.Z., et al., 2002.

3. Palmieri, L., et al., 2005.

Grants: KSCDR/RAC#2180004.

Conflict of Interest: None declared.

EP07.019 A bibliometric and VOSviewer analysis of molecular mechanisms of obesity

Austėja Letukienė 1, Valentina Ginevičienė1

1Department of Human and Medical Genetics, Biomedical Science Institute, Faculty of Medicine, Vilnius University, Lithuania, vilnius, Lithuania

Background/Objectives: Obesity has been a field of intense research in recent years. Our study aimed to identify the current research hotspot, status and trends in molecular mechanisms of obesity by using bibliometric analysis.

Methods: The bibliometric analysis using VOSviewer software (version 1.6.19) was applied to assess global literatures about obesity, scanned from the Web of Science database (February 2023) using relevant keywords “obesity”, “molecular mechanism”, “gene expression”.

Results: Globally, 442,108 publications on the topic of obesity were identified. The mean citation count of the top 100 most cited articles was 8,3 (range 5-28). Most of them were descriptive studies and 1,151 clinical trials. The “obesity” with a total link strength (TLS) of 3,370 appeared as the most frequent keyword which had a strong link to “inflammation” (TLS = 1,207, and associated with “insulin resistance”, “high-fat diet”, “metabolic syndrome”), and “adipose tissue” (TLS = 904, associated with “adipogenesis”, “diabetes”). Keyword “gene expression” had 358 occurrences (TLS = 461, associated with “epigenetics”, “diabetes”). In total, 23,271 publications on gene expression related to obesity were identified from 2000–2023, 80.6% of them was published in the last decade and the number of publications continues to increase gradually every year.

Conclusions: The current growth trends predict a significant increase in the number of global publications on obesity and its molecular mechanisms. USA made the most outstanding contribution within this field. Research hotspots focused on inflammation, lipid metabolism, insulin resistance, diabetes, metabolic syndrome, adipose tissue and adipogenesis. Gene expression, epigenetics, sarcopenic obesity and adipokines may be the frontiers of future research.

Conflict of Interest: None declared.

EP07.020 uAUG-creating variant in the LDLR gene causes mild familial hypercholesterolemia

Alexandra Filatova 1, Petr Vasiliev1, Olga Ivanova1, Natalia Semenova1, Mikhail Skoblov1

1Research Centre for Medical Genetics, Moscow, Russian Federation

Background/Objectives: Variants that create upstream AUG codons at the 5`UTRs are under strong negative selection and could lead to the Mendelian disorders.

Methods: For patient with clinical diagnosis of familial hypercholesterolemia (FH) we performed custom panel sequencing. Experimental analysis of the effect of the variants on translation efficiency was conducted using dual luciferase assay and qPCR.

Results: A family with a 4-year-old girl applied for genetic counseling due to mild clinical features of hypercholesterolemia. Panel sequencing revealed the c.-8C > A variant in the LDLR 5`UTR. This variant leads to the formation of the upstream AUG codon and could create an overlapping uORF that suppresses the translation of the downstream LDLR coding sequence. Using experimental approaches, we showed that the c.-8C > A variant did not influence on transcription, but reduced main protein translation efficiency by about two times. Such partial reduction is in good agreement with the mild clinical picture of the patient, which is not typical for familial hypercholesterolemia caused by loss-of-function variants of the LDLR gene. We also analyzed three previously published variants in the 5’UTR of the LDLR gene. These variants were registered as pathogenic in HGMD and as uncertain significance in ClinVar, however they did not lead to the creation of uAUG. Our experimental analysis showed that these variants did not affect the level of transcription and translation efficiency. Thus, we suggest that these variants are not associated with FH.

Conclusion: Experimental analysis helps to determine the pathogenicity of rare variants and thereby explain the clinical features of patients.

Conflict of Interest: None declared.

EP07.021 Genotype–phenotype correlation of 2q21microdeletions: report of a 2.5 Mb microdeletion associated with metabolic acidosis and literature review

Nasser Almobadel 1, Fadia Almohaisen1, Mishal Alsulami1, Inesse Abdallah1, Hamoud Alanazi1, Abdulmalik Alenezy1, Fatma Alshahrani1, Aeyesh Almazead1, hatem elghezal1

1Prince Sultan Military Medical City, Riyadh, Saudi Arabia

Background/Objectives: 2q21 microdeletions are rarely reported with an unclear phenotype/genotype correlation because of the variability of clinical manifestations according to the size of deletions and their locations in the limited reported cases. To elucidate this correlation we report here a case of 2q21.2q21.3 microdeletion associated with severe congenital malformation syndrome and metabolic acidosis.

Patient & Methods: The proband is a termed baby boy of cousin Saudi parents died at the age of 6 months because of lactic acidosis, hyperammonemia, cardiac malformation and bilateral hydronephrosis.

Genetic investigation was performed using karyotype and 180K Agilent oligonucleotides array CGH analysis according to the manufacturer’s instructions.

Results: CGH Array revealed the presence 2.5 Mb deletion on the long arm of chromosome 2 at q21.2q21.3 including DARS, MCM6, LCT, and RAB3GAP1 genes. Result was reported as: arr[GRCh37] 2q21.2q21.3(134208726_136736954)x1

Conclusion: We describe here a novel interstitial deletion encompassing 2q21.2-q21.3, which was diagnosed by CGH array. The deletion of approximately 2.5Mb includes four genes. Among them, MCM6 and LCT genes are liked respectively to autosomal dominant and recessive lactase deficiency. We suggest that lactic acidosis is most likely caused by the loss of these contiguous genes. Cardiac malformation and hydronephrosis were observed in previous reported cases with largest deletions including 2q21 region. However, reported cases of 2q21.1 deletions were with mild phenotype and associated only with mental retardation. The comparison between our patient and previously reported cases in the literature contributes to a better definition of genotype–phenotype correlation of 2q21microdeletions.

Conflict of Interest: None declared.

EP07.022 Genetic predisposition for several complex diseases

Lavinia Mariana Berca1, Robert-Mihail Sionel 1;2, Catalina Zenoaga-Barbarosie2, Danut Cimponeriu2, Mihai Toma3, Matei Ioan Nica4, Ortansa Csutak2

1National Research and Development Institute for Food Bioresources – IBA Bucharest, Molecular Biology, Bucharest, Romania; 2University of Bucharest, Faculty of Biology, Genetics, Bucharest, Romania; 3Central Military Emergency Hospital Dr. Carol Davila, Bucharest, Romania; 4University of Medicine and Pharmacy Carol Davila, Bucharest, Romania

Background/Objectives: The predisposition for type 2 diabetes mellitus (T2DM), metabolic syndrome (MetS), obesity (Ob) and arterial hypertension (AH) present intricated elements. We aim to investigate the predisposition for these diseases in a case control study.

Methods: A total of 400 patients (213 men/187 women) were equal distributed in these four groups. 100 healthy subjects (HC) were considered as control. The ACE rs4646994 (I/D), eNOS rs1799983 (VNTR 4a/b), OXTR rs53576 (A > G), ATR1 rs5186 (A1166C), CAT rs7943316 (A > T) and SOD1 rs2234694 (+35A/C) polymorphisms were genotyped by PCR based methods for all samples. Fifteen variables related with life style, paraclinical and anthropometrical date were recorded for each subject.

Results: The distribution frequency of genotypes met Hardy-Weinberg equilibrium law. rs2234694AA genotype were protective for T2DM (p < 0.05). and was associated with triglyceride levels in the T2DM and Ob lots. The presence of both rs4646994 I and rs53576 A alleles was associated with T2DM. The rs4646994 DD was associated with hypertension in patients from T2DM and AH lot. Patients who reported drinking alcohol had increased frequency of rs4646994 DD and rs53576 G (p < 0.05) compared to nondrinkers from T2DM or Ob lots.

Conclusions: The investigated SOD1, ACE, OXTR polymorphisms seems to be associated with T2DM, obesity or arterial hypertension. No significant association was detected for sublot of subjects stratified according to the gender.

Grant References: This research work was carried out with the support of Ministry of Research and Education, under the Core Program, project PN 23 01 03 03.

Conflict of Interest: Lavinia Mariana Berca National Research and Development Institute for Food Bioresources – IBA Bucharest, project PN 23 01 03 03, Robert-Mihail Sionel National Research and Development Institute for Food Bioresources – IBA Bucharest, project PN 23 01 03 03, Catalina Zenoaga-Barbarosie AMS Laborator Genetic, Danut Cimponeriu University of Bucharest, Faculty of Biology, Department of Genetics, project PN 23 01 03 03, Mihai Toma Central Military Emergency Hospital Dr. Carol Davila, Bucharest, Matei Ioan Nica: None declared, Ortansa Csutak University of Bucharest, Faculty of Biology, Department of Genetics.

EP07.023 The genetic spectrum of maturity-onset diabetes of the young in the Lithuanian diabetic population

Eglė Mazgelytė 1;2, Laima Ambrozaitytė1;2, Gintarė Naskauskienė2;3, Birutė Tumienė1;2, Zydrune Visockiene2;3

1Institute of Biomedical Sciences, Faculty of Medicine, Vilnius University, Vilnius, Lithuania; 2Vilnius University Hospital Santaros klinikos, Vilnius, Lithuania; 3Institute of Clinical Medicine, Faculty of Medicine, Vilnius University, Vilnius, Lithuania

Background/Objectives: The most common type of monogenic diabetes is maturity-onset diabetes of the young (MODY) that affects 1–5% of all patients with diabetes mellitus. Identification of the MODY subtype is important due to subtype-related differences in clinical course and progression, response to treatment and for the genetic counseling. The aim of the study was to determine frequency and genetic spectrum of MODY in the Lithuanian diabetic population selected according to the criteria.

Methods: The study included 55 patients from the diabetic population. Patients were referred for genetic testing based on the results of MODY probability calculator developed by Exeter University. For each individual with the diagnostic criteria for MODY, a panel of 14 MODY genes was screened using targeted next generation sequencing assay.

Results: The (likely) pathogenic variants confirming a diagnosis of MODY were identified in 17 (30.9%) individuals. The majority of patients (88.2%) were found to harbour (likely) pathogenic variants in the GCK gene, one patient (5.9%) had pathogenic variant in ABCC8 gene and one patient (5.9%) carried (likely) pathogenic variants in both GCK and ABCC8 genes. Also, a variant of uncertain significance in PAX4 gene was detected in one individual.

Conclusion: The most prevalent MODY subtype in the selected Lithuanian diabetic population is GCK-MODY and only few patients were found having mutations in ABCC8 and PAX4 genes. Translational biology, integrative genomic research and studies on both monogenic and polygenic forms of diabetes are required, which will broaden our understanding in terms of pathophysiology and treatment of diabetes.

Conflict of Interest: None declared.

EP07.024 Detailed genetic and clinical analysis of a novel de novo variant in HPRT1: Case report of a female patient from Saudi Arabia with Lesch–Nyhan syndrome

Albandary Al-Bakheet 1, Hanan AlQudairy1, Joud Alkhalifah2, Sheikhah Almoaily1, Namik Kaya1, Zuhair Rahbeeni3

1King Faisal Specialist Hospital & Research Centre, Department of Translational Genomics Center for Genomic Medicine, Riyadh, Saudi Arabia; 2King Saud University, Medicine college, Riyadh, Saudi Arabia; 3King Faisal Specialist Hospital & Research Centre, Department of Medical Genomics Center for Genomic Medicine, Riyadh, Saudi Arabia

Hypoxanthine-guanine phosphoribosyltransferase (HPRT1) deficiency is an inborn error of purine metabolism responsible for Lesch–Nyhan syndrome (LNS). The disease is inherited in an X-linked recessive manner and predominantly affects male individuals. Female individuals can carry a mutation as heterozygotes, but typically, they are asymptomatic because of the random inactivation of the affected allele. Nevertheless, although rare, heterozygote female individuals may manifest LNS with full characteristics. Herein, we describe a female patient from Saudi Arabia with LNS. The patient (a 4-year-old girl) presented with typical characteristics of the disease, which include global developmental delay, self-mutilation, hyperuricemia, hypotonia, speech delay, spasticity, and seizures. Her general biochemical laboratory results were normal except for high levels of uric acid. The abdominal MRI\MRS, mostly unremarkable, showed bilateral echogenic foci within the renal collecting system. Genetic testing (whole-exome sequencing, iterative variant filtering, segregation analysis, and Sanger sequencing) pointed a novel de novo frameshift variant in HPRT1. X-inactivation assay using HpaII showed the presence of a 100% skewed X chromosome carrying the affected allele. RT-PCR of the cDNA indicated complete loss of the expression of the normal allele. Our study presents a female patient who has a severe case of LNS and found to be the 15th female patient with the disease in the world. The study emphasizethe need for a streamlined protocol that will help an early and accurate diagnosis of female LNS patients to avoid unnecessary interventions that lead to costly patient care.

Conflict of Interest: None declared.

EP07.025 Metabolomic profiling to resolve complicated case with MMADHC gene mutation

Viktoriya Yordanova 1, Mariya Ivanova1;2, Alexey Savov1, Ralitsa Yordanova3, Katerina Gaberova3

1University Hospital of Obstetrics and Gynecology “Maichin Dom”, National Genetic Laboratory, Sofia, Bulgaria; 2Faculty of Chemistry and Pharmacy, Sofia University “St. Kl. Ohridski”, Department of Analytical Chemistry, Sofia, Bulgaria; 3University Hospital "St. George", Department of Pediatrics and Medical Genetics, Plovdiv, Bulgaria

Introduction: Сombined methylmalonic aciduria and homocystinuria cblD type

(MAHCD) is a rare disorder of vitamin B12 (cobalamin) metabolism. It is characterized by decreased levels of the coenzymes adenosylcobalamin (AdoCbl) and methylcobalamin (MeCbl) and extremely variable clinical manifestation. MAHCD is an autosomal recessive disorder caused by mutation in MMADHC gene.

Materials and Methods: We report a 5-year-old boy with an uneventful premorbid history with current onset of subacute encephalopathy including dementia syndrome, increased sleepiness and ataxia. Differential diagnoses as a progressive cavitating leukoencephalopathy, Canavan disease, vanishing white matter, mitochondrial leukoencephalopathy, primary and secondary CNS vasculitis, subacute sclerosing panencephalitis, HIV- encephalopathy were discussed based on different imaging, hematological and biochemical studies. Metabolic profiling was performed by LC-MS/MS and GC/MS methods. Molecular genetic testing was performed by NGS of genes associated with a defect in cobalamin metabolism.

Results: Metabolic profiling of acylcarnitines in blood and organic acids in urine showed significantly increased levels of propionylcarnitine and extremely elevated excretion of methylmalonic acid and methylcitrates. This profile combined with the high total serum homocysteine found, suggests a defect in cobalamin metabolism. The molecular genetic testing showed a homozygous pathogenic variant с.748С>Т (p.Arg250Ter) in MMADHC gene which confirmed the biochemical findings.

Conclusions: Metabolomics is an important step to clarify the diagnosis in rare genetic diseases with highly variable phenotype even in cases with a slight clinical suspicion of inherited metabolic disease. Тhe patient received adequate therapy with improvement in speech development but with persistent behavioral abnormalities.

Conflict of Interest: None declared.

EP07.026 Improvement of the genetic diagnosis of severe hypertiglyceridemia through new genes associated with this pathology

Javier Sanguino1;2, Carmen Rodriguez-Jimenez1;2, Francisco Arrieta Blanco3, Pedro Luis Martinez-Hernandez4, Daniel Martínez-Jimenez5, Ana Carazo1;2, Maria Victoria Del Pozo-Gomez6, Cristina Ortega-Patrón6, Juan Manuel Montejo7, Sonia Rodriguez Novoa 2;8

1La Paz University Hospital, Metabolic Disease Laboratory, Genetic Department, Madrid, Spain; 2Fundación Para La Investigación Biomedica Del Hospital Universitarios La Paz, Group of dislipemias of genetic origin and metabolic diseases, Madrid, Spain; 3Ramón y Cajal Hospital, Endocrinology and Nutrition Department, Madrid, Spain; 4La Paz University Hospital, Internal Medicine Department, Madrid, Spain; 5Hospital Germans Trias i Pujol, Genetics, Badalona, Spain; 6La Paz University Hospital, Next Generation Service Department, Madrid, Spain; 7La Paz University Hospital, Genetics, Madrid, Spain; 8La Paz University Hospital, Metabolic Disease Laboratory, Genetic Department., Madrid, Spain

Background/Objetive: Severe hypertriglyceridemia is a disorder of lipid metabolism characterized by elevations in triglycerides and high risk of acute pancreatitis. About 2% of patients present familial- chylomicronemia-syndrome caused by biallelic variants of loss of function in LPL, APOA5, APOC2, GPIHBP1, GPD1 and LMF1. Some studies have shown that other genes could be associated with a polygenic hypertriglyceridemia.

The aim of this study was to identify variants in these new genes to increase the genetic diagnosis of hypertriglyceridemia.

Methods: Forty-eight patients with severe hypertriglyceridemia (>10-mmol/L) were studied by next-generation sequencing with a customized panel of 500 genes. A total of eighteen genes were studied, primary genes: LPL, APOA5, APOC2, GPIHBP1, GPD1 and LMF1 and secondary genes: ANGPLT3, ANGPLT4, ANGPLT8, APOC3, APOE, CREB3L3, GALNT2, GCKR, LIPC, LIPI, MLXIPL and TRIB1. Variants with allele frequency <0.005 were analyzed in silico and classified according to the guidelines of American College of Medical Genetics.

Results: Fifteen variants were found in 18 patients in primary genes, two of these in homozygous. In addition, thirteen variants were found in 10 patients in secondary genes. Overall, 52% of the patients were carriers of one of these variants.

Episodes of acute pancreatitis were found in the 55% of patients who carried at least one variant in primary genes and in 30% of patients with variants in secondary genes.

Conclusions: Secondary genes analysis increased the genetic diagnostic resulting about 20%. However, carriers of primary gene variants showed a greater association with acute pancreatitis.

Grants: Work was supported by PI21/01239-ISCIII.

Conflict of Interest: Javier Sanguino full, Carmen Rodriguez-Jimenez full, Francisco Arrieta Blanco full, Pedro Luis Martinez-Hernandez part-time, Daniel Martínez-Jimenez part-time, Ana Carazo part-time, Maria Victoria Del Pozo-Gomez part-time, Cristina Ortega-Patrón part-time, Juan Manuel Montejo part-time, Sonia Rodriguez Novoa Full, This work was supported by grant PI21/01239, Instituto Salud-Carlos-III.

EP07.027 Cytosolic PEPCK deficiency caused by a novel homozygous frame-shift variant presenting as hypoglycemia and acute liver failure at birth

Dalit May 1, gheona Altarescu1, Stanley Korman1, Eyal Shteyer1

1Shaare Zedek Medical Center, Jerusalem, Israel

Here we describe a homozygous frameshift mutation in PCK1, manifested in transient hypoglycemia and acute liver failure with extreme hyperferritinemia, presented on the first days of life.

The patient, a child of non-consanguineous parents from a Jewish Yemeni origin, was born following an uneventful pregnancy. At day 3, she presented acute liver failure with extreme hyperferritinemia of >40,000ng/ml, hyperammonemia and coagulopathy that improved gradually. At the age of 7 months, she was admitted to the hospital following an acute illness with severe hypoglycemia. Whole-exome sequencing (WES) revealed a homozygous frameshift mutation in Cytosolic phosphoenolpyruvate carboxykinase (PEPCK, PCK1). This enzyme plays a rate-limiting step in gluconeogenesis occurring mainly in the liver during prolonged fasting. Biallelic deficiency of this enzyme results in a rare inborn error of metabolism disorder (OMIM #261680).

Typical main Manifestations include fasting hypoglycemia and lactic acidosis with urinary excretion of TCA cycle metabolites. The initial presentation varies between individuals in terms of age and clinical manifestations, however clinical information is lacking as it was diagnosed so far in <30 patients with a total of 6 different mutations, all either missense or splice variants.

To the best of our knowledge, here we describe the first homozygous frameshift mutation in PEPCK causing a severe very early-onset liver failure that was not previously described. This case expands our clinical and genetic understanding of this rare metabolic disorder and emphasizes the need to consider PEPCK deficiency in the differential diagnosis of neonatal liver failure.

Conflict of Interest: None declared.

EP07.028 X‐Linked Creatine Transporter Deficiency in Two Saudi Brothers with Autism

Mohammed Almatrafi 1, Zehour Al-Sabban2, Soher Balkhy3, Iman Sabri Abumansour1;4

1Department of Medical Genetics, Faculty of Medicine, Umm Al-Qura University, P.O. Box 127, Makkah 21961, Saudi Arabia, Makkah, Saudi Arabia; 2Department of Radiology, King Faisal Specialist Hospital and Research Center, Jeddah, Saudi Arabia, Jeddah, Saudi Arabia; 3General Pediatric Section, Department of Pediatrics, King Faisal Specialist Hospital and Research Center, Jeddah, Saudi Arabia, Jeddah, Saudi Arabia; 4Pediatric Neurology Section, Department of Pediatrics, King Faisal Specialist Hospital and Research Center, P.O. Box 40047, Jeddah 21499, Saudi Arabia, Jeddah, Saudi Arabia

Background/Objectives: X-linked creatine transporter deficiency (CTD) was first described in 2001. A previous genotype-phenotype correlation study of CTD revealed that all affected males have an intellectual disability together with delayed speech development of variable severity. Other frequent clinical features include neurobehavioral disorders such as attention-deficit/ hyperactivity disorder (ADHD), autism spectrum disorder (ASD), seizures, hypotonia, spasticity, and dystonia. There are no previous case reports of CTD in Saudi Arabia. Here, we report X-linked creatine transporter deficiency in two Saudi brothers who presented with autism.

Methods: Case A and B are siblings who were born to a consanguineous Saudi couple. Their father had adult-onset focal seizures and visual hallucinations on Depakene. Their mother and sister were healthy at the time of the presentation. There is a family history of febrile seizures, global developmental delay, and intellectual disability in different cousins.

Results: Case A: Brain MRI showed diffuse cerebral atrophic changes in form of sulcal prominence and ventricular distension. MRS study showed decreased creatinine levels with normal choline and NAA levels. Case B: Brain MRI showed nonspecific periventricular foci of bright T2/FLAIR signal intensity. MRS showed an absence of creatinine peak with normal choline and NAA levels

Conclusion: This case proposes the need to consider creatine deficiency disorders in the differential diagnoses of children with autism spectrum disorder and neurological symptoms in multiplex autism families. Clinicians should have a low threshold for investigation of this possibility, employing brain MRS, genetic testing, and biochemical screening tests for creatine disorders where these are available.


Conflict of Interest: None declared.

EP07.029 Case reports of Crigler-Najjar syndrome type 1 in Czech patients

Daniela Zahorakova 1, Tomas Honzik1, Pavel Martasek1

1Department of Pediatrics and Inherited Metabolic Disorders, General University Hospital in Prague and First Faculty of Medicine, Charles University, Prague, Czech Republic

Background: UDP-glucuronosyltransferase 1A1 is an essential enzyme in bilirubin metabolism. Mutations in the UGT1A1 gene cause hereditary unconjugated hyperbilirubinemias, which include Gilbert syndrome, Crigler-Najjar syndrome type 1 (CN1) and type 2 (CN2). These disorders differ in level of serum unconjugated bilirubin, severity of symptoms and response to treatment. Gilbert syndrome is the mildest phenotype with only slightly raised bilirubin level. Its clinical presentation sometimes overlaps with CN2, in which the UGT1A1 enzyme activity is moderately reduced and can be restored by phenobarbital administration. CN1 is extremely rare and the most severe phenotype with total or nearly total absence of enzyme activity and not responding to phenobarbital treatment. Here we present case reports of two Czech patients with CN1.

Methods: UGT1A1 gene was analyzed by Sanger sequencing. Analysis of mutations in parents was performed to confirm compound heterozygosity in both patients.

Results: Patient 1 had shown persistent unconjugated hyperbilirubinemia since day 3 of life. Diagnosis of CN1 was confirmed at the age of 10 weeks. The present age is 3.5 years. Genotype NM_000463.3:[-41_-40dupTA; 840C > A];[1220delA]. Patient 2 had significant hyperbilirubinemia requiring phototherapy from day 8 of life. The diagnosis was confirmed at the age of 2 months. He is currently almost 3 months old. Genotype NM_000463.3:c.[-41_-40dupTA;722_723delAG];[1021C > T]. Both patients require several hours of daily phototherapy.

Conclusion: Identification of causative mutations confirmed clinical diagnosis and enable future prenatal genetic testing in affected families. Patients with untreated CN are at risk of kernicterus and early death, therefore early diagnosis is critical.

Grant references: RVO-VFN 64165.

Conflict of Interest: None declared.

EP07.030 Quantification of genotype-phenotype relationships in Pompe disease by enzyme activity: a case for graded variant classification

Reet Mishra 1, Zhiqiang Hu1, Dona Kanavy2, Jennifer L. Goldstein2, Deeksha S. Bali2, Yuanbin Ru3, G. Karen Yu4, Jonathan H. LeBowitz5, Constantina Bakolitsa1, Wyatt T. Clark5

1University of California, Berkeley, Berkeley, United States; 2University of North Carolina at Chapel Hill, Chapel Hill, United States; 3Alara Biotherapeutics, South San Francisco, United States; 4Global Blood Therapeutics, South San Francisco, United States; 5BioMarin Pharmaceutical Inc., Novato, United States

Background: Pompe disease (Pompe) is a rare autosomal disorder caused by pathogenic variants in acid α-glucosidase (GAA). Two different clinical forms have been described, depending on age of onset: infantile (IOPD, <1 year of age), and late (LOPD, juvenile or adult). Early diagnosis of Pompe and initiation of enzyme replacement therapy are critical for symptom improvement and survival. However, the broad clinical spectrum of Pompe and the large number of GAA variants of unknown significance (VUS) can complicate and delay diagnosis, especially for LOPD.

Methods: We generated a Pompe patient database (n > 1,750), and applied a regression model to determine disease severity (phenotype) from GAA variant combinations (genotypes) through enzyme activity. We further characterized 400 rare GAA VUS taken from a healthy population (ExAc) through an in vitro enzymatic assay, the largest such study to date, and applied a clinical evaluation to determine their pathogenicity.

Results: Overall, our study enabled the clinical characterization of over 600 GAA variants, including 300 VUS. Integration with gnomAD allele frequencies, revealed that 99% of individuals carrying the most common pathogenic variant do not develop Pompe in their lifetime. Conversely, 75 GAA VUS from ExAc showed potential disease relevance. Our model provides a graded classification of variant pathogenicity and is highly reliable, with a predicted activity of 15% suggesting a lifespan disease risk of 1:100.

Conclusion: Our results challenge the binary model of pathogenicity found in variant databases, such as ClinVar, allowing for improved diagnostic potential in Pompe and potentially other monogenic diseases.

Grant references: U24HG007346.

Conflict of Interest: Reet Mishra: None declared, Zhiqiang Hu Illumina Inc., U24HG007346, PI Steven E. Brenner, Illumina Inc., Dona Kanavy ClinGen Lysosomal Storage Disorders Variant Curation Expert Panel, Jennifer L. Goldstein ClinGen Lysosomal Storage Disorders Variant Curation Expert Panel, Deeksha S. Bali ClinGen Lysosomal Storage Disorders Variant Curation Expert Panel, Yuanbin Ru Alara Biotherapeutics, Alara Biotherapeutics, G. Karen Yu Global Blood Therapeutics, Global Blood Therapeutics, Jonathan H. LeBowitz BioMarin Inc., Constantina Bakolitsa University of California, Berkeley, U24HG007346, PI Steven E. Brenner, Wyatt T. Clark BioMarin Inc., BioMarin Inc.

EP07.032 Estimating quality of life in adults with Mitochondrial disease and their carers

Deborah Schofield 1, Joshua Kraindler1, Owen Tan1, Sameen Haque2, Rupendra Shrestha1, Sarah West1, Natalie Hart1, Jayamala Parmar1, Adam Percival2, Karen Crawley2, Carolyn Sue2

1GenIMPACT: Centre for Economic Impacts of Genomic Medicine, Macquarie University, Australia; 2Kolling Institute, St Leonards, Australia

Introduction: Mitochondrial disease is a serious and debilitating neurometabolic disorder causing significant disease burden and mortality, with great heterogeneity in presentation of symptoms. Currently, there are no curative therapies, with treatments focused on managing and reducing the impacts of symptoms. There are limited studies on the Quality of Life (QoL) impacts of the disease and to our knowledge, no study using health utilities to quantify the impact. With advances in genetic testing and the hope of breakthroughs in treatments, QoL data using health utilities will be required for use in cost-effectiveness analysis for publicly funded interventions.

