Abstracts from the 55th European Society of Human Genetics (ESHG) Conference: e-Posters

Volume 31 | Supplement S1.

Vienna, Austria.

June 11–14, 2022.

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 Correlations between cytogenetic findings and spermatogenic failure in Bulgarian infertile men

Svetlana Yovinska ¹, Kalina Belemezova², Mariela Hristova-Savova², Tanya Milachich², Petya Andreeva², Yuri Buchvarov², Maria Yunakova², Tanya Timeva², Atanas Shterev², Ivanka Dimova³

¹Medical University - Sofia, Department of Pharmacology and Toxicology, Sofia, Bulgaria; ²SAGBAL “Dr. Shterev”, Sofia, Bulgaria; ³Medical University - Sofia, Department of Medical Genetics, Sofia, Bulgaria

Background/Objectives: Chromosomal aberrations have a great impact on spermatogenesis, semen quality, and successful conception. The objective of our study was to determine the type and frequency of chromosomal aberrations and polymorphisms in men with different degrees of spermatogenic failure in comparison to men with normozoospermia, in order to find some correlations between cytogenetic findings and the abnormal results of semen analysis.

Methods: In our study, we have performed cytogenetic analysis in 901 infertile men, divided into 5 groups according to semen analysis—normozoospermia, asthenozoospermia, oligoasthenozoospermia, severe male factor and azoospermia.

Results: The frequency of polymorphisms was similar in all groups (11–16%, without significant differences). The frequency of numerical and structural aberrations increases with the degree of the spermatogenic failure (3.5% in normozoospermia, 5.6% in asthenozoospermia, 9.8% in oligoasthenozoospermia, 9% in severe male factor and 13.5% in azoospermia). We have found significantly higher incidence of numerical chromosome aberrations in severe male factor (7%) and azoospermia (9.3%). Oligoasthenozoospermia was associated with chromosomal translocations, as it occurs in 45% of cases with translocation, compared to 20% in the group with normal karyotype.

Conclusion: We revealed that chromosomal translocations are significantly associated with oligoasthenozoospermia, whereas numerical chromosomal aberrations—with severe male factor and azoospermia. These are important aspects of genetic counseling for those cytogenetic findings. Chromosome polymorphisms don’t seem to disturb significantly spermatogenesis and their impact should be studied in regard to unsuccessful pregnancy achievement, even in patients with normozoospermia.

References:

Grants:

Conflict of Interest: None declared.

EP01.002 Comparison of carrier status among patients with or without family history of disease using targeted and expanded panels

Karen Phaik Har Lim ¹, Bridgette Meyers¹, Ezen Choo¹, Trevor Smart¹, Sindhu Arun¹, Nina Sanapareddy¹, Dianne Keen-Kim¹

¹Natera Inc., San Carlos, United States

Background/Objectives: Targeted carrier screening (CS) based on ethnicity or family history (FH) is common. However, many families lack background ethnic or genetic information. In this study, we compared the impact of using expanded versus targeted disease panels on patients with and without patient reported FH.

Methods: Results from Horizon^TM CS with 27-genes (N = 19,397) or 274-genes (N = 58,327) performed between January 2020–June 2021 were analyzed. FH among carriers was determined by ICD-10 code Z84.81. Cases without an ICD10 code were excluded.

Results: Among 77,724 individuals undergoing CS, those with FH were more likely to be carriers (59.3%; 2598/4,379) than those without FH (55.4%; 40,623/73,345). However, the 274-gene panel was more likely to identify carriers with FH (67.6%; 2236/3309) or without FH (65.0%; 35,745/55,018) than the 27-gene panel (33.8%; 362/1,070 and 26.6%; 4,878/18,327, respectively). Interestingly, patients without FH were more likely to be carriers of variants in a single gene (34.3%; 18,862/55,018) than patients with FH (31.9%; 1055/3309) (p < 0.05).

Conclusion: In this cohort, FH was associated with an increased positive rate. However, the expanded panel was better at identifying carriers than the targeted panel regardless of FH, and was more effective at identifying variants in a single gene without FH. Taken together, these results indicate that the use of expanded CS is more effective than targeted CS.

References:

Grants:

Conflict of Interest: Karen Phaik Har Lim Full-time employee of Natera Inc., Option to own stock in Natera Inc., Bridgette Meyers Full-time employee of Natera Inc., Option to own stock in Natera Inc., Ezen Choo Full-time employee of Natera Inc., Option to own stock in Natera Inc., Trevor Smart Full-time employee of Natera Inc., Option to own stock in Natera Inc., Sindhu Arun Full-time employee of Natera Inc., Option to own stock in Natera Inc., Nina Sanapareddy Full-time employee of Natera Inc., Option to own stock in Natera Inc., Dianne Keen-Kim Full-time employee of Natera Inc., Option to own stock in Natera Inc.

EP01.003 Chromosomal heteromorphisms as a risk factor for reproductive failure

Mariya Levkova ^1;2, Maria Tsvetkova^1;2, Tsanka Ruseva², Mari Hachmeriyan^1;2, Milena Stoyanova^1;2, Valentina Miteva^1;2, Dinnar Yahya^1;2, Lyudmila Angelova¹

¹Medical University Varna, Medical genetics, Varna, Bulgaria; ²University Hospital St Marina Varna, Laboratory of Medical genetics, Varna, Bulgaria

Background/Objectives: Chromosomal heteromorphisms are normal variations, which could be detected at specific chromosomal regions. Despite being considered normal findings, it is possible that these heteromorphisms are associated with reproductive failure (RF).

Methods: A total of 661 women with unexplained RF (primary infertility and recurrent miscarriages) and 139 female control subjects, who had at least one child, were investigated by conventional cytogenetic analysis. GTG differential banding technique was applied, and approximately 10 metaphases were analyzed per patient. A t-test was performed.

Results: Mean age of the patients with RF was 33.00 years, and of the control subjects—29.95 years. 61 (9.38%) of the case subjects had a chromosomal heteromorphism, and of the control subjects—seven (5.03%). The distribution of the chromosomal heteromorphisms between the two groups showed a statistically significant difference (p ≤ 0.05).

The most common heteromorphism among the women with RF was 21ps+ - 12 (19.67%), followed by inv(9)(qh) – 11 (18.03%), 16qh+ - 10 (16.39%), 9qh+ - 7 (11.47%), 22ps+ - 7 (11.47%). The rest of the polymorphisms involved the heterochromatin of chromosomes 1, 13, 14, and 15. For the control group, 21ps+ was also the most common polymorphism – 3 (42.86%), followed by 9qh+ - 2 (28.57%). There was one inv(9)(qh) and one 1qh+.

Conclusion: Our results showed a statistically significant difference between the distribution of chromosomal heteromorphisms among the two groups. These chromosomal heteromorphisms could be considered a risk factor for reproductive failure. This illustrates the importance of cytogenetic analysis in the investigation of patients with infertility.

References: Not applicable.

Grants: Not applicable.

Conflict of Interest: None declared.

EP01.004 Development of a nationwide study to identify monogenic causes of female idiopathic reproductive failure

Anu Valkna ¹, Kristiina Rull^1;2;3, Ülle Jakovlev⁴, Laura Kasak¹, Maris Laan¹

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

Background/Objectives: In Europe, the mean maternal age at the time of childbirth has increased and the proportion of women experiencing reproductive failure is growing. Premature ovarian insufficiency (POI) refers to menopause before the age of 40 due to diminished ovarian reserve. Often, recurrent miscarriages (RM) represent a prelude to POI due to possible overlapping molecular etiologies¹. Although rather common conditions (~1% of women), knowledge on (mono)genic causes of POI/RM is limited, hindering patient management.

Methods: We aim to develop a nationwide cohort of idiopathic POI and RM cases to be systematically analyzed for genetic causes, using exome sequencing of patients and their affected family members. Inclusion criteria follow international guidelines: menopause <40 yrs/3 or more miscarriages; anamnesis, family history, questionnaire, relevant laboratory findings (e.g. FSH > 25 IU/l).

Results: The study pipeline to recruit idiopathic POI/RM patients has been developed at the Women’s Clinics of Tartu University Hospital and East-Tallinn Central Hospital. Additional affected cases were identified from the Estonian Biobank (EstBB) database. From 768 EstBB participants with the ICD-10 diagnosis of POI, only 21 fit our stringent study criteria.

Conclusion: Research of idiopathic POI and RM is challenged due to insufficient clinical data to distinguish genetic vs non-genetic causes, and highly intimate nature of these conditions. Genetic research is alerted as timely counselling of women with high risk to POI/RM will have immediate impact to their reproductive decision making.

References: 1. Dean et al. (2018) J Assist Reprod Genet 35:2121–8.

Grants: PRG 1021 (Estonian Research Agency).

Conflict of Interest: None declared.

EP01.005 Aberrant hypomethylation of imprinted differentially methylated regions is involved in biparental placental mesenchymal dysplasia

Saori Aoki^1;2, Ken Higashimoto¹, Hidenori Hidaka¹, Yasufumi Ohtsuka³, Shigehisa Aoki⁴, Hiroyuki Mishima⁵, Koh-ichiro Yoshiura⁵, Kazuhiko Nakabayashi⁶, Kenichiro Hata⁶, Hitomi Yatsuki¹, Satoshi Hara¹, Takashi Ohba², HIDETAKA KATABUCHI², Hidenobu Soejima ¹

¹Faculty of Medicine, Saga University, Department of Biomolecular Sciences, Saga, Japan; ²Faculty of Life Sciences, Kumamoto University, Department of Obstetrics and Gynecology, Kumamoto, Japan; ³Faculty of Medicine, Saga University, Department of Pediatrics, Saga, Japan; ⁴Faculty of Medicine, Saga University, Department of Pathology and Microbiology, Saga, Japan; ⁵Nagasaki University Graduate School of Biomedical Sciences, Department of Human Genetics, Nagasaki, Japan; ⁶National Research Institute for Child Health and Development, Department of Maternal-Fetal Biology, Tokyo, Japan

Background/Objectives: Placental mesenchymal dysplasia (PMD) is often associated with androgenetic/biparental mosaicism (ABM), and is frequently complicated by Beckwith–Wiedemann syndrome. These phenomena suggest an association between PMD and aberrant genomic imprinting, likely involving CDKN1C and IGF2. Another type of PMD involving the biparental genome has also been reported. However, the frequency and etiology of biparental PMD are still not fully understood.

Methods: Twenty-five macroscopic PMD specimens were genotyped by DNA microarray or short tandem repeat analysis. DNA methylation analysis using bisulfite pyrosequencing was performed on 15 placenta-specific imprinted differentially methylated regions (DMRs) and 36 ubiquitous imprinted DMRs. Allelic expression of imprinted genes was examined by RT-PCR using single nucleotide polymorphisms. Whole exome sequencing (WES) was performed on four biparental PMD specimens.

Results: Genotyping revealed that approximately 30% of macroscopic PMD specimens were biparental, while the rest exhibited ABM. DNA methylation of most DMRs in PMD specimens with ABM displayed the paternal epigenotype. Importantly, biparental PMD specimens exhibited aberrant hypomethylation at six placenta-specific DMRs. Five imprinted genes associated with these DMRs were biallelically expressed. Aberrant hypomethylation was also observed at eight ubiquitous DMRs, including GRB10, but not ICR1 or ICR2, which regulate the expression of IGF2 and CDKN1C, respectively. WES did not reveal any pathological genetic abnormalities.

Conclusion: Our data clarify the prevalence of biparental PMD and ABM-related PMD. The results strongly implicate that the hypomethylation of DMRs, including placenta-specific DMRs and GRB10, but not ICR1 or ICR2, plays a role in the pathogenesis of biparental PMD.

References:

Grants: JSPS, AMED, MHLW.

Conflict of Interest: None declared.

EP01.006 Different IVF culture media do not affect the methylome of IVF children

Rebekka Koeck ^1;2, Jorg Tost³, Florence Busato³, Dimitri Consten⁴, Jannie van Echten-Arends⁵, Sebastiaan Mastenbroek⁶, Yvonne Wurth⁴, Heleen Zandstra², Ron van Golde², John Dumoulin², Han Brunner^2;7, Aafke van Montfoort², Masoud Zamani Esteki^1;2

¹Maastricht University, Maastricht, Netherlands; ²Academic Hospital Maastricht, Maastricht, Netherlands; ³CEA - centre National de Recherche en Genomique Humaine, Évry, France; ⁴St. Elisabeth-TweeSteden Hospital, Tilburg, Netherlands; ⁵University Medical Center Groningen, Groningen, Netherlands; ⁶Amsterdam UMC, Amsterdam, Netherlands; ⁷Radboud University Medical Center, Nijmegen, Netherlands

Background/Objectives: A growing number of children born are conceived through in vitro fertilization (IVF) procedures that have been linked to increased risks of adverse perinatal outcomes and altered growth profiles in the resultant children. These outcomes are also influenced by the media used for embryo culture and this effect is hypothesized to be mediated epigenetically, e.g. through DNA methylation. Therefore, we investigated the methylome in IVF offspring who underwent embryo culture in different media.

Methods: We profiled the umbilical cord blood (UCB) methylome of 106 IVF-neonates cultured in G5 or HTF, and the saliva methylome of 120 9-year-old IVF children, cultured in G3 or K-SICM, using the Infinium Human Methylation EPIC BeadChip. Analyses were carried out separately on UCB and saliva samples using empirical Bayes moderated mixed effects linear models adjusted for potential confounders. Methylation outliers represent values more than three interquartile ranges from the upper or lower quartiles.

Results: In both comparisons (UCB and saliva) we identified no significant methylation differences between the culture medium groups in terms of: (i) systematic differences at single CpG sites or regions, (ii) imprinted sites/genes or birth weight associated sites, (iii) stochastic differences presenting as DNA methylation outliers or differentially variable sites, and (iv) epigenetic gestational age acceleration (UCB samples only).

Conclusion: The IVF culture media investigated did not lead to methylome difference in the resultant neonates/children, suggesting that any culture medium induced epigenetic alterations resolve prenatally. To investigate environmental-epigenetic interactions occurring earlier during human development, epigenetic profiling of preimplantation embryos is required.

References:

Grants: March of Dimes (6-FY13-153).

Conflict of Interest: None declared.

EP01.007 Mosaic runs of homozygosity in first trimester spontaneous abortions with normal karyotype

Gleb Drozdov ¹, Igor Lebedev¹, Tatyana Nikitina¹, Anna Kashevarova¹, Elizaveta Fonova¹, Ekaterina Tolmacheva¹, Elena Sazhenova¹

¹Tomsk National Research Medical Center of Russian Academy of Sciences, Research Institute of Medical Genetics, Tomsk, Russian Federation

Background/Objectives: Runs of homozygosity (ROH) are considered as pathogenic factor for human health. It’s deleterious effect can be realized through the homozygotization of recessive mutations or imprinting disorders, highlighting the need to map such regions. Given the high frequency of mosaicism in embryos and the limitations of aCGH in detecting it, we decided to analyze different tissues of embryonic and extraembryonic origin, including parental samples.

Methods: Eleven trios (mother, father, SA) were investigated by SurePrint G3 Human CGH+SNP 4 × 180K microarrays (Agilent Technologies, USA). Euploid placental tissues were separated into chorionic villi (CV) and extraembryonic mesoderm (EM).

Results: Twenty-four ROHs were found in 10 samples. Seventeen ROHs were present in both tissues, while two were confined to CV (5q14.3, 8p23.3-23.1) and five to EM (4q28.2-28.3, 8q11.21-q11.23, 8q24.11-q24.12, 18q12.3, 18q12.1-12.2). Since none of the ROHs were inherited, we hypothesized that they resulted from a combination of identical parental haplotypes. SNP-analysis showed no uniparental disomy within homozygous regions. We hypothesize that a trisomy rescue could have occurred before divergence of embryonic and extraembryonic lineages. As a result, in one cell lineage a chromosome with a different haplotype was lost leading to ROH, and in another a chromosome with the same haplotype was lost keeping heterozygosity within the region. Therefore, ROH would be found in one tissue and heterozygosity of the same region in another.

Conclusion: This study highlights the underestimated level of mosaic ROHs in abnormal human reproduction.

References: non.

Grants: Russian Science Foundation №21-65-00017, https://rscf.ru/project/21-65-00017.

Conflict of Interest: None declared.