Materials and methods: Patients were recruited from Neurogenetic clinics in Sydney, Australia. 96 participants and 24 carers completed the tailored questionnaire, which collected information on QoL and health utilities through the Assessment of Quality of Life (AQoL)-8D, a validated measure of QoL.

Results: AQoL-8D utility values for patients and carers were significantly lower than the general population. We will present scores for each of the AQoL-8D domains and bivariate and multivariate regressions analysing the drivers of patient QoL.

Conclusion: Mitochondrial disease has substantial impacts on QoL. This paper will present the first analysis of QoL in Mitochondrial disease patients using health utilities. This will be crucial to inform cost-effectiveness of new interventions, including genomic testing, for patients with Mitochondrial disease.

Funding: National Health and Medical Research Council (NHMRC) (Grant ID: 1151906).

Conflict of Interest: Deborah Schofield CI NHMRC Grant APP1151906, Joshua Kraindler: None declared, Owen Tan: None declared, Sameen Haque: None declared, Rupendra Shrestha CI on NHRMC Grant 1151906, Sarah West: None declared, Natalie Hart: None declared, Jayamala Parmar: None declared, Adam Percival: None declared, Karen Crawley: None declared, Carolyn Sue CI on NHMRC Grant 1151906.

EP08 Immunology and Hematopoietic System

EP08.001 Genomics role at the identification of patients with inborn errors of immunity

Nathalie Yepes1, Lina Moreno-Giraldo 2

1Universidad Libre Cali - Valle Del Lili Campus, Pediatrics, Cali, Colombia; 2Universidad Libre Cali - Valle Del Lili Campus, Genetics, Cali, Colombia

Background: Inborn errors of immunity or primary immunodeficiencies are humoral or cell-mediated immunity disorders with a genetic etiology that in the absence of a precipitating predisposes to recurrent infections, autoimmune and oncological diseases. In Colombia, it is considered a mandatory notification disease (Law 1392 of 2010, Decree 1954 of 2012, Decree 780 of 2016, Resolution 5265 of 2018 and Resolution 946 of 2019) having a prevalence of 1: 2000 to 10000 in the general population.

Case report: 5-year-old female patient with a clinical history of repeated infections and oral thrush, Juvenile Dermatomyositis since lactation, and low height and weight for age; in multidrug management with little clinical improvement so a New Generation Sequencing (NGS) genetic study with a panel of 228 genes for immunodeficiencies was taken. A pathogenic heterozygous variant was found in the TNFRSF13B gene, associated with common variable immunodeficiency with atypical clinical presentation and the need for genomic diagnosis to establish a specific diagnosis.

Conclusion: Current genomic diagnostic methods allow to establish phenotype/genotype correlation, making a specific diagnosis, to establish directed therapeutic options, predict complications, develop curative target therapies, such as gene therapy, follow-up, genetic counseling, bringing closer to preventive, anticipatory and personalized medicine.

Grant Reference: Not applicable.

Conflict of Interest: None declared.

EP08.002 Genetic variant in complement receptor 1 (CR1, CD35) is associated with a cluster of severe fatal coronavirus disease 2019 (COVID-19) in a Family

marah khalaila1, nada farran2, moran avraham-klabert3, Naiel Bisharat 4

1Emek Medical Center, Department of Medicine D, Afula, Israel; 2Emek Medical Center, Genetics, Afula, Israel; 3Emek Medical Center, Flow Cytometry Laboratory, Afuka, Israel; 4Emek Medical Center, Clalit Health Services. The The Ruth and Bruce Rappaport Faculty of Medicine. Technion- Israel Institute of Technology, Department of Medicine D, Afula, Israel

Fatal coronavirus disease 2019 (COVID-19) clustering within a family could indicate a genetic predisposition to contract severe acute respiratory syndrome corona virus 2 (SARS-CoV-2). This study investigated whether such predisposition existed in a family that was severely affected by COVID-19 with four deaths and 3 suffering near-fatal disease. We applied whole exome sequencing and segregation analysis to identify unique genetic variants among those affected by SARS-CoV-2. A novel splice mutation in CR1 (complement receptor 1) gene was identified [c.5302+1G > A in (rs756221326, 1-207755349 -G-A (GRCh37)] (NM_000651.6). We found a high correlation between those severely affected by COVID-19 and heterozygote alteration. RNA expression analysis identified two isoforms expressed for carries: one with full exon 32 inclusion (wild-type allele) and second with exon skipping (without exon 32). A major outcome of exon skipping is abolishing normal reading frame and creating early stop gain, leading to truncated protein (31 exons compared to a wild-type protein that includes 47 exons). Quantitative allele expression (qPCR) targeting the wild-type allele revealed significantly decreased levels of CR1 among carriers compared to wild-type samples. Given its central role in regulation of complement activity acting on all three complement pathways as a membrane-bound receptor of C3b/C4b, C3/C5 convertase decay accelerator, and cofactor for factor I-mediated cleavage of C3b and C4b, it’s intriguing to hypothesize that reduced CR1 expression may have had a significant impact on disease severity and outcome among those carrying the genetic variant.

Conflict of Interest: marah khalaila Full time, nada farran Full time, moran avraham-klabert Full time, Naiel Bisharat Full time.

EP08.003 Describing the bone microenvironment of monoclonal gammopathies patients by single cell RNAseq analysis

Cristina Manferdini1, Benedetta Dalla Palma2, Tommaso Torelli3;4, Emanuela Aleo5, Matteo Scita2, Matia Bernardi2, Valentina Marchica6, Denise Toscani6, Jessica Burroughs Garcia2, Nicolas Thomas Iannozzi6, Vincenzo Raimondi6, Oxana Lungu6, Arianna Di Bernardo4, Andrea Devecchi3, Rosanna Vescovini6, Gina Lisignoli1, Luca Agnelli3;4, Nicola Giuliani2;6, Paola Storti 6

1IRCCS Rizzoli Orthopedic Institute, Laboratory of Immunorheumatology and Tissue Regeneration, Bologna, Italy; 2University of Parma Hospital, Hematology, Parma, Italy; 3IRCCS Istituto Nazionale dei Tumori, Department of Advanced Diagnostics, Milano, Italy; 4IRCCS Istituto Nazionale dei Tumori, Tumor Genomics Unit, Milano, Italy; 5IGA Technology Services, Udine, Italy; 6University of Parma, Medicine and Surgery, Parma, Italy

Background/Objectives: Multiple myeloma (MM) is a plasma cell dyscrasia, characterized with the occurrence of bone lesions, that may be preceded by pre-malignant condition as monoclonal gammopathy of undetermined significance (MGUS) and smoldering MM (SMM). Up to date we are not able to identify exactly the patients that will progress to active MM. The aim is to study the bone microenvironment (BM) in patients with MGUS/SMM to identify the mechanisms involved in the progression to MM.

Methods: From the bone biopsy of 4 MGUS, 4 SMM, 4 MM patients, we isolated and enriched the CD45-CD138-CD235a-CD31- non-hemopoietic cells. Single-cell RNAseq data were generate on 10X Genomics platform. Cell Ranger 6.1.2 have been used to obtain feature and barcode count matrices. In R software version 4.1.0, the predicted cell populations were calculated using singleR and the HumanPrimaryCellAtlasData. Differential transcript analysis was performed using the linear mixed effects model method in the MAST R package. Pathway analysis was performed using the decoupleR package.

Results: A total of 51672 cells were profiled, and 3 different types of mesenchymal stromal cells(MSCs), osteoblasts(OBs), MSCs-derived cells and a small amount of hemopoietic cells were identified. In MM samples, there was a significant reduction of OBs number, and MSCs-derived cells. Moreover, the BM cells of MM showed an alteration in the expression of genes of WNT, hypoxia, NF-kB and PI3K pathways.

Conclusions: The study at single cells level could be a new method to describe the alterations of BM cells in patients with monoclonal gammopathies.

Grant: AIRC MFAG 2020(ID:24932).

Conflict of Interest: None declared.

EP08.004 Exosomal specificity of microRNAs associated with disease activity and renal damage in systemic lupus erythematosus

Ana Flores-Chova 1, Olga Martinez-Arroyo1, Carlos Bea2, Ana Ortega1, Maria J Forner2;3, Raquel Cortés1

1Biomedical Research Institute Hospital Clinico - INCLIVA, Cardiometabolic and Renal Risk Research Group, Valencia, Spain; 2Hospital Clinico, Internal Medicine, Valencia, Spain; 3University of Valencia, Department of Medicine, Valencia, Spain

Background and objectives: Little is known about the biofluid specificity of microRNAs (miRNAs) in systemic lupus erythematosus (SLE). Our aim was to identify a specific miRNA profile in plasma exosomes and assess biological functions in response to lupus activity and renal damage using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis.

Methods: Plasma samples were obtained from 96 SLE patients and 25 controls. RNA was extracted from both plasma and plasma-exosomes and miRNAs were identified using SmallRNA sequencing analysis. Results were validated in a higher cohort by qPCR.

Results: From the small RNA-sequencing, the 25 miRNAs with the highest fold-change expression between biofluids were identified. Nine miRNAs were validated in a larger cohort, and were found to be increased in exosomal fraction and patient groups. Further analysis revealed that two panels combining three miRNAs [panel 1: LN (miR-101-3p, miR-144-5p and miR-15a-5p); panel 2: non LN (miR-144-3p, miR-101-3p and miR-19b-3p)] gave an area under the curve that improves the readout of the single miRNAs (0.964 and 0.828, p < 0.0001, respectively). Finally, biology system analysis showed that exosomal miRNAs panels target critical pathways regulating immune response (ubiquitination, TFG-β1-SMAD and VEGF signalling)

Conclusion: Biofluid origin influences on plasma miRNA expression and identified specific exosomal miRNA profiles which target critical pathways in SLE-associated activity and renal damage progression. This finding could contribute to advances in SLE diagnosis and as promising therapeutic targets.

Grant: Health Institute Carlos III [PI18/01405 and PI21/00249; FI20/00096 FI22/00032] IJC2020-045308-I, by MCIN/AEI. European Regional Development Fund (ERDF).

Conflict of Interest: None declared.

EP08.005 TREX1: The varying and overlapping manifestations of disease

Tracy Briggs 1;2, David McKee3, Sarah McGlasson4, Pietro D’Urso5, Tristan Hardy6, Amit Herwadkar7, Sadia Jahan8, Gillian Rice1;2, David Hunt4, Patrick Toby H Coates8

1University of Manchester, Evolution, Infection & Genomics, School of Biological Sciences, Faculty of Biology, Medicine and Health, Manchester, United Kingdom; 2Manchester Foundation Trust, Genomic Medicine, Manchester, United Kingdom; 3Salford Royal Hospital, Neurology, Salford, United Kingdom; 4UK Centre for Clinical Brain Sciences, UK Dementia Research Institute, Edinburgh, United Kingdom; 5Salford Royal Hospital, Neurosurgery, Salford, United Kingdom; 6Monash IVF Group, Genetics, Repromed, Dulwich, Australia; 7Salford Royal Hospital, Neuroradiology, Salford, United Kingdom; 8Royal Adelaide Hospital, Nephrology, Adelaide, Australia

TREX1 variants are associated with Aicardi-Goutières syndrome (AGS), Familial chilblain lupus (FCL) and Retinal vasculopathy with cerebral leukodystrophy (RVCL). Heterozygous variants are also described in Systemic lupus erythematosus (SLE).

In AGS, TREX1 variants are generally biallelic and result in dysfunctional exonuclease activity leading to an upregulation of type I interferon and interferon stimulated genes (ISG). Elevated ISGs are also noted in FCL and SLE and variants may be in the C terminus. In RVCL causative variants are heterozygous frameshift mutations, occur at the C terminal end of the protein and impede localization of the protein to the endoplasmic reticulum, rather than impact exonuclease function; in reported cases type I interferon levels have been normal and the phenotype distinct.

We describe a family with features of RVCL, including adult onset vasculopathy, cerebral white matter and contrast-enhancing lesions causing neuropsychiatric sequale and progressive neurological decline. We note however autoimmune manifestations in the kindred, including SLE-like symptoms and Polyarteritis nodosa and an elevated ISG in the proband. We identified a previously unpublished TREX1 frameshift C terminal pathogenic variant in the family. Functional studies demonstrated that the variant did not impact exonuclease activity, but resulted in mislocalisation of the protein, comparable to that seen in RVCL.

We highlight the diverse manifestations of RVCL and the diagnostic difficulties overlap cases can cause. The correct diagnosis here is pertinent given that different clinical trials with different modes of action are available for each of AGS, SLE and RVCL.

Funding: MRF (TAB), Clayco foundation (SM), WT (DG)

Conflict of Interest: None declared.

EP08.006 Using whole exome sequencing for detecting genetic causes of autoinflammatory diseases in families without common MEFV gene. mutation

Niloofar Bazazzadegan1, Shima Salehi2, Kimia Kahrizi1, Mojgan Babanejad1, Sussan Banihashemi1, Hossein Najmabadi1, Seyedeh Sedigheh Abedini 1;1;3

1Genetics Research Center, University of Social Welfare and Rehabilitation Sciences, Tehran, Iran; 2Department of Pediatrics, School of Medicine, Aliasghar children hospital, Iran University of Medical Sciences, Tehran, Iran; 3The University of New South Wales, BioMedical Machine Learning Lab, The Graduate School of Biomedical Engineering, Sydney, Australia

Background/Objectives: Autoinflammatory diseases (AIDs), often known recurrent fever syndromes, are a heterogeneous group of rare conditions characterized by innate immune dysregulation. It is thought that mutations in the Mediterranean fever (MEFV) gene are the most common cause of AIDs, but other genetic factors may also contribute. Up to now, more than 30 different genes have been identified as casual genes with autosomal or dominant inheritance patterns for AIDs. Therefore, in this study, we aimed to uncover the genetic causes of AIDs in five Iranian families without common MEFV gene mutations using whole exome sequencing (WES).

Methods: Peripheral blood was taken from the core family after clinical evaluation and consent forms were signed by all family members. Then, DNA was extracted and analyzed for quality and quantity. We performed WES on DNA samples from the proband of each family. The WES data were analyzed using different bioinformatics tools and compared to reference databases to identify candidate variants that could be associated with AIDs.

Results: Each family was analyzed multiple times to identify possible candidate genes. The candidate genes were either involved in the inflammation process or known genes associated with AIDs. Segregation analysis is performing to detect probable gene and variants as well as their inheritance pattern in each family.

Conclusion: The potential functional impact of the identified variants will be investigated through further analysis. Furthermore identified candidate genes with their probable causative variants would be useful to accelerate diagnostic processes.

Grant references: USWR: 2920.

Conflict of Interest: Niloofar Bazazzadegan: None declared, Shima Salehi In UIMS, Kimia Kahrizi In GRC, USWR, Mojgan Babanejad In GRC, Sussan Banihashemi In GRC, Hossein Najmabadi In GRC, Seyedeh Sedigheh Abedini In GRC, In GRC, Grant from USWR.

EP08.007 Mendelian susceptibility to mycobacterial disease: clinical and molecular findings of a familial case with variants in IL12RB1 gene

natalia velez tirado1, Shirley Iza2, lina castaño jaramillo1, Silvia Maradei 1;2

1fundación hospital pediátrico La Misericordia, bogotá, Colombia; 2biotecnología y genética s.a.s. biotecgen, bogotá, Colombia

Background: Clinical disease caused by weakly pathogenic mycobacterial species is a rare entity secondary to a defective immune response with IL-12RB1 deficiency being the most common genetic etiology. In recent years a number of mutations in related genes have been described with variable clinical and immunological findings.

Methods: We describe clinical and molecular findings in an 10-year-old patient with multiple salmonellosis infections, multidrug-resistant intestinal tuberculosis and pulmonary aspergillosis who required multidisciplinary medical treatment. Molecular analysis was indicated, and the study was extended to the nuclear family.

Results: Two variants were reported in IL12RB1 gene c.1456C>T (p.Arg486*) and c.1420T>A (p.Cys474Ser), with pathogenic and uncertain significance classification, respectively, in the index patient and one brother.

Conclusion: Two variants are reported in a patient with clinical diagnosis of mendelian susceptibility to mycobacterial disease. The importance of determining the allelic phase of variants and familial clinical features arises in classifying variants and determining recurrence counseling.

Grant references: cero grants.

Conflict of Interest: None declared.

EP08.008 Functional characterization of genetic variants associated with deficiency of adenosine deaminase II

pu chen1, Yasaman Pakdaman 2, noemie julie marie brissi2, tuva sundell2, lassi paavolainen1, monika szymanska2, Janna Saarela1;2

1Institute for Molecular Medicine, University of Helsinki, Helsinki, Finland; 2Norwegian Centre for Molecular Medicine, University of Oslo, Oslo, Norway

Background: Deficiency of Adenosine deaminase 2 (DADA2) is a rare, monogenic disorder characterized by reduced or absent activity of extracellular adenosine deaminase 2 (ADA2) enzyme. DADA2 patients suffer from vasculitis as the primary phenotype, as well as other immunological and hematological manifestations including hypogammaglobulinemia, pure red cell aplasia and lymphoproliferation. ADA2 deficiency is associated with several recessive mutations on the ADA2 gene. However, the biochemical effect of each defect, as well as the pathophysiological role of ADA2 related to various phenotypes of the disease is still largely unknown.

Methods: The expression pattern and catalytic activity of ADA2 were studied in the lysates and culturing media of HEK293 cells following transfection with 9 selected pathogenic variants. Subcellular co-localization of ADA2 with the Golgi marker TGN45 was also determined for these variants by immunofluorescence assay.

Results: We observed reduced or lack of protein expression in HEK293 media for 6 out of 9 examined ADA2 variants, which were associated with significantly lower levels or absence of enzyme activity in both media and cell lysates. Immunostaining results indicated that 8 ADA2 variants co-localized less within the Golgi apparatus than the wild type protein, suggesting an altered protein secretion pathway in cells under the effect of these variants.

Conclusion Our study suggests impaired protein secretion as a contributing factor behind pathogenesis of some DADA2-associated variants. We further aim to explore possible affected pathways of ADA2 secretion, and understand the biochemical significance of these selected variants on the structural properties and stability of the protein itself.

Conflict of Interest: None declared.

EP08.009 Association toll-like receptor TLR 2, TLR 4 and TLR9 gene polymorphisms with COVID-19 severity

Ali Al-Jawadi 1, Inna Pokudina1, Tatiana Shkurat1

1Southern Federal University, Rostov-on-Don, Russian Federation

Background/Objectives: Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has led to a pandemic. Toll-like receptors (TLRs) play an important role in the activation of innate immunity, regulation of cytokine expression, indirect activation of the adaptive immune system. TLRs are key sensors that recognize of pathogen-associated molecular patterns of SARS-Cov-2. This study aimed to explore the association between the of TLR2, TLR 4, TLR9 polymorphisms with COVID-19 severity.

Methods: The polymorphisms TLR2 (rs5743708), TLR 4(rs4986791) and TLR9 (rs5743836) were genotyped in 315 COVID-19 patients: 118 mild disease, 114 non-severe and 83 severe, age and sex-matched Genomic DNA was extracted from blood of participants, genotyping was performed using RT-PCR.

Results: No differences were found between groups regarding the allelic and genotype distribution of the TLR 4 and TLR 9. For the polymorphism of TLR2, statistically significant differences were found in the distribution of allele (χ2 = 6.322; p = 0.043) and genotypes (χ2 = 6.595; p = 0.0037) frequencies. Сarriage of dominant homozygous Arg/Arg ТLR2 trait increases the likelihood to protect against severe COVID-19 in participants mild disease (OR = 0.373; 95 % CI 0.14-0.993, p = 0.042) and non-severe (OR = 0.329; 95 % CI 0.118-0.916 p = 0.027). Participants with Arg/Gln genotype of TLR2 had 2.68 - and 3.049-times higher odds severe, than mild disease and non-severe (95 % CI 1.007-7.131, p = 0.042 and 95 % CI 1.092-8.477, p = 0.027 respectively).

Conclusion: Our study indicated, that the polymorphisms TLR2 (rs5743708), may be possible risk factors for severe COVID-19.

Conflict of Interest: None declared.

EP08.010 PCSK3 overexpression in Sjögren’s Syndrome patients may be regulated by rs4932178 SNP in the promoter region and correlates with IFN-γ expression

Andrea Latini1, Giada De Benedittis 1, Serena Colafrancesco2, Lucia Novelli3, Roberta Priori2;3, Giuseppe Novelli1;4;5, Cinzia Ciccacci3, Paola Borgiani1

1University of Rome “Tor Vergata”, Biomedicine and Prevention, Rome, Italy; 2Sapienza Univerisity, Clinical Internal, Rome, Italy; 3UniCamillus—Saint Camillus International University of Health Sciences, Rome, Italy; 4IRCCS Neuromed, Pozzilli, Italy; 5University of Nevada, Pharmacology, School of Medicine, Reno, United States

Sjögren’s Syndrome (SS) is an inflammatory autoimmune disease, characterized by chronic lymphocytic infiltrates in the exocrine glands. SS etiology is still partially unknown and the identification of new genes associated with this disease could therefore help to gain a more complete view of the mechanisms involved in its development. Several studies have suggested a possible involvement of PCSK3 gene in the pathogenesis of chronic inflammatory diseases. This gene encodes for the protease enzyme Furin, which promotes proteolytic maturation of important regulators of the immune response and enhance also the secretion of interferon-γ (IFN).

We investigated, by RT-qPCR, the PCSK3 gene expression level in peripheral blood mononuclear cells isolated from SS patients and healthy controls and we evaluated a possible correlation with IFN-γ gene expression. Moreover, we also explored the variability of two PCSK3 genetic polymorphisms (rs4932178 and rs4702) by direct sequencing, to evaluate a possible association between these polymorphisms and the expression levels of PCSK3 gene.

We observed that PCSK3 expression level was significantly higher in SS patients, compared to the controls (P = 0.028) and that it showed a positive correlation with IFN-γ expression level (P < 0.001). Moreover, we reported that the variant homozygous genotype of rs4932178 SNP is associated with a higher expression of PCSK3 gene (P = 0.038). The homozygous variant genotype of this SNP is also associated with a higher risk of SS development (P = 0.016).

Our data suggest that Furin could play a role in SS development, also promoting IFN-γ secretion.

Conflict of Interest: None declared.

EP08.011 Clinical characteristics of patients with Alpha-1-antitrypsin deficiency

Matija Rijavec 1;2, Julij Šelb1;3, Peter Korošec1;4, Katarina Osolnik1

1University Clinic of Respiratory and Allergic Diseases Golnik, Golnik, Slovenia; 2Biotechnical Faculty, University of Ljubljana, Ljubljana, Slovenia; 3Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia; 4Faculty of Pharmacy, University of Ljubljana, Ljubljana, Slovenia

Background: Alpha-1-antitrypsin (AAT) deficiency is an underdiagnosed autosomal recessive disease. The burden of under-diagnosis is high since risk factor modification (primarily smoking cessation), because of diagnosis awareness, can profoundly alter the natural course of the disease. The current study aimed to illuminate clinical aspects of the disease by characterising the cohort of patients with AAT deficiency managed at Golnik University Clinic.

Methods: Genetic testing for the two most common pathogenic variants, PI*Z (c.1096G>A, p.Glu366Lys) and PI*S (c.863A>T, p.Glu288Val), in SERPINA1 gene followed by sequencing was performed, and clinical information of patients was obtained from the hospital’s information system: i.) age, ii.) the serum concentration of AAT, iii.) pulmonary function parameters (FEV1 (%), TI, DLCO (%)), iv.) liver function parameters (AST, ALT, yGT), v.) presence of emphysema, and vi.) smoking status.

Results: Out of 700 patients with a genetic test, 210 had one (187 PI*MZ, 23 PI*MS), 39 had two pathogenic variants (1 PI*SS, 6 PI*SZ, 32 PI*ZZ genotype). Serum AAT concentration differed significantly between the groups (single-allele impairment/bi-allele non PI*ZZ/bi-allele PI*ZZ) with the lowest concentration present in PI*ZZ group. Pulmonary function tests, namely FEV1(%), and DLCO(%) were likewise the lowest in the PI*ZZ group. A similar trend was apparent with emphysema. There were no signs of clinically meaningful liver disease in the PI*ZZ group.

Conclusions: Among adult patients with AAT deficiency, lung disease – manifested as a decrease in pulmonary function and emphysema – was substantial. As expected, lung disease was more pronounced among smokers, even in patients with only single-allele impairment.

Conflict of Interest: Matija Rijavec Full-time, ARRS J3-2532, Julij Šelb Part-time, ARRS, Peter Korošec Full-time, ARRS J3-3072, J3-6787, J3-3626, J3-2234., Katarina Osolnik Full-time.

EP08.012 An exome-based, genotype-driven approach improves the genetic characterization of thrombocytopenias with or without predisposition to myeloid malignancies

Federica Isidori 1, Federica Melazzini2;3, Flavia Palombo4, Roberta Bottega5, Elena Nardi6, Valeria Bozzi2, Michela Faleschini5, Serena Barozzi2;3, Tania Giangregorio1, Pamela Magini1, Balduini Carlo2;3, Anna Savoia7, Marco seri1;6, Patrizia Noris2;3, Alessandro Pecci2;3, Tommaso Pippucci1, Caterina Marconi1

1IRCCS Azienda Ospedaliero Universitaria di Bologna, Medical Genetics unit, Bologna, Italy; 2IRCCS Policlinico San Matteo Foundation, Pavia, Italy; 3University of Pavia, Department of Internal Medicine, Pavia, Italy; 4IRCCS Istituto delle Scienze Neurologiche di Bologna, Bologna, Italy; 5IRCCS Burlo Garofolo, Institute for Maternal and Child Health, Trieste, Italy; 6University of Bologna, Department of Experimental, Diagnostic and Specialty Medicine, Bologna, Italy; 7University of Verona, Department of Neurosciences, Biomedicine and Movement Sciences, Verona, Italy

Inherited thrombocytopenias (ITs) are extremely diverse diseases with wide genetic heterogeneity.

We applied Exome Sequencing (ES) and targeted analysis of 43 known IT genes to a large cohort of 187 cases (135 families) presenting with non-syndromic IT. Most cases have undergone a validated diagnostic algorithm including phenotypic investigation and sequencing of suspected genes.

ES achieved an overall diagnostic yield of 32.5%, with 44 pathogenic variants occurring more frequently in ACTN1, ETV6, GP1BA and RUNX1. Moreover, ES-based Copy Number Variant analysis disclosed an unexpected high prevalence of RUNX1 large deletions (2.2%). Overall, 8% cases carried variants in genes potentially predisposing to hematological malignancies (ETV6, RUNX1).

When compared to the validated diagnostic algorithm, ES gained 16% yield. This could be explained by events of atypical inheritance, sex-related effects and phenocopies for which the candidate-gene approach hindered the diagnosis or false negative laboratory results. One of the major challenges of ES application to IT is the high proportion VUS (25% cases in our study). Since IT-associated variants are generally highly penetrant, segregation data even in small pedigrees can be useful to change variant classification, especially downgrading VUS to B/LB status, as occurred in 20/135 of our cases.

In conclusion, we report that an exome-based, genotype-driven approach contributes to the accurate diagnosis of ITs and to the surveillance over hematological malignancies. We propose the application of ES to overcome the limitations of current candidate-gene approaches. Simultaneous NGS of multiple family members is recommended to reduce the impact of VUS and therefore time-to-diagnosis.

Conflict of Interest: None declared.

EP08.013 CD26+ Leukemic Stem Cells identification in Tunisian chronic myeloid leukemia patient : a preliminary study

Fatma Turki 1, nour louati2, moez elloumi3, leila keskes4, tarek rebaii4, Hassen Kamoun1, Rim Frikha1

1Hedi Chaker Hospital, Medical Genetic Department, Sfax, Tunisia; 2Regional Blood Transfusion Center of Sfax, Sfax, Tunisia; 3Hedi Chaker Hospital, Clinical Hematology Department, Sfax, Tunisia; 4Medecine university, Sfax, Tunisia

Objectives: Chronic myeloid leukemia (CML) is a myeloproliferative disorder characterized by an acquired genetic alteration of the hematopoietic stem cells behaved as leukemic stem cells (LSC). CD26 is a highly specific marker expressed in CML LSC. Assessment of CD26+LSCs may be an optimal biomarker for the monitoring of CML patients.