EP01.008 A significant drop in sperm DNA fragmentation in a complete teratozoospermic patient post smoking cessation and its impact on ART outcome: case report

Dina Faris ^1;2, Suher Zada¹, Mohamed Faris²

¹The American University in Cairo, cairo, Egypt; ²Dr Faris Medical Center, cairo, Egypt

Background/Objectives: We assessed Sperm DNA Fragmentation in patients with 100% abnormal sperm morphology and its correlation with live birth rate post Intracytoplasmic Sperm Injection. One of our initial cases did not conceive after treatment. During his first visit, we advised him to quit smoking in addition to a previous treatment that included oral antioxidant was already being administrated. After three months of this combined approach, Sperm DNA fragmentation and semen parameters were re-evaluated. A second round of ICSI was performed that resulted in a healthy live birth. We are reporting this finding as a case report.

Methods: We evaluated the Sperm DNA fragmentation using sperm chromatin dispersion test and sperm structure using MSOME test prior the second round of Intracytoplasmic Sperm Injection.

Results: Sperm DNA fragmentation dropped after smoking cessation from 49% to 29%. Abnormal sperm morphology changed from complete teratozoospermia (100%) to severe teratozoospermia (97%). A live birth was achieved.

Conclusion: Although routine Sperm DNA fragmentation testing is not yet recommended in clinical practice, we concluded that it is of critical value in complete teratozoospermia patients. A combined approach of antioxidants and life style changes has a significant impact on sperm morphology, sperm DNA integrity and live birth post Intracytoplasmic Sperm Injection.

References:

Grants:

Conflict of Interest: None declared.

EP01.009 FOXL2 gene deletion in a patient from Assisted Reproduction Unit detected by the a-CGH technique

María Luz Bellido ¹, Teresa De Haro², Maria del Mar del Aguila³, Antonio Miguel Poyatos-Andújar¹

¹Hospital Virgen de las Nieves, Unidad de Genética, Granada, Spain; ²Hospital Universitario San Cecilio, Servicio de Análisis Clínicos., Granada, Spain; ³Hospital Virgen de las Nieves, Granada, Spain

Background/Objectives: Assisted reproduction guidelines consider the genetic evaluation of patients with reproductive difficulties by karyotyping, however, after a first evaluation of other techniques in patients with striking characteristics, a-CGH can be a technique of great value. We present the case of a 19-year-old woman with primary infertility of 5 years of evolution, with low ovarian reserve detected by transvaginal ultrasound and biochemical determination for AMH of 0.94 ng/ml (0.96–13.34 ng/ml).

Methods: GTG-banding karyotype was performed according to standard protocols. Twenty metaphases chromosomes were examined. PCR screening of CGG repeats in FMR1 gene. Array-CGH analysis was performed by a CGXTM HD v1,1 4-plex array 180k (PerkinElmer), with an average resolution of 40 kb in the backbone and 20 kb in the regions of interest.

Results: Karyotype result: female, 46,XX. CGG repeats in FMR1 gene normal profile. A-CGH: arr[GRCh37] 3q22.3(138662657_138665307)x1; 2.65 kb deletion in chromosomal region 3q22.3 that includes the FOXL2 gene described as crucial in ovarian development. Mutations in this gene, such as truncating mutations, have been associated with female infertility with an autosomal dominant inheritance pattern.

Conclusion: a-CGH can be considered as a screening technique for genome analysis in patients whose gynaecological and biochemical parameters may indicate the presence of genetic factors not assessed by karyotyping and can assist patients and physicians in making more appropriate and effective reproductive decisions.

References:

Grants:

Conflict of Interest: None declared.

EP01.010 A novel heterozygous variant in the DNA binding DM domain of the DMRT1 associated with Sertoli cell-only syndrome

Tihana Maric ¹, Antun Barišić², Mark W Murphy³, Monika Logara Klarić⁴, Lucija Žunić⁴, Ana Merkler⁵, Davor Jezek¹, Oliver Vugrek⁶, Robert Belužić⁶, Branimir Bertoša², Ana Katušić Bojanac¹, Maja Barbalić^4;7

¹School of Medicine, University of Zagreb, Zagreb, Croatia; ²Faculty of Science, University of Zagreb, Department of Chemistry, Zagreb, Croatia; ³University of Minnesota, Department of Genetics, Cell Biology, and Development, Minneapolis, United States; ⁴Genom Ltd, Zagreb, Croatia; ⁵University Hospital Centre Zagreb, Zagreb, Croatia; ⁶Ruđer Bošković, Zagreb, Croatia; ⁷University of Split, School of Medicine, Department of Medical Biology, Split, Croatia

Background/Objectives: Sertoli cell-only syndrome (SCOS) is the most severe form of male infertility accompanied by complete spermatogenic failure. It may arise as a consequence of genetic abnormalities, especially if detected in the family. The aim of this study was to determine the potential genetic cause of infertility in two SCOS brothers that had no common infertility-related genetic abnormalities.

Methods: Two brothers were subjected to testicular biopsy and diagnosed with SCOS. DNA was isolated from the peripheral blood and whole-exome sequencing was performed. Variants were called, annotated, while those variants with a frequency higher than 1% in databases, intron, and synonymous were removed. The remaining variants were assessed by the Exomiser software and researched in literature. Selected variants were confirmed in brothers and their parents by Sanger sequencing.

Results: A novel heterozygous missense variant was found and confirmed in the DNA binding DM domain of the DMRT1 gene (p.Pro74Leu; chr9:g.842059C>T) shared by brothers. Their mother was heterozygous for the same variant, while the father was not.

Conclusion: The DMRT1 gene is already associated in the literature with gonadal dysgenesis and male infertility. However, the novel P74L variant shared by the brothers is the first one reported in a familial case of male infertility and in the DM domain that directly interacts with the DNA.

References: Murphy MW, Lee JK, Rojo S, Gearhart MD, Kurahashi K, Banerjee S, et al. An ancient protein-DNA interaction underlying metazoan sex determination. Nature structural & molecular biology. 2015;22(6):442-51.

Grants: No. KK.01.1.1.01.0008.

Conflict of Interest: None declared.

EP01.013 Validation analysis of a non-invasive pre-implantation genetic test for aneuploidy (niPGT-A)

Michaela Blankenburg ¹, Joanna Stefaniak², Matthias Bloechle², Markus Stumm³

¹Medicover Genetics GmbH, Zytogenomik, Berlin, Germany; ²Kinderwunschzentrum Berlin, Berlin, Germany; ³Medicover Genetics GmbH, Berlin, Germany

Background/Objectives: Invasive techniques as polar body-, blastomer-, trophectodermbiopsy or blastocentesis are used in preimplantation genetic diagnosis to obtain embryonic DNA. The finding of cell-free embryonic DNA (cfDNA) in culture media samples from blastocysts (BCM) opened new possibilities, since invasive interventions on the embryo could be avoided and false findings due to mosaic constellation could be reduced.

Methods: Non-invasive PGT-A test based on MALBAC amplification, Analyses of 1.10 genomic DNAs with known complete and partial aneuploidies diluted to single cell level, 2.5 prenatal samples from native amniotic fluid supernatants, 3.20 BCM samples.

Results: In genomic DNAs as well as in amniotic fluid supernatants all aberrations could be confirmed. The concordance rate was 100%. In the analysis of BCM, the cfDNA showed a complete / partial concordance rate of ~88% with regard to the aberrant chromosomes to the corresponding DNA from biopsied TE cells.

Conclusion: The technical and analytical reliability of the analysis platform, kit and software have been tested. In the first analyses (genomic DNA and amniotic fluid), all aberrations could be reliably detected. The final aim was to determine the efficiency and concordance rate between the cfDNA from BCM samples and the DNA from TE cell biopsies of the same embryo. Our analyses results show a high level of concordance in comparison to results of TE analysis with regard to the affected chromosomes. niPGT-A is a promising new method for non-invasive aneuploidy screening in the context of artificial reproductive treatment.

References:

Grants:

Conflict of Interest: None declared.

EP01.015 Telomere length in spermatogenic cells from azoospermic patients is characterized by intercellular, but not interindividual variability

Mikhail Krapivin ¹, Olga Efimova¹, Yanina Sagurova¹, Irina Aleksandrova², Irina Mekina¹, Evgeniia Komarova¹, Aleksandr Gzgzyan¹, Igor Kogan¹, Anna Pendina¹

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

Background/Objectives: To analyze the intercellular and interindividual variability of telomere lengths (TLs) in the chromosomes from human spermatogenic cells: mitotic spermatogonia and meiotic spermatocyte I at the diplotene/diakinesis stage.

Methods: Testicular samples were obtained by open biopsy from 22 azoospermic patients aged 24–60 years, mean 38.5. TLs were measured in the chromosomes from 353 mitotic spermatogonia and 281 meiotic spermatocytes I at the diplotene/diakinesis stage. For chromosome preparations, testicular tissues were treated with colchicines, hypotonic solution and then fixed on the glass slides. TLs were assessed using quantitative fluorescence in situ hybridization (Q-FISH) as relative values: by dividing the telomeric fluorescence by the subtelomeric fluorescence measured in ImageJ 1.51. Intercellular variability was estimated as the fold change between the TL measured in each spermatogonia/spermatocyte and the mean TL value across all of the studied spermatogonia/spermatocytes of an individual. Interindividual variability was estimated as the fold change between the mean TL in all of the studied spermatogonia/spermatocytes of every individual and the mean TL in the whole group of patients (22 individuals).

Results: Intercellular TL variability both among spermatogonia and among spermatocytes I significantly exceeded interindividual variability (p = 0.03 and p = 0.01, respectively, Mann-Whitney U-test).

Conclusion: High intercellular TL variability both among spermatogonia and spermatocyte I could be explained by spermatogenic stem cell generations with different TLs in each testis. These cells differentiate and give rise to spermatozoa with different TLs. Spermatozoa with different TLs, in turn, could contribute to developmental potential of embryos.

References:

Grants:

RSF №18-75-10046.

Conflict of Interest: Mikhail Krapivin RSF №18-75-10046 (collaborator), Olga Efimova RSF №18-75-10046 (principal investigator), Yanina Sagurova RSF №18-75-10046 (collaborator), Irina Aleksandrova: None declared, Irina Mekina: None declared, Evgeniia Komarova RSF №18-75-10046 (collaborator), Aleksandr Gzgzyan: None declared, Igor Kogan: None declared, Anna Pendina RSF №18-75-10046 (collaborator).

EP01.016 Immunological incompatibility of a married couple as a cause of repeated abortions

Kateryna Sosnina ¹, Danuta Zastavna¹, Oresta Terpyliak¹, Bohdan Tretiak¹, Yaryna Zahanyach¹

¹Institute of Hereditary Pathology NAMS of Ukraine, Diagnosis of hereditary pathology, Lviv, Ukraine

Background/Objectives: Intricate KIR–HLA interactions occur between maternal NK cells and fetal extravillous trophoblasts in the decidua and are key for maintaining tolerance to the fetus, implantation and placentation. We assume that the immunological incompatibility of a married couple with KIR/HLAC genes leads to implantation and fetal development disorders. The aim of this study was to perform KIR/HLA-C genotyping to determine “good” compatibility between uterine KIR receptors and embryonic HLA-C.

Methods: KIR/HLA-C genotyping was performed by SSP-PCR. The experimental group consisted of 117 couples with ≥2 consecutive miscarriages and/or unsuccessful IVF.

Results: The spectrum of KIR genes was analyzed and the frequency of KIR genotypes in women with recurrent spontaneous abortion (RSA) was established. The AB genotype is the most common (70.09%), the AA genotype was established in 33 women out of 117 surveyed (28.21%), and the BB genotype was established in 1.71% women. HLAC genotyping of couples with RSA showed the C2/C2 genotype of the HLAC gene in 17.95% of women, 20.51% of men and 33.33% of embryos. According to the results of KIR-HLAC analysis of genotyping of couples with RSA, a significant risk of reproductive losses of immunological origin was found in 52.99% of cases.

Conclusion: The immunological incompatibility of the couple affects reproductive processes and causes recurrent miscarriages and KIR-HLAC genotyping is allows to assess the risks of the embryo being rejected by the maternal immune system.

References:

Grants:

Conflict of Interest: None declared.

EP01.017 Title: Mosaic mutation in androgen receptor gene in a patient with partial androgen insensitivity syndrome

Mohammed Almatrafi ¹, Zohor Azher², abdulaziz baazeem³, Abdullatif Almarashi⁴

¹umm Al Qura University, makkah, Saudi Arabia; ²umm AlQura University, medical genetics, makkah, Saudi Arabia; ³Umm AlQura University, surgery, makkah, Saudi Arabia; ⁴health affairs of jeddah province, jeddah, Saudi Arabia

Background/Objectives: Androgen insensitivity syndrome (AIS) is the most frequent etiology of 46, XY disorders of sex development (DSDs). It is characterized by evidence of feminization of the external genitalia at birth, abnormal secondary sexual development in puberty, and male infertility. AIS patients classified into complete, partial, and mild forms. It is caused by hemizygous pathogenic variant in Androgen receptor gene (AR).

There are more than 500 different AR gene allelic variants reported to be linked to AIS, but the mosaic mutations have been rarely identified.

Here, we describe the clinical and molecular features of male patient presented with Partial androgen insensitivity syndrome.

Methods: A 36-year-old male presented with primary infertility, sever oligospermia, bilateral gynecomastia, decreased body hair distribution, normal male external genitalia except for hypospadias. Whole exome sequencing with deletion/duplication analysis was performed.

Results: hemizygous likely pathogenic variant c.649del was found in AR gene with significantly reduced allele ratio compared with a normal hemizygous allele in male. This result is consistent with mosaicism in this patient.

Conclusion: AIS with AR gene sequencing can be considered in male patients with infertility. Mosaic mutations in AR are rare and their detection is still a great technical challenge. WES offer an opportunity to detect lower levels of mosaicism more readily than other traditional methods. This will improve the clinical management, and counselling.

References: 1. Singh S, Ilyayeva S. Androgen Insensitivity Syndrome. StatPearls [Internet]. 2021.

2. Androgen insensitivity syndrome: MedlinePlus Genetics [Internet].

3. Gottlieb B, Trifiro MA. Androgen Insensitivity Syndrome. GeneReviews® [Internet]. 2017.

Grants:

Conflict of Interest: Mohammed Almatrafi: None declared, Zohor Azher full, medical genetics, abdulaziz baazeem full, urology, Abdullatif Almarashi full, phd.

EP01.018 The Digital Genetic Assistant for expanded carrier screening: efficiency and effectiveness

adi reches^1;2;3, Michal Berkenstadt⁴, Vered Ofen Glassner⁵, Yael Furman⁶, Galit Delmar⁶, Amit Weinstein⁵, Doron Behar⁶, Nurit Goldstein⁴, Liat Abu-Gutstein⁴, Karin Alperin⁴, haike reznik wolf ⁴, Nofar Mimouni⁴, Odeya Kazimirski⁴, Anat Bar-Ziv⁴, Shlomit Eisenberg-Barzilai⁴, yuval yaron^2;5, elon pras^2;4, Hagit Baris Feldman^2;3

¹Lis Maternity Hospital, Tel Aviv Sourasky Medical Center, Department of Obstetrics and Gynecology, tel aviv, Israel; ²Tel Aviv University, Sackler Faculty of Medicine, tel aviv, Israel; ³Tel Aviv Sourasky Medical Center, Genetics Institute, tel aviv, Israel; ⁴Sheba Medical Center, Tel-Hashomer, The Danek Gertner Institute of Human Genetics, tel aviv, Israel; ⁵, Tel Aviv Sourasky Medical Center, Genetics Institute, tel aviv, Israel; ⁶Igentify inc., Israel, tel aviv, Israel

Background/Objectives: Paucity of genetic counsellors led to the development of the Digital Genetic Assistant (DGA) by Igentify. In enables online enrolment, education and consent for expanded carrier screening (ECS). Triaged low versus high-risk results are returned by personalized videos. Usability of DGA was ascertained for ECS.

Methods: Couples undergoing ThermoFisher CarrierScan® by ECS (1487 variants, 357 genes) at Genetics Institutes of Tel-Aviv and Sheba Centers from 12.2020 to 8.2021 utilizing DGA were included. Comprehension was assessed by interactive questions, followed by online consent. Results of the DGA algorithm were approved or rejected by the medical team. Low risk couples without mutations in the same gene received an personalized video and report, while high risk participants, carriers of a mutation in the same gene or x-linked conditions, underwent a face-to-face (F2F) counselling.