This study investigated the role of CD26+ LSCs in the follow-up of Tunisian CML patients and its correlation with molecular response in the area of tyrosine kinase inhibitors.

Methods: Flow cytometry and standard qRT-PCR technique were performed to evaluate CD26 + LSC and BCR-ABL1 transcript levels in peripheral blood (PB) in CML patients, respectively.

Results: A total of 48 patients with CML were enrolled in this study. The CD26 + LSC was higher in patients with failure than those with an optimal molecular response.

CD26+LSCs were significantly correlated with the molecular response expressed as a BCR-ABL1 ratio(p < 0.05).

Conclusions: To the best of my knowledge this is the first study that investigated CD26+ LSCs in Tunisian CML patients. CD26+LSCs identification can be a useful marker for monitoring patients with CML when a BCR-ABL1 quantitative assay is not available.

Grant references: Bocchia M and Raspadori D. Residual Peripheral Blood CD26+ Leukemic Stem Cells in Chronic Myeloid Leukemia Patients During TKI Therapy and During Treatment-Free Remission. Front. Oncol 2018. 8:194.

Raspadori D and Bocchia M. Flow Cytometry Assessment of CD26+ Leukemic Stem Cells in Peripheral Blood: A Simple and Rapid New Diagnostic Tool for Chronic Myeloid Leukemia. Cytometry Part B 2019; 00B: 1–6.

Conflict of Interest: None declared.

EP08.014 The results of the pilot project on primary immunodeficiencies in Sverdlovsk region (Russian Federation)

Svetlana Deryabina 1;2, Dmitry Cheremokhin2, Olga Lagutina1, Irina A Tuzankina2

1Institute of medical cell technologies, Ekaterinburg, Russian Federation; 2Institute of Immunology and Physiology of the Russian Academy of Sciences, Ekaterinburg, Russian Federation

Background: Newborn screening (NBS) for primary immunodefciency (PID) is based on the detection of T-cell receptor excision circles (TRECs) and kappa-deleting recombination excision circles (KREC). They are sensitive biomarkers for T- and B-cell lymphopenia, but not specific and usually can let to secondary findings and false-positive results. That’s why a choice for correct cut-off is a very important factor.

Methods: In 2021 we initiated a pilot study on newborn screening for PID in Sverdlovsk region. The analysis of TREC and KREC was performed by real-time polymerase chain reaction in neonatal dry blood spots by test system “Immunobit” (Generium). The DBS samples were collected between July and September 2021.

Results: In total, 5,044 newborns were screened. Three blood DNA samples were used for control: 1 – with X-linked agammaglobulinemia, 2 – with SCID (ADA-SCID and ILR2G deficiency). At first we used the cut off from instruction: 500 copies for TREC and 300 copies for KREC (both in 100000 leukocytes). Retest from the first DBS was provided in 6.1% of patients. New sample test was needed in 158 (3.1%) of newborns, referral to immunologist was used in 57 cases (two abnormal values successively). Then we recalculated cut-off (99.9 percentile) retrospective. In fine the group with risk for primary immunodeficiency reduced to 1.0% and all 57 newborns who was referred to immunologist stayed in it. Wherein a number of false-positive was reduced significant.

Conclusions: Using of own data to form a risk group for PID is more effective in preventing false-positive referrals.

Conflict of Interest: None declared.

EP08.015 Сomparison of two methods for TREС, KREC molecules measuring for inborn errors of immunity NBS

Ivanna Shymanska 1;2, Iryna Polishchuk2, Volodymyr Kravets1, Bohdan Tretiak2, Kateryna Sosnina2, Oksana Boyarchuk3, Halyna Makukh1;2

1Scientific medical genetic center LeoGENE, Lviv, Ukraine; 2Lviv Regional Clinical Perinatal Centre, Lviv, Ukraine; 3I. Horbachevsky Ternopil National Medical University, Ternopil, Ukraine

Background/Objectives: The level of T-cell receptor excision rings (TRECs) and kappa-deleting excision rings (KRECs) could be used for newborn screening patients with inborn errors of immunity. The comparison and unification of the data obtained with measurement by two different methods are essential.

Methods: The analysis of TREC and KREC was performed by real-time PCR followed by analysis of melting curves (HRM) and with using the “Biocore® SMA/TKID plus” diagnostic kit. HRM analysis was performed among 9 000 DBS. Measuring of TREC, KREC levels was performed among 18 000 DBS.

Results: The pilot project was launched to determine the levels of TREC, KREC molecules in DBS of children from the Ternopil region of Ukraine were carried out by the method of quantitative PCR, using plasmids with known concentrations of TREC, KREC, and albumin as standards. The reactions were carried out in three separate tubes followed with HRM to determine the specificity of low-copy products. TREC, KREC and RNAsP as internal control levels involve the detection of three products in one reaction with Biocore® SMA/TKID. We compare Ct and ∆Ct (TREC, KREC, albumin) and (TREC, KREC, RNASaP) for both methods. According to the results, ∆Ct (T-A) = 6,47, ∆Ct (K-A) = 6,46 and for second method ∆Ct (T-R) = 4,69, ∆Ct (K-R) = 7,10.

Conclusion: Both methods were sensitive to detect T- and/or B-lymphopenia. The changing internal control from RNASaP to albumin gene is needed to find out the TREC, КRЕС molecules absolute number.

Grant References:

Conflict of Interest: None declared.

EP08.017 Down syndrome phenotype associated with TRNT1 gene

Mihaela Bataneant 1;2, Adela Chirita-Emandi3;4, Estera Boeriu1;2, Mihaela Baica5, Patricia Urtila1;2

1“Victor Babes” University of Medicine and Pharmacy, IIIrd Pediatric Clinic, Timisoara, Romania; 2“Louis Turcanu” Clinical Emergency Hospital for Children, IIIrd Pediatric Clinic, Timisoara, Romania; 3“Victor Babes” University of Medicine and Pharmacy, Genetic, Timisoara, Romania; 4“Louis Turcanu” Clinical Emergency Hospital for Children, Genetic, Timisoara, Romania; 5“Louis Turcanu” Clinical Emergency Hospital for Children, Laboratory, Timisoara, Romania

Introduction: Biallelic mutations in TRNT1 gene cause SIFD syndrome-Sideroblastic anemia, B-cell Immunodeficiency, periodic Fevers and Developmental delay.

The aim: s to present two cases of SIFD syndrome, both of them with Down syndrome phenotype, association that wasn’t reported.

Case presentation: Patient 1 (P1)-female, patient 2 (P2)-male come from non-consanguineous parents with insignificant disease family history. In P1 disease started at 3 months and in P2 at 3 weeks, with fever every 2-3 weeks, lasting 5-10 days accompanied by diarrhea and vomiting. Both patients have a Down phenotype with psychomotor retardation and microcephay in P1. There were excluded Down syndrome (karyotype, microarray), celiac disease, hypothyroidism, cystic fibrosis, Schwachmann-Diamond syndrome, inflammatory bowel disease. Laboratory tests showed sideroblastic anemia, CRP > 100 mg/L, elevated serum amyloid, low IgA, IgG and IgM, poor response to vaccination at P1, B lymphopenia and decreased switched memory B cells. We performed WES in P1, revealing a heterozygous double missense pathogenic mutation c.608+1G > T/c.1246A > G in the TRNT1 gene. In P2 the diagnosis was much faster, being suggested by the Downian phenotype associated with periodic fever and proven by gene panel sequencing-heterozygous double mutation c.428_431del /c.1246A > G. Corticosteroid therapy and immunoglobulin replacement were administered to both patients.

Conclusions: SIFD is a rare immunodeficiency, with 46 patients reported in the literature. It should be suspected in any case of recurrent fever associated with hypogammaglobulinemia and Downian phenotype. Early diagnosis will allow the early establishment of treatment and even the optimal time for bone marrow transplantation.

Conflict of Interest: None declared.

EP08.018 Identification of a splice variant in PSMB10 gene in a multiply affected family with undiagnosed autoinflammatory syndrome

Muhammed Gecgel1, Alper Bulbul2, Dora Sigli3, Inci Onat1;3, Serdal Ugurlu4, Eda Tahir Turanlı 1;3

1Acibadem University, Graduate School of Natural and Applied Science, Molecular and Translational Biomedicine, Istanbul, Turkey; 2Acibadem University, Graduate School of Health Sciences, Biostatistics and Bioinformatics, Istanbul, Turkey; 3Acibadem University, Faculty of Engineering and Natural Sciences, Molecular Biology and Genetics, Istanbul, Turkey; 4Istanbul University-Cerrahpasa, Department of Internal Medicine, Division of Rheumatology, Istanbul, Turkey

Background/Objectives: Monogenic autoinflammatory syndromes are heterogenous group of disorders that do not always follow simple genetic inheritance patterns. Here we present exome sequencing and interferon signature analysis in a family diagnosed with autoinflammatory syndrome who were clinically and genetically not fulfilling FMF, CAPS, TRAPS, HIDS, and MVK diagnoses.

Methods: Whole exome sequencing is performed in a family with 3 affected siblings, father and unaffected mother using Illumina TrueSeq capture kit. Broad GATK germline short variant calling pipeline was used. Variants were filtered based on frequency (MAF < 0.01), tissue expressions, variant effect predictions (CADD, SpliceAI) and family segregation patterns. PBMC’s were isolated from all the family members and healthy controls fresh blood samples. Selected interferon signature genes (IFI27, IFI44L, IFIT1, ISG15, RSAD2, SIGLEC1, IFIT3, OASL, IFI35) were analyzed by RT-qPCR. Delta Ct fold changes relative to housekeeping gene (HPRT1) were calculated. Independent T-test was used to calculate the significance in expression levels.

Results: A rare splice variant (rs201451622, NM_002801:exon1:c.56+1G > A) in Proteasome 20S Subunit Beta 10 (PSMB10) gene (heterozygous) was found in affected father and 3 affected siblings. This variant is predicted to cause a loss at the donor-splice site at exon 1. Among the interferon signature genes ISG15 was significantly upregulated (2.24-fold, p = 0.0108) in patients compared to healthy controls.

Conclusion: c.56+1G > A variant in PSMB10 gene is a candidate variant in the present family and future work will explore more interferon gene expressions and in vitro cell culture models of splicing.

Grant: This work is supported by ACU, ABAPKO, 2022/07-16

Conflict of Interest: None declared.

EP08.019 Genetic evaluation of inborn errors of immunity

Cristina-Loredana Pantea 1;2, Maria Puiu1;2, Mihaela Bataneant1;2, Adela Chirita-Emandi2;3

1University of Medicine and Pharmacy Victor Babes, Timisoara, Romania; 2Emergency Hoospital for Children “Louis Turcanu”, Timisoara, Romania; 3University of Medicine and Pharmacy Victor Babes, Center for Genomic Medicine, Timisoara, Romania

Background/Objectives: Inborn errors of immunity (IEI) constitute a heterogeneous group of over 450 different disorders, polygenic pathologies with a wide variability of clinical manifestations.

We aim to establish the optimal test to perform for the diagnosis of IEI and to compare diagnostic rate using WES (whole exome sequencing) or WGS (whole genome sequencing) versus gene panels analysis (including different number of genes from 300 to 4813 genes).

Methods: The medical data collected from the patients who fulfilled the IEI criteria were: personal and family history, clinical picture and paraclinical investigations.

In total, 131 unique index patients were studied with genetic tests. We performed 56 gene panels, 22 WES and 53 WGS tests to determine a conclusive diagnosis.

Results: A total number of 176 tests was performed. The majority of patients (104 patients) have done 1 test, 24 patients had 2 tests, 3 patients had 3 tests and 1 patient had 4 tests.

27 patients had a positive primary immunodeficiency NGS Panel, with a 48% rate of positive result identified; 9 patients had a WES positive result (41%), and 41 patients had a positive WGS result (77%).

Conclusion: A larger number of patients is needed to establish which molecular genetic test has optimal diagnostic yield in the identification of disease-causing variants of IEI.

Conflict of Interest: Cristina-Loredana Pantea Emergency Hospital for Children “Louis Turcanu”, Maria Puiu Emergency Hospital for Children “Louis Turcanu”.

Genetic Department, “Victor Babes’ University of Medicine- professor, Mihaela Bataneant Emergency Hospital for Children “Louis Turcanu”

“Victor Babes” University of Medicine Timisoara, Adela Chirita-Emandi Associate Professor, Genetics Department

University of Medicine and Pharmacy “Victor Babes”

Timisoara, Romania

EP08.020 When a routine blood test can change it all- Hereditary hyperferritinemia cataract syndrome

Iulia Simina 1, Florin-Ioan Jurca2, Florina Stoica3, MEDA ADA BUGI4;5, Maria Puiu1;6, Adela Chirita-Emandi1;6

1“Victor Babes” University of Medicine and Pharmacy, Department of Genetics, Center of Genomic Medicine, Timișoara, Romania; 2Nefroped Team, Alba-Iulia, Romania; 3Municipal Clinical Emergency Hospital of Timisoara, Department of Ophthalmology, Timișoara, Romania; 4“Louis Turcanu” Emergency Hospital for Children, Timișoara, Romania; 5Victor Babeş University of Medicine and Pharmacy, Timișoara, Romania; 6Clinical Emergency Hospital for Children “Louis Turcanu”, Regional Center of Medical Genetics Timis, Timișoara, Romania

Introduction: Hereditary hyperferritinemia cataract syndrome (HHCS) is an autosomal dominant disorder consisting of high levels serum ferritin and juvenile bilateral cataracts. It is often caused by mutations in the ferritin L-subunit (FTL) gene. We report a 17-year-old adolescent who presented with persistently hyperferritinemia.

Methods: The clinical workup was completed for the affected individual. Sequence analysis and deletion/duplication testing of the 27 genes listed in the NGS iron related disorders panel was performed, using Illumina technology; reads were aligned to the reference sequence GRCh37. Moreover, Sanger sequencing was performed to validate the mutation.

Results and discussions: The patient aged was diagnosed with hyperferritinemia (average 1300 ng/ml) in May 2019. For 2 years, she undergone multiple clinical and laboratory investigations to exclude the presence of iron overload and secondary causes of hyperferritinemia and even overloading with vitamin and iron supplements was taken into consideration by the medical team. A detailed personal history revealed the presence of early-onset cataract by the age of 7, when a specific literature search has oriented us towards HHCS. Two pathogenic heterozygous variants were identified in the patient: (c.-161C > T,rs139325959) of the FTL gene and (c.187C>G,rs1799945) of the HFE gene. HHCS was therefore confirmed, after ruling out the other 2 possible diagnoses, and our young patient can receive a proper care by only visiting the ophthalmologist, instead of a lifetime of searching reasons for hyperferritinemia.

Conclusion: HHCS should be considered in the differential diagnosis of hyperferritinemia, especially in the presence of cataracts, normal serum iron concentration and transferrin saturation.

Conflict of Interest: None declared.

EP09 Intellectual Disability

EP09.001 Developmental and epileptic encephalopathy 87, caused by CDK19 mutation, could include two distinct phenotypes

Tomoko Uehara 1, Mie Inaba1, Seiji Mizuno1

1Aichi Developmental Disability Center, Department of Clinical Genetics, Aichi-ken, Japan

Background: CDK19 is a paralog of CDK8, which constitutes the CDK/cyclin module of the mediator complex. Recently, CDK19 was identified as a causative gene for developmental and epileptic encephalopathy 87 (MIM618916; DEE87). The main symptoms are generalized developmental delay, hypotonia, and recurrent epilepsies, but the number of reported DEE87 patients is small and most of the details are yet unknown.

Methods: We compared 16 patients with DEE87 reported in previous papers, including one outpatient of our hospital, in terms of the details of symptoms and mutations.

Results: All of the 16 patients’ mutations were located within the kinase domain of CDK19. Of these, 11 patients had (1) mutations in the ATP-binding pocket and 5 patients had (2) mutations in the activation segment. Patients of (1) had no short stature greater than -2.5 SD, whereas two of the five patients of (2) had severe growth retardation (-9.7 and -4.6 SD). Patients of (1) commonly showed hypertelorism, blephaophimosis, wide nasal bridge, wide nasal base, thick nasal wings, and wide mouth. Arched eyebrows, prominent nasal bridge, narrow nasal wings, and upturned nostrils were common in patients of (2).

Conclusion: Patients with DEE87 could be divided into two major clinical groups: those with mutations in the ATP-binding pocket and those with mutations in the activation segment. Regarding the pathogenesis of DEE87, it has been shown that both loss-of-function and gain-of-function patterns are possible. Further elucidation of the symptoms and pathomechanisms of the disease is needed by collecting informations of patients with DEE87.

Conflict of Interest: None declared.

EP09.002 Cost of exome analysis in patients with intellectual disability: a micro-costing study in a French setting

Anne-Laure Soilly1;2, Charley Robert-Viard 2;3, Celine Besse4, Ange-Line Bruel5, Benedicte Gerard6, anne boland4, Amelie Piton6, Yannis Duffourd5, Jean Muller6;7;8, Charlotte Poe5, Antonio Vitobello5, Arthur Sorlin5, Christophe Philippe5, Frederic Tran Mau Them5, Denommé-Pichon Anne-Sophie5, Thibaud Jouan5, Samy El-Doueiri9, Laurence Faivre5;10, Delphine Bacq4, Bertrand Isidor11, David Geneviève12, Sylvie Odent13;14, Nicole Philip15, Martine Doco-Fenzy16;17, Didier LACOMBE18, Marie-Laure Humbert3, Jean-François Deleuze4, Christine Binquet3, Christel Thauvin-Robinet5;10, Catherine Lejeune3

1CHU Dijon Bourgogne, Délégation à la Recherche Clinique et à l’Innovation, USMR, F-21000, Dijon, France; 2CHU Dijon Bourgogne, Délégation à la Recherche Clinique et à l’Innovation, Unité Innovation, F-21000, Dijon, France; 3CHU Dijon Bourgogne, Inserm, Université de Bourgogne, CIC 1432, Module Épidémiologie Clinique, F21000, Dijon, France; 4Université Paris-Saclay, CEA, Centre National de Recherche en Génomique Humaine (CNRGH), Evry, France; 5Inserm, Université Bourgogne-Franche-Comté, UMR1231, équipe GAD, Dijon, France; 6Laboratoires de Diagnostic Génétique, Hôpitaux Universitaires de Strasbourg, Institut de Génétique Médicale d’Alsace (IGMA), 67000, Strasbourg, France; 7Unité Fonctionnelle de Bioinformatique Médicale appliquée au diagnostic (UF7363), Hôpitaux Universitaires de Strasbourg, Strasbourg, France; 8Inserm UMRS_1112, Institut de Génétique Médicale d’Alsace, Université de Strasbourg, France et CHRU, Strasbourg, France; 9CHU Dijon Bourgogne, Service financier, 21000, Dijon, France; 10CHU Dijon-Bourgogne, CRMR « Anomalies du Développement et syndromes malformatif de l’Est » et Déficiences intellectuelles de causes rares, Fédération Hospitalo-Universitaire Médecine Translationnelle et Anomalies du Développement (TRANSLAD), Dijon, France; 11Service de Génétique Médicale, CHU de Nantes, Nantes, France; 12Département de Génétique Médicale, Centre de Référence Maladies Rares Anomalies du Développement et Syndromes Malformatifs Sud-Languedoc Roussillon, Hôpital Arnaud de Villeneuve, Montpellier, France; 13Service de Génétique Clinique, Centre Hospitalier Universitaire Rennes, F-35203, Rennes, France; 14Centre National de la Recherche Scientifique Unité Mixte de Recherche 6290, Institut Génétique et Développement de Rennes, Université de Rennes 1, F-35203, Rennes, France; 15Département de Génétique Médicale, Hôpital d’Enfants de La Timone, Marseille, France; 16Service de Génétique, CHU de Nantes, Nantes, France; 17CRMR Anddi-Rares constitutif, CLAD-EST, CHU Reims, Reims, France; 18CHU de Bordeaux, Génétique Médicale, INSERM U1211, Laboratoire MRGM, Université de Bordeaux, Bordeaux, France

Aim: To determine an adequate tariff for exome sequencing (ES) in individuals with intellectual disability (ID), we assessed the unit cost per ES diagnostic test from the preparation of the pre-analytical step until the report writing step and identified its main cost drivers.

Methods: A micro-costing approach was conducted for the year 2018 in a French setting as part of the DISSEQ study, a cost-effectiveness study funded by the Ministry of Health and performed in collaboration with the genetic GAD team from the Dijon University Hospital, and a public sequencing platform (CNRGH). All the resources (labor, equipment, disposables and reagents, reusable material) required to analyze samples were collected and valued. Several sensitivity analyses were performed.

Results: The unit cost per ES diagnostic test was estimated to be €2,019.39 Labor represented 51% of the total cost. The analytical step represented 88% of the cost. Sensitivity analyses suggested that a simultaneous price decrease of 20% for the capture kit and 50% for the sequencing support kit led to a cost estimation of €1,769 per ES diagnostic test.

Conclusion: This is the first estimation of ES cost to be done in the French setting of ID diagnosis. The estimation is especially influenced by the price of equipment kits, but more generally by the organization of the centers involved in the ES analysis. This information can now be used to define an adequate tariff and assess the efficiency of ES.

Grant: French Ministry of Health as part of the 2015 Medico-Economic Research Program.

Conflict of Interest: None declared.

EP09.003 Male patient with a novel likely pathogenic variant in HDAC4 causing neurodevelopmental disorder with central hypotonia and dysmorphic facies

Juliane Köhler 1, Nadine Hornig2, Malte Spielmann3

1University of Kiel, Human Genetics, Kiel, Germany; 2University of Kiel, Human Genetics, Kiel, Germany; 3University of Kiel and University of Lübeck, Human genetics, Lübeck, Germany

Background/Objectives: Neurodevelopmental disorder with central hypotonia and dysmorphic facies (NEDCHF; #619797) is a rare autosomal dominant disorder, caused by heterozygous pathogenic variants in HDAC4. So far there are 7 patients described. Here we report a 6 year old patient with NEDCHF and a stop gain variant in HDAC4.

Methods: We acquired a patient’s family history and clinical data. Chromosome banding analysis, array-CGH and exome sequencing were performed using blood samples. The disease’s severity of our patient was assessed and we did literature research for similar patients.

Results: The patient presented with mental retardation, behavioral abnormalities and typical facial features of NEDCHF, such as such as a mild ptosis, deeply set eyes, low-set ears, thin upper lip and pointed chin. Furthermore, he displayed with autism spectrum disorder and eyebrows fanned out medially, features so far not associated with NEDCHF. Chromosome banding analysis and array-CGH showed unremarkable findings. Whole-exome sequencing revealed a heterozygous likely pathogenic variant in HDAC4 (NM_006037.4:c.3208G>T; p.Glu1070*) predicted to lead to a translational stop at position 1070 of in total 1080 amino acids. The variant was not present in the gnomAD database. A segregation analysis is pending.

Conclusion: Here we report an additional patient with NEDCHF and a novel stop gain variant in HDAC4. The variant has so far not been described. Our case report emphasizes the genetic heterogeneity and the variability of the phenotype of variants.

Conflict of Interest: None declared.

EP09.004 The elucidation of the intrafamilial phenotypic heterogeneity of neurodevelopmental disorders by trio-based exome sequencing

Marketa Wayhelova 1;2, Vladimira Vallova1;2, Jan Smetana2, Petr Broz2;3, Aneta Mikulasova4, Dominika Machackova2, Renata Gaillyova5, Petr Kuglik1;2

1University Hospital Brno, Centre of Molecular Biology and Genetics, Brno, Czech Republic; 2Masaryk University, Department of Experimental Biology, Faculty of Science, Brno, Czech Republic; 3Charles University and Motol University Hospital, Department of Biology and Medical Genetics, 2nd Faculty of Medicine, Prague, Czech Republic; 4Newcastle University, Biosciences Institute, Newcastle upon Tyne, United Kingdom; 5University Hospital Brno, Department of Medical Genetics and Genomics, Brno, Czech Republic

The intrafamilial phenotypic heterogeneity of neurodevelopmental disorders represents a challenging issue in the molecular diagnostics of paediatric NDDs and subsequent genetic counselling. Trio-based ES serves as the unique solution to identify multiple genetic hits and explain their contribution to the clinical manifestation of NDDs.

Our study included 89 paediatric patients with NDDs and MCA and their parents. The strategy of trio-based ES utilized the commercial kit Human Core Exome (Twist Biosciences), Illumina NovaSeq 6000 and in-house bioinformatic pipeline.

Trio-based ES has elucidated 49% of paediatric NDDs (44/89), including recurrent and novel causative gene variants affecting the central nervous system development and functioning.

The detailed variant prioritization and analysis uncovered the familial occurrence of causative variants in “OMIM-morbid” genes in fourteen paediatric patients from twelve families. Familial causative variants in “OMIM-morbid” genes or CNVs were detected in seven patients from six families whereas the variable combinations of causative variants in “OMIM-morbid” genes and rare CNVs encompassing “OMIM-morbid” genes were identified in seven patients from six families. The segregation analyses were performed in other familial members, including those with the clinical manifestation of NDDs.

The intrafamilial phenotypic heterogeneity of NDDs may be explained by “two-“ or “multiple-hit” model for NDDs or by dual diagnosis.

Our results shed light on the genetic basis of this phenomenon and demonstrate how trio-based ES can facilitate the molecular diagnostics of paediatric NDDs.

Supported by Ministry of Health of the Czech Republic, grant nr. NU20-07-00145, and conceptual development of research organization (FNBr, 65269705).

Conflict of Interest: None declared.

EP09.005 Ultra-rare causes of growth and speech development delay – new cases of Alazami syndrome and Rauh-Steindl syndrome

Katarzyna Połatyńska 1, Tadeusz Kałużewski2, Magdalena Kacprzak3, Olga Drgas3, Małgorzata Piotrowicz2, Agnieszka Gach2, Łukasz Kępczyński2

1Polish Mother’s Memorial Hospital Research Institute, Department of Neurology, Łódź, Poland; 2Polish Mother’s Memorial Hospital Research Institute, Department of Genetics, Łódź, Poland; 3MedGen Medical Center, MedGen Diagnostic Laboratory, Warszawa, Poland

Background: Alazami syndrome and Rauh-Steindhal syndrome are two ultra-rare monogenic disorders sharing clinical features. Both syndromes are characterized by intellectual impairment, severe growth restriction, and similar, but subtle, facial phenotype. Alazami syndrome (ALAZS, OMIM #615071) is an autosomal recessive disorder caused by biallelic disruption of LARP7 gene and Rauh-Steindhal syndrome (RAUST, OMIM# 619695) is an autosomal dominant disorder resulting from heterozygous pathogenic variant in NSD2 gene.

Material and methods: Here we report two unrelated females of Polish origin (individual 1 and 2), identified by the search for monogenic causes of speech development delay. Both girls presented severe growth restriction, facial dysmorphism and speech development delay. Initial genetic assessment included negative testing for Silver-Russell syndrome, negative SRCAP sequencing for Floating-Harbor syndrome, negative testing for uniparental disomy of 7 and 14 chromosomes, and normal aCGH.

Results: Whole exome sequencing detected compound heterozygosity for in trans LARP7 frameshift variants: Asn252ArgfsTer7 (maternal origin) and Glu286ArgfsTer4 (paternal origin) in individual 1, and heterozygosity for missense NSD2 variant Phe344Val in individual 2.

Conclusions: To our best knowledge LARP7 and NSD2 genes variants identified in our study are novel and were not reported to date, hence this work expands the molecular spectrum of both disorders. Given the shortage of data regarding both conditions, clinical findings described here expand the knowledge of both diseases variability.

Conflict of Interest: None declared.

EP09.006 NSD1 Mutations in Sotos Syndrome Induce Differential Expression of Long Noncoding RNAs, miR646 and Genes Controlling the G2/M Checkpoint

Giuseppina Conteduca1, Davide Cangelosi2, simona coco3, Michela Malacarne1, Chiara Baldo1, Alessia Arado1, rute pinto1, Barbara Testa 1, Domenico Coviello1

1IRCCS Istituto Giannina Gaslini, Laboratory of Human Genetics, Genova, Italy; 2IRCCS Istituto Giannina Gaslini, Clinical bioinformatics unit, Genova, Italy; 3IRCCS Ospedale Policlinico San Martino, Lung Cancer Unit, Genova, Italy

Background/objectives: NSD1 gene (nuclear-receptor-binding-set-domain-protein) encodes a methyltransferase [1] implicated in the transcription and methylation of histone H3 at lysine 36 (H3-K36), but the molecular mechanisms involved in these processes remain largely unknown. An increasing amount of evidence indicates the critical role of the NSD1 gene in Sotos syndrome (SoS), a rare genetic disease, and in tumors [2].

Methods: In order to assess the impact of NSD1 haploinsufficiency in the pathogenesis of SoS, we analyzed the gene expression profile of fibroblasts isolated from the skin samples of 15 SoS patients and of 5 healthy parents.

Results: We identified seven differentially expressed genes and five differentially expressed noncoding RNAs. The most upregulated mRNA was stratifin (SFN) (fold change, 3.9, p < 0.05), and the most downregulated RNA was goosecoid homeobox (GSC) (fold change, 3.9, p < 0.05). The most upregulated lncRNA was lnc-C2orf84-1 (fold change, 4.28, p < 0.001), and the most downregulated lncRNA was Inc-C15orf57 (fold change, -0.7, p < 0.05). A gene set enrichment analysis reported the enrichment of genes involved in the KRAS and E2F signaling pathways, splicing regulation and cell cycle G2/M checkpoints.

Conclusion: Our results suggest that NSD1 is involved in cell cycle regulation and that its mutation can induce the down-expression of genes involved in tumoral and neoplastic differentiation.


1. Douglas J et al., 2003 Am J Hum Genet

2. Mohanty S et al 2020 Cancers

Grants: AssiGulliver Association, Fondazione Sardegna and Banca d’Italia

Conflict of Interest: None declared.

EP09.007 Gonadal mosaicism in FOXP1 related neurodevelopmental syndrome

Anna Zsigmond 1, Kinga Hadzsiev2, Agnes Till1, Judit Bene1, Laszlo Barati1

1University of Pécs, Department of Medical Genetics, Pécs, Hungary; 1University of Pécs, Department of Medical Genetics, Pécs, Hungary

Background: Mutations in FOXP1 gene are responsible for a well characterised neurodevelopmental syndrome, the Intellectual developmental disorder with language impairment with or without autistic features (OMIM 613670). FOXP1 gene encodes the fokhead box protein 1, which is a transcription factor important for the early embryonal development. The main features of the condition are intellectual disability, language deficits, autism spectrum disorder, hypotonia, and congenital anomalies, including mild dysmorphic features, and brain, cardiac, urogenital abnormalities.

Methods, results: Here, we present two siblings with de novo heterozygous FOXP1 variant. The childrens were born from a healthy non-consanguineous couple. Both the four years old boy and the 10 months old girl have delayed psychomotor development, hypotonia, inappropriate laughter and very similar facial features. The four years old boy has no expressive speech. We performed whole exome sequencing for the boy, which identified a pathogenic heterozygous c.1541G>A FOXP1 mutation. His sister’s targeted mutation analysis also showed the same heterozygous FOXP1 variant. The segregation analysis revealed de novo origin of the mutation, thus it suggests gonadal mosaicism.

Conclusion: De novo variants are common causes of rare intellectual disability syndromes, associated with low recurrence risk. However, when such variants occur in parental germ cells, the recurrence risk is much higher. The prevalence of gonadal mosaicism is usually unknown. To the best of our knowledge this is the first report in medical literature of gonadal mosaicism in case of FOXP1 related neurodevelopmental syndrome.

Conflict of Interest: None declared.

EP09.009 Further phenotypical delineation of DLG3 related Intellectual developmental disorder: description of 9 new cases

Marlène Malbos 1, Estelle Colin2, Xavier Le Guillou3, Oana Caluseriu4, Bertrand Isidor5, Benjamin Cogne5, Cyril Mignot6, Boris Keren6, Sacha Weber7, Christel Thauvin-Robinet8;9, Frederic Tran Mau Them8;10, Ange-Line Bruel8;10, Antonio Vitobello8;10, Hana Safraou8;10, Yannis Duffourd8, Christophe Philippe8;10, Denommé-Pichon Anne-Sophie8;10, Laurence Faivre1;9

1Centre de Référence Maladies Rares “Anomalies du Développement et syndromes malformatifs”, Centre de Génétique, FHU-TRANSLAD, CHU Dijon Bourgogne, France, Dijon, France; 2Service de Génétique Médicale, CHU d’Angers, Angers, France, Angers, France; 3Service de Génétique Médicale, Centre Hospitalier Universitaire de Poitiers, BP577, 86021, Poitiers, France, Poitiers, France; 4Department of Medical Genetics, University of Alberta, Edmonton, AB T6G 2H7, Canada, Edmonton, Canada; 5Service de Génétique Médicale, Centre Hospitalier Universitaire de Nantes, Nantes, Pays de la Loire, France, Nantes, France; 6Département de Génétique, Centre de Référence Déficiences Intellectuelles de Causes Rares, Groupe Hospitalier Pitié Salpêtrière et Hôpital Trousseau, APHP, Sorbonne Université, Paris, France, Paris, France; 7Service de Génétique Médicale, Centre Hospitalier Universitaire de Caen, Caen, France, Caen, France; 8Unité Fonctionnelle “Innovation diagnostique dans les maladies rares” laboratoire de génétique chromosomique et moléculaire, Plateau Technique de Biologie, CHU Dijon, Dijon, France, Dijon, France; 9Centre de Référence Maladies Rares “Déficiences Intellectuelles de causes rares”, FHU-TRANSLAD, CHU Dijon Bourgogne, France, Dijon, France; 10Inserm UMR 1231 GAD, Université de Bourgogne-Franche Comté, FHU TRANSLAD, Dijon, France, Dijon, France

Objectives: DLG3 encodes a member of the membrane-associated guanylate kinases protein family, SAP102, expressed in brain and involved in synaptic function. Loss-of-function (LoF) variants in DLG3 are a known cause of non-syndromic X-linked intellectual developmental disorder (ID) and to date, only 13 families have been reported. Limited clinical descriptions do not permit to precise the developmental trajectories of affected individuals. Also, the pathogenicity of missense variants has to be discussed. In this context, we proposed to constitute an international clinical series of novel patients in order to better define the clinical and molecular spectrum of this condition.

Methods: Recruitment of probands was made by using GeneMatcher.

Results: We report 9 new male patients from 2- to 23-year-old presenting ID and developmental delay in association with other clinical features including ocular (strabismus 2/9 and visual impairments 2/9), psychiatric and behavioral disorders (auto- and hetero-aggressiveness, intolerance to changes and anxiety 5/9), but also some other previously unreported features, including cleft palate (1/9), sacrococcygeal cyst (2/9) and pain hypoesthesia (2/9). Variants were generally inherited from the mother (8/9). Four variants were loss-of-function, one is a deletion encompassing the last exon and the stop codon of the gene and one is a delins leading to a predicted splice site effect. Two variants were missense and classified as VUS.

Conclusion: These descriptions broaden the phenotypic spectrum of the X-linked intellectual developmental disorder induced by DLG3 variants. Additional patients with missense variants are required to help in their reclassification.

Conflict of Interest: None declared.

EP09.010 Neurocognitive and neurobehavioral characterization of two frequent forms of neurodevelopmental disorders: the DYRK1A and the Wiedemann-Steiner syndromes

Benjamin Durand 1, Elise Schaefer1, Pauline Burger2, Sarah Baer3, Jean Louis Mandel2, Amelie Piton2;4, Romain Coutelle5;6

1Service de Génétique Médicale, Institut de Génétique Médicale d’Alsace, Hôpitaux Universitaires de Strasbourg, Strasbourg, France; 2Institut de Génétique et de Biologie Moléculaire et Cellulaire, Institut National de la Santé et de la Recherche Médicale U964, Centre National de la Recherche Scientifique UMR7104, Illkirch, France; 3Service de Pédiatrie Spécialisée et Générale, Unité de Neurologie Pédiatrique, Hôpitaux Universitaires de Strasbourg, Strasbourg, France; 4Laboratoire de diagnostic génétique, Hôpitaux Universitaires de Strasbourg, Strasbourg, France; 5Service de psychiatrie de l’enfant et de l’adolescent, Hôpitaux Universitaires de Strasbourg, Strasbourg, France; 6Clinique psychiatrique, INSERM U-1114, Strasbourg, France

DYRK1A and Wiedemann–Steiner syndromes (WSS) are two genetic conditions associated with neurodevelopmental disorders (NDDs). Although their clinical phenotype has been well described, their behavioral phenotype has been little studied and not systematically using standardized assessment tools. To characterize the latter, we conducted a retrospective study, collecting data on developmental history, autism spectrum disorder (ASD) (using the Autism Diagnostic Interview-Revised and the Social Communication Questionnaire), adaptive functioning (using the Vineland Adaptive Behavior Scales II), behavioral assessments (using the Aberrant Behavior Checklist, the Screen for Child Anxiety Related Emotional Disorders and the Conners’ Parent Rating Scale) and sensory processing (using the Sensory Profile) of individuals with these syndromes (n = 14;21). In addition, we analyzed information collected from families (n = 20;20) using the GenIDA database, an international patient-driven data collection aiming to better characterize natural history of genetic forms of NDDs. In the retrospective study, individuals with DYRK1A syndrome showed lower adaptive behavior scores compared to those with WSS, whose scores showed greater heterogeneity. An ASD diagnosis was established for 57% (8/14) of individuals with DYRK1A syndrome and 24% (5/21) of those with WSS. Language and communication were severely impaired in individuals with DYRK1A syndrome, which was also evident from GenIDA data, whereas in WSS patients, exploration of behavioral phenotypes revealed the importance of anxiety symptomatology and attention deficit hyperactivity disorder signs, also flagged in GenIDA. This study, describing the neurobehavioral profiles of individuals with WSS and DYRK1A syndrome, highlighted some specificities important to be considered for patients’ management.

Conflict of Interest: None declared.

EP09.011 Identification of a rare homozygous variant in the gene GPT2, causing a neurodevelopmental disorder in a consanguineous Pakistani family

Zafar Ali1, Ambrin Fatima2, Saadia Maryam Saadi3, Muhammad Jameel4, Shahid M Baig2, Mathias Toft5;6, Zafar Iqbal 6

1University of Swat, Centre for Biotechnology and Microbiology, Swat, Pakistan; 2The Aga Khan University, Department of Biological and Biomedical Sciences, Karachi, Pakistan; 3National Institute for Biotechnology & Genetic Engineering, PIEAS, Human Molecular Genetics Laboratory, Health Biotechnology Division, Faisalabad, Pakistan; 4The Aga Khan University, Centre for Regenerative Medicine and Stem Cells Research, Karachi, Pakistan; 5University of Oslo, Institute of clinical Medicine, Oslo, Norway; 6Oslo University Hospital, Department of Neurology, Oslo, Norway

BACKGROUND: GPT2 is also known as ALT2 enzyme that catalyzes the reversible addition of an amino group from glutamate to pyruvate, producing alanine and α-ketoglutarate, pathogenic variants in GPT2 result in clinically heterogeneous neurodevelopmental disorder (OMIM#616281). The clinical phenotype is variable and extremely heterogeneous and includes the symptoms such as intellectual disability, speech impairment, postnatal microcephaly, spastic paraplegia, delayed developmental milestones.

METHODS: A consanguineous Pakistani family consisting of two affected and two normal individuals along with their phenotypically healthy parents were analysed using whole exome sequencing (WES). The data obtained were filtered against control data bases to remove the polymorphisms. The prioritized variant testing was performed by Sanger sequencing.

RESULTS: The patients were presented with delayed developmental milestones, intellectual disability, microcephaly, speech impairment, aggressive behaviour and behavioural abnormalities. WES data analysis revealed a novel homozygous variant, c.601G>A (p.G201R) in GPT2 (NM_133443.4) gene. The identified variant is highly conserved and was predicted to be “deleterious” by various bioinformatic prediction tools. The predicted pathogenicity score using CADD was 22.7. The variant was also not found in homozygous form in public variant databases such as gnomAD, 1000G and dbSNP. Sanger sequencing confirmed the segregation of the GPT2: c.601G>A (p.G201R) with the phenotype.

CONCLUSIONS: This study does not not only expand the genetic variability associated with this phenotype but also demonstrate the usefulness of WES in clinical heterogeneous disorders.

Conflict of Interest: None declared.

EP09.012 Case report of a hemizygous intragenic deletion of WDR13 - Further evidence for the role of WDR13 in X-linked intellectual disability

Kirsten Cremer 1, Tim Bender1;2, Hartmut Engels1, Stefanie Heilmann-Heimbach1, Hela Hundertmark1, Pietro Incardona3, Claudia Perne1, Axel Schmidt1, Jessica Trautmann1, Sophia Peters1

1Institute of Human Genetics, School of Medicine, University of Bonn - University Hospital Bonn, Bonn, Germany; 2Center for Rare Diseases Bonn, School of Medicine, University of Bonn - University Hospital Bonn, Bonn, Germany; 3Core Unit for Bioinformatics Data Analysis, Medical Faculty, University of Bonn, Bonn, Germany

Introduction: X-linked intellectual disability (XLID) accounts for over 10% of all cases of ID in males. Next generation sequencing techniques have enabled the detection of a multitude of novel causative and candidate genes for ID over the last years. To date, more than 150 causative genes for XLID and several candidate genes have been reported.

Case Report: We report on an 8-year-old boy with global developmental delay, significantly delayed speech and language development, intellectual disability (IQ 50), mild postnatal macrocephaly, obesity, behavioural abnormalities and bilateral leg spasticity with pointed foot position. Furthermore, he showed minor facial dysmorphism and brachyphalangy.

Methods and Results: Conventional cytogenetic analysis, microarray CGH analysis and testing for fragile X syndrome gave normal results. Trio exome sequencing revealed a hemizygous deletion of exons 6-10 of the WDR13 gene (NM_001347217.2: c.523+68_*2393del;p.(Val175Glyfs*21)) in our patient, which was confirmed by quantitative PCR. The patient’s healthy mother is a heterozygous carrier of the variant.

Conclusion: WDR13 (OMIM *300512) is a poorly characterized gene previously proposed as a candidate gene for XLID. Here we present an intragenic deletion of WDR13 in a boy with intellectual disability and his healthy mother. To our knowledge, there are only four descriptions of WDR13 variants as a potential cause of learning disability and ID in the literature so far (PMIDs: 20655035, 20662849, 29302074, 34946860). Our case is a further indication that WDR13 is a causative gene for XLID. A GeneMatcher search to identify additional patients is ongoing.

Conflict of Interest: None declared.

EP09.013 Identification of a novel de novo missense variant in FBXO11 and a known variant in TECTA

Sinje Geuer 1, Victoria Strohhäcker1, Silke Hartmann1

1MVZ genetikum GmbH - Center for Human Genetics, Neu-Ulm, Germany

Background: We report on a 7 year old female child with speech delay, intellectual disability, congenital hearing impairment, recurrent infections of the lung with febrile convulsions. Her father as well as her paternal grandmother suffer from progressive hearing impairment.

Methods: Array-CGH (180k Agilent, design 086332) and trio whole exome sequencing (TWES, Twist Human Core Exome with additional customized probes and NGS with NovaSeq Illumina)

Results: Array-CGH of the child gave normal results. By trio analysis the heterozygous novel de novo missense variant c.1715C>T p.(Ala572Val) in FBXO11 (NM_001190274.2) was identified in the child.

Missense variants in FBXO11 cause syndromic neurodevelopmental delay with variable degree of intellectual disability. Rarely patients have seizures. Additional common features include short stature and distal mild skeletal malformations. Most patients have behavioral abnormalities and dysmorphic features (Gregor et al., 2018). In comparison to this reported phenotypical spectrum, our patient has a rather mild manifestation without skeletal or behavioral abnormalities. Noteworthy, to our knowledge, susceptibility to recurrent infections was not reported so far.

In the father, the known pathogenic heterozygous variant c.5597C>T p.(Thr1866Met) in TECTA (NM_005422.4) was identified, which was present in the daughter as well. This variant is known to cause autosomal dominant hearing impairment with pre- or postlingual manifestation. Therewith, this variant in TECTA explains the hearing impairment in father and daughter.

Conclusion: This case report highlights the heterogeneity FBXO11 related neurodevelopmental delay. Furthermore, this example emphasizes the need of considering multiple genetic diseases as causal for different symptoms in one family.

Conflict of Interest: None declared.

EP09.014 Clinical exome sequencing for diagnosis of patients with intellectual disability, autism and psychomotor delay

Helena Paszeková 1, Kristýna Hanuláková1, Tomáš Píš1, Věra Hořínová2, Zděnka Vlčková1, Renáta Michalovská1

1GHC GENETICS, s.r.o., Prague, Czech Republic; 2Jihlava Hospital, Jihlava, Czech Republic

Background/Objectives: Intellectual disability, autism and psychomotor delay are common features seen in several rare inherited syndromes and diseases, which often exhibit a wide range of severity and accompanying symptoms. The aim was to use clinical exome sequencing to identify the underlying genetic cause of these disorders.

Methods: We utilized the Clinical Exome Solutions (CES v3) panel by Sophia Genetics to perform massive parallel sequencing on NextSeq550. CES v3 covers the coding regions (± 5bp of intronic regions) of 4,728 genes, the entire mitochondrial genome and non-coding variants known to be associated with rare and inherited disorders. The SOPHiA DDM™ platform was used to analyze the data and identify variants, including SNVs, Indels, and CNVs.

Results: A 14-year old girl had a personal medical history of atypical autism and suspected Rett syndrome. Using CNV analysis, we have reported a de novo heterozygous deletion in PHIP gene affecting exons 10-15. The patient was then diagnosed with the autosomal dominant syndrome Chung-Jansen. Similarly, in a 8-year old girl with psychomotor delay, we identified a de novo frameshift heterozygous variant c.552_553delTA (p.Asp184GlufsTer14) in WAC gene, leading to a diagnosis of the autosomal dominant syndrome Desanto-Shiwani. Both syndromes are characterized by varying degrees of developmental delay, impaired intellect, learning disabilities and behavioral abnormalities, such as autistic features, ADHD, anxiety, aggression, impulsive behavior and mood swings.

Conclusion: The use of clinical exome sequencing proved to be beneficial in determining the final diagnosis for patients with intellectual disability of different clinical manifestations caused by rare inherited disorders.

Conflict of Interest: Helena Paszeková research associate, Kristýna Hanuláková research associate, Tomáš Píš research associate, Věra Hořínová clinical geneticist, Zděnka Vlčková clinical geneticist, Renáta Michalovská head of laboratory.

EP09.016 Patient with characteristic features of Lamb- Shaffer syndrome. Case study

Katarzyna Wojciechowska 1, Whitley Zie2, Joanna Rusecka3, Monika Lejman4

1Medical University of Lublin, Independent Laboratory of Genetic Diagnostic, Lublin, Poland; 2Medical University of Lublin, Independent Laboratory of Genetic Diagnostic, Lublin, Poland; 3MedGen Medical Centre, Warsaw, Poland; 4Medical University of Lublin, Independent Laboratory of Genetic Diagnostic, Lubin, Poland

Background: Lamb-Shaffer syndrome is a neurodevelopmental disorder characterized by global developmental delay, intellectual disability, poor expressive speech, and mild dysmorphic facial features.

Our aim is to discuss the case of a patient with a new heterozygous mutation in SOX5 gene.

Methods: Molecular analysis showed a new, de novo heterozygous mutation p.Arg426Ter in SOX5 gene. The genetic analysis was performed by whole-exome sequencing. The library was prepared using Agilent Sure select VI exome Kit and analyzed with a NovaSeq sequencing platform. The presence of the detected variant was confirmed by Sanger sequencing.

Results: Our patient was born on time of healthy, nonconsanguineous parents. Her birth weight was -3250g, length- 55 cm, OFC-34 cm. Array CGH testing showed normal balanced female karyotype. The whole-exome sequencing showed mutation in SOX5 gene. Among the main health problems of our patient at the age of five years were: speech delay, delayed motor development (she started sitting without support at the age of 9 months, walking at the age of 22 months), dysmorphic facial features, strabismus, wide toes and hypoplasia of cerebellar vermis.

Conclusion: The case of our patient contributes to the studies of the phenotypes of patients with Lamb-Shaffer syndrome which occur as a result of SOX5 mutation.

Conflict of Interest: Katarzyna Wojciechowska full, Whitley Zie Student, Joanna Rusecka full, Monika Lejman Full.

EP09.017 Co-occurrence of Vissers-Bodmer syndrome and a likely pathogenic CHD8 variant: a case report

André Travessa 1, Marcello Niceta2, Carla Mendonça3, Noémia Rosado da Silva3, Ana Berta Sousa1

1Medical Genetics Department, Hospital de Santa Maria, Centro Hospitalar Universitário Lisboa Norte, Lisbon, Portugal; 2Molecular Genetics and Functional Genomics Research Unit, Ospedale Pediatrico Bambino Gesù, IRCCS, Rome, Italy; 3Centro de Neuropediatria e Desenvolvimento, Hospital de Faro, Centro Hospitalar Universitário do Algarve, Faro, Portugal

Background/Objectives: In 2014, heterozygous CHD8 variants were found to cause intellectual disability (ID)/developmental delay (DD) with autism and macrocephaly. In 2020, the eponym “Vissers-Bodmer syndrome” was adopted to characterize a neurodevelopmental disorder caused by disease-causing CNOT1 variants. Here, we report a patient with co-occurrence of pathogenic variants in CNOT1 and CHD8 genes.

Case Report: We report a 5-year-old boy with unremarkable family history. The prenatal and the neonatal periods were uneventful. He evolved with severe DD and autism. On physical examination, he has relative microcephaly, mild dysmorphic features, and asymmetry of the calves. Electroencephalogram, head magnetic resonance imaging and extensive metabolic studies were normal. While genetic workout including array-CGH and fragile-X testing was normal, whole-exome sequencing identified two heterozygous likely pathogenic variants: a de novo variant in CNOT1, c.6916C>T, p.(Arg2311Trp), and a variant in CHD8, c.5393G>A, p.(Trp1798*) inherited from his healthy father.

Conclusion: Herein, we report a case of co-occurrence of variants in CNOT1 and CHD8 genes. Both genes codify for proteins that are believed to modify the epigenetic machinery. The patient’s father, who harbors the variant in CHD8, is healthy, suggesting that even truncating CHD8 events can be not sufficient to promote disease. In our subject, the contribution of the CHD8 variant on top of the Vissers-Bodmer syndrome phenotype, however, is difficult to ascertain. Multi-omics approaches also including the methylome analysis are required to confirm the occurrence of more than one causal variant in the same patient, complicating the (reverse) phenotyping and making genetic counselling very challenging.

Conflict of Interest: None declared.

EP09.018 Two novel mutations in ANKRD11 gene in KBG syndrome patients and incomplete penetrance of missense mutations

Nada Amllal 1;2, siham chafai elalaoui1;2, maria zerkaoui3, amal chiguer1;2, lamia afif1;2, amal thimou izgua3, abdelaziz sefiani1;2, lyahyai jaber1;2

1Faculty Of Medicine And Pharmacy Of Rabat, Rabat, Morocco; 2National Institute of Hygiene, Medical Genetics, Rabat, Morocco; 3Chu Ibn Sina, Center of consultations and external explorations, Rabat, Morocco

Background: KBG syndrome is a rare genetic disorder caused by either pathogenic variants in ANKRD11 gene or the 16q24.3 microdeletion involving this latter. It is characterized by a distinctive facial appearance, typically macrodontia of upper central incisors, intellectual disability, seizures, developmental delay, skeletal abnormalities and short stature. The severity of KBG syndrome vary greatly, even among individuals with the same genetic mutations. In this work, we report two Moroccan children with typical KBG syndrome, and discuss the incomplete penetrance of missense ANKRD11 mutations by comparing the phenotypes of patients with inherited pathogenic variants and their transmitting parents.

Methods: Both patients have typical KBG syndrome phenotype. Clinical Exome Sequencing was performed on both patients. Segregation analysis was done via conventional Sanger sequencing in both patients’ parents. KBG syndrome cases with inherited pathogenic mutations were collected from literature. Our cases were compared to those reported in the literature.

Results: The first patient harbors a monoallelic missense mutation that was inherited from her mother while the second one harbors a monoallelic de novo frameshift mutation. The analysis of collected clinical data shows that most of the transmitting parents tend to have mild features. Our first patient’s parent does not have any relevant clinical sign, which may be explained by the location of the mutation on protein level.

Conclusion: By reporting new cases and analysing clinical data of patients with inherited ANKRD11 pathogenic variants that are increasingly observed in KBG syndrome, we confirm the variable expression and incomplete penetrance of this syndrome.

Conflict of Interest: None declared.

EP09.019 Characterization of blood and urinary levels of markers of elastin metabolism in 7q11.23 imbalances (Williams and 7q11.23 microduplication syndromes)

Severine Ruet1, William Dufour2, Cecile Acquaviva1, Massimiliano Rossi 2

1CHU Lyon, Service Biochimie et Biologie Moléculaire Grand Est, UF Maladies héréditaires du métabolisme, Bron, France; 2CHU Lyon, Service de génétique, Centre de référence des anomalies du développement, Bron, France

Williams syndrome (WS), due to 7q11.23 microdeletion, is characterized by arterial stenoses, congenital abnormalities and a typical neurocognitive profile. The vasculopathy is the main cause of mortality. 7q11.23 duplication syndrome (7qDS) is characterized by aortic dilatation and variable neurodevelopmental involvement. The 7q11.23 critical region encompasses 26-28 genes including ELN, whose haploinsufficiency causes the WS vasculopathy; the pathogenesis of aortic dilatation in 7qDS is not known. ELN encodes tropoelastin, which undergoes post-translational modifications of specific lysine residues into desmosine and isodesmosine (D&I), which can be considered as specific biomarkers of elastin metabolism. Our study aims to characterize the blood and urinary levels of D&I in WS (1 ELN copy), normal controls (2 ELN copies) and 7qDS (3 ELN copies).

We set up D&I analysis by Liquid Chromatography/tandem Mass Spectrometry, not previously available in France in a routine setting. Statistical analysis was performed by Mann-Witney non-parametric test.

We recruited 23 WS (0.9-32y), 7 7qDS (4-43y), and 39 unaffected individuals (gender/age matched). Urines D&I/creatinine levels varied depending on age and were significantly lower in WS patients aged <2y (7.1[3.9-8.6] vs controls: 29,4[8.4-97.6], p < 0.05) and >15y (1.7[0.9-2.4] vs controls: 2,4[1.2-8.3], p < 0.05). D&I levels were normal in WBS serum and in 7qDS serum/urine samples. No obvious correlation was noted with vascular involvement severity.

This study suggests that urinary D&I might be considered as potential biomarkers of elastin metabolism for WS patients (<2y and >15y), in the perspective of the development of future therapeutic trials. Further studies are needed to characterize other markers of elastin biosynthesis.

Conflict of Interest: None declared.

EP09.020 A novel pathogenic mutation in ARID1B gene in a child with syndromic diabetes mellitus - presentation of Coffin-Siris syndrome

Kalina Belemezova 1, Ivamina Baltova1, Dzhaner Bashchobanov1, Margarita Archinkova1, Radka Savova1;2, Ivanka Dimova1

1Medical University - Sofia, Sofia, Bulgaria; 2Specialized Hospital of Pediatry, Sofia, Bulgaria

Background/Objectives: We present a case of a 6-year-old girl with insulin-dependent diabetes mellitus developed at 2 years of age; intellectual deficit/autism, with a severe delay in speech; muscle hypotonia and difficult eating. Parents are interested in the genetic etiology of the condition.

Methods: Clinical exome sequencing by NGS was performed covering the coding regions of about 5000 genes.

Results: A genetic variant c.2025C>G, p.(Tyr675*) was detected in ARID1B gene. This variant is described for the first time and generates a premature stop codon, resulting in a shortened and damaged protein – we defined it as possibly pathogenic. Mutations in ARID1B are connected to the development Coffin-Siris syndrome (CSS). Differentiation of cranial neural crest cells is disturbed, thus explaining the expressed intellectual deficit.

Discussion: CSS is a rare autosomal dominant genetic disorder with de novo arising mutations. It is characterized by retardation in mental and physical development, microcephaly, thick lips, flat nasal root, hypoplasia of the distal phalanges (most commonly the 5th finger) and other facial features. The disease is associated with neurological problems (EEG changes, epilepsy, low IQ) and endocrinological changes; visual and hearing problems.