Results: 197 couples underwent ECS via DGA. Eight high-risk couples (4%) underwent F2F counselling. Of the low-risk couples (189, 96%), 46 (23%) did not carry any mutation. DGA saved traditional F2F or telephonic counselling in 143 (73%) of couples that were not carriers of a mutation in the same gene.

An online survey provided feedback, completed by either one or both partners in 136 couples (69%). The consent video was clear for 91% of responders and 95% of felt comfortable receiving their results this way and were satisfied (88%) with the DGA.

Conclusion: Digitalized platform saved 72.6% of F2F interactions. Understanding and Satisfaction were high. This platform may be utilized to facilitate accessibility for further genetic services.

References:

Grants: Israel Innovation Authority.

Conflict of Interest: adi reches fugene genetics, Michal Berkenstadt: None declared, Vered Ofen Glassner: None declared, Yael Furman: None declared, Galit Delmar: None declared, Amit Weinstein: None declared, Doron Behar: None declared, Nurit Goldstein: None declared, Liat Abu-Gutstein: None declared, Karin Alperin: None declared, haike reznik wolf: None declared, Nofar Mimouni: None declared, Odeya Kazimirski: None declared, Anat Bar-Ziv: None declared, Shlomit Eisenberg-Barzilai: None declared, yuval yaron medical consultant to Genoox, elon pras: None declared, Hagit Baris Feldman Sanofi Genzyme, Sanofi Genzyme Protalix Pfizer Taked Shire, Sanofi-Genzyme Igentify, in the past Shire and Regeneration.

EP01.019 The ability of human oocytes to develop into blastocysts depends on telomere length and TERT content in cumulus cells

Irina Aleksandrova ¹, Mikhail Krapivin², Olga Efimova², Irina Mekina², Evgeniia Komarova², Mariia Ishchuk², Aleksandr Gzgzyan², Igor Kogan², Anna Pendina²

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

Background/Objectives: The present study has examined whether the ability of human oocytes to undergo preimplantation development depends on the content of catalytic telomerase subunit (TERT) and telomere length (TL) in cumulus cells.

Methods: The studied sample included cumulus cells obtained from 32 oocyte-cumulus complexes retrieved from 31 patients in IVF cycles. TERT content and TLs were measured in 32-50 cumulus cells from each studied oocyte-cumulus complex using immunohistochemistry with specific antibodies, and Q-FISH, respectively. A total of 1342 cumulus cells were analyzed.

The oocytes from the studied oocyte-cumulus complexes were fertilized using standard IVF procedures and then were cultured individually. The oocytes were divided into two groups: the first group included oocytes that developed to the blastocyst stage and the second group included those that demonstrated developmental arrest. The TERT content and TLs were compared between the cumulus cells of the oocytes from two groups.

Results: Both TERT content and TLs were significantly higher in cumulus cells of oocytes that were able to develop into blastocysts compared to those of the arrested ones (Mann-Whitney U test, p = 0.0052 and p = 0.0058, respectively). TERT content and TLs correlated positively in cumulus cells (r = 0.4577).

Conclusion: The ability of human oocytes to develop into blastocysts depends on TERT content and TLs in cumulus cells.

References:

Grants: RSF № 18-75-10046.

Conflict of Interest: Irina Aleksandrova: None declared, Mikhail Krapivin RSF № 18-75-10046 (collaborator), Olga Efimova RSF № 18-75-10046 (principal investigator), Irina Mekina: None declared, Evgeniia Komarova RSF № 18-75-10046 (collaborator), Mariia Ishchuk RSF № 18-75-10046 (collaborator), Aleksandr Gzgzyan: None declared, Igor Kogan: None declared, Anna Pendina RSF № 18-75-10046 (collaborator).

EP01.020 A retrospective overview in female patients prediagnosed with FMR1 associated disorders

Duygu Gamze Aracı ¹, Alp Peker¹, Özden Altıok Clark¹, Esra Manguoğlu², Murat Özekinci³, Hilmi Uysal⁴, Feyza Altunbaş¹, Mert Coşkun¹, Gokcen Karamik⁵, nuray ozturk⁵, Öznur YILMAZ BAYER⁵, Aslı Toylu¹, banu nur⁵, ercan mihci⁵

¹Akdeniz University School of Medicine, Medical Genetics, Antalya, Turkey; ²Akdeniz University School of Medicine, Medical Biology and Genetics, Antalya, Turkey; ³Akdeniz University School of Medicine, Obstetric and Gynecology, Antalya, Turkey; ⁴Akdeniz University School of Medicine, Neurology, Antalya, Turkey; ⁵Akdeniz University School of Medicine, Pediatric Genetics, Antalya, Turkey

Background/Objectives: Repetitions of trinucleotide “CGG” in FMR1 gene account for diseases such as Fragile-X syndrome (FXS), Fragile-X Tremor Ataxia Syndrome (FXTAS), Fragile-X associated primary ovarian insufficiency (FXPOI) & diminished ovarian reserve(FXDOR), intellectual disability, subfertility or infertility. In this study, we aim to make retrospective reviews on FMR1 mutation in female patients forwarded to our clinic for genetic analysis between 2014 & 2021.

Methods: PCR based fragment analysis of genomic DNA from a total of 401 patients’ blood samples was performed. Resulting data and collected clinical information were matched.

Results: Only 3 patients with prediagnosis of FXPOI & FXDOR, out of 64, was identified with premutations in FMR1 gene (%4.7) with no full mutation found. These patients were %20 of the total amount(15) of patients with premutation.

1 patient out of 28 with prediagnosis of FXTAS was found to carry premutation (%3.6).

13 of patients with developmental delay out of 138 (%9.4), were carrying mutations, 9 full mutation(%6.5), 3 intermediate form(%2.1), 1 grey zone(%0.7).

Abnormal chromosomal analysis results were found in 2 of the patients prediagnosed with FXPOI (47,XXX & 46,X,del(X)(q26)). Noting that 88 out of 203 patients with FMR1 associated disorders prediagnosis’ weren’t forwarded to cytogenetics evaluations.

Conclusion: Female patients reported with POI & DOR must be evaluated for mutations in FMR1 gene. We suggest the cut-off limits for repeat size length responsible for FXPOI & FXDOR should be further investigated as they might show differences from FXS and FXTAS. Cytogenetic testing shouldn’t be neglected with patients of FMR1 associated prediagnosis.

References: https://doi.org/10.1002/ajmg.a.36511.

Grants: None.

Conflict of Interest: None declared.

EP01.021 CYP21A2 gene pathogenic variants in the women of reproductive age with hyperandrogenism

Natalia Osinovskaya ^1;2, Yulia Nasykhova¹, Maria I. Yarmolinskaya^1;2, Olga Glavnova¹, Iskender Sultanov¹, Andrey Glotov¹

¹D. O. Ott’s Research Institute of Obstetrics, Gynecology and Reproductology, St. Petersburg, Russian Federation; ²I.I. Mechnikov Northwestern State Medical University, Russian Ministry of Health, St. Petersburg, Russian Federation

Background/Objectives: One of the causes of hyperandrogenism in women of reproductive age is a non-classical form of congenital adrenal hyperplasia (NC CAH). NC diagnosis presents difficulties due to the absence of specific clinical manifestations.To determine the significance of the using CYP21A2 gene analysis of the in women with symptoms of hyperandrogenism.

Methods: DNA from 82 patients with hyperandrogenism and 43 controls was analyzed for the presence of pathogenic variants in the CYP21A2 gene using the PCR-RFLP, NGS, real-time PCR.

Results: The compound mutations were identified in five patients (6%), and the diagnosis of NC was confirmed. Heterozygous carriage of pathogenic variants was revealed in 28 patients (34%). Both “heavy” and “light” variants are identified. Deviations of the CYP21A2 gene genotype from the “wild” type were detected in 40% of patients. The 2 substitutions of unknown significance were also identified. In controls the heterozygous pathogenic variants were detected in two patients (4.5%). The frequency of pathogenic variants in patients’ groups with infertility, miscarriage and without them was almost the same (45%, 37%, and 37.5%, respectively).

Conclusion: The frequency of pathogenic variants in the CYP21A2 gene is significantly higher in individuals with hyperandrogenism compared to controls. This fact requires a personalized approach to therapy and further examination of spouses at the planning stage of pregnancy for estimation of the risk of CAH in the child.

References:

Grants: Supported by the Ministry of Science and Higher Education of the Russian Federation within the Applied Science Research Program, project number № AAAA-A20-120041390028-0.

Conflict of Interest: None declared.

EP01.023 Genetic map of reproductive health like a base for restoring demographic balance in Ural population

Svetlana Deryabina ^1;2, Elena Kudryavceva¹, Alex Rezaikin¹

¹Institute of medical cell technologies, Laboratory of molecular genetic methods, Ekaterinburg, Russian Federation; ²Medical Center “Health Care of Mother and Child”, Ekaterinburg, Russian Federation

Background/Objectives: In the last decade in Russia, the general trend is the expansion of the geography of reproductive centers and the growing of services Assisted Reproductive Technologies. However, the use of ART cannot prevent giving birth to ill children automatically. In our region we know 2 cases of this sad scenario at least: a child with cystic fibrosis and a child with phenylketonuria were born after ART. Since 2022 we have been ready to propose for future parents a carrier testing for severe inherited pathology as an optimal solution.

Methods: By our research, we plan to test 1000 Ural family couples for spinal muscular atrophy (SMA)-carrier by multiplex ligase amplification probes and carrier some monogenic diseases by next generation sequencing (NGS).

Results: We have the first 200 samples of DNA. It is couples who came for preconception genetic consultation and have karyotype normal. Our actual genetic map of reproductive health includes 52 genes associated with known inherited diseases, among them are CFTR, PAH, SMN1, GALT, LAMA2, MMUT, BTD, and others.

Conclusion: The widespread introduction of such a genetic map into medical practice can be of decisive importance in reducing child and infant disability and mortality in the region, in the long term - restoring the demographic balance of the population of the Middle Urals.

References:

Grants:

Conflict of Interest: None declared.

EP01.025 Assessment of spermatogenic progression in men with structural chromosomal aberrations

Eva Pohl ^1;2, Yvonne Stratis², Sabine Kliesch³, Albrecht Röpke², Frank Tüttelmann¹

¹Institute of Reproductive Genetics, University of Münster, Münster, Germany; ²Institute of Human Genetics, University of Münster, Münster, Germany; ³Centre of Reproductive Medicine and Andrology, Department of Clinical and Surgical Andrology, University Hospital Münster, Münster, Germany

Background/Objectives: Cytogenetic aberrations are well-established causes for male infertility. Besides aberrations of sex chromosomes, structural autosomal aberrations are associated with infertility, i.e. low sperm counts. Meiosis in translocation carriers results in chromosomally normal/balanced or unbalanced gametes. There is evidence that presence of translocations impairs spermatogenetic/meiotic processes. However, studies on spermatogenic progression in translocation carriers are scarce.

Methods: In order to investigate associations between cytogenetic aberrations and spermatogenesis status, we retrospectively analysed 4663 males from one fertility centre and assessed cytogenetics, semen parameters, and testicular histology.

Results: We found cytogenetic aberrations in 9.3% (432/4663) with 1.3% (60/4663) being structural autosomal aberrations. Carriers of reciprocal translocations (RT, n = 28) and Robertsonian translocations (ROB, n = 15) showed large variation in sperm counts, ranging from normozoospermia to azoospermia. Testicular histology in azoospermic patients with RT (n = 5) and ROB (n = 2) revealed marked differences in spermatogenic progression. We observed that some RT and ROB carriers showed meiotic arrest in most seminiferous tubules and a smaller proportion with full spermatogenesis. Interestingly, in other carriers with the same aberration type, we found in the majority of seminiferous tubules a complete absence of germ cells. To exclude further genetic causes for the spermatogenic failure in translocation carriers, we currently perform exome sequencing.

Conclusion: Translocation carriers, even of the same aberration, i.e. the common der(13;14)(q10;q10), show large variation in sperm counts and spermatogenic progression. Whether additional genetic variants influence the testicular phenotype, remains to be elucidated.

References:

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

Conflict of Interest: None declared.

EP01.026 Deep RNA sequencing of decidual cells identifies widespread isoform diversity and alternative splicing

Ekaterina Trifonova ^1;2, Anastasia Babovskaya³, Maria Swarovskaja³, Maria Gavrilenko³, Viktoria Serebrova³, Ekaterina Izhoykina², Aleksei Zarubin⁴, Stepanov Vadim³

¹Tomsk National Research Medical Center of the Russian Academy of Sciences, Research Institute of Medical Genetics, Tomsk, Russian Federation; ²Siberian State Medical University, Tomsk, Russian Federation; ³Tomsk National Research Medical Center of the Russian Academy of Sciences Research Institute of Medical Genetics, Tomsk, Russian Federation; ⁴Tomsk National Research Medical Center of the Russian Academy of Sciences Research Institute of Medical Genetics, Tomsk, Russian Federation

Background/Objectives: Alternative splicing (AS) is crucial for the regulation of human genes expression. Global screening for changes in AS has not been previously carried in the placental cells despite a comprehensive analysis of pre-mRNA AS in many human tissues and cells in normal and pathological conditions. The decidual stromal cells (DSCs) play a key role in maintaining the function of placenta during human pregnancy. The aim of this study was to investigate genes and isoforms involved in functioning of the human DSCs during uncomplicated pregnancies.

Methods: We applied deep RNA sequencing of DSCs obtained by Laser capture microdissection. AS events are identified in existing genome annotations using the R-package “SGSeq”.

Results: Analyses of AS isoforms detected 151233 isoforms in DSCs. 373 genes with two or more transcripts have been identified. Of these, 130 genes meet criteria (CPM > 10; the proportion of transcript ranges from 0.5 to 0.95). GO enrichment analysis revealed that these genes are associated with canonical wnt-signaling pathway, regulation of substrate-dependent cell migration, mRNA splicing, stem cell proliferation. The network of gene interactions revealed a cluster of co-expression of 24 genes. The CTNNB1, EIF4A2, EIF4G1, EIF4G2, FLNA, FN1, HNRPA2B1, HNRNPH1, HNRNPK, HNRNPU, RBFOX2, RBM25 and SRSF5 genes are central in the network with the largest number of interactions. A maximum of 10 of alternative transcripts corresponds to the FN1 gene.

Conclusion: Obtained data confirm the importance of AS in DSCs, which significantly increases transcriptional diversity and plays a key role in regulatory functions.

References:

Grants: The reported study was funded by RFBR №18-29-13045.

Conflict of Interest: None declared.

EP01.027 MCM-domain containing 2 (MCMDC2) - a novel candidate gene for male infertility

Nadja Rotte ¹, Birgit Stallmeyer¹, Sabine Kliesch², Frank Tüttelmann¹, Corinna Friedrich¹

¹Institute of Reproductive Genetics, University of Münster, Münster, Germany; ²Centre of Reproductive Medicine and Andrology, University Hospital Münster, Münster, Germany

Background/Objectives: Infertility affects 10–15% of all couples, and although frequently suspected, underlying genetic causes often remain unidentified. Recently, Mcmdc2, a minichromosome maintenance (MCM) paralog, was published in the context of murine sterility and meiotic arrest. Members of the MCM family are important for DNA replication, repair, and meiotic recombination, which suggests that this also applies to human MCMDC2.

Methods: Whole exome sequencing data from the MERGE cohort of >1600 men, mostly affected by azoospermia and infertility, was screened for bi-allelic rare (MAF <1%, gnomAD) coding variants in MCMDC2.

Results: Two azoospermic, otherwise healthy men from non-consanguineous parents were identified with bi-allelic, loss-of-function variants in MCMDC2. Hormonal parameters (FSH, LH, testosterone) and testicular volumes were within reference ranges for M1226, who carried two compound heterozygous frameshift variants inherited from each parent. His fertile sister was wildtype for both variants. M1762 was homozygous for a stop-gain variant likely disturbing the MCM family domain. His parents were heterozygous carriers. Decreased testosterone and increased FSH levels indicate testicular disturbance, accordingly, M1762’s histology revealed meiotic arrest on spermatocyte level, with apoptotic spermatocytes and occasional round spermatids; testicular sperm extraction (TESE) was negative.

Conclusion: This is the first report of recessively inherited MCMDC2 deficiency in the context of male infertility. The testicular phenotype of meiotic arrest seen in mutation carriers is in accordance with the phenotype observed in two independently described infertile Mcmdc2 knockout mice and further supports a function of MCMDC2 in meiosis.

References: PMID 27986806/27760146.

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

Conflict of Interest: None declared.

EP02 Prenatal genetics

EP02.001 Prenatal tests and pregnancy termination amongst Moslem women living at home with a child afflicted with a genetic disease or syndrome

Aliza Amiel ¹, Mahadi Tarabeih¹

¹The Academic College of Tel-Aviv-Yaffa, Israel, School of Nursing Sciences, Tel-Aviv, Israel

Background/Objectives: According to the Islamic religion, Moslem women are not permitted to terminate a pregnancy after the first 120 days. Prenatal diagnosis tests are divided into screening and invasive tests which are low risk for abortions. Consanguinity is very common in Moslem society. Herein, we raised the question as to whether a Moslem woman with a child afflicted with a genetic disease, living at home, would perform more prenatal tests and pregnancy terminations versus women with normal children living at home.

Methods: 771 Moslem women participated in the study; 37.1% had a child afflicted with a genetic disease; 62.9% did not; 52% were city-dwellers and 48% lived in villages.

Results: Moslem women with an abnormal child afflicted with a genetic disease living at home will undergo more prenatal tests including invasive tests and will undergo more pregnancy terminations (p < 0.001). More city-dwellers underwent different prenatal tests and pregnancy terminations. Village-dwellers were more religious and counselled more by a religious authority. City-dwellers received more emotional support from their husbands, family, and friends. Non-invasive prenatal testing (NIPT) was performed in direct correlation with a higher socioeconomic status and academic education and was performed more in the cities (p < 0.001).

Conclusion: Families with a child afflicted with a genetic disease received more genetic counseling. Families living in cities received more genetic counselling. Women living in the villages were more religious and abided with religious rulings. NIPT was performed more in the cities due to higher economic status and academic education.

References:

Grants:

Conflict of Interest: None declared.

EP02.002 Type and frequency of copy number aberrations in prenatal samples detected by karyotyping and array CGH analysis in Bulgarian patients

Kalina Belemezova ¹, petya chaveeva², Mariela Hristova-Savova¹, Radka Kaneva³, Gergana Stancheva³, Violeta Stratieva⁴, Maria Yankova⁴, Petya Andreeva^2;5, Maria Yunakova², Tanya Milachich^2;6, Tanya Timeva^2;7, Atanas Shterev², Ivanka Dimova^2;3

¹Medical Complex Dr. Shterev, Genetics Laboratory, Sofia, Bulgaria; ²Medical complex Dr. Shterev, Sofia, Bulgaria; ³Molecular Medicine Center, Medical University of Sofia, Sofia, Bulgaria; ⁴OSCAR clinic, Sofia, Bulgaria; ⁵South-West University Neofit Rilski Blagoevgrad, Blagoevgrad, Bulgaria; ⁶Institute of Biology and Immunology of Reproduction, Sofia, Bulgaria; ⁷Angel Kanchev University of Ruse, Ruse, Bulgaria

Background/Objectives: Chromosome analysis by microarrays (array CGH) is a high-resolution technology capable of detecting microdeletions and microduplications throughout the genome that are missed by conventional karyotyping. In the present study we aimed to summarize the results of prenatal diagnosis by karyotyping and chromosome microarray analysis to make a comparison and determine the frequency of aberrations in different indications, which will allow us to create an algorithm for array CGH analysis in prenatal diagnostics.

Methods: The study included 63 amniotic samples subjected to conventional karyotyping and 145 prenatal samples for microarray analysis.

Results: Pathogenic aberrations were found in 9.5% of karyotyping cases, as 83.3% of them could be diagnosed by QF-PCR for the most common aneuploidies (rapid aneuploid test - RAT). Array CGH analysis revealed 23 pathogenic aberrations (15.75% of cases after RAT). In 5 cases (21.7%) the size of the found aberrations is below 1 Mbp, in 11 cases (47.8%) the size is between 1 and 6 Mbp. Seven of the pathogenic aberrations (30.4%) could be diagnosed by karyotyping due to size over 10 Mbp—4.8% of all samples. We found variants of unclear significance (VOUS) in 19% of cases—between 8% and 28% in different indications.

Conclusion: Our results show that array CGH should be used after the exclusion of common aneuploidies by QF-PCR and it increases the diagnostic rate of karyotyping more than three times—15.75% vs 4.8%. The lowest was the diagnostic rate in isolated elevated nuchal fold, and the highest—in a family history for disorder.

References:

Grants:

Conflict of Interest: None declared.

EP02.003 AnDDI-prenatome - the French national project of prenatal trio exome sequencing: 43% of diagnostic yield in 28 days with 80% pregnancy care changes

Frédéric Tran Mau-Them ^1;2, Anne-Sophie Denommé-Pichon^1;2, Hana Safraou^1;2, Ange-Line Bruel^1;2, Antonio Vitobello^1;2, Sophie Nambot³, Julian Delanne³, Aurore Garde³, Caroline Racine³, Arthur Sorlin^1;2;3, Sebastien Moutton^1;2;3, Chloe Quelin⁴, Marine Legendre^5;5, Cindy Colson⁶, Anne-Claire Brehin⁷, Alban Ziegler⁸, Audrey Putoux⁹, Alinoe Lavillaureix⁴, Anne-Marie Guerrot⁷, Jeanne Amiel¹⁰, Caroline Rooryck-Thambo¹¹, Carine Abel¹², Patricia Blanchet¹³, Magali Gorce⁸, Godelieve Morel⁴, Alice Goldenberg⁷, nicolas gruchy¹⁴, Melanie FRADIN⁴, Agnes Guichet¹⁵, Odile Boute⁶, Elise Schaefer¹⁶, Gabriella Vera⁷, Catherine Delorme⁶, Rodolphe Dard¹⁷, Christine Francannet¹⁸, Estelle Colin¹⁵, Emilie Tisserant², Marie Vincent¹⁹, Bertrand Isidor¹⁹, Sylvie Odent⁴, Marie-Laure Humbert²⁰, Yannis Duffourd^1;2, Christinne Binquet²⁰, Christophe Philippe^1;2, Laurence Faivre^2;3, Christel Thauvin-Robinet^1;2;3

¹CHU Dijon, UF6254 Innovation en Diagnostic Génomique des Maladies Rares, Dijon, France; ²CHU Dijon, Service de Génétique des Anomalies du Développement, Dijon, France; ³CHU Dijon, Centre de génétique, Hôpital d’enfants, Dijon, France; ⁴CHU Rennes, Service de génétique clinique, Rennes, France; ⁵CHU Poitiers, Service de génétique médicale, Poitiers, France; ⁶CHU Lille, Clinique de Génétique “Guy Fontaine”, Lille, France; ⁷CHU Rouen, Service de génétique - Unité de génétique clinique, Rouen, France; ⁸CHU Angers, Biochemistry and Genetics Department, Angers, France; ⁹CHU Lyon, Service de génétique, Lyon, France; ¹⁰Hôpital Necker-Enfants Malades, Service de Génétique Médicale et Clinique, Paris, France; ¹¹CHU Bordeaux, Service de génétique médicale, Bordeaux, France; ¹²CHU Lyon, Service de génétique et centre de diagnostic anténatal, Lyon, France; ¹³CHU Montpellier, Equipe Maladies Génétiques de l’Enfant et de l’Adulte, Montpellier, France; ¹⁴CHU Caen, Laboratoire de génétique moléculaire, Caen, France; ¹⁵CHU Angers, Centre Robert Debré, Angers, France; ¹⁶CHU Strasbourg, Service de génétique médicale, Strasbourg, France; ¹⁷CH Poissy, Unité fonctionnelle de génétique médicale, Poissy, France; ¹⁸CHU Clermont-Ferrand, Service de génétique médicale, Clermont-Ferrand, France; ¹⁹CHU Nantes, Service de génétique médicale - Unité de Génétique clinique, Nantes, France; ²⁰CHU Dijon, CIC 1432 Module Épidémiologie Clinique, Dijon, France

Background/Objectives: Etiological prenatal diagnosis (PD) of congenital abnormalities is a real challenge since abnormal ultrasound signs are discovered in 5–10% of pregnancies. The large deployment of exome/genome sequencing (ES/GS) appears as a strong opportunity to improve PD but faces the difficulties of returning diagnosis in a short turnaround time and interpreting genomic data with incomplete clinical/imaging data. We present the French multicentric AnDDI-prenatome study of prenatal trio-ES.

Methods: Rapid trio-ES (delay maximum 42 days) was performed, in parallel or after array-CGH, in pregnancies (10–34 weeks of gestation) with 2 ultrasound abnormalities. Only ACMG class 4 and 5 variants were considered for positive diagnosis.

Results: 19 different centers included 150 pregnancies. ES was performed in first intention in 81/150 and after normal array-CGH in 60/150. The median delay was 28 days for positive diagnosis and 26 days for negative ones. The diagnostic rate was of 42% in first intention and of 27% after normal array-CGH. The concordance rate between ES and array-CGH was of 100% to detect CNV. Altogether, a causal diagnosis was identified in 50/150 foetuses, rising to 54/150 after VUS investigation. The pregnancy management could have been modified in 79% of the cases.

Conclusion: The AnDDI-prenatome study shows that prenatal ES is feasible with acceptable turnaround in France, with a major interest in pregnancy management. Despite prenatal incomplete clinical/imaging data, its diagnostic yield appears similar to postnatal ES. Its application on a larger scale, notably its impact on healthcare system and organization, should now be carried to consider its routine implementation.

References:

Grants:

Conflict of Interest: None declared.

EP02.005 Noninvasive prenatal testing for cystic fibrosis using circulating trophoblasts in maternal blood

Line Dahl Jeppesen ^1;2, Lotte Hatt², Dorte Lildballe^1;3, Ida Vogel^1;4

¹Center for Fetal Diagnostics, Department of Clinical Medicine, Aarhus University, Aarhus, Denmark; ²Arcedi Biotech, Vejle, Denmark; ³Department of Molecular Medicine, Aarhus University Hospital, Aarhus, Denmark; ⁴Department of Clinical Genetics, Aarhus University Hospital, Aarhus, Denmark

Background/Objectives: Cystic fibrosis is the most common autosomal recessive disorder and prenatal screening has been recommended (1). This study aimed to develop cell-based noninvasive prenatal testing (NIPT) for cystic fibrosis using circulating trophoblasts in maternal blood.

Methods: Blood samples were collected from pregnant women with either high risk for CF (group 1, N = 8) or invasive sampling performed for other reasons (group 2, N = 21). Circulating trophoblasts were enriched and isolated by FACS single cell sorting. The cell origin was identified by DNA profiling using STR analysis. Cystic fibrosis analysis for detection of 50 pathogenic variants in CFTR was conducted by fragment analysis or next-generation sequencing and the results were compared between trophoblasts and invasive samples.

Results: Circulating trophoblast cells (range 1–5, median 4) were tested from eight pregnancies at risk of cystic fibrosis in group 1. The cell-based NIPT results were in concordance with the result of invasive testing in seven cases but one cell-based NIPT gave an inconclusive result. From the second group, an average of circulating trophoblast cells (range 1–10, median 5) were tested from 21 pregnancies. Among these, two previously unidentified maternal carriers of cystic fibrosis were found and all cell-based NIPT results were in concordance with those of invasive testing for the 50 CFTR variants. In one sample, no trophoblast cells were found.

Conclusion: This study provides a proof-of-concept for detection of cystic fibrosis using cell-based NIPT.

References: (1) ACOG, webpage accessed 9th Feb 2022, https://www.acog.org/womens-health/faqs/cystic-fibrosis-prenatal-screening-and-diagnosis.

Grants: IV: Novo Nordisk Foundation (NNF16OC0018772), LJ: Innovation Fund Denmark (0153-00004B).

Conflict of Interest: Line Dahl Jeppesen Full time employed by Arcedi, Lotte Hatt Full-time employed by Arcedi, Dorte Lildballe: None declared, Ida Vogel: None declared.

EP02.006 Can the phenotype of Down syndrome be predicted at the combined first trimester screening for trisomy 21?

Ellen Steffensen ^1;2, Lars Henning Pedersen^3;4, Ida Vogel^1;2

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

Background/Objectives: The ability of the combined first trimester screening (cFTS) to identify Down syndrome pregnancies is well established, but can cFTS for trisomy 21 predict the Down syndrome phenotype? We aimed to investigate the potential association between cFTS result and phenotype in Down syndrome.

Methods: We performed a register based cohort study including all cases of trisomy 21 in Denmark during 2005-2018 diagnosed in pregnancy or before 13.5 years of age. We compared screen negative (<1:300) and screen positive (≥1:300) trisomy 21 cases with respect to phenotypic characteristics: congenital malformations, anthropometry, and childhood morbidity.

Results: Of 2,167 trisomy 21 cases (pre- or postnatally detected), 1,672 (77%) were screen positive, 242 (11%) screen negative and 253 (12%) not screened. Prenatally, more screen positive cases had a recorded malformation (table); potentially due to detection bias. In liveborn children, comparable proportions of screen negative and positive cases had severe congenital heart disease (CHD) and non-CHD malformations (table).

	Screen positive, % (95% CI)	Screen negative, % (95% CI)
Prenatal CHD	28.6% (20.0-38.6%)	10.0% (6.2-14.9%)
Prenatal non-CHD malformation	32.7% (23.5-42.7%)	9.5% (5.8-14.4%)
Postnatal severe CHD	24.1% (15.4-34.7%)	20.5% (14.8-27.2%)
Postnatal non-CHD malformation	8.4% (3.5-16.6%)	13.1% (8.5-19.0%)

Fetal and newborn anthropometry as well as duration of childhood hospital admissions was similar among screen negative and positive cases.

Conclusion: In a 14-year nationwide cohort, we did not observe a consistent phenotypic difference between screen negative and screen positive trisomy 21 cases.

References:

Grants: Aarhus University. Health Research Foundation of Central Denmark Region (A2602). Health Foundation (20-B-0065).

Conflict of Interest: None declared.

EP02.007 Undiagnosed arthrogryposis: further expanding the molecular and phenotypic spectrum

G. Tutku Turgut ¹, Umut Altunoglu^2;3, Tugba Sarac-Sivrikoz⁴, Tugba Kalayci², Guven Toksoy², Sahin Avci^2;3, Birsen Karaman², Cagri Gulec², Gozde Yesil Sayin², Seher Basaran², Hülya Kayserili^2;3, Zehra Oya Uyguner²

¹Istanbul University, Istanbul Medical Faculty, Medical Genetics, İstanbul, Turkey; ¹Istanbul University, Istanbul Medical Faculty, Medical Genetics, İstanbul, Turkey; ³Koç University School of Medicine (KUSoM), Medical Genetics, İstanbul, Turkey; ⁴Istanbul University, Istanbul Medical Faculty, Perinatology Unit, Department of Obstetrics and Gynecology, İstanbul, Turkey

Background/Objectives: Arthrogryposis multiplex congenita (AMC) is a phenotypic descriptor implying congenital contractures in two or more body areas. Exome sequencing provides an effective approach in elucidating the molecular basis, considering the presence of genetic and phenotypic heterogeneity. We aimed to identify the molecular etiology in undiagnosed fetuses within the AMC spectrum, using exome sequencing.

Methods: 14 patients from ten families displaying AMC were enrolled. Array-CGH analysis was performed in all fetuses, followed by exome analysis. Sanger sequencing was performed to validate variants.

Results: We identified known or novel candidate variants in COG6, NEB, RAPSN, KIAA1109, DOK7, HSPG2, PSAT1, and PIEZO2. Three patients were shown to harbor additional biallelic variants in VPS13B, DNAH9, and USH2A, complicating the phenotype. Moreover, we identified a frameshift variant in USP14 in three similarly affected fetuses. The resemblance of the fetal findings to the previously reported Usp14 mouse models supported that our variant likely led to a novel intrauterine-onset human AMC phenotype (1).