Conclusion: Next-generation sequencing is a powerful tool in making an accurate diagnosis in children suffering from diabetes in combination with symptoms from other organs and systems and dysmorphism. Our case presents syndromic diabetes mellitus due to Coffin-Siris syndrome and supports subsequent clinical behavior in the child.

Grant References: The study was supported by Grant No7400/19.11.2021 of Council of Medical sciences of Medical University Sofia.

Conflict of Interest: None declared.

EP09.021 Noonan-like phenotype with pathogenic variant in FBXW11

Olga Levchenko 1, Sabina Nagieva1, Alexander Lavrov1

1Research Centre for Medical Genetics, Moscow, Russian Federation

Background/Objectives: FBXW11 is a member of the ubiquitin ligase complex. It’s involved in the Wnt and Hh signaling. Its role in human disorders is poorly known. Only 7 patients were described with pathogenic variants in FBXW11 which cause autosomal dominant development disorders. One patient had Noonan-like phenotype. Noonan syndrome is associated with the RAS-MAPK pathway. Ubiquitination is involved in Ras metabolism and FBXW11 may play a role in its signalling.

Methods: Analysis of the Whole exome sequencing (WES). Sanger sequencing for variant confirmation and trio analysis.

Results: The patient is a Caucasian 5-years old boy. He was born to healthy parents. At birth: weight 3300 g, length 50 cm, Apgar score 8/8. His motor development was delayed. He spoke his first words at 2 years, simple sentences appeared at 4.5 years. Self-care skills are almost formed. His current height is 102.5 cm (-1.47SD), weight is 17 kg (-0.72SD), OD is 54.5 cm (+2.44SD). Phenotype features: low-set protruding ears, strabismus, crease of the lower eyelid, flat nose bridge, wide nose tip, long smooth philtrum, talipes valgus. High degree of hyperopia. MRI revealed Arnold’s-Chiari malformation type I, EEG was normal, no variant was found by WES. Reanalysis of the WES data revealed a heterozygous missense variant NM_012300.2:c.793T>C(Trp265Arg) in FBXW11. De novo origin of the variant was confirmed by trio Sanger sequencing. The variant was classified as likely pathogenic according to ACMG recommendations.

Conclusions: This is the second case of the Noonan-like syndrome caused by a likely pathogenic de novo variant in FBXW11.

Conflict of Interest: None declared.

EP09.022 Case report of rare neurodevelopmental Menke–Hennekam Syndrome

Rasa Traberg 1, Rasa Ugenskiene1, Kristina Aleknavičienė1

1Lithuanian University of Health Sciences, Medical academy, Department of Genetics and Molecular medicine, Kaunas, Lithuania

Background: Menke–Hennekam Syndrome (MHS) is rare genetic multiple congenital anomalies/dysmorphic disorder characterized by variable intellectual disability, developmental delay, autistic behaviour, short stature, and microcephaly. MHS type 1 is caused by missense variants of CREBBP gene, located in exons 30 or 31 and it is allelic disorder for Rubinstein–Taybi syndrome.

Methods: The proband is 7-year-old female who was born at 39 weeks of gestation from normal pregnancy, birth weight 2495 g (<3‰), birth hight 48 cm. Her mother received growth hormone during adolescence. Global developmental delay was noticed in the first year of life. At 5 years of age, she was assessed by scale of Diagnostic inventory for screening children, 1984 (DISC). Test value was 38-61%. Later, she had learning disabilities and special school needs. Paediatric endocrinologist assessed her for short stature multiple times and growth hormone deficiency diagnosis was rejected. At 4 years of age sleep EEG showed bilateral frontotemporal epileptic activity and epilepsy diagnosis was made. Dysmorphic facial features were noticed: strabismus, short and upslanted palpebral fissures, telecantus, depressed nasal ridge, short nose, anteverted nares, short columella, and long philtrum.

Result: Whole exome sequencing (WES) showed likely pathogenic missense variant in CREBBP (NM_004380.3) gene c.5128T>C (p.Cys1710Arg) (PP3 strong, PM1 moderate, PM2 moderate, PP2 supporting) in the 30th exon. Only the mother was available for genetic testing and she was negative for the variant.

Conclusions: The proband’s phenotype and genotype was compatible with diagnosis of rare neurodevelopmental Menke–Hennekam Syndrome. Appropriate genetic counselling was provided.

Conflict of Interest: None declared.

EP09.023 ITGB8 is a candidate disease gene for recessive trait form of neurological disease

Scott Barish1, Elisa Lorefice 2, Chiara Passarelli3, Monia Ginevrino3, Diego Martinelli4, Antonio Novelli4

1Baylor College of Medicine, Department of Molecular and Human Genetics, Houston, United States; 2Children Hospital Bambino Gesù, Rome, Italy is the institution, Rome, Italy; 3Bambino Gesù Children Hospital, IRCCS, 00146 Rome, Italy, Laboratory of Medical Genetics, Translational Cytogenomics Research Unit, Rome, Italy; 4Bambino Gesù Children Hospital, IRCCS, 00146 Rome, Italy, Division of Metabolism, Rome, Italy

Background/Objectives: Integrins are a family of cell adhesion molecules that facilitate cell-cell and cell-extracellular matrix (ECM) contact. Integrins form heterodimers of an alpha and a beta subunit. There are 26 integrins in the human genome (18 alpha, 8 betas), nine of which have been associated with Mendelian conditions. Integrins have been primarily associated with skin diseases such as epidermolysis bullosa (MIM #226730) and none have been associated with neurological disease to date.

Methods: Whole exome sequencing was performed on proband and parents’ genomic DNA, when possible, and sequenced on the Illumina NovaSeq6000 platform. The variants were filtered by in silico analysis considering the frequency in the public databases and the prediction of deleterious non-synonymous SNVs for human diseases. Global minor allele frequency (MAF) was calculated according to gnomAD. The variants were evaluated by VarSome and categorized in accordance with the ACMG recommendations.

Results: We identified two siblings with putatively pathogenic variants in ITGB8 (NM_002214.3). The probands are compound heterozygous for the start-loss c.1A>C (p.Met1?) and the splicing variant c.1056+1G > A. Functional studies on cDNA of both patients confirmed the impairment of the transcription, with the loss of an allele and the skipping of exon 7. These probands present with neurological phenotypes including developmental delay, intellectual disability, microcephaly, and dysmorphic features, and an altered Interferon-signature.

Conclusions: Together, our data suggest that variants in ITGB8 could be associated with a recessive neurodevelopmental rare disease trait.

Conflict of Interest: Scott Barish: None declared, Elisa Lorefice: None declared, Chiara Passarelli Biologist in Laboratory of Medical Genetics, Children Hospital Bambino Gesù, Rome, Monia Ginevrino: None declared, Diego Martinelli: None declared, Antonio Novelli: None declared.

EP09.024 Extension of the phenotypic manifestation of the Shukla-Vernon syndrome with renal and cardiac malformations?

Predrag Noveski 1, Ivana Maleva Kostovska1, Velibor Tasic2, Dijana Plaseska-Karanfilska1

1Research Center for Genetic Engineering and Biotechnology “Georgi D. Efremov”, Macedonian Academy of Sciences and Arts, Skopje, Macedonia; 2University Children’s Hospital, Medical Faculty Skopje, Skopje, Macedonia

Background/Objectives: Pathogenic variants in the transcriptional corepressor BCORL1 were recently associated with Shukla-Vernon syndrome (SHUVER), X-linked disorder manifested with intellectual disability, dysmorphic features and behavioural abnormalities. BCORL1 is highly constrained for the loss-of-function (LoF) mutations (pLI=1, o/e = 0.05), and so far, only hemizygous missense (Z = 2.06, o/e = 0.79) mutations were reported in the affected patients. Here, we present a case with LoF mutation with additional clinical presentation, not reported previously in SHUVER patients.

Patient and Methods: Eleven-month male infant with psychomotor delay, hypotonia, dysmorphic features, polythelia, digital hand contractures, crossed fussed ectopia of the left hypoplastic kidney with hydronephrosis of the right kidney, atrial septal defect and patent ductus arteriosus, was admitted for genetic diagnosis. Conventional karyotyping, aCGH with CytoScan 750K Array, clinical exome sequencing with Illumina TruSight One kit and Sanger sequencing were performed.

Results and Discussion: After normal findings of 46,XY karyotype and aCGH result, clinical exome analysis revealed presence of novel pathogenic hemizygous frameshift mutation BCORL1:c.2706dupT (p.Thr903TyrfsTer14). Sanger sequencing showed that mutation was a de novo event (not present in proband’s healthy mother; biological relatedness confirmed). Renal and cardiac malformations were additional symptoms to those previously described in SHUVER. Interestingly, pathogenic mutations in BCOR, another transcriptional corepressor and paralog of the BCORL1, are associated with similar cardiac malformations and laterality disorders, which implies possible overlap in disease manifestation in patients with LoF mutations in these two genes.

Conclusion: Recognizing and reporting genotype-phenotype correlation in patients with pathogenic BCORL1 mutations will further improve the SHUVER characterization.

Conflict of Interest: None declared.

EP09.025 Co-occurrence of two rare autosomal recessive disorders due to double heterozygosity in RYR1 and BCS1L genes in a patient from non-consanguineous family

Emilija Shukarova Stefanovska 1, Gjorgji Bozhinovski2, Gordana Kiteva-Trenchevska3, Dijana Plaseska-Karanfilska2

1Macedonian Academy of Sciences and Arts, Research Center for Genetic Engineering and Biotechnology “Georgi D. Efremov”, Skopje, Macedonia; 1Macedonian Academy of Sciences and Arts, Research Center for Genetic Engineering and Biotechnology “Georgi D. Efremov”, Skopje, Macedonia; 3University Neurology Clinic, Medical Faculty, Skopje, Macedonia

Pathogenic variants in RYR1 gene are the most common cause of congenital muscle weakness, congenital myopathies and malignant hyperthermia. On the other hand, BCS1L pathogenic variants cause widely different clinical phenotypes, such as mild Björnstad syndrome, characterized with abnormal hair growth and sensorineural hearing loss, severe GRACILE syndrome, characterized by growth restriction, aminoaciduria, iron overload, lactic acidosis and early death, and a heterogenous condition of complex III deficiency, involving hepatic pathologies, generalized hypotonia, hyperreflexia, cerebral atrophy and global developmental delay.

Here we present a 17 years old girl of Macedonian origin, the first child born from non-consanguineous parents, presented at infancy with severe hypotonia/hemiparesis, congenital deafness and no hair. As growing, severe form of intellectual disability arose. Epileptic seizures started at five years of age, tonic-clonic seizures presented mainly during sleeping. The Whole Exome Sequencing analysis revealed pathogenic variants in two genes: c.548G>A; p.Arg183His inherited from the father and c.871C>T, p.Arg291*, inherited from the mother in BCS1L gene; and c.13013_13032del, p.Ala4338fs inherited from the father, and c.3128G>A, p.Arg1043His, inherited from her mother in RYR1 gene. Our patient presented symptoms associated with both BCS1L and RYR1 genes; while deafness, brittle hair, seizures and intellectual disability are most probably due to only BCS1L gene defects, the generalized muscular weakness might be caused by overlapping effect of both genes. To the best of our knowledge, this is a first presentation of a patient with co-occurrence of RYR1 and BCS1L related disorders.

Conflict of Interest: Emilija Shukarova Stefanovska Macedonian Academy of Sciences and Arts, Gjorgji Bozhinovski Macedonian Academy of Sciences and Arts, Gordana Kiteva-Trenchevska University Neurology Clinic, Medical Faculty, Skopje, Dijana Plaseska-Karanfilska Macedonian Academy of Sciences and Arts.

EP09.026 First description of likely pathogenic variants in ACTB causing Baraitser-Winter cerebrofrontofacial syndrome without intellectual disability in two generations

Juliette Coursimault 1, severine drunat2, Stéphane Marret3, François Lecoquierre1, Alain Verloes4, Alice Goldenberg1

1Normandie Univ, UNIROUEN, Inserm U1245 and CHU Rouen, Department of Genetics and Reference Center for Developmental Disorders, F-76000 Rouen, France, Rouen, France; 2Genetics Department, APHP-Robert Debré University Hospital, Paris, France, paris, France; 3Department of Neonatal Pediatrics, Intensive Care and Neuropediatrics, Rouen University Hospital, 76000 Rouen, France, rouen, France; 4Genetics Department, APHP-Robert Debré University Hospital, Université de Paris, Paris, France, Paris, France

Background: Baraitser–Winter cerebrofrontofacial syndrome (BWCFF) (BRWS; MIM #243310, 614583) is a rare developmental disorder characterized by distinctive facial features, intellectual disability, brain malformations, seizures, short stature, coloboma and sensorineural deafness. BWCFF is caused by de novo heterozygous missense variants, with a suggested gain-of-function effect, in ACTB (MIM 102630) or ACTG1 (MIM 102560), both genes encoding cytoplasmic actin proteins. BWCFF patients commonly show global developmental delay and/or intellectual disability, of variable severity, related to the severity of brain malformations. There are no report of individuals presenting without intellectual disability associated with ACTB variants. In addition, almost all reported cases were sporadic with only three descriptions of family inheritance.

Methods: Gene panel sequencing including ACTB and ACTG1 was performed in three children. Their symptomatic parents benefited from a targeted sequencing. We collected clinical data for all five individuals.

Results: We describe here for the first time the case of five patients, from two different families, presenting with typical craniofacial features and malformations, but no developmental delay nor intellectual disability. Both adult patients had a normal autonomous life. A Noonan syndrome was initially suspected in two individuals. Gene panel sequencing detected two distinct probably pathogenic, heterozygous, missense variants in ACTB. Variants were inherited from their symptomatic parents also presenting with a mild phenotype, conferring a diagnosis of BWCFF in these individuals.

Conclusion: Our observation broadens the phenotypic spectrum associated with ACTB and underlines the existence of milder forms without disabilities.

Conflict of Interest: None declared.

EP09.027 Two new cases with HMGB1 loss-of-function variants

Marlene Rio 1, Thomas Courtin1, Sophie Rondeau1, marion lesieur-sebellin1, Tania Attie-Bitach1, Valérie Cormier-Daire1

1Necker Hospital, APHP Center, University Paris Cité, Genomic Medicine of Rare Disorders, PARIS, France

Background/Objectives: Heterozygous HMGB1 loss-of-function variants are assocated with a newly neurodevelopmental disorder characterized mainly by developmental delay and microcephaly To date, only 6 patients have been reported. We report two new cases in order to better characterize the phenotype of patients with pathogenic variants of HMGB1 gene.

Methods: We present clinical and molecular characterisation of two unrelated patients with de novo variants in HMGB1. Clinical data was obtained by retrospective file analysis, clinical exam and formal neuropsychological evaluation

Results: Case 1, a 14-year-old female, had speech delay, short stature, microcephaly and overweight. Neuropsychological assessment showed cognitive score in the lower limit. She had a history of feeding difficulties in neonatal period and aortic coarctation with bicuspid aortic valve.

Her brain MRI showed cerebral atrophy with simplified gyral pattern. Case 2, a 7-year-old boy, had motor and speech delay, and autism spectrum disorder. His growth was normal, except microcephaly. His brain MRI was normal. The two patients exhibited dysmorphic features with thin upper lip.

Conclusion: An extensive revision of the clinical features of these two patients revealed high concordance with the 6 cases previously reported, including developmental delay with speech delay, feeding difficulties, microcephaly, short stature and overweight. Cerebral atrophy with simplified gyral pattern present in one case expand the spectrum of this new condition.

Conflict of Interest: None declared.

EP09.028 Novel truncating variant reinforces the involvement of ZNF148 in autism spectrum disorder with normal neuroimaging

Lourdes Vega Hanna 1, Clara Serra-Juhé1, Susana Boronat2, Benjamin Rodríguez-Santiago1, Manel Baena1, Ivon Cuscó1

1Hospital de la Santa Creu i Sant Pau, Nou Hospital, Genetics, Barcelona, Spain; 2Hospital de la Santa Creu i Sant Pau, Nou Hospital, Pediatrics, Barcelona, Spain

Background: Heterozygous variants in ZNF148 gene, a Krüppel-type transcription factor that has transcriptional regulatory functions, cause an intellectual disability syndrome characterized by global developmental delay, abnormalities of corpus callosum and dysmorphic facial features. Such variants have only been described in five patients worldwide, of which only one presented with autism spectrum disorder (ASD).

Case report: We report a patient with a novel, de novo, heterozygous truncating variant c.1160delC resulting in a premature termination signal in the protein sequence (p.P387Hfs*4) in the ZNF148 gene.

The patient is a 9 year-old female that presents with mild intellectual disability, ASD, short stature, strabismus and mild facial dysmorphic traits, including long palpebral fissures, anteverted nares, thick lips and prominent chin. Brain magnetic resonance imaging showed normal brain development, without abnormalities of the corpus callosum. Abdominal and cardiac ultrasounds were normal.

Conclusions: During the last decade, Next Generation Sequencing techniques have greatly advanced the identification of new genes related with neurodevelopmental disorders. However, in many cases a thorough clinical description is lacking. Case reports help to better characterize the clinical manifestations.

Here, we describe a novel ZNF148 heterozygous truncating variant in a patient with distinct phenotype of ASD and normal brain imaging which expands the genotype-phenotype spectrum of ZNF148, reinforcing its role as a potential target gene for ASD.

Grant references: not applicable.

Conflict of Interest: None declared.

EP09.029 Dual genetic diagnosis in a patient with complex ocular-neurobehavioral phenotype

Aleksandra Pietrzyk 1, Małgorzata Stawicka- Niełacna1, Agnieszka Łobodzińska2, Olga Drgas2, Anna Lipińska2, Agnieszka Sobczyńska-Tomaszewska2, Tomasz Huzarski1

1Institute of Medical Sciences, Collegium Medicum, University of Zielona Góra, Department of Clinical Genetics, Zielona Góra, Poland; 2Medgen Medical Centre, Warsaw, Poland

Objectives: The advent of genome-wide analyses based on next generation sequencing (NGS) enabled discovery of novel mutations, disease causing genes as well co-existence of two or more Mendelian disorders in one patient known also as dual/ multiple genetic diagnoses (DGD/MGD). MGD are identified in 2-5% patients tested with NGS methods but the data regarding its specifics is scarce. In this report we present a patient diagnosed with SETBP1- and CNGB3- related disorders.

Methods: A 28-year-old female was referred for a consultation due to moderate intellectual disability (ID), behavioural abnormalities and visual impairment. Upon examination unspecific dysmorphic features were observed. Firstly, array-CGH was performed, giving negative result. Then NGS analysis was ordered, revealing two heterozygous frameshift variants of known pathogenicity in CNGB3 gene, correlated with achromatopsia. Family testing confirmed carrier status in parents and biallelic nature of detected variants in patient. However the result did not explain the cause of ID observed in patient. Further extensive analysis of NGS data revealed a novel heterozygous frameshift variant in SETBP1 gene, which was of de novo origin.

Conclusion: MGD should be considered in patients with complex phenotypes, involving many organs or manifestations that are not clearly related. NGS based methods are of preference. Symptoms, which are not precisely explained by detected variants should be treated with caution, especially in the context of phenotype broadening and require more extensive NGS data analysis.

Conflict of Interest: None declared.

EP09.031 A Novel ZBTB20 Variant In A Patient With Primrose syndrome: A rare clinical entity

Merve Soğukpınar 1, beren karaosmanoglu2, Gulen Eda Utine1, Koray Boduroğlu1, Pelin Simsek-Kiper1

1Hacettepe University, Department of Pediatrics, Division of Pediatric Genetics, Ankara, Turkey; 2Hacettepe University, Department of Medical Genetics, Faculty of Medicine, Ankara, Turkey

Background/Objectives: Primrose syndrome (PS) is a rare syndrome characterized by developmental delay, intellectual disability, behavior abnormalities, macrocephaly, characteristic dysmorphic features (high anterior hairline, downslanting palpebral fissures, frontal bossing, prominent ears), calcification of the external ear cartilage, sparse body hair, and distal muscle wasting. It is an autosomal dominant disorder caused by pathogenic variants in ZBTB20. Herein, we present a 10-year-old female patient who was referred to our centre with the findings of intellectual disability, autism spectrum disorder, macrocephaly and dysmorphic facial fetaures.

Methods: Exome sequencing was performed after informed consent was obtained from the parents.

Results: Physical examination revealed macrocephaly (>+2 SD) and facial features including telechantus, long palpebral fissures, and deep set eyes. Mildly limited extension on interphalangeal joints, scoliosis, and pes planus were noted as well. She also had bilateral deafness, hypothyroidism, mitral valve prolapse, and mildly cerebral atrophy. Abdominal ultrasound was normal. Chromosomal microarray analysis was normal. Exome sequencing revealed a novel, de novo heterozygous variant c.1948A>C (p.Asn650His) in ZBTB20 gene, which is classified as “VUS” according to the ACMG criteria.

Conclusion: Primrose syndrome is a rare genetic syndrome that should be kept in mind in patients presenting with macrocephaly, intellectual disability, and dysmorphic facial features.

Conflict of Interest: Merve Soğukpınar Full, beren karaosmanoglu Full, Gulen Eda Utine Full, Koray Boduroğlu Full, Pelin Simsek-Kiper Full.

EP09.032 Three novel de novo variants in TAOK1 associated with intellectual disability

Ariana Mendes 1, Catarina Silva Rosas1, Mafalda Saraiva Santos1, Ana Carvalho1;2, Lina Ramos1;3, Jorge M Saraiva1;4;5

1Centro Hospitalar e Universitário de Coimbra, Medical Genetics Unit, Coimbra, Portugal; 2University Clinic of Genetics, Faculty of Medicine, Universidade de Coimbra, Coimbra, Portugal; 3Faculty of Health Sciences, Universidade da Beira Interior, Covilhã, Portugal; 4University Clinic of Pediatrics, Faculty of Medicine, Universidade de Coimbra, Coimbra, Portugal; 5Clinical Academic Center of Coimbra, Coimbra, Portugal

Background/Objectives: The relationship between pathogenic variants in the TAOK1 gene and neurodevelopment disorders, namely development delay with or without intellectual impairment or behavioural abnormalities (MIM #619575), was reported for the first time in 2019 in 28 patients and in more 13 since then. The molecular spectrum of the identified TAOK1 variants comprises mainly truncanting and nonsense variants, but also missense variants and deletions. Here we describe three additional cases of de novo TAOK1 variants and the associated phenotype.

Methods: During the etiological investigation of three unrelated probands with intellectual disability and macrocephaly, we performed whole exome sequencing (WES), followed by parent segregation studies. We identified three de novo TAOK1 variants, one nonsense, one intronic, and a deletion involving at least the second exon of the gene. All three variants were absent from gnomAD (Genome Aggregation Database) and were classified as probably pathogenic according to the American College of Medical Genetics and Genomics variant classification guidelines.

Results: The patients described here had mild to moderate intellectual disability, with behavioural problems in two of them. They had non-specific dysmorphic features, macrocephaly and additional history of developmental delay, namely in gross motor and language areas. Two of them also had history of hypotonia. Brain MRI performed during childhood did not show relevant abnormalities.

Conclusion: The phenotype of these three additional patients not only supports the clinical features already associated with TAOK1 (developmental delay/intellectual disability with or without behavioural problems, hypotonia and non-specific dysmorphic features), but also expands the molecular spectrum of this disorder.

Conflict of Interest: None declared.

EP09.033 A patient with intellectual disability and a de novo missense variant identical to that reported in six previous patients helps to define the phenotype of SCAMP5 syndrome

Šárka Bendová 1, Marketa Vlckova1, Miroslava Hančárová1, Darina Prchalová1, Viktor Stranecky2, Zdeněk Sedláček1

1Charles University 2nd Faculty of Medicine and University Hospital Motol, Department of Biology and Medical Genetics, Prague, Czech Republic; 2Charles University 1st Faculty of Medicine and General University Hospital, Department of Paediatrics and Adolescent Medicine, Diagnostic and Research Unit for Rare Diseases, Prague, Czech Republic

Mammalian genomes contain five types of SCAMP genes which encode secretory carrier membrane proteins (SCAMPs). SCAMPs play an important role in vesicle cycling, fusion of vesicles and cell membranes, and regulation of exocytosis and endocytosis. Among them, SCAMP5 is brain-specific and highly abundant in synaptic vesicles. We describe a six-year-old boy with severe intellectual disability, seizures, and delayed speech and fine motor skills. His phenotype includes rough facial features, small head circumference, receding forehead, small chin, and reduced visual acuity. His growth is normal and he shows no autistic features. Trio exome sequencing identified a de novo missense SCAMP5 variant NM_001178112.2:c.538G>T, p.(Gly180Trp) which was confirmed using Sanger sequencing. The same de novo variant in the C-terminal domain of SCAMP5 was identified previously in six unrelated patients with severe developmental delay, seizures, problems with gait, variable dysmorphic features, and with or without autism (Hubert 2020, Jiao 2020). Another missense variant located in the E-peptide domain of SCAMP5 was revealed in homozygous state in a consanguineous family where two affected siblings had paediatric epilepsy and juvenile Parkinson’s disease, but not intellectual disability or autism (Zhang 2020). The phenotype of our patient is relatively consistent with the six previous cases carrying the same heterozygous de novo SCAMP5 variant. Because this variant is apparently a very rare cause of neurodevelopmental disorders, each additional patient will be of value for the definition of the full phenotypic spectrum and prognosis of the affected individuals. Supported by AZV NU22-07-00165 and IPE 6980364.

Conflict of Interest: None declared.

EP09.034 Chung-Jansen Syndrome – a new overweight and developmental delay syndrome caused by haploinsufficiency of PHIP – a case report

Anne Sørensen 1, Lise Lotte Bjerregaard2, Anja Lisbeth Frederiksen1;3

1Aalborg University Hospital, Department of Clinical Genetics, Aalborg, Denmark; 2Aalborg University Hospital, Department of Pediatrics, Aalborg, Denmark; 3Aalborg University, Clinical Institute, Aalborg, Denmark

Background/Objectives: Monogenetic causes of obesity in children are rare. Chung-Jansen syndrome, a newly described phenotypic entity including overweight and developmental delay/intellectual disability, is caused by haploinsufficiency of PHIP (MIM#612870). At present, around 60 patients have been described in the literature – most of them with unique variants. Here, we report of a child, who since early childhood had been followed because of developmental delay and intellectual disability. Initially weight was normal, but from around age 4 years he presented hyperphagia and subsequently obesity. At age 6 years, he was referred to the department of clinical genetics. Subtle dysmorphic features included a flat facial profile, large ears with fleshy earlobes and bilateral clinodactyly of the 5th finger. Weight was 44,8 kg (+4,4SD), height 131 cm (+1,7SD), head circumference 55 cm (+2SD). Behavioural issues and attention deficit disorder was reported. Chromosomal microarray and screening for Prader-Willi syndrome were normal.

Methods/Results: Trio whole exome sequencing was performed and revealed a de novo pathogenic variant (C5) in PHIP: NM_017934.6: c.919_923delATAAA, p.(Ile307Profs*22).

Conclusion: This patient share some of the characteristic features previously reported in other patients with pathogenic PHIP alterations, and a distinguishable phenotype is emerging. Obesity is present in many, but not all patients, though there seems to be an age-dependent relationship. PHIP interacts with Proopiomelanocortin (POMC) and extending knowledge about the biological effects of PHIP may bring novel targets of treatment of obesity. When genetic screening for Prader-Willi syndrome is negative, Chung-Jansen syndrome should be considered in children with developmental delay and obesity.

Conflict of Interest: None declared.