Conclusion: Our results provide new insights into the clinical and molecular characterization of a small AMC cohort; to contribute to phenotype-genotype correlation, expand the clinical spectrum, report novel variants, and present the first human phenotype that appears to be attributable to a truncating variant in USP14.

References:

(1) Turgut, Gozde Tutku et al. “Functional loss of ubiquitin-specific protease 14 may lead to a novel distal arthrogryposis phenotype.” Clinical genetics, https://doi.org/10.1111/cge.14117. 23 Jan. 2022, https://doi.org/10.1111/cge.14117.

Grants: This project is granted by the Scientific Research Projects Coordination Unit of Istanbul University (Project ID 37765).

Conflict of Interest: G. Tutku Turgut This project is granted by the Scientific Research Projects Coordination Unit of Istanbul University (Project ID 37765), Umut Altunoglu: None declared, Tugba Sarac-Sivrikoz: None declared, Tugba Kalayci: None declared, Guven Toksoy: None declared, Sahin Avci: None declared, Birsen Karaman: None declared, Cagri Gulec: None declared, Gozde Yesil Sayin This project is granted by the Scientific Research Projects Coordination Unit of Istanbul University (Project ID 37765)., Seher Basaran: None declared, Hülya Kayserili: None declared, Zehra Oya Uyguner: None declared.

EP02.008 Polymorphisms of the MTHFR gene and maternal risk of offspring aneuploidy

Olivera Miljanovic ¹, Jelena Jovanovic², Sladjana Teofilov², Dragan Likic³, Miljana Andjelic², Tatjana Ostojic², Milena Bulatovic², Gordana Stojanovic²

¹Clinical Center of Montenegro, Faculty of Medicine, Podgorica, Montenegro 2, Center for Medical Genetic and Immunology, Podgorica, Montenegro; ²Clinical Center of Montenegro, Podgorica, Montenegro, Center for Medical Genetic and Immunology, Podgorica, Montenegro; ³Institute for public health of Montenegro, Center for non-communicable diseases, Podgorica, Montenegro

Background/Objectives: Chromosomal aneuploidies cause about 50% of early pregnancy losses and affect 0.3–0.5% of childbirths, with still insufficiently understood underlying mechanisms of chromosomal malsegregation. Folate metabolism gene polymorphisms have been shown to lead to DNA hypomethylation and damage, with possible consequent abnormal chromosomal segregation during meiosis. The objective was to investigate the association between maternal 5,10-methylentertahydrofolate reductase (MTHFR) gene polymorphisms, crucial for DNA methylation, and risk of offspring aneuploidy.

Methods: MTHFR gene polymorphisms 677C>T and 1298A>C were determined by polymerase chain reaction based method, in 163 women with offspring aneuploidy and 155 women with healthy children. Five genetic models were used to assess risk, according to the type of aneuploidy and the age of women at conception.

Results: MTHFR 677TT genotype and T allele were significantly more prevalent among women with offspring aneuploidy, with an increased risk of aneuploidy demonstrated under a recessive (OR 3.499), homozygote (OR 3.456) and allele contrast model (OR 1.574). The more prominent association was found with sex chromosome aneuploidies and trisomy 13/18, and also in women ≤35 years at conception. No association was observed between 1298A>C polymorphism and risk of offspring aneuploidy, although synergistic effect of two polymorphisms increase the risk of aneuploidy, primarily amplifying the 677T allele effects (p < 0.001).

Conclusion: Maternal MTHFR 677C>T gene polymorphism, alone or in combination with another 1298A>C polymorphism, appears to be a substantial risk factor for offspring aneuploidy in Montenegro population, especially for sex chromosome aneuploidies and trisomy 13/18, and among younger women.

References:

Grants:

Conflict of Interest: None declared.

EP02.009 Non-invasive fetal sex determination in maternal plasma using chip-based digital PCR

Anna Nykel ¹, Agnieszka Gach¹

¹Polish Mother’s Memorial Hospital—Research Institute, Department of Genetics, Lodz, Poland

Background/Objectives: Clinical indications for fetal sex determination include risk of X-linked disorders and the management of fetuses at risk of congenital adrenal hyperplasia. Currently fetal sex diagnosis could be obtained using invasive procedures such as amniocentesis and chorionic villus sampling, which are associated with a 1% risk of miscarriage. Non-invasive prenatal diagnosis based on circulating cell-free fetal DNA created new possibilities of early detection of fetal sex. The aim of this study was to design and validate a rapid, reliable and low-cost method for non-invasive fetal sex determination based on the assessment of cell-free fetal DNA in maternal plasma.

Methods: To determine fetal sex, blood samples from 35 pregnant women at weeks 11 to 17 of gestation were analysed. Cell-free DNA was isolated from the plasma samples using a commercially available kit. A multiplex PCR was performed for the simultaneous amplification of target sequences of Y chromosome using a chip-based QuantStudio 3D Digital PCR system. The results were validated by results from invasive diagnostics.

Results: All maternal plasma samples based on the detection of Y chromosome-specific sequences were determined correctly.

Conclusion: Chip-based digital PCR is a reliable method with high accuracy for non-invasive fetal sex determination. Our results demonstrate that fetal sex determination by detecting Y chromosome sequences in maternal plasma using chip-based digital PCR platform is clinically applicable and could be used in clinical practice.

References:

Grants:

Conflict of Interest: None declared.

EP02.010 Prenatal diagnosis of holoprosencephaly associated with a de novo balanced t(4;7)(p14;q36) translocation

Isabel Ochando^1;2;3, Antonio Urbano^1;2;3, Rosa Bermejo⁴, Rosario Vazquez^1;3, Marina Sánchez ^1;3, Laura del Junco⁴, Estefanía Montoya^1;3, JOAQUIN RUEDA^1;2;3

¹Unidad de Genética, Hospital HLA Vistahermosa, Alicante, Spain; ²Departamento de Histología y Anatomía, UMH, Alicante, Spain; ³Cátedra de Biomedicina Reproductiva Vistahermosa, UMH, Alicante, Spain; ⁴Hospital Universitario de Sant Joan, Servicio de Ginecología y Obstetricia, Alicante, Spain

Background/Objectives: Holoprosencephaly (HPE) is a complex brain malformation resulting from incomplete division of the forebrain. Approximately 25–50% of individuals with HPE have a chromosome abnormality. Analysis of recurrent chromosomal anomalies led to the identification of 12 candidate regions (HPE1 to HPE12). SHH, the major gene implicated in holoprosencephaly, was isolated from the critical region HPE3 on chromosome 7q36. We report a prenatal case with holoprosencephaly and a balanced de novo translocation t(4;7)(p14;q36) without any loss of the distal part of chromosome 7q confirmed by array-CGH.

Methods: Chorionic villi culture and GTG banding were done following standard protocols. Array-CGH was performed using 60K KaryoNIM Prental®. Maternal contamination was ruled out prior to the study.

Results: At 12 weeks of gestation, ultrasound examination of the fetus showed holoprosencephaly. Cytogenetic studies showed a balanced translocation between the long arms of chromosomes 4 and 7, 46,XX,t(4;7)(p14;q36). Parental karyotypes were normal. CGH-array study was normal, without any loss of the distal part of chromosome 7q.

Conclusion: Two breakpoint de novo rearrangement has a 6.7% empiric risk of phenotypic abnormality. Abnormal phenotypes are thought to result from gene disruption, position effect, or deletion at breakpoints. Balanced translocations involving 7qter have been reported previously related with HPE[1]. We present a prenatal case of holoprosencephaly with altered karyotype and normal array-CGH, which highlights the importance of performing a karyotype study in cases of HPE.

References: [1] Benzacken, B. et al. Different proximal and distal rearrangements of chromosome 7q associated with holoprosencephaly. J. Med. Genet. 1997; 34: 899-903.

Grants: UMH-Citolab1.19A.

Conflict of Interest: None declared.

EP02.011 Deletion of PAX3 gene in aborted fetus with spina bifida and meningomyelocele

Šárka Trávníčková ^1;2;3, Jana Duchoslavová^1;3, Václava Curtisová¹, Andrea Štefeková¹, Pavlína Čapková¹, Zuzana Čapková¹

¹Olomouc, Department of Medical Genetics, Olomouc, Czech Republic; ²Palacký University Olomouc, Department of Neurology, Olomouc, Czech Republic; ³Palacký University Olomouc, Department of Medical Genetics, Olomouc, Czech Republic

Background/Objectives: PAX3 gene is localized on chromosome 2 (q36.1) and its product (transcription factor Pax3) is a key regulator of embryogenesis involved in the development of nervous and muscle system. Heterozygous mutations of PAX3 gene are linked to autosomal dominant Waardenburg syndrome (WS) 1 and 3, characterized by hearing loss, loss of pigmentation and eye abnormalities. We present a case of PAX3 deletion detected in a fetus with multiple congenital abnormalities.

Methods: We examined a tissue of a fetus aborted at 20+4 week gestation for US abnormalities which included an absent nasal bone, an abnormal finding in posterior cranial fossa, dilatation of left lateral ventricle, sacral spina bifida with meningomyelocele, and left club foot.

Results: Karyotype was normal 46,XX. This deletion (size 3 Mb) on chromosome 2 including PAX3 was detected by aCGH – 2q35q36.1(221148359_224433293) x1. A complete loss of one copy of PAX3 was confirmed by MLPA. The CNV was not found in healthy parents.

Conclusion: Arnold-Chiari malformation (cerebellar abnormalities), spina bifida and meningomyelocele are described as rare features of WS. While most cases of spina bifida and meningomyelocele are determined multifactorially, we hypothesize that a combination of an absent nasal bone and spina bifida may indicate a loss of function of PAX3 gene. The simultaneous presence of these abnormalities can guide investigation strategies during prenatal diagnosis.

References:

Grants: This abstract was supported by MH CZ - DRO (FNOL, 00098892) and Palacký University Olomouc.

Conflict of Interest: None declared.

EP02.012 Prenatal diagnosis of a novel pathogenic HNF1B variant: phenotypic variability between monozygotic twins

Maria Chiara Baroni ^1;2, Chiara Locatelli³, Cristina Bertulli⁴, Claudio La Scola⁴, Andrea Pasini⁴, Enrico Ambrosini¹, Paola Battaglia¹, Antonella Tanzariello¹, Raffaella Morandi⁵, Ginevra Salsi⁵, Vilma Mantovani¹, Marco seri^1;2, Giulia Lanzoni¹

¹IRCCS Azienda Ospedaliero-Universitaria di Bologna, Medical Genetics Unit, Bologna, Italy; ²University of Bologna, Department of Medical and Surgical Sciences, Bologna, Italy; ³IRCCS Azienda Ospedaliero-Universitaria di Bologna, Neonatal Intensive Care Unit, Bologna, Italy; ⁴IRCCS Azienda Ospedaliero-Universitaria di Bologna, Nephrology and Dialysis Service. Pediatric Unit, Bologna, Italy; ⁵IRCCS Azienda Ospedaliero-Universitaria di Bologna, Obstetric Unit, Bologna, Italy

Background/Objectives: HNF1B mutations are associated with autosomal dominant tubulointerstitial kidney disease (OMIM 137920). Although the severity of renal phenotype is extremely variable, HNF1B intragenic mutations result in more severe renal function impairment than copy number variants encompassing the entire gene (17q12 microdeletions). HNF1B-related disease often manifests with fetal hyperechogenic kidneys; however, reports of prenatal diagnosis of intragenic variants are rare.

Methods: We performed an NGS renal disease gene panel in a bichorial biamniotic pregnancy, in which renal anomalies were detected by prenatal ultrasounds.

Results: The analysis identified a novel de novo heterozygous splice-site variant c.1046-2A>T (IVS4) in the HNF1B gene in both twins. Karyotype was normal (46,XX) and QF-PCR analysis determined monozygosity. Ultrasounds revealed hyperechogenic multicystic kidneys and severe oligohydramnios in fetus 2, already at 16+2 weeks. At birth, she showed flattened head with frontal bossing, unilateral cataract, clubfeet and bilateral pneumothorax; she died on the second day of life. Instead, fetus 1 showed no abnormalities until 20 weeks, when ultrasound detected bilateral renal enlargement and hyperechogenicity with right kidney cysts; amniotic fluid was normal. She had normal birth parameters and at last assessment (5 months) creatinine was 1.34 mg/dL.

Conclusion: We have described a case of prenatal diagnosis of a novel pathogenic HNF1B variant associated with a severe renal disease in a twin pregnancy, confirming that intragenic variants are associated with a poorer renal prognosis, but with variability even between identical twins. We have also deeply described the prenatal and postnatal presentation of the disease, which can be useful for prenatal genetic counseling.

References:

Grants:

Conflict of Interest: None declared.

EP02.013 The diagnostic utility of genome sequencing for fetal congenital heart defects

Ye Cao^1;2;3, Matthew Hoi Kin Chau^2;3;4, Yu ZHENG ², Yilin Zhao², Shuk Yi, Annie Hui², Hoi Wan, Angel Kwan², Zirui Dong^2;3;4, Kwong Wai Choy^2;3;4

¹The Chinese University of Hong Kong, Department of Paediatrics, HONG KONG, China; ²The Chinese University of Hong Kong, Department of Obstetrics and Gynaecology, HONG KONG, China; ³The Chinese University of Hong Kong, Hong Kong Hub of Paediatric Excellence, HONG KONG, China; ⁴Shenzhen Research Institute, The Chinese University of Hong Kong, Key Laboratory for Regenerative Medicine, Ministry of Education (Shenzhen Base), Shenzhen, China

Background/Objectives: Compelling data in support of prenatal genetic diagnoses by exome sequencing in fetuses with structural abnormalities detected by ultrasonography is well recognized¹. Congenital heart defects are genetically heterogeneous and contributed wide range of genetic mutation types. This is a pilot study to investigate the diagnostic utility of genome sequencing (GS) to detect a broader range of causative genetic variants in a prenatal diagnosis setting.

Methods: Genome sequencing (>30X) was prospectively performed 13 prenatal trios with heart defects for which karyotyping and/or chromosomal microarray results were undiagnostic.

Results: 4/13 (30.8%) of trios received diagnostic genetic findings by genome sequencing. They included pathogenic or likely pathogenic variants in DNAH5, COL4A1, PTPN11, and KRAS. Notably, a balanced translocation [46,XX,t(14;22)(q32.33;q13.31)mat] detected by GS in the trio with the DNAH5 mutations. The translocation also explained the adverse pregnancy histories of presumably de novo Phelan-McDermid syndrome of this trio. GS also detected medically actionable findings in BRCA2 and carrier statues in GJB2, HBB, USH2A, HBA1 and HBA2.

Conclusion: Genome sequencing provided additional yield and timely results of diagnostic variants, including a wide spectrum of mutation types. Not only did genome sequencing facilitate genetic diagnoses, it also provided clinically relevant medical actionable findings and carrier statuses. We provide evidence to support the application of genome sequencing for fetuses with congenital heart defects.

References: 1. Lord, Jenny, et al. “Prenatal exome sequencing analysis in fetal structural anomalies detected by ultrasonography (PAGE): a cohort study.” The Lancet 393.10173 (2019): 747–757.

Grants: Partially supported by Funding: CUHK Direct Grant (2020.074), HMRF (07186576) and ITF (MHX/003/19).

Conflict of Interest: Ye Cao Research Assistant Professor, Department of Paediatrics & Department of Obstetrics and Gynecology, The Chinese University of Hong Kong, Shatin, Hong Kong, China, partially support by Funding source: CUHK Direct Grant (2020.074), Matthew Hoi Kin Chau Postdoctoral Fellow, Department of Obstetrics and Gynaecology, The Chinese University of Hong Kong, Hong Kong, China, Yu ZHENG: None declared, Yilin Zhao: None declared, Shuk Yi, Annie Hui Consultant & Clinical Associate Professor (honorary), Department of Obstetrics and Gynaecology, The Chinese University of Hong Kong, Hong Kong, China, Hoi Wan, Angel Kwan Resident, Clinical Tutor (honorary), Department of Obstetrics and Gynaecology, The Chinese University of Hong Kong, Hong Kong, China, Zirui Dong Assistant Professor, Department of Obstetrics and Gynaecology, The Chinese University of Hong Kong, Hong Kong, China, HMRF (07186576), Kwong Wai Choy Professor, Department of Obstetrics and Gynaecology, The Chinese University of Hong Kong, Hong Kong, China.