EP09.035 Co-Occurrence of Fragile X Syndrome with a Second Genetic Condition

Roberta Polli 1;2, Giorgia Beffagna3, Elisa Bettella1;2, Marilena Cameran1;2, Valentina Liani1, Simona Agata3, Elisa Di Giorgio4, Giulia Parmeggiani5, Alberta Leon3, Alessandra Murgia1;2

1University of Padua, Department of Women’s and Children’s Health of Padua, Padua, Italy; 2Istituto di Ricerca Pediatrica Foundation-Città della Speranza, Padua, Italy, Padua, Italy; 3R&I Genetics SRL, Padova, Italy, Padua, Italy; 4University of Padua, Department of Developmental and Social Psychology, Padua, Italy; 5Medical Genetics Unit, AUSL della Romagna, Ospedale Bufalini, Cesena (FC), Italy

Fragile X syndrome (FXS; MIM 300624) is a genetic disorder whose phenotype is, beyond intellectual disability, characterized by a spectrum of psychiatric impairments and pediatric comorbidities.In more than 99% of cases, FXS is due to expansions of a CGG trinucleotide repeat in the 5′- UTR of the FMR1 gene exceeding 200 units, leading to DNA methylation of the promoter and transcriptional silencing, although FMRP loss of function leading to the FXS phenotype can also result from other FMR1 sequence modifications. We present the results of Whole Exome Sequencing (WES) analysis performed in a 7- year-old girl with clinical symptoms consistent with FXS, associated with distinct dysmorphic features including thick arched eyebrows, broad nasal bridge with bulbous nose, thin upper lip, widened thumbs and first toes. After negative FMR1 CGG-repeat analysis, and CGH array, WES analysis revealed the presence of a FMR1 de novo canonical splice-site mutation (NM_002024.5:c.1275+1G > A), predicted in silico as pathogenic. Further analysis of WES data, based on phenotypic features inconsistent with FXS, revealed a KIRREL3 missense variant segregating in the maternal family with limb alterations. Autosomal dominant KIRREL3 mutations are associated with a neurodevelopmental condition and possible limb malformations. FMR1 transcript analysis, family segregation of the KIRREL3 variant, and clinical evaluations of carrier relatives are in course. Our data provide further evidence of the possibility that the FXS phenotype variability may at least partly depend by combined effects of possible additional genetic factors. We also show a distinct pattern of dysmorphic features associated with KIRREL3 alterations.

Conflict of Interest: Roberta Polli: None declared, Giorgia Beffagna: None declared, Elisa Bettella: None declared, Marilena Cameran: None declared, Valentina Liani: None declared, Simona Agata: None declared, Elisa Di Giorgio: None declared, Giulia Parmeggiani: None declared, Alberta Leon: None declared, Alessandra Murgia SHIONOGI B.V.

EP09.036 Clinical and molecular characterization of FOXP1 variants in subjects with neurodevelopmental disorders

Elisa Bettella 1;2, Denis Drongitis3, Roberta Polli1;2, Lucia Verrillo3, Emanuela Leonardi1, Marilena Cameran1;2, Matteo Della Monica4, Roberta Cernetti5, Chiara Spitalieri5, Franco Stanzial6, Demetrio Baldo7, Licia Turolla7, Donatella Milani8, Giuseppe Cossu9, Maria Giuseppina Miano3, Alessandra Murgia1;2

1University of Padua, Department of Women’s and Children’s Health, Padua, Italy; 2Pediatric Research Institute, Città della Speranza, Padua, Italy; 3Institute of Genetics and Biophysics “Adriano Buzzati-Traverso”, CNR, Naples, Italy; 4Antonio Cardarelli Hospital, Medical and Laboratory Genetic Unit, Naples, Italy; 5ULSS 7 Pedemontana, Childhood Adolescence Family Unit, Bassano, Italy; 6Regional Hospital of Bolzano, Genetic Counseling Service, Department of Pediatrics, Bolzano, Italy; 7ULSS 2 Treviso Hospital, Unit of medical genetics, Treviso, Italy; 8Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy; 9Centro Medico di Foniatria, Children’s Cognitive Neurorehabilitation Unit, Padua, Italy

Background: FOXP1 (forkhead-box protein P1) is a member of the FOX transcription factor family essential for embryonic development. FOXP1, highly expressed in the nervous system, regulates molecular pathways required for proper brain development and function. FOXP1 heterozygous mutations and deletions are associated with “Intellectual developmental disorder with language impairment with or without autistic features” (OMIM#613670), a neurodevelopmental condition characterized by intellectual disability, speech and language delay, motor delay, ASD, and mild dysmorphic features.

Methods: Two different customized targeted gene panels, one including 74 ID/ASD and a second including the FOXP1/FOXP2/CNTNAP2 genes, were used to screen respectively a cohort of 844 pediatric subjects with Neurodevelopmenatl Disorders (NDDs) and a selected group of 31 pediatric individuals with Specific Language Impairments (SLIs).

Results: 10 FOXP1 variants were detected: 4 novel de novo protein truncating and 1 novel frameshift, all predicted to cause haploinsufficiency, 1 novel paternal missense, 1 maternal and previously reported missense, and 3 ultra-rare missense. Segregation analysis is underway for all the missense variants. Even if more than 100 FOXP1 variants with likely clinical significance are reported in literature, for only few of them the actual impact on protein expression and function has been dissected. We are currently performing a functional characterization of the FOXP1 variants we have identified in order to evaluate their effects on protein expression and transcription activity.

Conclusion: Our work promises to provide new data to improve genotype-phenotype correlation and elucidate the pathogenic mechanisms underlying FOXP1-related conditions.

Conflict of Interest: Elisa Bettella: None declared, Denis Drongitis: None declared, Roberta Polli: None declared, Lucia Verrillo: None declared, Emanuela Leonardi: None declared, Marilena Cameran: None declared, Matteo Della Monica: None declared, Roberta Cernetti: None declared, Chiara Spitalieri: None declared, Franco Stanzial: None declared, Demetrio Baldo: None declared, Licia Turolla: None declared, Donatella Milani: None declared, Giuseppe Cossu: None declared, Maria Giuseppina Miano: None declared, Alessandra Murgia Shionogi B.V.

EP09.038 Revealing novel variants associated with chromatinopathies

Asuman Koparir 1, Eva Grauer2, Paulina Bahena Carbajal3, Konstantinos Kolokotronis4, Erkan Koparir3, Yvonne Jelting5, Michaela AH Hofrichter5, Jörg Klepper6, Thomas König7, Eva Runkel6, Magdalena Schreglman7, Juliane Spiegler7, Nicole Stachelscheid6, Erdmute Kunstmann5, Thomas Haaf5, Eva Klopocki5

1Julius Maximilians University, Institute of Human Genetics, Wuerzburg, Germany; 2MVZ genetikum GmbH, Neu-Ulm, Germany; 3Julius Maximilians University, Institute of Human Genetics, Wuerzburg, Germany; 4University of Zurich, Insitute of Medical Genetics, Zurich, Switzerland; 3Julius Maximilians University, Institute of Human Genetics, Wuerzburg, Germany; 6Klinikum Aschaffenburg-Alzenau, Department of Pediatrics and Neuropediatrics, Aschaffenburg, Germany; 7University Hospital Würzburg, Department of Pediatrics, Wuerzburg, Germany

Background: Chromatinopathies are a class of neurodevelopmental disorders caused by mutations in chromatin regulators, comprising more than 50 disorders. Patients affected by chromatinopathies share clinical features, including developmental delay, intellectual disability, facial dysmorphism, and behavioral disturbances. Cornelia de Lange syndrome (CdLS), Rubinstein-Taybi syndrome (RSTS), KBG syndrome (KBGS), Coffin-Siris syndrome (CSS), and Wiedemann-Steiner syndrome (WDSTS) are prominent examples of chromatinopathies.

Methods: In this study, we evaluated 231 individuals with neurodevelopmental delay and additional clinical features. We performed whole-exome sequencing to identify the underlying molecular pathology.

Results: 40 of 231 affected individuals carry 38 different variants in chromatinopathy genes. We detected eight ANKRD11 and four SETD5 variants which belong to the most frequently mutated genes associated with neurodevelopmental delay. Furthermore, we found eight variants in genes that encode histone lysine methylases and demethylases (KMT2A, KMT2B, KMT2C, KMT2D, KDM5C) in nine patients with overlapping phenotypes. Seven more patients have been diagnosed with CSS carrying variants in ARID2, BICRA, SMARCB1, SMARCD1, and SMARCA4. CREBBP and EP-300 variants have been identified in patients with RSTS syndrome. Five patients displayed variants in genes encoding chromodomain-helicase-DNA-binding proteins, CHD3, CHD4 und CHD5. We identified two variants in BRPF1 encoding a multivalent chromatin reader. In addition, two variants have been detected in other chromatinopathy genes such as DNMT3A and SIN3A.

Conclusion: In the current study, 17.3 % of our patients with neurodevelopmental delay harbor variants in chromatinopathy genes. We identified 33 novel variants in genes encoding chromatin remodelers and transcriptional regulators, and expand the genotypic spectrum of chromatinopathies.

Conflict of Interest: None declared.

EP09.039 KMT2C pathogenic variants result in a neurodevelopmental disorder with distinct clinical and DNA methylation features

Dmitrijs Rots 1, Sanaa Choufani2, Víctor Faundes3, Alexander Dingemans4, Lisenka Vissers4, Tjitske Kleefstra1;5;6, Siddharth Banka7, Rosanna Weksberg2

1Contributed equally, Department of Human genetics and Donders Center for Medical Neuroscience, Radboudumc, Nijmegen, Netherlands; 2Contributed equally, Program in Genetics and Genome Biology, The Hospital for Sick Children, Toronto, Canada; 3Contributed equally, Laboratorio de Genética y Enfermedades Metabólicas, Instituto de Nutrición y Tecnología de los Alimentos Doctor Fernando Monckeberg Barros (INTA), Universidad de Chile, Santiago, Chile; 4Radboudumc, Department of Human genetics and Donders Center for Medical Neuroscience, Nijmegen, Netherlands; 5Center for Neuropsychiatry Vincent van Gogh, Venray, Netherlands; 6ErasmusMC, Department of Clinical Genetics, Rotterdam, Netherlands; 7Contributed equally, Manchester Centre for Genomic Medicine, Division of Evolution and Genomic Sciences, School of Biological Sciences, Faculty of Biology, Medicine and Health, University of Manchester, Manchester, United Kingdom

Introduction: KMT2C haploinsufficiency initially was shown to result in a syndromic neurodevelopmental disorder overlapping with Kleefstra syndrome. Moreover, KMT2C (histone-3 lysine-4 (H3K4) methyltransferase) has strong biological interactions with EHMT1 (H3K9 methyltransferase). Therefore, KMT2C-related NDD was named Kleefstra syndrome type 2. As the number of diagnosed cases has increased considerably, we sought to characterize this disorder and compare it to the molecularly related Kleefstra (EHMT1) and Kabuki 1 (KMT2D) syndromes.

Methods: We ascertained individuals with rare KMT2C variants through international collaboration, systematically collected and analysed clinical features, performed DNA methylation (DNAm) studies, and used PhenoScore to objectively study facial features.

Results: We ascertained 76 individuals with pathogenic, and 22 with variants of uncertain significance (VUS) in KMT2C. Using 16 cases with pathogenic KMT2C variants and 50 age- and sex-matched controls, we identified a KMT2C-specific DNAm signature. We found this signature to be distinct from those of Kleefstra and Kabuki 1 syndromes. Using the DNAm signature, we re-classified 4/22 KMT2C VUS as pathogenic, and, thereby, established a cohort of 80 individuals with pathogenic KMT2C variants. We found that KMT2C-related NDD is characterized by developmental delay/intellectual disability (~85%), behavioral/psychiatric problems (including autism spectrum disorder (77%) and ADHD (62%)), short stature (36%), hypotonia (33%), recurrent infections (30%), and various congenital anomalies. PhenoScore showed that KMT2C-related NDD has a typical facial gestalt (p < 0.001) which is different from Kleefstra (p < 0.001) and Kabuki 1 (p = 0.04) syndromes.

Conclusions: KMT2C pathogenic variants result in a unique neurodevelopmental disorder which is distinct from Kleefstra and Kabuki 1 syndromes.

Grant references: SFARI;KNAW(KNAWWF/1327/TMB202120)

Conflict of Interest: None declared.

EP09.040 Biallelic intragenic tandem duplication of CPLANE1 in Joubert syndrome: a case report

Francisco Martínez-Granero1, Elena Martinez-Cayuelas2, Cristina Rodilla1;3, Gonzalo Nuñez Moreno3;4, Marta Rodriguez de Alba1;3, Fiona Blanco-Kelly1;3, Raquel Romero3;4, Pablo Mínguez3;4, Carmen Ayuso1;3, Isabel Lorda1;3, Marta Corton1;3, Berta Almoguera Castillo 3;5

1Fundación Jiménez Díaz University Hospital, Department of Genetics and Genomics, Madrid, Spain; 2Fundación Jiménez Díaz University Hospital, Department of Pediatrics, Madrid, Spain; 3Center for Biomedical Network Research on Rare Diseases (CIBERER), ISCIII, Valencia, Spain; 4Fundación Jiménez Díaz University Hospital, Bioinformatics Unit, Madrid, Spain; 5Fundación Jiménez Díaz University Hospital. Madrid, Spain, Department of Genetics and Genomics., Madrid, Spain

Background/objectives: Joubert syndrome (JS) is a genetic disorder with both neurological and extraneurological manifestations. Of the 40 JS-causing genes currently reported, CPLANE1 is one of the most frequently mutated, with biallelic pathogenic missense and truncating sequence variants explaining up to 14% of JS cases. We present a case of JS diagnosed after the identification of a novel biallelic intragenic duplication in CPLANE1.

Methods: The case is a 3-year-old female patient with developmental delay. We used a clinical exome sequencing (CES) and aCGH for the identification, confirmation and segregation of the duplication. A long PCR and a nanopore-based long-read sequencing (LRS) of the PCR product were used to confirm whether the duplication was in tandem and to identify the breakpoints. The presence of a common haplotype was explored with haplotyping analysis and homozygosity mapping of the CES.

Results: CES and aCGH identified a quadruplication of exons 20-46 of CPLANE1 and confirmed the duplication was inherited from both unrelated parents. LRS allowed the fine mapping of the duplication with proximal and distal junctions located on intron 19 and intron 46 and a size of 75,559bp. Based on the genetic findings, a brain MRI was ordered, evidencing the molar tooth sign, which confirmed the diagnosis of JS in the patient. A region of homozygosity was identified in the CES data of the patient supporting the duplication being in a common haplotype.

Conclusions: This is the first report of an intragenic duplication in CPLANE1 as the molecular mechanism of JS.

Grants: ISCIII,FEDER (JR17/00020;PI18/01098;PI20/00851);CIBERER (06/07/0036);CM (PEJ-2020-AI/BMD-18610).

Conflict of Interest: None declared.

EP09.041 Exome sequencing in patients with intellectual disability in a large Indian cohort

Neerja Gupta 1, Mounika Endrakanti1, Kamal Naini1, Savita Sapra1, Sheffali Gulati1, Madhulika Kabra1

1All India Institute of Medical Sciences, Pediatrics, New Delhi, India

Background/Aims: Global developmental delay (GDD)/intellectual disability (ID) is etiologically diverse and has an identifiable genetic etiology in approximately 50% of the patients. We aimed to evaluate the clinical spectrum of patients with ID and the diagnostic yield of exome sequencing (ES).

Methods: Data from patients with ID/GDD from 2016 to 2022 were analysed retrospectively. Patients with chromosomal disorders, triplet repeat disorders, and imprinting disorders were excluded. Clinical geneticists evaluated all the enrolled patients. They were categorized into isolated ID, ID plus (ID with/ without dysmorphism and/or other systemic abnormalities), and ID with neuroregression. Either clinical exome sequencing (CES), whole exome sequencing (WES), or whole genome sequencing (WGS) was performed.

Results: We assessed 411 families with 491 affected individuals (ages 3 months to 46 years) and classified them as ID plus (358/411,87%), ID with neuroregression (46/411,11.2%), and isolated ID (7/411,1.7%). Fifteen percent (64/411) were multiplex while 14% (57/411) had consanguinity. Out of 411, proband-ES, trio-ES, and WGS were performed in 403, 7, and 1 respectively. The overall yield for pathogenic/likely pathogenic variants was 47.7% (196/411), with CES yielding more (51%, 140/274) than WES (36.8%, 56/152). There were 111 novel and 6 copy number variants discovered. Autosomal recessive disorders were more common than others.

Conclusion: In the study cohort, ID plus phenotype had a higher diagnostic yield. It broadens the phenotypic and genotypic spectrum of rare neurodevelopmental disorders.

Grant references: The study received partial financial support from the DBT grant (BT/PR13921/MED/12/704/2015).

Conflict of Interest: Neerja Gupta Full-time, The study received partial financial support from the DBT grant (BT/PR13921/MED/12/704/2015), Mounika Endrakanti: None declared, Kamal Naini: None declared, Savita Sapra: None declared, Sheffali Gulati: None declared, Madhulika Kabra: None declared.

EP09.042 GNB5-related neurodevelopmental disorder: A novel variant detected in a large consanguineous family

Norah Alsaleh 1;2, Aziza Mushiba3, wafa eyaid1;2;4

1King Abdullah Specialized Children’s Hospital, King Abdulaziz Medical City, Ministry of National Guard-Health Affairs (NGHA), Department of Genetics and Precision Medicine, Riyadh, Saudi Arabia; 2King Abdullah International Medical Research Center (KAIMRC), Riyadh, Saudi Arabia; 3King Fahd Medical City, Division of Medical Genetics, Department of Pediatrics, riyadh, Saudi Arabia; 4King Saud bin Abdulaziz University for Health Sciences, Ministry of National Guard-Health Affairs (NGHA), Riyadh, Saudi Arabia

Background: GNB5-related neurodevelopmental disorders occur due to homozygous or compound heterozygous pathogenic variants in the GNB5 gene. They are characterized by a wide range of intellectual disabilities (ID), developmental delays, language delays, seizures, and visual impairment, and the majority present with bradycardia due to sick sinus syndrome.

Method: We did a retrospective chart review of three affected children from two consanguineous families. A positive family history of four other affected children with a similar phenotype has not been confirmed molecularly. All affected children had overlapping features of global developmental delay, moderate to severe ID, absent speech, and visual impairment. Two behavioral problems and conductive hearing loss, and one with a seizure disorder. One with documented bradycardia and long sinus pause. All had an unremarkable brain MRI.

Results: Chromosomal microarray for all affected children were negative. Proband 1 had inconclusive exome sequencing and genome sequencing. Genome reanalysis later revealed a homozygous variant of unknown significance (VOUS) in GNB5. Molecular testing for probands 2 and 3 also detected the same variant in a homozygous state.

Conclusion: Animal studies have shown that GNB5 plays an essential role in neuronal signaling and autonomic function. So far, around 11 pathogenic, five likely pathogenic, and 17 VOUS have been reported in GNB5. To our knowledge, this variant has not been previously reported in any database; however, it segregated well with the disease and fits the phenotypic description of this disorder. Segregation analysis of the remaining affected members and functional studies to be followed.

Conflict of Interest: None declared.

EP09.043 Paralogous annotation of disease-causing variants and somatic mutations in CREBBP/EP300 can improve variant interpretation in Rubinstein-Taybi syndrome

Marta Codina 1;2, Berta Campos1;2, Irene Valenzuela Palafoll1;2, Paula Fernandez1;2, Anna Maria Cueto-González1;2, Amaia Lasa-Aranzasti1;2, Laura Trujillano1;2, Jordi Leno1;2, Elena García-Arumí1;2, Eduardo Tizzano1;2

1Vall d’Hebron University Hospital, Department of Clinical and Molecular Genetics, Barcelona, Spain; 2VHIR - Vall d’Hebron Institut de Recerca, Medicine Genetics Group, Barcelona, Spain

Background/Objectives: Rubinstein-Taybi syndrome (RTS) is a rare syndrome caused by loss-of-function mutations in two paralogous tumour-suppressor genes: CREBBP and EP300. The description of milder cases and the paucity of missense variants reported, make the interpretation of novel changes challenging. We hypothesize that paralogous annotation of disease-causing variants and of tumour somatic mutations can improve variant interpretation in RTS.

Methods: We constructed multiple protein sequence alignments for CREBBP/EP300 using Clustal-W. We mapped each paralogue residue with a known missense or indel pathogenic variant (n = 87, from the literature our own data) or a somatic mutation (COSMIC) onto the equivalent residue of the paralogous gene. Chi-square test was used to calculate if pathogenic variants occurred more often in residues with paralogous pathogenic variants or somatic mutations.

Results: Seven of 29 residues with pathogenic variants in EP300 (24%) affected residues with a mutation in CREBBP (χ2 = 0.03, OR = 7). Regarding correlation between germinal and somatic mutations, 8 (27%) of the 29 RTS pathogenic variants in EP300, affected a recurrently (>4) somatic mutated residue in COSMIC (χ2 = 0.02, OR = 8). For CREBBP, 16 (27%) of 58 RTS pathogenic variants in EP300, affected a recurrently somatic mutated (>4) residue in COSMIC (χ2 = 0.02, OR = 8). Using these criteria, we could reclassify 4/185 and 12/266 variants of unknown significance from ClinVar in EP300 and CREBBP respectively.

Conclusions: Our results show that annotation of somatic and paralogous disease-causing variants in CREBBP and EP300 can facilitate variant interpretation. This method is applicable to other paralogous and tumour-suppressor genes.

Conflict of Interest: None declared.

EP09.044 IQSEC2-associated epileptic encephalopathy: case report

Maria Revkova1, Ekaterina Domoratskaya 1, Denis Chernevskiy1, Aleksey Antonenko1, Ruslan Belov1, Anna Panferova1, Victoria Aydarova1, Svetlana Safina1, Milana Gubona1, Maria Golovanova1, Natalia Sokolova1, Olga Chesnokova1, Polina Ulanova1, Elizaveta Bondarchuk2;3, Natalia Doroshchuk1, Maria Makarova1, Anastasia Krinitsina1, Olga Groznova2;3, Maxim Belenikin1;4

1Evogen LLC, Moscow, Russian Federation; 2Academician Yu. E. Veltishchev Research Clinical Institute of Pediatrics, N.I. Pirogov Russian National Research Medical University, Ministry of Health of the Russian Federation, Moscow, Russian Federation; 3Charity foundation for medical and social genetic aid projects “Life genome”, Moscow, Russian Federation; 4Lomonosov Moscow State University, Moscow, Russian Federation

Background: Whole-genome sequencing (WGS) can detect rare and novel genetic variants associated with hereditary neurodegenerative diseases. We report the case of 3 boys (9, 7 and 2 years old) from the same family with similar clinical features: impaired speech and motor development after the first year of life, muscle hypotension. The older brothers show delayed myelination on MRI and generalized epileptiform discharges on the EEG. The phenotypes of the older and younger children included anomalies of the skull and skeleton, delayed physical development. They have a half-sibling girl without pathological symptoms.

Methods: WGS (PE150, enzymatic fragmentation-based and PCR-free protocol, DNBseq-T7 (MGI, China)) with Sanger sequencing (ABI3500, Applied Biosystems) was performed for siblings and their parents.

Results: Genetic analysis revealed novel hemizygous variant c.1118C>G (p.Ser373Ter) in IQSEC2 gene in each siblings and heterozygous variant in their mother. In silico algorithms predict the likely pathogenic status for this variant.

Conclusion: Detected by WGS variant IQSEC2: c.1118C>G (p.Ser373Ter) can be associated with X-linked intellectual developmental disorder (OMIM 309530) and can cause epileptic encephalopathy in the described siblings.

Grant References: The research was supported by non-profit organization Charity Fund for medical and social genetic aid projects «Life Genome».

Conflict of Interest: None declared.

EP09.045 Keloid Lesions in Intellectual Developmental Disorder with Cardiac Defects and Dysmorphic Facies:A Case Report

Noor Bawahab 1, Mohammed Almannai2, Rawabi Al-Ghamdi3

1King Abdulaziz University Hospital, Jeddah, Saudi Arabia; 2King Abdulaziz Medical City, Genetics and Precision Medicine, Riyadh, Saudi Arabia; 3King Abdulaziz Medical City, Riyadh, Saudi Arabia

Background: Biallelic variants in TMEM94 were recently reported to cause an autosomal recessive intellectual developmental disorder with cardiac defects and dysmorphic facies -IDDCDF- (OMIM:618316). As the name implies, the main phenotype of this disorder is characterized by intellectual disability/developmental delay, CHD, and dysmorphic features. In the initial cohort reported in 2018, one of the subjects was reported to have boggy subcutaneous lesions. In addition, an adult female patient had a cutaneous lesion in the form of dermal type nevi. In this case report, we describe a 20 year old male who presented with intellectual disability, Ventricular septal defect, refractive error and hypospadias. On exam, he has dysmorphic features with an anterior low hairline, large nails, long eyelashes, protruding ears. In addition to the clinical findings described he has multiple keloids, the largest being in the upper back. And the rest are in the chest, lower abdomen and left arm.Whole exome sequencing showed homozygous splice site variants in TMEM94 (c.2729-2A > G).

Methods: case report

Conclusion: This case report shows that variants in TMEM94 could result in cutaneous manifestations and their presence could help in suspecting this rare syndrome and also in classifying variants in TMEM94.

Conflict of Interest: None declared.

EP09.046 Exploring the phenotype and genotype of Menke-Hennekam syndrome 2: case report and literature review

Susana Lemos Ferreira 1, Ana Peres2, Ana Laura Fitas3, Inês Carvalho1

1Centro Hospitalar Universitário Lisboa Central, Serviço de Genética Médica - Hospital Dona Estefânia, Lisboa, Portugal; 2Hospital Vila Franca de Xira, Serviço de Pediatria, Lisboa, Portugal; 3Centro Hospitalar Universitário Lisboa Central, Unidade de Endocrinologia Pediátrica, Departamento de Pediatria Médica - Hospital Dona Estefânia, Lisboa, Portugal

Introduction: Menke-Hennekam syndrome 2 (MKHK2) is a recently recognized neurodevelopmental disorder that results from heterozygous missense mutations in exons 30/31 of EP300 gene, mainly known as a cause of Rubinstein-Taybi syndrome 2 (RSTS2). MKHK2 is characterised by developmental delay/intellectual disability, microcephaly, short stature, autism, epilepsy, feeding difficulties, vision and hearing impairment. Dysmorphims are largely different from RSTS2 and include short palpebral fissures, telecanthus, depressed nasal bridge, short nose, anteverted nares, short columella, and long philtrum. Here, we present a case that contribute to an increasing understanding of MKHK2.

Case presentation: A 1-year-old female was first referred to our outpatient genetic department with global developmental delay. At our observation, she showed facial dysmorphisms, syndactyly, short stature and post-natal microcephaly. Microarray was normal. A WES-based gene panel identified a missense variant in the EP300 gene, NM_001429.4:c.4783T>C p.(Phe1595Leu) in exon 30. The variant was not previously described in literature, nor in gnomAD population. It occurs at a conserved position across species, at the same position as another pathogenic missense change, and in silico analysis support a deleterious effect. Segregation studies revealed a de novo origin. Reverse phenotyping highlighted that our patient did not have typical RSTS2 characteristics, but rather shared the clinical features of MKHK2.

Conclusions: This case contributes to the expansion of the genotypic spectrum of MKHK2. It also emphasizes the wide clinical phenotype associated with EP300, and how reverse phenotyping is an important tool in the interpretation of Next-Generation Sequencing data.

Conflict of Interest: None declared.

EP09.047 An integrated approach to the diagnosis of mental retardation linked to a fragile X chromosome

Vita Antsupova 1, Iryna Lastivka2, Larysa Sheiko3, Ljudmila Brisevac3, Ludmila Khlunovska2, Iryna Malieieva1, Iana Ushko1, Anastasiya Babintseva2

1Bohomolets National Medical University, Kyiv, Ukraine; 2Bukovinian State Medical University, Chernivtsi, Ukraine; 3Shupyk National Healthcare University of Ukraine, Kyiv, Ukraine

The relevance of studying X-linked forms of mental retardation is due to their prevalence and the importance of medical genetic counseling for such families. The most frequent disease of this group is FraX syndrome. The diagnosis of fragile X syndrome is based on clinical criteria and paraclinical examination methods. The technology of complex DNA diagnostics includes high-throughput parallel DNA sequencing, multiplex ligated probe amplification, and multiplex methyl-sensitive PCR. Here is our own clinical observation of a 5-year-old boy suffering from epilepsy and mental retardation. The child was born at a gestational age of 38 weeks, weighing 3600 g, body length 53 cm. He held his head from 7 months, sat from 1 year, walked from 1 year 6 months. At 4 months strabismus appeared. At the age of 4, he suffered from acute glomerulonephritis, epilepsy attacks began. Phenotype: macrocephaly, high forehead, protruding ears with soft cartilage, inferior prognathia. Based on clinical data and the result of portrait diagnostics using the Face2Gene computer program, a suspicion of mental retardation linked to a fragile X chromosome was established. Molecular genetic analysis (MLPA) revealed an allele of the FMR1 gene containing a complete mutation (200 CGG repeats), which made it possible to verify the diagnosis of Martin-Bell syndrome. Thus, an integrated approach to the examination of patients with epilepsy and mental retardation makes it possible to identify the cause, which is important in the treatment and socialization of the patient, as well as in the prevention of new similar cases in the family.