Deputy Director, Prenatal Genetic Diagnosis Centre, CUHK.

Director, Pre-implantation Genetic Diagnosis Laboratory, CUHK.

Deputy Director, CUHK-Utrecht Joint Centre for Language, Mind and Brain, CUHK.

Scientific Officer (Medical), New Territories East Cluste r, Hospital Authority.

Deputy Programme Director, MSc in Medical Genetics, CUHK, ITF (MHX/003/19).

EP02.014 Familial form of Milroy disease with incomplete penetrance creates diagnostic challenges in a family with recurrent fetal lymphedema

Irena Bradinova ^1;2, Kunka Kamenarova^3;4, Radostina Raynova², Radoslava Vazharova⁵, Andrey Andreev², Kalina Mihova^3;4, Darina Kachakova-Yordanova^3;4, Dimitar Markov^6;7, Radka Kaneva^3;4, Alexey Savov^1;2

¹Medical University Sofia, Department of Obstetrics and Gynecology, Sofia, Bulgaria; ²UHOG “Maichin dom”, National Genetic Laboratory, Sofia, Bulgaria; ³Molecular Medicine Center, Medical University - Sofia, Department of Medical Chemistry and Biochemistry, Sofia, Bulgaria; ⁴Laboratory of Genomic Diagnostics, Medical University - Sofia, Department of Medical Chemistry and Biochemistry, Sofia, Bulgaria; ⁵SU “St. Kl. Ohridski”, Faculty of Medicine, Department of Biology, Medical genetics and Microbiology, Sofia, Bulgaria; ⁶Markov’s Medical Center, Sofia, Bulgaria; ⁷Medical University Sofia, Faculty of Public Health, Sofia, Bulgaria

Background/Objectives: Milroy disease is an autosomal dominant condition caused by mutations in the FLT4 gene that affects the normal function of the lymphatic system resulting in congenital lymphedema. Intrafamilial variability and reduced penetrance are observed in this condition. About 10–15% of people with a mutation in the FLT4 gene do not develop the features of Milroy disease. Incomplete penetrance can sometimes obscure autosomal dominant inheritance pattern.

Methods: We present a family of healthy parents with two consecutive pregnancies with fetal lower limbs edema detected prenatally at second trimester ultrasound examination. Karyotyping was performed on amniotic fluid samples from the two pregnancies and blood samples of both partners. Clinical exome sequencing of DNA obtained from uncultered amniocytes from the second pregnancy was performed on MiSeq (Illumina). Sanger sequencing in fetal and parental DNA was applied for confirmation and segregation analysis.

Results: Prenatal diagnosis of the first pregnancy revealed mosaic fetal karyotype: 46,XX,-10,+mar (del10q?)[2]/46,XX[25]. The family decided to terminate the pregnancy. Both partners were carriers of normal karyotypes. Amniocentesis performed on the second pregnancy (ten years later) detected normal fetal female karyotype (46,XX) and a heterozygous pathogenic variant in FLT4 gene - c.3341C>T (p.Pro1114Leu). This variant was inherited by the father.

Conclusion: The phenomenon of incomplete penetrance can make it challenging for genetics professionals to interpret a person’s family medical history and predicting the risk of passing a genetic condition to future generations. This should be taken into account in counselling for autosomal dominant conditions.

References: None.

Grants: None.

Conflict of Interest: None declared.

EP02.015 High-resolution chromosomal microarray analysis in the prenatal setting

Anastasios Mitrakos ¹, Konstantina Kosma¹, MARIA TZETIS¹

¹National and Kapodistrian University of Athens, Department of Medical Genetics, Athens, Greece

Background/Objectives: Prenatal chromosomal microarray analysis (CMA), traditionally indicated for high-risk pregnancies, is nowadays frequently applied preventively. Here we report on the outcomes of the application of high-resolution CMA in a series of prenatal cases.

Methods: 219 couples were referred to the Laboratory of Medical Genetics of the National and Kapodistrian University of Athens for prenatal CMA. Indications included ultrasonographic findings, previous pregnancy or offspring with chromosomal abnormality, a parent carrier of a balanced chromosomal translocation, advanced maternal age, and preventive testing due to parental anxiety. DNA was extracted from either amniotic fluid (AF) or chorionic villus sample (CVS) (DNA mini kit, Qiagen) and was subsequently tested for chromosomal aberrations with the 4x180K G3 CGH+SNP microarray (Agilent Technologies). Maternal cell contamination was excluded for all samples.

Results: A total of 250 samples were studied (143 AF, 107 CVS). 141 of all embryos were male and 109 female. 198 embryos (79.2%) had a normal karyotype and 52 (20.8%) showed one or more chromosomal abnormalities. The findings were classified as pathogenic or likely pathogenic in 38 cases (15.2%) and as Variants of Uncertain Clinical significance (VUS) in 14 cases (5.6%).

Conclusion: High-resolution CMA is a powerful tool in the prenatal setting, with high diagnostic yield regardless of indication. VUS appear with relatively high frequency, however this is expected to further decline as knowledge on the clinical significance of findings increases.

References: Oneda B, et al. High-resolution chromosomal microarrays in prenatal diagnosis significantly increase diagnostic power. Prenat Diagn. 2014;34:525–33.

Grants: N/A

Conflict of Interest: None declared.

EP02.016 Prenatal diagnosis of Saethre-Chotzen syndrome caused by TWIST1 microdeletion and complex chromosomal rearrangement involving chromosomes 5, 7 and 11

Ivana Joksic ¹, Mina Toljic¹, Nela Maksimovic², Dijana Perovic², Aleksandar Jurisic³

¹Gynecology and Obstetrics Clinic “Narodni front’, Genetic laboratory department, Belgrade, Serbia; ²InsInstitute of human genetics, Faculty of medicine, University of Belgrade, Belgrade, Serbia; ³Gynecology and Obstetric Clinic “Narodni front”, Faculty of Medicine, University of Belgrade, Belgrade, Serbia

Background/Objectives: Saethre-Chotzen (SC) is syndromic craniosynostosis rarely diagnosed prenataly. It is caused by mutations, or less frequently deletions of TWIST1 gene leading to its haploinsufficiency. Clinical presentation is variable, with coronal craniosynostosis, facial dysmorphism, hand and foot anomalies being the most common manifestations. We present a case of prenataly diagnosed SC-like syndrome due to complex chromosomal rearrangement involving chromosomes 5, 7, and 11.

Methods: A 32-year-old women was referred to genetic counselling in 28th gestation week of her third pregnancy due to presence of ultrasound anomalies. Brachicephaly, hypertelorism, flat face, micrognathia, relative macroglossia and small posterior fossa were noted.

Results: Karyotype analysis was performed on blood sample obtained by cordocenthesis. De novo translocation involving chromosomes 7 and 11 was found- 46,XY,t(7;11)(p15.5;q21)dn. Subsequent chromosomal microarray analysis (Agilent Sureprint G3 Human 8x60K) revealed presence of three microdeletions on chromosome 7: 7p21.1-p15.3 (4,82 Mb including TWIST1 gene), 7p12.1-p11.2 (1.3Mb), 7q21.11 (2,9 Mb), and one on chromosome 5 (5p12-p11, 581Kb).

Conclusion: Complex chromosomal rearrangements involving TWIST1 gene are very rare cause of SC syndrome. Prenatal manifestations in our case were consistent with TWIST1 haploinsufficiency, however, additional clinical manifestation can be expected due to presence of multiple microdeletions in proband. This case confirms utility of chromosomal microarray in prenatal cases of suspected syndromic craniosynostosis.

References: Spaggiari E, Aboura A, Sinico M, Mabboux P, Dupont C, Delezoide AL, Guimiot F. Prenatal diagnosis of a 7p15-p21 deletion encompassing the TWIST1 gene involved in Saethre-Chotzen syndrome. Eur J Med Genet. 2012 Aug-Sep;55(8-9):498-501.

Grants: None.

Conflict of Interest: None declared.

EP02.017 Further delineation of prenatal presentation in congenital dyserythropoietic anemia type II (CDAII)

Anna Abulí ¹, María Ángeles Sánchez², Carlota Rodo², Eulàlia Rovira¹, Marta Codina-Sola¹, Irene Valenzuela¹, Elena García-Arumí¹, Eduardo Tizzano¹

¹University Hospital Vall d’Hebron, Department of Clinical and Molecular Genetics, Barcelona, Spain; ²University Hospital Vall d’Hebron, Department of Obstetrics, Barcelona, Spain

Background/Objectives: Nonimmune hydrops fetalis (NIHF) is a fetal abnormality with high genetic heterogeneity including genes associated to congenital anemias. Congenital dyserythropoietic anemia type II (CDAII) is caused by pathogenic variants in the SEC23B gene following an AR inheritance. Patients usually manifest with mild to moderate anemia, although in some cases blood transfusion may be required. Prenatal clinical manifestation as hydrops foetalis and intrauterine death is barely described. We present here two new cases of severe CDAII presenting with hydrops foetalis.

Methods: Two pregnant women were referred to the Foetal Medicine Unit of our Hospital at 26 and 18 weeks of gestation, respectively. Ultrasound findings showed fetal ascites and pericardial effusion resulting in severe hydrops foetalis in both cases. They underwent prenatal exome sequencing in fetal DNA.

Results: Molecular results showed pathogenic and probably pathogenic variants c.325G>A (p.Glu109Lys) and c.1648C>T; (p.Arg550*) in SEC23B (fetus 1), and c.2101C>T (p.R701C) and c.2102G>A (p.R710H) in SEC23B (fetus 2). The variants were in heterozygous state in their respective healthy parents. Although some of these variants have previously been reported, there is no clear genotype-phenotype correlation considering the type of variant.

Conclusion: Challenges in prenatal exome include the unknown fetal presentation of several genetic conditions that are recognized in postnatal settings. Indeed, our study provides further evidence of the presentation of CDAII as hydrops fetalis, expanding the clinical spectrum of this condition. Moreover, a categorical molecular diagnosis enables an early and adequate prenatal management to improve the prognosis of CDAII and to offer an appropriate genetic counseling to the families.

References:

Grants:

Conflict of Interest: None declared.

EP02.018 Non-indicated invasive tests yield higher percentage of Ashkenazi Jewish patients

Moran Echar ¹, Amir Peleg¹, Jumana Haddad-Halloun¹, Lena Sagi-Dain^1;2

¹carmel medical center, Haifa, Israel; ²The Ruth and Bruce Rappaport Faculty of Medicine Technion, Haifa, Israel

Background/Objectives: The risk for clinically significant copy number variants in uneventful pregnancies is estimated at about 1%. Numerous factors influence the uptake of prenatal invasive testing, and the objective of our study was to explore the effect of maternal origin on the willingness to undergo non-indicated testing.

Methods: This retrospective cohort study included data of all prenatal microarray tests during 2019–2021 in genetic laboratory of Carmel Medical Center, Haifa, Israel. The reasons for invasive testing were categorized as: 1) indicated tests (e.g., abnormal ultrasound and high-risk serum screening), 2) maternal age ≥35 years and 3) younger women. The percentage of Ashkenazi Jewish patients in each indication was compared to non-Ashkenazi Jewish and to Israeli Arab population.

Results: Of the overall 1400 prenatal microarray tests, 676 (48.3%) were performed due to a medical indication, 392 (28.0%) in women older than 35 years, and 332 (23.7%) in younger women. The percentage of Ashkenazi patients performing invasive testing due to maternal age over 35 years (76.0%) and in younger women (81.0%) was significantly higher compared to the rate of Ashkenazi patients undergoing indicated testing (61.4%). A significant decline in patients from other origins was noted in non-indicated vs. indicated tests.

Conclusion: Higher rates of Ashkenazi patients performing non-indicated invasive tests can be explained by various factors, including socio-economic status, religious beliefs, language barriers, and geographic factors. These potential influences should be taken into consideration during prenatal genetic counseling.

References:

Grants:

Conflict of Interest: None declared.

EP02.019 Retrospective analysis of fetal magnetic resonance imaging (feMRI) examinations in the last 10 years at a tertiary center: experience of a single radiologist and a single perinatologist

Beril Ay ¹, Efe Sarı¹, Deniz Can Alis², Ercan Karaaslan², İbrahim Bildirici³, Yasemin Alanay⁴

¹Acibadem Mehmet Ali Aydinlar University, School of Medicine, Istanbul, Turkey; ²Acibadem Mehmet Ali Aydinlar University, School of Medicine, Department of Radiology, Istanbul, Turkey; ³Acibadem Mehmet Ali Aydinlar University, School of Medicine, Department of Obstetrics and Gynecology, Istanbul, Turkey; ⁴Acibadem Mehmet Ali Aydinlar University, School of Medicine, Department of Pediatrics, Istanbul, Turkey

Background/Objectives: To evaluate the role of fetal magnetic resonance imaging (feMRI) in high-risk pregnancies and the concordance between feMRI and ultrasonography (US).

Methods: Retrospective analysis of US and feMRI in 97 fetuses in the last 10 years at a tertiary center. US was performed by a single perinatologist, feMRI images were analyzed by a single radiologist.

Results: Mean gestational age at US was 26.0 ± 4.2 weeks, and feMRI was within 3 days after US. Indications for feMRI were CNS (n = 80, 82%) and non-CNS (n = 17, 18%). CNS-related indications, ventriculomegaly (n = 41, 42%) had the greatest ratio. In 51 fetuses (53%), feMRI and US were completely concordant. In 11 fetuses (12%), feMRI demonstrated extra findings. In 33 fetuses (34%), feMRI excluded US indication completely. There was no instance where US had additional value to feMRI. For fetuses without additional value, the kappa score was 0.69, indicating a substantial agreement.

Conclusion: This study is the first in the literature to present data from a single center, involving a single perinatologist and a single radiologist. We claim that feMRI and USG have low concordance in prenatal imaging (<0.8 kappa score). However, this low concordance still helps to navigate the pregnancy follow-up. The couple is counselled for invasive prenatal studies when additional findings are reported. Exclusion of the findings suspected in the US examination serves to decrease anxiety the remaining 34%.

References: Davidson J, Brennan K, Matthew J et al. Fetal magnetic resonance imaging (MRI) enhances the diagnosis of congenital body anomalies. J Pediatr Surg. 2022;57(2):239–244.

Grants:

Conflict of Interest: None declared.

EP02.021 Re-analysis of exome sequencing data of prenatal cases presenting with fetal structural anomalies

Rand Dubis ¹, John Filby², Andrea Haworth², Natalie Trump², Helen Savage², Nick Lench², Basky Thilaganathan³, John Short⁴, Charlene Crosby⁴, Sahar Mansour⁴, Esther Dempsey⁴, Tessa Homfray⁴, Suzanne Drury²

¹Congenica Ltd, Cambridge, United Kingdom; ¹Congenica Ltd, Cambridge, United Kingdom; ³Fetal Medicine Unit, St George’s Hospital, London, United Kingdom; ⁴South West Thames Regional Genetics, St George’s Hospital, London, London, United Kingdom

Background/Objectives: Exome sequencing for fetuses with structural anomalies detected on ultrasound has been implemented into clinical practice in the UK Genomics Medicine Service, demonstrating diagnostic rates between 6.2% and 80%. Prenatal diagnosis provides information for prenatal and postnatal management and treatment (Best et al. 2018).

Re-analysis of exome data of previously unsolved cases can increase diagnostic yield by ~12% (Ji et al. 2021).

The objective of this study is to evaluate the diagnostic potential of exome sequence re-analysis in previously unsolved fetal anomaly cases.

Methods: DNA was extracted from CVS, amniotic fluid, fetal blood or post-mortem fetal tissue, and enriched using Agilent SureSelect CRE V2 or Nonacus ExomeCG and sequenced on Illumina NextSeq 500 or NovaSeq. Review of SNVs and CNVs was undertaken using the Congenica clinical decision platform. Re-analysis was performed using updated fetal anomaly panels and gene agnostic prioritisation.

Results: Through original exome sequencing analysis, diagnosis was achieved in 59/180 fetuses. Of 82 cases assessed for CNVs, 2 had pathogenic variants. Re-analysis was performed on 123 unsolved cases. Updated figures will presented.

Conclusion: This study illustrates the diagnostic utility of re-analysing exome sequencing data in unsolved cases with fetal structural anomalies.