Conflict of Interest: None declared.

EP09.048 Triplication of PCDH19 gene as a potential novel disease mechanism associated to epilepsy phenotype resembling loss-of-function mutations

Francisco Martinez 1, Carmen Orellana1, Mónica Roselló Piera1, Sandra Monfort Membrado1, Juan Silvestre Oltra Soler1, Carla Martín-Grau2, Alba Gabaldon Albero2

1La Fe University and Polytechnic Hospital, Genetics Unit, València, Spain; 2Institut d’Investigació Sanitària La Fe de València, Grupo de Investigación Traslacional en Genética, València, Spain

Introduction: Loss-of-function variants in PCDH19 cause an early-onset seizure, autism and neurocognitive disorder in heterozygous females or in males with somatic mutations, presumably by a cellular interference mechanism. However, the phenotype associated with a gain of dosage have not been described so far.

Results: One female patient with seizures, including focal, generalized atonic and tonic-clonic seizures, with fever as the triggering factor, cluster presentation and drug resistance. Comorbidities include global developmental delay with impaired socialization (autistic traits), language delay, and difficulties in gross motor skills. Dysmorphic features are absent.

Metabolic, MRI and exome sequencing showed no pathological findings. In the array study (Affymetrix CytoScan 750K) a de novo triplication of 12.2 megabases of the chromosomal region Xq21.3-q22.1 was found (chrX:89355579-101615553; Hg19). The triplicated segment included 41 genes, of which only PCDH19 has been associated with a compatible clinical phenotype. The pattern of inactivation of the X chromosome in blood cells showed a preferential, but not complete, inactivation of the maternal X chromosome, showing some functional mosaicism.

Discussion: A similar pathogenicity mechanism has been reported in the only gene with a similar pattern of inheritance (EFNB1), for which the duplication associated familial hypertelorism only in heterozygous carriers, supported by mouse model studies. Further, this is compatible with the “cellular interference” hypothesis proposed as molecular basis of PCDH19-related disease.

Conclusion: The dose gain in the PCDH19 gene may be a potential novel disease mechanism, which causes a clinical phenotype resembling that caused by loss-of-function variants.

Grant: PI22/00272 funded by ISCIII.

Conflict of Interest: Francisco Martinez PI22/00272, funded by Instituto de Salud Carlos III (ISCIII) and co-funded by the European Union, Carmen Orellana: None declared, Mónica Roselló Piera: None declared, Sandra Monfort Membrado: None declared, Juan Silvestre Oltra Soler: None declared, Carla Martín-Grau: None declared, Alba Gabaldon Albero: None declared.

EP09.049 From autism spectrum disorder to monogenic neurodevelopmental disorder

Gordana Stojanovic1, Aleš Maver2, Alenka Hodžić2, Borut Peterlin2, Olivera Miljanovic1, Jelena Jovanovic 3

1Clinical Center of Montenegro, Centre for Medical Genetics and Immunology, Podgorica, Montenegro; 2University Medical Centre Ljubljana, Clinical Insitute of Genomic Medicine, ljubljana, Slovenia; 1Clinical Center of Montenegro, Centre for Medical Genetics and Immunology, Podgorica, Montenegro

Introduction: Intellectual developmental disorder, autosomal dominant 41 (MRD41) is disorder with variable phenotype including nonspecific dysmorphia, delayed psihomotor development, intellectual disability with variable severity, speech delay, epileptic seizures and behavioral manifestations (autistic features). It occurs as a consequence of heterozygous mutation in the TBL1XR1 gene on chromosome 3q26.

The aim: We report a patient with MKRD41 in order to increase the rate of identification of patients with a rare inherited disease of actually unknown prevalence.

Results: We present a child with clinical diagnosis of autism spectrum disorder with nonspecific dysmorphia (low-set ears, prominent nasal bridge, protruding ear, long eyelashes), hyperactivity, short attention span, delayed speech and language development, atria septal defect. After aCGH result of normal molecular karyotype, we performed whole exome sequencing. A “de novo” patogenic mutation c.58G>T in TBL1XR1 gene was detected, which is a known causative to MRD41. Mutation c.58G>T in TBL1XR1 has not yet been reported in association with human diseases in the biomedical literature, however, based on the evidence (PVS1, PM2, PM6_SUP), the identified variant is classified as pathogenic. Detected mutation in TBL1XR1 gene is responsible for loss of function and inability to synthesize the protein which plays an essential role in transcription activation mediated by nuclear receptors.

Conclusion: Taking into account the very rare occurrence of MRD41 with unknown prevalence, we hope that the presentation of the patient can contribute to the easier recognition of children with MRD41 and inclusion of NGS analysis in diagnostic protocols of children with autism spectrum disorder.

Conflict of Interest: None declared.

EP09.050 Ultra-rare neurodevelopmental disorder with degenerative course and progressivemovement disorders associated with biallelic ZBTB11 variants

Juan Darío Ortigoza-Escobar1;2;3, Mina Zamani4;5, Dorison Nathalie6, Saeid Sadeghian7, Reza Azizimalamiri7, Hamid Galehdari4, Gholamreza Shariati5;8, Alihossein Saberi5;9, Seyed MohammadSaleh Zahraei 4, Niloofar Chamanrou4, Andres Moreno-De-Luca10, Lisette Leeuwen11;12, Giovanni Zifarelli13, Peter Bauer13, Vincent d’Hardemare14, lydie BURGLEN15;16;17, Diane Doummar12, Cynthia curry18, Henry Houlden19, Reza Maroofian19

1Institut de Recerca, Hospital Sant Joan de Déu Barcelona, Movement Disorders Unit, Pediatric Neurology Department, Barcelona, Spain; 2Instituto de Salud Carlos III, U-703 Centre for Biomedical Research on Rare Diseases (CIBER-ER), Barcelona, Spain; 3European Reference Network for Rare Neurological Diseases (ERN-RND), Barcelona, Spain; 4Shahid Chamran University of Ahvaz, Department of Biology, Ahvaz, Iran; 5Narges Medical Genetics and Prenatal Diagnosis Laboratory, Ahvaz, Iran; 6Reference Center of inherited Metabolic Diseases, Necker-Enfants-Malades University hospital, APHP, Université de Paris, Inserm U1151, Institut Necker Enfants-Malades, MetabERN, Paris, France; 7Golestan Medical, Educational, and Research Centre, Ahvaz Jundishapur University of Medical Sciences, Department of Paediatric Neurology, Ahvaz, Iran; 8Ahvaz Jundishapur University of Medical Sciences, Department of Medical Genetics, Faculty of Medicine, Ahvaz, Iran; 9Ahvaz Jondishapur University of Medical Sciences, Diabetes Research center, Health Research Institute, Ahvaz, Iran; 10Autism & Developmental Medicine Institute, Genomic Medicine Institute, Department of Radiology, Geisinger, Danville, United States; 11University Medical Center Groningen, department of Genetics, Groningen, Netherlands; 12Reference Center of inherited Metabolic Diseases, Necker-Enfants-Malades University hospital, APHP, Université de Paris, Inserm U1151, Institut Necker Enfants-Malades, MetabERN, Paris, France; 13CENTOGENE GmbH, Rostock, Germany; 14Unité Dyspa, Neurochirurgie Pédiatrique, Hôpital Fondation Rothschild, Paris, France; 15Centre de Référence Maladies Rares “Malformations et Maladies Congénitales du Cervelet”, Paris, Lyon, Lille, France; 16Hospital Armand Trousseau Ap-Hp, Département de Génétique, Paris, France; 17Developmental Brain Disorders Laboratory, Imagine Institute, INSERM UMR 1163, Paris, France; 18UCSF Fresno, Deptartment of Pediatrics, Genetic Medicine, Fresno, United States; 19UCL Queen Square Institute of Neurology, Department of Neuromuscular Diseases, London, United Kingdom

Background/Objectives: Zbtb11 is a conserved zinc finger transcriptional regulator. Fifteen individuals with intellectual disability, bilateral cataracts, combined malonic and methylmalonic aciduria (CMMMA), and neuroradiologic abnormalities have previously been reported to have bi-allelic variants in ZBTB11. We present 8 additional individuals with ZBTB11-related neurodevelopmental disorder to further delineate the spectrum of clinical features and genetic variants contributing to this syndrome.

Methods: Phenotypic and molecular data from 8 unreported individuals with ZBTB11 variants were gathered through international collaboration and compared to the previously reported individuals.

Results: Six ultra-rare variants (NM_014415.4: c.2009T>C, c.2517G>C, c.2618A>G, c.2708G>A, c.3130G>A and c.999dup) were identified. Frequent phenotypic features include mild to moderate intellectual disability, motor developmental regression, bilateral cataracts, and complex movement disorders. Hypotonia was prevalent in childhood but evolved into hypertonia with limb contractures as individuals aged. Complex movement disorders included orofacial and limb dystonia, myoclonus, stereotypies, and coarse and resting tremor. One patient underwent deep brain stimulation for generalized dystonia with a good response. Six patients showed bilateral cataracts. One patient presented with CMMMA. MRI of the individuals showed cavum septum pellucidum et vergae, megacisterna magna, T2W hyperintensity and atrophy of the basal ganglia, and T2W hyperintensity of the posterior thalami.

Conclusion: Our description establishes bi-allelic ZBTB11 variants as a cause of disorder characterized by variable combinations of neurodevelopmental delay, intellectual disability, complex movement disorders, cataracts and CMMMA. Our study adds the phenotype and expands the causative genetic variant spectrum of this complex movement disorder seen in adult patients and the response to deep brain stimulation.

Conflict of Interest: None declared.

EP09.051 Koolen-de Vries syndrome: Two patients with 17q21.31 microdeletion and de novo KANSL1 variant

Sena Cetin1, Elif Yuksel Karatoprak2, Ceren Melis Ozkan3, Filiz Ozen 4, Elif Yilmaz Gulec1

1Istanbul Medeniyet University Medical School, Department of Medical Genetics, Istanbul, Turkey; 2Istanbul Medeniyet University Medical School, Department of Pediatric Neurology, Istanbul, Turkey; 3Istanbul Goztepe Prof. Dr. Suleyman Yalcin City Hospital, Pediatric Neurology Clinic, Istanbul, Turkey; 4Istanbul Goztepe Prof. Dr. Suleyman Yalcin City Hospital, Department of Medical Genetics, Istanbul, Turkey

Koolen-de Vries syndrome (KdVS; MIM#610443) is a clinically heterogeneous disorder characterized by developmental delay, neonatal hypotonia and facial dysmorphisms. Epilepsy, congenital heart defects, urogenital malformations and ectodermal anomalies were also reported. The syndrome either caused by a monoallelic single nucleotide variant, mostly truncating, in KANSL1 (KAT8 Regulatory NSL Complex Subunit 1, MIM#612452) gene or by haploinsufficiency or disruption of KANSL1 gene secondary to 17q21.31 microdeletion. This gene is responsible for encoding a nuclear protein called KAT8 regulatory NSL complex Subunit 1, which is involved in chromatin modification.

Two unrelated boys (seven and five years old) were referred to our clinic from pediatric neurology because of global developmental delay and intellectual disability. The seven-year-old one had cerebral palsy, epilepsy and corpus callosum agenesis, additionally. He was followed by pediatric endocrinology and nephrology due to hypothyroidism and bilateral vesicoureteral reflux. A de novo nonsense heterozygous variant c.2470C>T p. (Arg824*) in the KANSL1 gene was detected using a multigene panel based exon sequencing.

The five-year-old one had a history of neonatal hypotonia, unilateral cryptorchidism and laryngomalacia. He had typical features of the disorder such as bilateral epicanthal folds, hypertelorism, anteverted ears, long and slender fingers, asymmetric thorax and nasal speech. He also had horseshoe kidney and immunodeficiency due to low B-lymphocyte count. Molecular karyotyping revealed a 536.3 Kb deletion on chromosome 17q21.31 encompassing KANSL1.

KdVS is a rare autosomal dominantly inherited syndrome associated with global developmental delay. This report provides insight into clinical differences between KdVs patients with microdeletion and KANSL1 point mutations.

Conflict of Interest: None declared.

EP09.053 Multiple genetic disorders in one family - a new de novo frameshift variant disrupting ZBTB7A, familial hemiplegic migraine related to ATP1A2 and WNT10A linked hypodontia

Eva Grauer 1, Victoria Strohhäcker1, Dieter Gläser1, Gerlinde Funck2

1MVZ genetikum GmbH, Neu-Ulm, Germany; 2Alb FIls Kliniken, Social Pediatric Center, Goeppingen, Germany

Background: We report on a 5-year-old female index patient with global developmental delay, macrocephaly and overgrowth of adenoid tissue. Her 37-year-old mother suffered from epilepsy and migraine with hemiplegic episodes. Both daughter and mother have hypodontia. The father is healthy.

Methods: Chromosome analysis, array-CGH (180k Agilent, design 086332) and trio whole exome sequencing (Twist Human Comprehensive Exome with additional customized probes and NGS with NovaSeq Illumina) was performed. Validation of de novo variants was done by sanger sequencing.

Results: Chromosome analysis and array-CGH of the index gave normal results. Using trio analysis a heterozygous, de novo frameshift variant c.1073del, p.(Gly358Alafs*68) in ZBTB7A (NM_015898.4) was identified in the patient. Loss-of-function variants in ZBTB7A cause a newly discovered autosomal dominant intellectual disability syndrome with so far only 13 published cases by Oishi et al., in 2020 and von der Lippe et al., in 2022. Macrocephaly and lymphoid hyperplasia seem to be typical signs of ZBTB7A related disorder and hypodontia is also reported in published cases. In the mother known pathogenic heterozygous variants c.1643G>A, p.(Arg548His) in ATP1A2 (NM_000702.4) and c.682T>A, p.(Phe228Ile) in WNT10A (NM_025216.3) were identified, which were not present in the index, and can account for all symptoms of the mother.

Conclusion: This case report highlights the still growing heterogeneity of intellectual disability syndromes and the importance of using trio analysis to easily identify variants in newly discovered genes in diagnostic processes. Furthermore, this example emphasises the need of considering multiple genetic diseases as causal for different symptoms in one family.

Conflict of Interest: Eva Grauer MVZ genetikum GmbH, Victoria Strohhäcker MVZ genetikum GmbH, Dieter Gläser MVZ genetikum GmbH, Gerlinde Funck: None declared.

EP09.054 Health-related quality of life of carers, their spouse and patients affected by familial ID and change after genomic diagnosis

Deborah Schofield1, Joshua Kraindler 1, Katherine Lim1, Rupendra Shrestha1, Owen Tan1, Sarah West1, Natalie Hart1, Jackie Boyle2, Lucinda Murray2, Tony Roscioli3;4;5, Mike Field2

1GenIMPACT: Centre for Economic Impacts of Genomic Medicine, Macquarie University, Australia; 2Genetics of Learning Disability (GoLD) Service, Newcastle, Australia; 3New South Wales Health Pathology Genomics, Prince of Wales Hospital, Sydney, Australia; 4Centre for Clinical Genetics, Sydney Children’s Hospital, Sydney, Australia; 5Neuroscience Research Australia - NeuRA, Randwick, Australia

Background: Intellectual disability (ID) is associated with far reaching lifetime impacts on patients’ health and functioning, resulting in significant care needs. For families, genetic testing can inform an etiologic diagnosis, which can assist in access to appropriate health and social care services. Presently, little is known about the quality of life (QoL) of patients with familial ID or how a genetic diagnosis may impact the quality of life of carers and patients.

Methods: Data on quality of life and genetic testing was collected as part of the EPIC-ID study. 111 households (carers and patients) who had a range of genetic testing including whole genome sequencing were recruited and administered the EPIC-ID survey in New South Wales, Australia. QoL data was collected through validated measures of QoL: the Health Utilities Index (HUI) for children and the Assessment of Quality-of-Life 8D (AQoL-8D) for adult carers and their spouse.

Results: We will present results on QoL of carers and their spouses, measured through health utilities from the AQoL-8D. Results for the health-related quality of patients will be reported using health utilities based on the HUI. Change in quality of life before and after diagnosis will also be reported.

Conclusion: Health-related utility data for carers of those with familial ID should be used to inform cost-effectiveness studies for genetic testing and targeted therapies, supporting access to address the health and social care needs of families with ID.

Funding: Supported by the National Health and Medical Research Council (NHMRC) (Grant ID: 113895).

Conflict of Interest: Deborah Schofield PI on NHMRC Grant 1116360, Joshua Kraindler: None declared, Katherine Lim: None declared, Rupendra Shrestha CI APP1113895, Owen Tan: None declared, Sarah West: None declared, Natalie Hart: None declared, Jackie Boyle: None declared, Lucinda Murray: None declared, Tony Roscioli CI on NHMRC 1113895, Mike Field CI on NHMRC 1113895.

EP09.055 A novel candidate gene for neurodevelopmental disorders: JKAMP

Enise AVCI DURMUŞALİOGLU1, Esra Isik 1, Muzaffer Polat2, Ebru Canda1, Tahir Atik1

1Ege University, Medical Faculty, İzmir, Turkey; 2Manisa Celal Bayar University, Medical Faculty, Manisa, Turkey

Bakground/objectives: The widespread use of whole exome sequencing (WES) in children with developmental delay (DD) or intellectual disability (ID) allows to elucidate genetic etiology of neurodevelopmental disorders (NDDs). Herein, we presented a case with a homozygous variant in the JKAMP gene and aimed to show JKAMP could be a novel candidate gene for NDDs.

Case Report: A 4-year-old boy presented with ID, epilepsy and autistic features. He was the fourth child of consanguineous parents, one of his brothers had died due to prematurity and the other two brothers were healthy. Developmental delay had been noticed when he was 6 months old, he had diagnosed with epilepsy at the age of 1 year and had never gained the ability to speak and communicate. He had a narrow forehead, synophrysis, long eyelashes, depressed nasal bridge, short nose, broad nasal tip, long philtrum, and thin upper lip in dysmorphologic evaluation. In the WES, a homozygous frameshift variant (c.243dup, p.Lys82GlufsTer16) classified as “likely pathogenic” was detected in the JKAMP gene (ENST00000556985). His parents and both healthy brothers were heterozygous for the variant.

Discussion: The variant detected in our patient has been previously reported in a 9-year-old case with ID, epilepsy and autism by Strauss et al., The expression of JKAMP was shown in wide range of mouse tissues including brain but the gene does not have a confirmed disease association.

Conclusion: Based on these two cases, JKAMP should be considered as a candidate gene for a new NDD and supported by functional analyses.

Conflict of Interest: None declared.

EP09.056 Combined screening for genetic causes of idiopathic intellectual disability by chromosomal microarray analysis and trio whole exome sequencing in a large cohort of Russian patients

Elizaveta Fonova 1, Anna Kashevarova1, Maria Lopatkina1, Alexey Sivtsev1, Aleksei Zarubin1, Viktoriia Demeneva1, Andrew Zuev1, Gulnara Seitova1, Larisa Minaycheva1, Olga Salyukova1, Valeria Petrova1, Svetlana Fadyushina1, Dmitry Fedotov1, Elena Belyaeva1, Ekaterina Tolmacheva1, Nadezhda Babushkina1, Nikolay Skryabin1, Ludmila Nazarenko1, Igor Lebedev1

1Research Institute of Medical Genetics, Tomsk, Russian Federation

Background/Objectives: Chromosomal microarray analysis (CMA) and whole exome sequencing (WES) are powerful techniques for the search of genetic causes of intellectual disability (ID). The aim of the study was to evaluate the value of combined CMA and WES analysis for patients with ID due to congenital brain anomalies

Methods: CMA was performed by 60K Agilent microarrays. WES was performed with SureSelect Human All Exon V8 on NextSeq 2000 Sequencing system.

Results: Pathogenic or probably pathogenic CNVs were detected in 422 from 1175 patients with ID (36%). For 9 selected families with a one child and 3 families with two affected children (a total of 39 members including parents) with ID due to congenital brain anomalies, like polymicrogyria, pachygyria, lissencephaly, but balanced karyotype, WES was applied. As a result, 3 pathogenic variants in 3 families, 3 likely pathogenic in three other families, and 10 variants with uncertain clinical significance for 4 families were identified. Novel pathogenic missense variants were identified in the DYNC1H1, MACF1, CDKL5, MID1 genes. The majority of variants are arisen de novo.

Conclusion: Trio-based WES has been shown to be an important step in making a genetic diagnosis for families with balanced karyotype after microarray analysis. The most important step for the correct interpretation of WES results is a deep phenotyping of patient, which allows to establish the exact genetic cause of the disease if several variants with unclear clinical significance were previously identified.

Grant References: This study was supported by the Russian Science Foundation, grant 21-65-00017.

Conflict of Interest: None declared.

EP10 Neurogenetic and Psychiatric Disorders

EP10.001 A novel variant of the POLR3A gene in a patient with hypomyelinating POLR3-related leukodystrophy

Jeemin Kim 1, Ji Yoon Han1, Hyein Yeo1

1The Catholic University of Korea Daejeon St. Mary’s Hospital, Pediatric Neurology, Daejeon, Korea, Rep. of South

Background: Hypomyelinating POLR3-related leukodystrophy is a group of rare neurological diseases characterized by degeneration of the white matter of the brain with different combinations of major clinical findings. Here we report the first Korean POLR3-related leukodystrophy caused by bi-allelic POLR3A c.1771-6C > G and novel c.1650_1661del variants.

Methods: An 18-month-old girl was admitted for evaluation of a seizure-like activity with spasticity that affected her entire body. She showed dental abnormalities, but not suspicious facial dysmorphism. She was in a bed-ridden state with severe cognitive impairments and episodes of dystonic posturing for 1–2 min. Trio exome sequencing (ES) was performed to determine the potential genetic cause of severe developmental delay with leukodystrophy in our proband.

Results: Trio ES revealed that bi-allelic POLR3A deleterious variants, c.1650_1661del of the exon 13, and c.1771- 6C > G of the intron 13 were best candidate as causes of hypomyelinating POLR3-related leukodystrophy. Sanger sequencing confirmed the genetic origin of these POLR3A deleterious variants as autosomal recessive hereditary transmission.

Conclusion: Our report provides additional evidence for a phenotypic continuum of hypomyelinating POLR3- related leukodystrophy caused by bi-allelic POLR3A variants. Further genetic studies are required to understand underlying pleiotropic effects of different POLR3A variants.

Grant References: n/a.

Conflict of Interest: Jeemin Kim Daejeon St.Mary’s Hospital (Full time), Ji Yoon Han Daejeon St.Mary’s Hospital (Full time), Hyein Yeo Daejeon St.Mary’s Hospital (Full time).

EP10.002 Mutational spectrum of Greek patients with axonal Charcot-Marie-Tooth disease

Zoi Kontogeorgiou1, CHRISOULA KARTANOU 1, Michail Rentzos2, Thomas Zampelis3, Panagiotis Kokotis3, Georgios Koutsis1, Georgia Karadima1

1Neurogenetics Unit, 1st Department of Neurology, Eginitio University Hospital, National and Kapodistrian University of Athens, Athens, Greece; 21st Department of Neurology, Eginitio University Hospital, National and Kapodistrian University of Athens, Athens, Greece; 3Clinical Neurophysiology Unit, 1st Department of Neurology, School of Medicine, Eginition Hospital, National and Kapodistrian University of Athens, Athens, Greece

Background: Axonal forms of Charcot-Marie-Tooth disease (CMT) are classified as CMT2, dominant intermediate CMT (DI-CMT), hereditary sensory and autonomic neuropathy (HSAN) and distal hereditary motor neuropathy (dHMN). They are caused by mutations in over 30 genes. The aim of this study was to decipher the genetic landscape of axonal CMT in the Greek population.

Methods: Seventy-four index patients with axonal neuropathy, negative for CMT1A, were initially Sanger sequenced for GJB1 and, if negative, further screened by an NGS custom gene panel covering 24 of the most mutated genes in axonal CMT. The study was carried out in the Neurogenetics Unit of the 1st Department of Neurology, NKUA, Eginition Hospital.

Results: Overall, we identified 17 cases with GJB1 mutations and 12 cases with pathogenic or likely pathogenic variants in a further six genes (MPZ, MFN2, GDAP1, DNM2, BSCL2, LRSAM1), representing 39.2% of the cohort. One of these variants, originally characterized as variant of uncertain significance (VUS), was further evaluated through ACMG criteria and re-classified as likely pathogenic.

Conclusion: The present diagnostic algorithm had a total yield of 39.2%, while the NGS panel had a yield of 21.1%. Given the limited number of genes tested, these results compare favorably with studies in other European populations. Our study delineates the genetic and phenotypic variability of inherited axonal neuropathies in the Greek, while also contributing to the pathogenicity characterization of one VUS.

Grant: This study was partly supported by a grant from Genesis Pharma (Grant/Award Number: 13044, special account for research grants, NKUA).

Conflict of Interest: None declared.

EP10.004 A preliminary report on fragile X syndrome carrier screening in Thai women

Areerat Hnoonual 1;2, Sunita Kaewfai3, Oradawan Plong-On1, Pornsiri Sangmanee1, Pornprot Limprasert1;2

1Department of Pathology, Faculty of Medicine, Prince of Songkla University, Songkhla, Thailand; 2Genomic Medicine Center, Faculty of Medicine, Prince of Songkla University, Songkhla, Thailand; 3Graduate Program in Biomedical Sciences, Faculty of Medicine, Prince of Songkla University, Songkhla, Thailand

Background/Objectives: Fragile X premutation carriers have considerable public health significance because they are at increased risk of having a child with FXS and at risk of developing health problems. There have been no reported studies examining the prevalence of fragile X premutation carriers in Thai women. This study aimed to estimate the prevalence of premutation carriers and the distribution of the FMR1 alleles in Thai women.

Methods: Fragile X screening was performed in 369 Thai female blood donors who had no psychiatric or neurological disorders and no family history of psychiatric or neurological disorders for three generations by questionnaire testing. Fluorescent PCR and triplet repeat primed PCR was performed to determine the FMR1 CGG repeat. Southern blotting was used to confirm repeat numbers and methylation status of expanded allele.

Results: Of the 369 women, one individual carried a normal allele in whom a premutation allele (89 CGG repeats with one AGG interruption) was identified. We also found four individuals (1.08% or 1/92) with intermediate alleles (46,49,49,49 CGG repeats). The most common CGG repeat allele was 30, followed by 29 and 36.

Conclusion: This study provides the first evidence for study of fragile X carrier screening in Thai women with a prevalence of 0.27% (1/359). This finding can help to improve fragile X screening in reproductive-age and preconception women to prevent unwanted births of FXS-affected children. We also recommend that carrier screening to identify female carriers should be carried out on a large scale in our population.

Conflict of Interest: Areerat Hnoonual Lecturer (Full time) at Prince of Songkla University, Grant from Faculty of Medicine, Prince of Songkla University (REC62-018-5-2), Sunita Kaewfai: None declared, Oradawan Plong-On Medical scientists (Full time) at Faculty of Medicine, Prince of Songkla University, Pornsiri Sangmanee Medical scientists (Full time) at Faculty of Medicine, Prince of Songkla University, Pornprot Limprasert Professor (full time) at Faculty of Medicine, Prince of Songkla University, Grant from The Education&Public Welfare Foundation.

EP10.005 Differential expression of Alzheimer’s disease associated genes in rat brain with ischemic stroke

Svetlana A. Limborska 1, Ivan B. Filippenkov1, Alina E. Denisova2, Vasily V. Stavchansky1, Ivan V. Mozgovoy1, Leonid V. Gubsky2;3, Lyudmila V. Dergunova1, Andrey V. Khrunin1

1National Research Center “Kurchatov Institute”, Institute of Molecular Genetics, Moscow, Russian Federation; 2Pirogov Russian National Research Medical University, Moscow, Russian Federation; 3Federal Center for the Brain and Neurotechnologies, Federal Biomedical Agency, Moscow, Russian Federation

Background/Objectives: Alzheimer’s disease (AD) and ischemic stroke (IS) are among the most significant neuropathologies. The AD- and IS-related neurodegeneration profiles largely coincide. Moreover, IS served as a significant risk factor for AD. Therefore, both diseases are often considered as comorbidities[1]. Here, using CEU-based proxies for AD-related single nucleotide polymorphisms (SNPs), we created an extended list of candidate AD-associated genes. Then, RNA-Seq of penumbra-associated dorsolateral areas of the frontal cortex of rats at 24h after transient middle cerebral artery occlusion (tMCAO) was obtained, and rat orthologous of AD-associated genes were annotated by gene expression data.

Methods: Wistar rats, tMCAO, magnetic resonance imaging, RNA-Seq, bioinformatics.

Results: Human SNPs that had sqr(r)≥0.2 with known AD-related SNPs were found using LDlink tool. They were annotated with 198 genes. Then, 149 rat orthologous genes were found using g:Profiler and RGD tools. Twenty six of them were differentially expressed genes (DEGs) with cut-off>1.5 and padj<0.05 after tMCAO. They were mainly associated with antigen processing and presentation. Namely, 15 DEGs (e.g. RT1-Da, Psmb8, Tap1) were up-, and 11 (e.g. C1qtnf4, Apoe, Ptk2b) were downregulated in IS conditions.

Conclusion: The genetic relationship between AD and IS was found by exploration of human DNA variations, functional genomics, and bioinformatics. Perhaps, the identified genes are fragile nodes for the development of neurodegeneration. Our results contribute to the improvement of approaches to the analysis and prevention of complex brain comorbidities.

References: Rost et al. Stroke. 2021;52(8):e499-e516

Grant: The research was funded by the Russian Science Foundation, grant №19-14-00268, https://rscf.ru/project/22-14-35023/.

Conflict of Interest: None declared.

EP10.006 Interest of exome sequencing in non-syndromic specific learning disorders: a French pilot study

Eléonore Viora-Dupont 1;2, Julian Delanne1;2, Aurore Garde1;2, Sophie Nambot1;2, Estelle Colin1;2, Marie Bournez1, Clémence Fauconnier-Fatus1, Céline Bernard1, Aurélie Espitalier1, Christine Binquet3, Marion Bouctot3, Marie-Laure Humbert3, Anne-Sophie Briffaut3, Véronique Darmency4, Patricia Plumet4, Noémie Relin1;4, Patrick Callier2;5, Anne-Laure Mosca-Boidron2;5, Nathalie Marle2;5, Frederic Tran Mau Them2;5, Anne-Sophie Denommé-Pichon2;5, Hana Safraou2;5, Antonio Vitobello2;5, Ange-Line Bruel2;5, Christophe Philippe2;5, Christel Thauvin-Robinet1;2, Laurence Faivre1;2

1CHU Dijon-Bourgogne, Centre de Génétique et centre de référence des anomalies du développement et syndromes malformatifs de l’Est, Dijon, France; 2Université de Bourgogne, INSERM-Université de Bourgogne UMR1231 GAD “Génétique des Anomalies du Développement”, FHU-TRANSLAD, Dijon, France; 3Université de Bourgogne, CIC 1432 Module Épidémiologie Clinique, Dijon, France; 4CHU Dijon-Bourgogne, Centre Référent des Troubles du Langage et des Apprentissages, Dijon, France; 5CHU Dijon-Bourgogne, Unité Fonctionnelle d’Innovation diagnostique des maladies rares, Dijon, France

Background/Objectives: Specific learning disorders (SLD) (dyslexia/dysorthography, dysphasia, dyspraxia, dyscalculia and ADHD) affect about 5 to 10% of school-age children. In France, in the absence of a syndromic diagnosis, array-CGH can be proposed, associated in girls with the search for a X-fragile syndrome. Nevertheless, examples of genes described in ID and also described in patients with SLD are multiplying. Some international teams have identified by exome sequencing (ES) a certain percentage of patients, with severe SLD, associated with a monogenic disease.

Material and Methods: We initiated a pilot project in the care setting, aiming to propose ES in patient referred to genetic consultation for well-documented severe SLD.

Results: 72 patients have been included. Among the 64 patients analyzed, likely pathogenic variants were identified in 7 (CDK13, KCNN2, SMARCC2(x2), SET, TRIO and PKD1), 20 variants of uncertain significance (VUS) in epilepsy or ID genes were identified in 16 patients and 8 variants in genes of unknown significance (GUS). Thus, a diagnosis was obtained in 11% (36% with VUS). Families segregations and episignatures are in progress in order to help in the classification of some VUS.

Discussion: All patients in whom a likely pathogenic variant or variant of interest was identified have either multiple SLD or have an association with behavioral disorders. The fact that the selected genes are involved in other NND is consistent with a broadening of the clinical phenotype. The study will be continued in additional 100 patients in order to confirm this first results.

Conflict of Interest: None declared.

EP10.007 Analysis of intronic and exonic variants in patients with DEPDC5-related epilepsy and correction of splicing using modified snRNAs

Evgeniya Osipova 1, Igor Bychkov1, Alexandra Filatova1, Artem Borovikov1, Artem Sharkov2;3, Snezhana Sharkova4, Dmitriy Saushev5, Vyacheslav Tabakov1, Mikhail Skoblov1

1Research Centre for Medical Genetics, Moscow, Russian Federation; 2Veltischev Research and Clinical Institute for Pediatrics and Pediatric Surgery of the Pirogov Russian National Research Medical University, Moscow, Russian Federation; 3Genomed, Moscow, Russian Federation; 4Research Center of Neurology, Moscow, Russian Federation; 5Don state technical university, Rostov-on-Don, Russian Federation

Background/Objective: Considering different effects of splicing variants, it is necessary to perform functional assays to establish the exact molecular mechanism and to estimate the pathogenicity of the variant. It helps to make an accurate diagnosis for patients and develop possible strategies to personalized therapy.

Methods: The impact of the variants on splicing was evaluated using SpliceAI and SPiP tools. Functional analysis was carried out using RT-PCR and minigene assay. Correction of splicing was performed using U1 and U7 snRNAs in HEK293T cells.

Results: We carried out a functional assay for four previously unpublished exonic and intronic variants in patients with DEPDC5-related epilepsy using RT-PCR and presented their effect on splicing. Using minigene assay, we investigated seven previously described exonic variants and showed that three of them affect splicing. One exonic variant c.3264G>A was chosen for splicing correction. We created 22 constructions using modified U1 snRNAs and U7 snRNAs with different splicing regulatory elements: exonic silencers, exonic enhancers and intronic enhancers. The most effective and specific construction contained U7 snRNA with intronic enhancer and increased the amount of the wild-type transcript up to 55%.

Conclusion: This work shows the importance of functional assays to evaluate the effect of intronic and exonic variants on splicing. U7 snRNAs can be used as a promising approach for future therapy in patients with DEPDC5-related epilepsy.

Conflict of Interest: None declared.

EP10.009 Investigating the SNPs involved in metabolism in Cypriot ATTR patients

Christiana Christodoulou 1, elena panayiotou-worth2, Eleni Zamba-Papanicolaou1

1The Cyprus Institute of Neurology and Genetics, Neuroepidemiology, Nicosia, Cyprus; 2The Cyprus Institute of Neurology and Genetics, Neuropathology, Nicosia, Cyprus

Background/Objectives: Amyloidosis polyneuropathy (ATTR) is a rare, inherited and progressive disease, Val30Met is the most common mutation. ATTR is caused by an accumulation of amyloid fibrils, which contains the protein transthyretin. The protein deposits accumulate in several tissues such as the nerves, heart, kidneys and eye. The aim of this project was to investigate metabolic involvement in carriers and patients of V30M mutation, with varying symptoms and age of onset. The SNPs chosen have an involvement in the onset and progression of metabolic disorders.

Methods: The SNPs rs1183910 (HNF1A), rs1421085 (FTO), rs17782313 (MC4R) and rs2943634 (IRS1) were investigated. DNA samples from a cohort of 48 patients and 48 gender and age matched controls were used for the TaqMan SNP Genotyping Assay. PCR purification was performed through the MontageTM PCRm96 plate (Millipore). The Pearson’s chi-square test was used to determine if there was a statistically significant difference between expected and observed SNP frequencies of cases vs controls for the investigated SNPs.

Results: Regarding the HNF1A (p-value1), FTO (0.6632), and IRS1 (p-value 0.412244) did not show any statistically significant results. However, MC4R (p-value 0.000988) was found to be statistically significant.

Conclusion: To the best of our knowledge, this is the first study investigating the SNPs of NHF1A, FTO, MC4R and IRS1 in Cypriot ATTR patients. Our study identified that MC4R was strongly related to ATTR. In addition, bioinformatics analysis will be applied in future studies to investigate the role of metabolism SNPs and metabolic pathways in ATTR.

Conflict of Interest: Christiana Christodoulou Full time, elena panayiotou-worth Ful time, Eleni Zamba-Papanicolaou Full time.

EP10.010 De novo variant analysis of childhood-onset obsessive-compulsive disorder in the French-Canadian population

Kate Bornais 1;2, Jay Ross1;2, Zoe Schmilovich1;2, Miranda Medeiros1;2, Dan Spiegelman2;3, Patrick Dion2;3, Guy Rouleau1;2;3

1McGill University, Human Genetics, Montreal, Canada; 2Montreal Neurological Institute, Montreal, Canada; 3McGill University, Neurology and Neurosurgery, Montreal, Canada

Background: Obsessive-compulsive disorder (OCD) is a neuropsychiatric disorder characterized by cycles of intrusive thoughts or sensations (obsessions) and repetitive behaviours (compulsions). Despite an estimated heritability of 45-65%, known genetic factors are not sufficient to explain risk. De novo variants (DNVs) arise post-zygotically in individuals but are not present in biological parents and may contribute to OCD risk. However, the number of identified DNVs in OCD remains limited and the genetic contribution to OCD suggests more are likely to exist. Here, we identified DNVs in French-Canadian (FC) childhood-onset OCD trios.

Methods: Whole-exome sequencing was performed on 53 FC OCD trios (n = 159). Single-nucleotide protein-coding variants were prioritized. Candidate DNVs were selected based on estimated impact on gene function, population minor allele frequency < 0.1%, elevated deleteriousness (CADD > 20), and biological relevance. The candidate gene list was then used to identify enriched biological and molecular pathways underlying childhood-onset OCD. Regression analyses estimated the impact of DNVs on OCD symptom severity.

Results: 89 DNVs across 89 genes were observed in 42 probands (2.12 DNVs/proband, average CADD Score=26.5). 15 of these genes were identified in previous OCD publications (Halvorsen 2021; Cappi 2020). The top enriched Gene Ontology (GO) term was “regulation of neuron projection development” (p = 9.50E-05). The number of DNVs carried by a proband did not significantly explain OCD symptom severity.

Conclusion: Findings from this study shed light on the genetic and biological factors underlying childhood-onset OCD risk, further highlighting the ability to leverage the FC population to elucidate the genetics of complex neuropsychiatric disorders.

Conflict of Interest: None declared.

EP10.011 Mutations in H3.3 that cause a neurodevelopmental syndrome disrupt chaperone interactions

Kelly Clark 1;2, Laura Bryant2, Elizabeth Bhoj2;3;4

1University of Pennsylvania, Cell and Molecular Biology Graduate Group, Philadelphia, United States; 2Children’s Hospital of Philadelphia, Division of Human Genetics, Philadelphia, United States; 3University of Pennsylvania, Department of Genetics, Philadelphia, United States; 4Children’s Hospital of Philadelphia, Center for Applied Genomics, Philadelphia, United States

Background/Objectives: Bryant-Li-Bhoj syndrome is a disorder characterized by neurodegeneration and multiple congenital anomalies. This syndrome is caused by mutations in the genes H3-3A and H3-3B, which encode the replication-independent histone H3.3. Histone H3.3 is deposited on euchromatin by the chaperone complex HIRA and on heterochromatic regions by DAXX. Prior work investigating the pathogenesis of the causative variants underlying Bryant-Li-Bhoj syndrome has identified changes in post-translational modification profiles, transcript expression, and proliferation rate in cells with these H3.3 mutations compared to control cells. Notably, patient fibroblasts that harbor H3.3 mutations have a hyperproliferative phenotype. Based on these observed alterations and guided by the patient phenotypes, we are now investigating the effects of patient H3.3 mutations on chaperone interactions and telomere maintenance.

Methods: Using nuclear co-immunoprecipitation assays, we assessed changes to HIRA and DAXX interactions with H3.3 in H3.3-overexpressing HeLa cells harboring different patient mutations. To assess telomere length, we used a telomere restriction fragment (TRF) assay with patient-derived fibroblasts.

Results: The H3.3 patient mutations p.G90R and p.P121R both disrupt H3.3-DAXX interaction, suggesting that H3.3 deposition on heterochromatin may be altered in these cells. TRF analysis showed shortened telomeres in patient-derived cells harboring the p.G90R and p.P121R mutations.

Conclusion: Disrupted DAXX-H3.3 interaction is associated with either loss of establishment of telomere length in stem cells or accelerated telomere shortening. While loss of DAXX interaction is not seen with all Bryant-Li-Bhoj patient mutations, these data may indicate a broader molecular phenotype of convergent H3.3 dysregulation across mutations.

Grant References: Hartwell Foundation, Burroughs-Wellcome Fund.

Conflict of Interest: None declared.

EP10.012 The MAPT H1b haplotype as a risk factor for sporadic amyotrophic lateral sclerosis

Ivan Tourtourikov 1;2, Kristiyan Dabchev2;3, Tihomir Todorov2, Teodor Angelov4, Ivaylo Tournev4;5, Vanyo Mitev4, Albena Todorova1;2

1Medical University of Sofia, Medical Chemistry and Biochemistry, Sofia, Bulgaria; 2Genetic Medico‑Diagnostic Laboratory ‘Genica’, Sofia, Bulgaria; 3Sofia University St. Kliment Ohridski, Faculty of Biology, Sofia, Bulgaria; 4Medical University of Sofia, Department of Neurology, Faculty of Medicine, Sofia, Bulgaria; 5New Bulgarian University, Department of Cognitive Science and Psychology, Sofia, Bulgaria

Background: Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder that affects nerve cells in the brain and spinal cord, ultimately leading to death. The majority of ALS cases (90-95%) are sporadic (sALS) and present without a clear genetic cause or a family history. The microtubule-associated protein tau (MAPT) gene has been linked to sALS, with studies showing that altered tau protein is part of the overlap between sALS and the frontotemporal dementia (FTD) spectrum.

Methods: А total of 100 sALS patients from Bulgaria were genotyped for the H1 and H1 subtypes tagging SNPs (rs1467967, rs242557, rs1800547, rs3785883, rs2471738, and rs7521). Genotyping data for 2504 individuals from the 1000 Genomes Project Phase 3 was used to create a control group for the haplotype reconstruction. The most frequent haplotypes were reconstructed with Haploview 4.2 and SHEsisPlus; individual SNP association tests and haplotype analysis was performed with the same software.

Results: Haplotype H1b emerged as a risk factor for ALS, with close to a two-fold increased risk of developing sALS compared to other haplotypes (odds ratio 1.973, 95% CI 1.279-3.044). These results are statistically significant (Fisher’s p = 0.004, Pearson’s p = 0.001) after test correction methods and 500000 permutations in Haploview.

Conclusion: The MAPT H1b subhaplotype was associated with a two-fold increase in sALS risk. Further functional evaluations of the MAPT H1 subtypes are required to determine their contribution to an individual’s risk of sALS.

Grant References: The financial support of Medical University Sofia, Grant № D-186 /14.06.2022 is gratefully acknowledged.

Conflict of Interest: Ivan Tourtourikov The financial support of Medical University Sofia, Grant № D-186 /14.06.2022 is gratefully acknowledged., Kristiyan Dabchev: None declared, Tihomir Todorov: None declared, Teodor Angelov: None declared, Ivaylo Tournev: None declared, Vanyo Mitev The financial support of Medical University Sofia, Grant № D-186 /14.06.2022 is gratefully acknowledged., Albena Todorova The financial support of Medical University Sofia, Grant № D-186 /14.06.2022 is gratefully acknowledged.

EP10.013 The sensory neuropathy caused by pathogenic amplification in the RFC1 gene is not so frequent among the Czech neuropathy patients

Dana Safka Brozkova 1, Anna Uhrova Meszarosova1, Denisa Stanclova1, Petra Lassuthova1

12nd Faculty of Medicine, Charles University and University Hospital Motol, Neurogenetic laboratory, Prague, Czech Republic

Background/Objectives: Hereditary sensory neuropathies comprise a broad group of patients manifesting with sensory nerve affection. This genetically determined disorder can be caused by pathogenic variants in almost 20 known genes. Recently, the pathogenic pentanucleotide amplification in the intron of the RFC1 gene was reported in the homozygous state as a cause of the CANVAS syndrome. Except the sensory neuropathy, also the cerebellar ataxia and vestibular syndrome are other clinical manifestation of the disease.

Methods: In our laboratory, we focus on diagnostics and research of hereditary neuropathies. We selected 1519 patients without genetic diagnosis. The selection criteria were; neuropathy, preferably sensory type, recessive or sporadic inheritance and age of onset after 20 years of age. Firstly, we performed the short range PCR to amplify the intronic region of the RFC1 gene. Secondly, in 239 samples with no product or atypical length of the product, we performed the triple repeat primed PCR for three different alleles usually presented in this area.

Results: Finally, in six patients with no product from short range PCR we proved the pathogenic amplification of the AAGGG allele and we genetically proved the CANVAS disease.

Conclusion: The genetics correlated with the clinical findings of the patients, where later onset (30 years +) and primary sensory neuropathy dominated.

Grant reference: Supported by the MHCR AZV NU22-04-00097.

Conflict of Interest: Dana Safka Brozkova Principal investigator of grant Exploring causes of the neurogenetic disorders with the newest genomic methods, Anna Uhrova Meszarosova: None declared, Denisa Stanclova: None declared, Petra Lassuthova: None declared.

EP10.014 Potential associations between PER2, PER3, HCRTR2 and APOE genetic variants with neuropsychiatric symptoms in patients with Alzheimer’s disease

GLADYS SUSANA LOZANO TOVAR 1, Yaneth Rodríguez-Agudelo2, David José Dávila-Ortiz de Montellano3, Nancy Monroy-Jaramillo3, Alberto Ortega-Vázquez4, Blaca Estela Pérez-aldana5

1Universidad Nacional Autónoma de México (UNAM), Facultad de psicología, Ciudad d México, Mexico; 2Instituto Nacional de Neurología y Neurocirugía, “Manuel Velasco Suárez”, Laboratorio de Neuropsicología clínica, Ciudad de México, Mexico; 3Instituto Nacional de Neurología y Neurocirugía, “Manuel Velasco Suárez”, Departamento de genética, Ciudad de México, Mexico; 4Universidad Autónoma Metropolitana, unidad Xochimilco, Departamento de Sistemas Biológicos, Ciudad de México, Mexico; 5Universidad Autónoma Metropolitana, unidad Xochimilco, Departamento de Sistemas Biológicos, Ciudad de México, Mexico

Background/Objectives: Alzheimer’s disease (AD) presents multifactorial neuropsychiatric symptoms (NPS). NPS have been associated with APOE_Ɛ4 allele which is also the major genetic AD risk factor. Circadian genes dysregulation and orexins-HCRTR2 genes are two innovative proposals for BPSD in AD. Period genes and orexins have been associated with mood and sleep disturbances. There are no studies considering gene-gene interactions.

Methods: The association of seven variants in PER2, PER3, APOE and HCRTR2 genes was evaluated in 31 AD patients and 31 paired cognitively healthy controls. All participants signed an informed consent letter (protocol 11/20). Genotyping was performed from blood samples. Allelic-genotypic frequencies of variants were calculated for the sample study. We explored associations between allelic variants with NPS in AD patients based on NPI, PHQ-9 and sleeping disorders questionnaires. Gene-gene interaction analysis by MDR was included.

Results: APOE_Ɛ4 allele was confirmed as an AD risk variant (p = 0.03). The remaining variants did not reveal significant differences between patients and controls. The PER3_rs228697 variant showed an increased risk for circadian rhythm sleep-wake disorders in Mexican AD patients (OR = 9.736, p = 0.028). Gene-gene interaction analysis included PER2_rs2304672, PER3_rs228697 and APOE_ɛ4 variants in the best model.

Conclusions: PER3_rs228697 variant was associated with a nine-fold increased risk for circadian rhythm sleep-wake disorders in patients. We identified a novel PER2-PER3-APOE_ɛ4 interaction in AD patients; therefore, patients carrying APOE_ε4 allele might present a higher risk for circadian rhythm dysregulation due to this novel interaction. However, these findings need to be further confirmed in larger samples.

Grant References: UAMX, #34605034. CONACyT #1004932

Conflict of Interest: None declared.

EP10.015 Novel intragenic deletion within the FXN gene in a patient with Friedreich ataxia: are they more prevalent than we think?

Anna Esteve Garcia 1, Ariadna Padró Miquel2, Carlos Casasnovas3, Valentina Velez3, Laura Rausell4, Pablo Gargallo4, Javier Garcia Planells4, Pedro Alía2, Nuria Llecha1;2, Cinthia Aguilera2

1Bellvitge University Hospital, Clinical Genetics Unit. Laboratori Clínic Territorial Metropolitana Sud, L’Hospitalet de Llobregat, Spain; 2Bellvitge University Hospital, Genetics Laboratory. Laboratori Clínic Territorial Metropolitana Sud, L’Hospitalet de Llobregat, Spain; 3Bellvitge University Hospital, Neuromuscular Unit. Neurology Department, L’Hospitalet de Llobregat, Spain; 4Health in Code, València, Spain

Background: Friedreich ataxia (FRDA) is mainly caused by biallelic pathogenic expansions of the GAA trinucleotide repeat in intron 1 of the FXN gene. Rarely, affected individuals carry either an intragenic point mutation or a deletion of FXN in combination with the abnormally expanded GAA repeat. Here, we describe a novel intragenic deletion identified in a 32-year-old patient with classical clinical features of FRDA. Previous routine genetic analysis of the FXN intron 1 led to the assumption that the patient carried the common biallelic expansion.

Methods: PCR and TP-PCR fragment analysis of the polymorphic region in intron 1 of the FXN gene was carried out on samples from the proband and both parents. Subsequently, duplication/deletion analysis of FXN was performed in the mother and proband samples using the SALSA MLPA Probemix P316-B4 Recessive Ataxias kit (MRC Holland) following the manufacturer’s instructions.

Results: The maternal sample only showed a peak corresponding to 9 GAA repeats but no expansion was observed while the paternal sample showed an allele of 7 GAA repeats and an expansion, corresponding to a healthy carrier of the disease. Mother’s result did not correlate with a biallelic expansion in the proband. MLPA analysis allowed the identification of an intragenic deletion encompassing 5’UTR and exons 1-2 of FXN gene in both the proband and his mother.

Conclusion: With this case, we want to highlight that intragenic deletions may be more prevalent than first thought in patients with FRDA. Performing parental testing is crucial in order to provide accurate genetic counselling.

Conflict of Interest: Anna Esteve Garcia Full-time, Ariadna Padró Miquel Full-time, Carlos Casasnovas Full-time, Valentina Velez Full-time, Laura Rausell Full-time, Pablo Gargallo Full-time, Javier Garcia Planells Full-time, Pedro Alía Full-time, Nuria Llecha Full-time, Cinthia Aguilera Full-time.

EP10.016 Whole exome sequencing identifies a heterozygous TRPC3 missense variant in a patient with adult onset spinocerebellar ataxia

Tim Bender 1;2, Axel Schmidt1, Kirsten Cremer1, Claudia Perne1, Hartmut Engels1, Stefanie Heilmann-Heimbach1, Pietro Incardona3, Sophia Peters1

1University Hospital Bonn, Institute of Human Genetics, Bonn, Germany; 2University Hospital Bonn, Center for Rare Diseases Bonn, Bonn, Germany; 3University of Bonn, Core Unit for Bioinformatics Data Analysis, Bonn, Germany

Background/Objectives: Spinocerebellar ataxias (SCA) are a group of genetically heterogeneous neurological disorders with the main symptom gait ataxia. Other symptoms, age of onset, as well as mode of inheritance differ between the respective types of SCA.

Case report: We present the case of a sixty years old woman with slowly progressive gait ataxia, dysarthria, slurred speech and poor fine motor coordination. First symptoms appeared at the age of 38 years. Brain MRIs show a slowly progressive atrophy of the cerebellum. Genetic testing for the most common spinocerebellar ataxias (SCA 1, 2, 3, 6, 7, 11, 13, 14 and 17) gave negative results.

Methods and Results: Whole exome sequencing revealed a heterozygous missense variant (NM_001130698.2:c.2216T>C;p.(Leu739Pro)) in TRPC3 in the patient; no other (likely) pathogenic variant was identified. All applied in silico tools predict a damaging effect of this variant. The Z-score for TRPC3 is 3.84, indicating a high intolerance for missense variants of this gene.

Conclusion: To our knowledge, there is only one published case in which a pathogenic heterozygous missense variant in the TRPC3 gene was reported to cause adult onset SCA (Fogel et al., 2015). In vitro functional expression studies of the variant p.(Arg762His) indicated a toxic gain of function effect. A gain of function etiological mechanism is indirectly supported by the relatively frequent observation of loss of function variants in the general population (pLI=0).

Our findings support the hypothesis that heterozygous pathogenic missense variants in the TRPC3 gene are a possible rare cause of adult onset spinocerebellar ataxia.

Conflict of Interest: Tim Bender University Hospital Bonn, Axel Schmidt University Hospital Bonn, Kirsten Cremer University Hospital Bonn, Claudia Perne University Hospital Bonn, Hartmut Engels University Hospital Bonn, Stefanie Heilmann-Heimbach University Hospital Bonn, Pietro Incardona University of Bonn, Sophia Peters University Hospital Bonn.

EP10.017 Biallelic variants in HEATR5B associated with severe neurologic phenotypes in two additional families: further evidence for an autosomal recessive syndrome

Lauren Badalato 1, Elaine Chan2, Verena Kuret3, Julie Lauzon4

1Queen’s University, Pediatrics, Kingston, Canada; 2University of Calgary, Pathology and Laboratory Medicine, Calgary, Canada; 3University of Calgary, Obstetrics and Gynecology, Calgary, Canada; 4University of Calgary, Medical Genetics, Calgary, Canada

Background/Objectives: Biallelic splice site variants in HEATR5B have previously been reported in four children from two families with a neurological syndrome including pontocerebellar hypoplasia. Reduced HEATR5B protein expression was observed, and in mouse models, biallelic knockout variants were associated with embryonic lethality. In this case series, we report three additional patients from two families with severe neurologic phenotypes.

Methods: We performed a chart review of the clinical and genetic data of two families with biallelic variants in HEATR5B, and compared them to the cases reported in the literature.

Results: Family 1 consisted of two siblings with compound heterozygous frameshift variants in HEATR5B: c.3184_3185del; p.(Val1062Glnfs*4) and c.6163del; p.(Ala2055Profs*12). As the latter variant is quite distal we suspect it escapes nonsense mediated decay. Patient 1-A presented in the neonatal period with intractable seizures and died at 6 months of life. His sibling, patient 1-B, is currently 1.5 years old with severe global developmental delay and abnormal neurologic exam, and has evidence of pontocerebellar hypoplasia on MRI. Family 2 consisted of a pregnancy with arthrogryposis, hydrops, cystic hygroma, micrognathia and cleft palate; termination of pregnancy was pursued due to the severity of the findings. The fetus was found to have a more proximal homozygous variant, c.327dup (p.Pro110Thrfs*), expected to cause complete loss of function of HEATR5B.

Conclusion: This case series provides further evidence that partial loss of function of HEATR5B is associated with severe neurologic disorders (Family 1), whereas complete loss of function may be lethal in utero (Family 2).

Conflict of Interest: None declared.

EP10.018 Expanding the genotypic spectrum of TUBGCP2 related neurodevelopmental disease: The sixth patient in the literature

Büşranur Çavdarlı 1, Tuna Eren Esen1, Didem Ardıçlı2

1Ankara Bilkent City Hospital, Department of Medical Genetics, Ankara, Turkey; 2Ankara Bilkent City Hospital, Department of Child Neurology, Ankara, Turkey

Background/Objectives: TUBGCP2 gene (tubulin-gamma-complex-associated protein 2 (MIM*617817) encodes the γ-tubulin complex 2 (GCP2) protein which is an essential part of the γ-tubulin ring complex at microtubule-organizing-center. Bi-allelic mutations on TUBGCP2 have been associated with neurodevelopmental disease spectrum consisting of microcephaly, hypotonia, epilepsy, and abnormal brain magnetic resonance imaging (MRI) (lissencephaly, pachygyria, cerebral and cerebellar atrophy, heterotopia)1,2. We aim to expand the genotypic spectrum of TUBGCP2- delated neurodevelopmental-disease by presenting a patient with a novel homozygous TUBGCP2 variant.

Methods: 6 years-old male with neuromotor developmental-delay, hypotonia, epilepsy, microcephaly, and