References: Best, S., Wou, K., Vora, N., Ven der Veyver, I. B., Wapner, R., Chitty, L. S., 2018. Promises, pitfalls and practicalities of prenatal whole exome sequencing. Prenatal Diagnosis. 38(1):10–19.

Ji, J., Leung, M. L., Baker, S., Deignan, J. L., Santani, A. 2021. Clinical Exome Reanalysis: Current Practice and Beyond. Molecular Diagnosis and Therapy. 25(5):529–536.

Grants:

Conflict of Interest: None declared.

EP02.022 Preimplantation genetic testing of m.8344A>G MERRF mitochondrial DNA mutation: challenge and success

Julie Steffann^1;2, Nadine Gigarel², Joana Bengoa ², Roxana Borghese², Anne Mayeur³, nelly achour³, Benoit Funalot², arnold munnich¹, Jean-Paul BONNEFONT¹, sophie monnot²

¹Imagine Institute, Genetics of mitochondrial disease, Paris, France; ²Necker-Enfants Malades Hospital, APHP, Paris, France; ³Antoine-Beclere Hospital, APHP, Clamart, France

Background/Objectives: Mitochondrial DNA (mtDNA) mutations cause a wide range of serious genetic diseases with high transmission risk, due to their maternal inheritance. One of the most frequent mtDNA mutation, the m.8344A>G of the MT-TK gene, is the cause of MERRF syndrome (Myoclonic Epilepsy with Ragged-Red Fibers). Two patients, carrier of the m.8344A>G mutation, have requested PGT in our center, but their high mutation levels in blood (75%) questioned their chance to get at least one « transferable » embryo.

Methods: Mutant loads were assessed by semi-quantitative fluorescent PCR followed by enzymatic digestion on 56 single-cells sampled from 30 embryos.

Results: Embryonic mutant loads were ranging from 10 to 97%, in favor of random segregation of mutant mtDNA molecules during oogenesis. Except in one embryo, the mutant loads were stable among the different blastomeres of day-3 embryos (±2%) supporting the feasibility of PGT by single-blastomere analysis. Furthermore, embryos with low (<30%; n = 3/30) or intermediate mutant loads (>30% et <70%; n = 7/30) were obtained. Uterine transfers of embryos were performed in 3 cycles, resulting in a single pregnancy and one healthy baby was born. The remaining 8 healthy embryos have been vitrified.

Conclusion: Our data demonstrate the clinical utility of the PGT procedure even for patients who carry high mtDNA mutation levels, and have important consequences in terms of genetic counseling.

References:

Grants:

Conflict of Interest: None declared.

EP03 Sensory Disorders (Eye, Ear, Pain)

EP03.003 Pathogenicity prediction for novel genetic variants related to Hearing Loss in a cohort of patients from Argentina

Paula Buonfiglio ¹, Carlos David Bruque², Vanesa Lotersztein³, Sebastián Menazzi⁴, Liliana Francipane⁴, Bibiana Paoli⁵, Ana Belén Elgoyhen^1;6, Viviana Dalamón¹

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, Santa Cruz, Argentina; ³Hospital Militar Central “Dr. Cosme Argerich”, Servicio de Genética, CABA, Argentina; ⁴Hospital de Clínicas “José de San Martín”, División Genética, CABA, Argentina; ⁵Hospital de Clínicas “José de San Martín”, Sector de Otorrinolaringología Infantil, CABA, Argentina; ⁶Facultad de Medicina, Universidad de Buenos Aires, Instituto de Farmacología, CABA, Argentina

Background/Objectives: Hearing loss (HL) is the most common disorder affecting 1:500 newborn children. Identification of causative mutations is demanding due to the large number (more than 100) genes involved. Whole-exome sequencing (WES) has become a cost-effective alternative approach for molecular diagnosis of HL. However, the follow-up of novel variants, in particular missense changes, which can lead to a spectrum of phenotypes and unequivocal genotype-to-phenotype correlations, is not always straightforward. In this study, we investigated the genetic cause of sensorineural hearing loss in patients with severe/profound deafness.

Methods: After the exclusion of frequent GJB2-GJB6 mutations by Sanger Sequencing, we performed WES in 32 unrelated Argentinean families. To predict the effect of some novel variants, protein modelling and protein stability analysis were employed.

Results: Mutations were detected in 16 known deafness genes in 20 patients: ACTG1, ADGRV1 (GPR98), CDH23, COL4A3, COL4A5, DFNA5 (GSDDE), EYA4, LARS2, LOXHD1, MITF, MYO6, MYO7A, TECTA, TMPRSS3, USH2A and WSF1. Notably, 11 variants affecting 9 different non-GJB2 genes resulted novel. Structural protein analysis in LARS2 and MYO6 proteins provided strong evidence on the impact of the mutations on protein function, as well as a novel high confident protein modelling to be used in future analyses.

Conclusion: These results highlight the value of whole exome sequencing to identify candidate variants, as well as bioinformatic strategies to infer their pathogenicity.

References:

Grants:

Conflict of Interest: None declared.

EP03.004 Clinical exome sequencing reveals pathogenic mutations in Bulgarian patients with inherited retinal degenerations

Kunka Kamenarova ¹, Kalina Mihova², Nevyana Veleva³, Elena Mermeklieva⁴, Bilyana Mihaylova⁵, Galina Dimitrova³, Alexander Oscar³, Ivaylo Tarnev⁶, Silvia Cherninkova⁶, Radka Kaneva²

¹Medical University - Sofia, Bulgaria, Molecular Medicine Center, Sofia, Bulgaria; ¹Medical University - Sofia, Bulgaria, Molecular Medicine Center, Sofia, Bulgaria; ³Medical University - Sofia, Bulgaria, Department of Ophthalmology, University Hospital Alexandrovska, Sofia, Bulgaria; ⁴Sofia University “St. Kliment Ohridski”, Sofia, Bulgaria, Department of Ophthalmology, University Hospital Lozenets, Sofia, Bulgaria; ⁵Vision Clinic, Sofia, Bulgaria; ⁶Medical University - Sofia, Bulgaria, Department of Neurology, University Hospital Alexandrovska, Sofia, Bulgaria

Background/Objectives: Inherited retinal degeneration (IRD) is a group of retinopathies with more than 300 genes associated and more than 20 different clinical phenotypes described (RetNet). However, genetic diagnosis remains unclear in ~1/3 of cases. Clinical and genetic heterogeneity makes difficult identification of disease-causing mutations. Therefore, molecular diagnosis is important for genetic counseling and treatment. The aim of the present study was to identify the genetic diagnosis in a group of Bulgarian patients affected by IRDs.

Methods: In a selected group of 80 patients diagnosed with different forms of IRDs, we performed targeted sequencing of clinical exome (including 4813 OMIM genes) on MiSeq platform of Illumina. A detailed analysis, followed by Sanger sequencing and segregation analysis, was used to identify pathogenic variants.

Results: Pathogenic variants in IRD-related genes were found in 72 out of 80 patients analyzed. Seventy nine mutations were found in 33 IRD genes, explaining 90% of the cases—65 known and 14 novel. In 8 patients (10%) genetic cause of the disease was not identified, possibly due to low-covered region (ORF15) of a major gene for X-linked retinopathy, RPGR, or a mutation in an undescribed gene yet. Predominant clinical phenotype in the studied patient group was macular degeneration with mutations in the common gene responsible for this disease, ABCA4.

Conclusion: Targeted sequencing of clinical exome allowed detection of disease-causing mutations in 92% of our cases, a percentage which exceeds previously reported diagnostic yield of 60–70% for IRDs.

References: RetNet (https://sph.uth.edu/retnet/).

Grants: KP-06-N33/12/18.12.2019, D01-285/17.12.2019, D01-395/18.12.2020, D01-302/17.12.2021.

Conflict of Interest: None declared.

EP03.005 Estrogen and androgen related genes in the corneal epithelium in Keratoconus

Katarzyna Jaskiewicz ¹, Magdalena Maleszka-Kurpiel^2;3, Malgorzata Rydzanicz⁴, Justyna Karolak⁵, Rafał Płoski⁴, Marzena Gajecka^1;5

¹Institute of Human Genetics, Polish Academy of Sciences, Poznan, Poland; ²Optegra Eye Health Care Clinic, Poznan, Poland; ³Poznan University of Medical Sciences, Chair of Ophthalmology and Optometry, Poznan, Poland; ⁴Medical University of Warsaw, Department of Medical Genetics, Poznan, Poland; ⁵Poznan University of Medical Sciences, Chair and Department of Genetics and Pharmaceutical Microbiology, Poznan, Poland

Background/Objectives: Keratoconus (KTCN) is the most common corneal ectasia, affecting 1:2000 individuals worldwide, characterized by progressive thinning of the cornea, leading to pathological cone formation(1). Hormones play a critical role in regulating cell survival, proliferation, and differentiation(2). Since sex-dependency and pregnancy-caused progression were observed in KTCN, we aimed to analyze the transcriptomic profile of sex hormones-related genes in the corneal epithelium (CE) of KTCN.

Methods: RNA samples were extracted from CE of 15 male KTCN patients undergoing cross-linking procedure and 3 male mild myopia patients undergoing refractive error correction (RNA/DNA/Protein PurificationPlus MicroKit, NorgenBiotek). The NGS libraries were prepared using TruSeq Stranded TotalRNA LibraryPrep Gold (Illumina) and sequenced on Novaseq6000 platform (100M reads/sample). RNA-seq data was analyzed implementing previously established pipelines.

Results: We haven’t revealed any difference in the expression of sex hormones receptors (ESR1, ESR2, AR) between KTCN and control samples. However, the expression of genes responsible for the transduction of signals to the nucleus (RAF1, GRB2, JAK1, CREB1), and genes related to sex hormones’ synthesis (HSD17B2) was changed. Moreover, in the pathway analysis we have noticed ‘Activated PKN1 stimulates transcription of AR regulated genes KLK2 and KLK3 pathway’ as one of the most overrepresented in the CE of KTCN.

Conclusion: As abnormalities in estrogen and androgen related genes have been identified, these features should be considered in CE transcriptomic profiling in KTCN.

References: 1.https://doi.org/10.1007/s00438-016-1283-z.

2. https://doi.org/10.1038/srep25534.

Grants: The National Science Centre grant no.2018/31/B/NZ5/03280. Co-financed by the European Social Fund, Operational Programme Knowledge Education Development, project ‘International scholarship exchange of doctoral students and academic staff’, No.POWR.03.03.03.00-00-00-PN13/18.

Conflict of Interest: None declared.

EP03.006 Molecular findings in a Romanian case series of retinal dystrophies

Andreea Tutulan-Cunita ¹, Anca Pavel¹, Simona Buliga², VASILICA PLAIASU³, Ina Ofelia Focsa^1;4, Danai Stambouli¹

¹Cytogenomic Medical Laboratory, Bucharest, Romania; ²West Eye Hospital, Bucharest, Romania; ³Alessandrescu Rusescu National Institute for Mother and Child Health, Bucharest, Romania; ⁴Carol Davila University of Medicine and Pharmacy, Medical Genetics, Bucharest, Romania

Background/Objectives: Retinal dystrophies (RDs) are a group of heterogenous disorders with complex inheritance and variable clinical pictures, that include nyctalopia, colour blindness, central or peripheral vision loss up to complete blindness. Significant progress has been made to determine the molecular substratum, linked to more than 300 genes.

Methods: Six patients with retinal dystrophies were referred to our laboratory for WES or panel sequencing for genes associated with RDs, between November 2019–July 2021. Ion GeneStudio S5 System (Thermo Fisher Scientific) and Ion Reporter platform were used.

Three patients were clinically diagnosed with retinitis pigmentosa, two with cone-rod dystrophy, and one with unspecific retinal anomaly and cataract.

Results: Two of the patients diagnosed with retinitis pigmentosa and one of the patients with cone-rod dystrophy carried pathogenic variants in ABCA4 gene. The third retinitis pigmentosa patient had a pathogenic missense in RHO gene. An indel in CDHR1 gene and a missense variant in RGR gene were found in the other patient with the cone-rod dystrophy. Only a pathogenic variant in CRYBB2 gene, causative for cataract, was found in the last patient.

Conclusion: Molecular diagnosis of RDs is essential in order to clarify the clinical presentation and evaluate the prognosis of age-related visual impairment, as well as the genetic of these highly heterogenous group of diseases. Appropriate counselling and where available, a gene-based therapy depending on the nature of the disease pathology will help the management of the symptoms.

References:

Grants:

Conflict of Interest: Andreea Tutulan-Cunita Cytogenomic Medical Laboratory, Anca Pavel Cytogenomic Medical Laboratory, Simona Buliga: None declared, VASILICA PLAIASU: None declared, Ina Ofelia Focsa Cytogenomic Medical Laoratory, Danai Stambouli Cytogenomic Medical Laboratory.

EP03.007 Is the decreased methylation of the PCDHA10 gene causative in high myopia in children?

Joanna Swierkowska ¹, Justyna Karolak^1;2, Sangeetha Vishweswaraiah³, Malgorzata Mrugacz⁴, Uppala Radhakrishna³, Marzena Gajecka^1;2

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

Background/Objectives: High myopia (HM), an eye disorder with a refractive error of −6.0 D or higher, has a complex etiology involving environmental, genetic, and likely epigenetic factors. Here, we report the study of differentially methylated protocadherin alpha (PCDHA) gene cluster in children with HM.

Methods: From the genome-wide methylation data of blood DNA of 18 Polish children with HM and 18 matched controls, we retrieved CG dinucleotides with lower methylation levels in HM cases. Selected sequence variants were Sanger sequenced in the studied children and members of a Polish family affected with HM.

Results: The PCDHA gene cluster, located in myopia locus MYP25 (5q31), includes the CG dinucleotide with the most decreased methylation level in HM cases versus controls. The CG site is located in transcription start site-1500 region of PCDHA10 and intronic regions of PCDHA 1–9. A previous GWAS indicated that SNV rs246073 in this cluster, was associated with refractive error in Europeans (n = 542,934, p value = 2.0 × 10⁻¹⁴). Also, an exonic nonsense variant rs200661444 (c.2017C>T, p.(Q673X)) in CpG island of PCDHA10, predicted as disease causing, was detected in our exome sequencing in the HM family, but did not completely segregate with HM. The SNV overlaps binding sites of AP-2alphaA, essential for the morphogenesis of lens vesicle and regulating the transcription of genes involved in the eye development.

Conclusion: Alterations in the methylation pattern of specific CG dinucleotides could be linked to early-onset HM and therefore be used to develop non-invasive biomarkers of HM in children and adolescents.

References: PMID:30858441.

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

Conflict of Interest: None declared.

EP03.008 TCF4 trinucleotide repeat expansion in the Finnish patients with Fuchs’ endothelial corneal dystrophy

Inka-Tuulevi Pettinen ^1;2;3, aino jaakkola⁴, Kari Krootila⁴, maria kaukonen^1;2;3, Hannes Lohi^1;2;3, Tero Kivela⁴, Joni Turunen^1;4

¹Folkhälsan Research Center, Helsinki, Finland; ²University of Helsinki, Department of Medical and Clinical Genetics, Helsinki, Finland; ³University of Helsinki, Department of Veterinary Biosciences, Helsinki, Finland; ⁴University of Helsinki and Helsinki University Hospital, Department of Ophthalmology, Helsinki, Finland

Background/Objectives: Fuchs endothelial corneal dystrophy (FECD) causes a progressive loss of corneal endothelial cells, resulting in structural changes and loss of corneal clarity. FECD is the leading cause of corneal transplantation in western countries. Multiple genetic risk factors contribute to the disease, but the best-known risk variant is an expansion of intronic CTG-repeat in the TCF4 gene. An expansion of more than 50 repeats causes a significant risk for the disease.

Methods: One hundred and ten Finnish patients with a clinical diagnosis of FECD were collected from Helsinki University Hospital. The patients with FECD were examined by slit-lamp, corneal tomography, and endothelial specular microscopy. Short tandem repeat analysis and triplet repeat primed PCR were used to assess the repeat number in TCF4. The CTG-genotype was also analyzed in a control cohort of 202 individuals from the Finnish Red Cross Biobank.

Results: A CTG-expansion of >50 was observed in 92 of the 110 patients with FECD in homo- or heterozygous state (84%, 95% CI 0.75–0.90). One of the 92 cases had the expansion present in both alleles. Furthermore, only five of the 202 control individuals had >50 CTG-repeats, each in a heterozygous state (0.03%, 95% CI 0.007–0.05).

Conclusion: These results indicate that the CTG-repeat in TCF4 is a notable risk factor for FECD also in the Finnish population with more than 80% of the patients carrying the expansion. Further analysis is performed to clarify the role of the expansion in the clinical and pathological features of these patients.

References:

Grants:

Conflict of Interest: None declared.

EP03.009 The first study of the RPE65 gene in the Russian Federation

Anna Stepanova ¹, Vitaly Kadyshev¹, Olga Shchagina¹, Alexander Polyakov¹

¹Research Centre for Medical Genetics, Moscow, Russian Federation

Background/Objectives: Retinitis pigmentosa (RP) is a group of heterogeneous genetic diseases with a prevalence of ~1:4000 worldwide. LUXTURNA® (voretigene neparvovec) is a gene therapy approved for the treatment of patients with autosomal recessive retinal dystrophy caused by biallelic pathogenic variants in the RPE65 gene. Therefore, an accurate molecular diagnosis is critical to the approval of targeted therapy.

RPE65 mutations are thought to be responsible for ~0.6–16% of all RP or Leber congenital amaurosis (LCA) cases in various populations. Studies have not yet been conducted to assess the frequency of RPE65-dependent forms of RP in Russia.

Methods: Our study included 189 unrelated patients with different forms of hereditary retinal degeneration. Patients’ DNA was analyzed by targeted next‐generation sequencing (NGS) using an «Ophthalmo» disease‐specific panel, which included 211 genes.

Results: In 11 patients, variants in the RPE65 gene were identified in the homozygous (4 patients) or compound heterozygous (7 patients) state. The variants were confirmed by Sanger sequencing for all patients. Segregation analysis was performed for 9 families, and the variants were proved to be biallelic. Thus, the proportion of RPE65-patients in the current study was 5.8%.

We have identified 12 different variants in the RPE65 gene, including 8 missenses (c.1024T>C(p.Tyr342His), c.1307G>A(p.Gly436Glu), c.746A>G(p.Tyr249Cys), c.1451G >T(p.Gly484Val), c.272G>A(p.Arg91Gln), c.1451G>A(p.Gly484Asp), c.1340T>C(p.Leu447Pro), c.982C>T(p.Leu328Phe), 2 nonsense (c.370C>T(p.Arg124*), c.304G>T(p.Glu102*)) and 2 splicing site mutations (c.1451-1G>A, c.11+5G>A). Two mutations were novel (c.1024T>C(p.Tyr342His), c.1340T>C(p.Leu447Pro)). The most common mutation c.370C>T(p.Arg124*) was found on 5 chromosomes (22.7%).

Conclusion: All patients with confirmed biallelic mutations in the RPE65 gene are eligible for targeted therapy.

References:

Grants:

Conflict of Interest: None declared.

EP03.010 Leber’s hereditary optic neuropathy: case report

Loubna SOUFIAN ^1;2, Aymane Bouzidi³, Meriem El Qabli¹, Patrizia Bonneau⁴, Vincent Procaccio⁴, Abdelhamid Barakat³, Guy Lenaers⁴, Nisrine Aboussair^1;2

¹CENTRE HOSPITALO-UNIVERSITAIRE MOHAMMED VI MARRAKECH, Genetics, Marrakech, Morocco; ²Faculty of Medicine and Pharmacy in Marrakech, Marrakech, Morocco; ³Institut Pasteur Du Maroc, Human Molecular Genetics Laboratory, Casablanca, Morocco; ⁴Angers University Hospital Center, MitoLab team, Institut MitoVasc, UMR CNRS 6015, INSERM U1083, Angers, France

Background/Objectives: Leber’s hereditary optic neuropathy (LHON) is an inherited mitochondrial disorder that is typically present in young males with sequential visual loss due to optic neuropathy, characterised by acute or subacute failure of central vision. LHON is maternally inherited, with variable penetrance, typically an autosomal recessive condition, but rare dominant cases have been reported.(1,2).

In 95% of cases, LHON is caused by one of three primary mutations of the mitochondrial DNA (mtDNA), m.11778G>A in the MT-ND4 gene, m.14484T>C in the MT-ND6 gene, or m.3460G>A in the MT-ND1 gene.(3).

Methods: In this study, we report a Moroccan family with multiple affected individuals, suffering from visual impairment since childhood.

Results: Among the tested members of the family, 6 individuals were found harbouring one of the primary mutations, m.11778G>A in the MT-ND4 gene, with a 99.2% heteroplasmy rate.

Conclusion: Leber’s hereditary optic neuropathy is one of the most common mitochondrial diseases, secondary to three major mutations in three mitochondrial genes. In our study, we found the most common mutation, confirming its global distribution in the population.

References:

1. Erickson RP. Leber’s optic atrophy, a possible example of maternal inheritance. Am J Hum Genet. 1972 May;24(3):348–9.

2. Sadun AA, Morgia CL, Carelli V. Leber’s Hereditary Optic Neuropathy. Curr Treat Options Neurol. 2011 Feb;13(1):109–17.

3. Chun BY, Rizzo JF. Dominant Optic Atrophy and Leber’s Hereditary Optic Neuropathy: Update on Clinical Features and Current Therapeutic Approaches. Seminars in Pediatric Neurology. 2017 May;24(2):129–34.

Grants:

Conflict of Interest: None declared.

EP03.012 Variant c.394C>T in FGF3 is a recurrent cause of profound congenital deafness in Slovak Roma

Maria Giertlova ¹, Martin Mistrík², Petra Drenčáková¹, Veronika Lopáčková², Renáta Zemjarová Mezenská³, Katarína Čokášová³, Michaela Urminská³, Zuzana Zboňáková³, Viktor Stránecký⁴, Lenka Nosková⁴

¹Unilabs Slovakia, Medical Genetics Outpatient Service, Kosice, Slovakia; ²Unilabs Slovakia, Medical Genetics Outpatient Service, Spišská Nová Ves, Slovakia; ³Unilabs Slovakia, Laboratory of Medical Genetics, Bratislava, Slovakia; ⁴First Medical Faculty of Charles University and the General University Hospital, Laboratory for the Study of Rare Diseases, Department of Pediatrics and Inherited Metabolic Disorders Department of Pediatrics and Inherited Metabolic Disorders, Prague, Czech Republic

Background/Objectives: Recessive mutations of fibroblast growth factor 3 (FGF3) cause rare type of congenital deafness, with labyrinthine aplasia, microtia, and microdontia (LAMM syndrome, OMIM # 610706). We present two cases of unrelated Slovak children of Roma ethnicity with congenital deafness with homozygous c.394C>T p.(Arg132Trp) variant in FGF3.

Methods: NGS sequencing was performed (Illumina, Nextera Flex Pre-enrichment True Sight One Expanded; NextSeq 550 Illumina) with targeted bioinformatic analysis of genes associated with hearing loss (bcl2fastq v2.20 and SEQNEXT v5.2.0), ref. GRCh37/hg19.

Results: Newborn hearing screening was positive in both patients (boys) with absent OAEs and with profound bilateral mixed hearing loss confirmed by further audiology testing. In patient N^o1 microtia was present bilaterally in contrast to patient N^o2 without external ear abnormality. MRI examination of middle and internal ear structures was not available. Targeted NGS sequencing confirmed homozygosity for c.394C>T p.(Arg132Trp) variant in FGF3 gene (NM_005247.2) dbSNP: rs372402801, with variant frequency in gnomAD 0,004 % (0 homozygotes), not reported in ClinVar nor in HGMD. The variant was classified as variant of unknown significance (PM2, PP2, PP3) according to ACMG criteria.

We evaluated the frequency heterozygotes for the given variant in the in-house database of 167 exomes of healthy controls of self-determined Roma persons from Slovakia and Czech republic unrelated to our patients. We found 7/167 heterozygotes with the resulting carrier frequency 4,19%.

Conclusion: Though generally very rare, LAMM syndrome might be frequent cause of profound mixed congenital deafness in Roma population in our region. Further studies are necessary to confirm founder effect.

References:

Grants:

Conflict of Interest: None declared.

EP03.013 Unexpected Array CGH result in a patient with leading symptom ocular coloboma

Kirsten Cremer¹, Hela Hundertmark¹, Nuria Ortega Ibanez¹, Elisabeth Mangold¹, Jelena Maric-Biresev¹, Hartmut Engels ¹, Jessica Trautmann¹

¹Institute of Human Genetics, University of Bonn, School of Medicine & University Hospital Bonn, Bonn, Germany

Background/Objectives: Ocular colobomata are caused by a failure to close of the embryonic fissure during development. Occurrence may be isolated, but they are often associated with other systemic anomalies. Although many pathogenic variants in different genes have been identified, a large proportion of isolated cases remains idiopathic. Here we report on a 31-year-old male with bilateral retinal, choroidal and iris colobomata, mild facial dysmorphism, premature graying of the hair, aplasia of the left 12th rib and jaw cysts.

Methods: We performed cytogenetic banding and microarray CGH analyses followed by qPCR.

Results: Cytogenetic banding analysis gave a normal male karyotype (46,XY) and no evidence of an isodicentric derivative chromosome 22 (Cat Eye syndrome). Microarray CGH and qPCR analyses in the parents identified a de novo heterozygous ~3.47 Mb deletion in 6p24.3-p24.1 encompassing 25 genes, i.a. TFAP2A. Pathogenic TFAP2A mutations cause the rare autosomal dominant Branchiooculofacial syndrome (BOFS, MIM # 113620). The clinical presentation is extremely variable and typically includes branchial skin defects, eye abnormalities (e.g. colobomata), nasolacrimal duct stenosis / atresia and facial dysmorphism. Other reported abnormalities include subcutaneous cysts, dental abnormalities, and premature hair graying. Intellect is usually normal.

Conclusion: More than 95% of pathogenic variants in patients with BOFS are SNVs or indels. Here we present a de novo 6p24.3-p24.1 deletion including TFAP2A as a rare cause of BOFS. Our case shows the value of chromosomal microarray diagnostics in patients without the “classical” indication neurodevelopmental disorder.

References:

Grants:

Conflict of Interest: None declared.

EP03.014 Detection of two novel splicing mutations associated to deafness by whole exome sequencing

Juan Manuel Ruiz Robles ^1;2;3, Nerea Gestoso-Uzal^1;2;3, Victoria A Reyes-Sánchez¹, Juan José Tellería^4;5, Fernando Benito-González⁶, Luis Antonio Corchete^2;3, Ana-Belén Herrero^1;2;3, Rogelio González-Sarmiento^1;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, Spain

Background/Objectives: Point mutations affecting splicing may result in the formation of aberrant transcripts of the gene. Recent research has underlined the abundance and importance of splicing mutations in the etiology of inherited diseases. The myosin gene family is associated with different diseases including deafness, although few splicing mutations in MYO genes have been described.

The aim of this study was to identify mutations associated with deafness by whole exome sequencing (WES).

Methods: Genomic DNA was extracted from peripheral blood leukocytes in a cohort of 12 patients. WES was performed to identify germinal mutations that could be responsible for the disorder.

Results: In one patient, we identified a mutation in heterozygosis in the MYO7A gene (c.4852G>A; p.Ala1618Thr, rs772542296), which affects both splicing and the protein sequence. This mutation has been classified as an uncertain significance variant in ClinVar, but it is considered likely pathogenic in Varsome. In other case, we found a splicing mutation in heterozygosis in MYO3A gene (c.2506-1G>A, rs201023600), reported as conflicting interpretations of pathogenicity in ClinVar but predicted as pathogenic in Varsome. MYO7A and MYO3A have been associated with nonsyndromic progressive deafness, which is compatible with both clinical histories. This is the first time that these mutations are reported as pathogenic and associated with deafness. Implications of other mutations found, mainly missense, is being investigated in the same cohort of patients.

Conclusion: WES is a useful approach to identify splicing mutations associated with deafness, as c.4852G>A; p.Ala1618Thr in MYO7A and c.2506-1G>A in MYO3A.

References:

Grants: This study was funded by FIS-FEDER: PI18/01476.

Conflict of Interest: None declared.

EP03.015 NTRK1-associated congenital insensitivity to pain with anhidrosis – two novel cases from Polish population

Łukasz Kępczyński ¹, Kinga Sałacińska¹, Katarzyna Połatyńska², Tatiana Chilarska¹, Tadeusz Kałużewski¹, Paweł Flont³, Agnieszka Gach¹

¹Polish Mother’s Memorial Hospital - Research Institute, Department of Genetics, Łódź, Poland; ²Polish Mother’s Memorial Hospital - Research Institute, Department of Developmental Neurology and Epileptogy, Łódź, Poland; ³Polish Mother’s Memorial Hospital - Research Institute, Department of Orthopedics and Traumatology for Children, Łódź, Poland

Background/Objectives: NTRK1-associated congenital insensitivity to pain with anhidrosis (NTRK1-CIPA, OMIM #256800) is severe neurological disorder of autosomal recessive inheritance, characterized by sensory and autonomic neuropathy leading to self-injuries and hyperthermia episodes, with high risk of neurodevelopmental dysfunctions including intellectual disability and epilepsy. It is extremely rarely reported outside the Japanese and Israeli Bedouin population.

Methods: Here we report two unrelated individuals of Polish origin, presenting features consistent with hereditary sensory and autonomic neuropathy diagnosis. Individual 1: 4 years of age female, insensitivity to pain, anhidrosis, hyperthermia episodes, drug-resistant epilepsy, tendency to self-injuries, dental self-extractions, neurodevelopmental delay. Individual 2: 12 years of age male, insensitivity to pain, anhidrosis, recurrent hyperthermia episodes, palmar and plantar hyperkeratosis, recurrent joints injuries and inflammation.

Results: Direct sequencing of NTRK1 coding region detected compound heterozygosity in both patients: individual 1: c.574+1G>A (maternal origin) and c.845delT (paternal origin), individual 2: c.574+1G>A (paternal origin) and c.850+6T>A (de novo).

Conclusion: c.850+6T>A is a novel variant detected in NTRK1 gene. Both individuals share common variant c.574+1G>A, described previously and associated with NTRK1-CIPA. Given the shortage of data regarding the disorder outside the populations with higher prevalence, clinical description and molecular findings described here expand the knowledge of disease variability.

References:

Grants:

Conflict of Interest: None declared.

EP03.016 Novel COL9A1 mutation causes autosomal recessive isolated high myopia

ofek freund ¹, ohad wormser¹, yonathan perez¹, libe gradstein², Ohad Shmuel Birk^2;3

¹Ben-Gurion University of the Negev, beer sheva, Israel; ²Ben-Gurion University of the Negev, Clalit Health Services, beer sheva, Israel; ³Soroka Medical Center, Genetics institute, beer sheva, Israel

Background/Objectives: Identifying the genetic cause of isolated high myopia affecting two siblings in consanguineous Bedouin family.

Methods: Affected individuals underwent thorough ophthalmologic examination, including fundus photography and optical coherence tomography (OCT). Whole-exome sequencing (WES) of the proband and homozygosity mapping of the studied kindred (750k SNP arrays) were done, assuming autosomal recessive heredity. WES variants which passed our filtering cascade were screened using our database of 500 ethnically-matched controls and validated using Sanger sequencing.

Results: The two siblings were affected since infancy, and recently, ages 18 and 21 years, their refractive errors ranged from -8 to -20 dioptres. One sibling had anisometropia (9 dioptres), amblyopia, and strabismus. Fundus and OCT findings were compatible with high myopia. They had no facial deformities, hearing loss or skeletal anomalies. Linkage analysis unravelled several homozygous loci shared only by two affected individuals. Using our variant analysis pipeline, all WES variants within these loci were ruled out except for one: COL9A1 (NM_001851.4): c.1550G>A, p.G517E, segregating in the kindred as expected for recessive heredity and not found in 500 ethnically-matched controls or public databases.

Conclusion: We report a novel COL9A1 mutation causing autosomal recessive high myopia without systemic abnormalities. COL9A1 takes part in assembly of type IX collagen molecules, and is expressed in various tissues including the eye. COL9A1 mutations were previously reported mainly in association with Stickler syndrome, hearing loss and multiple epiphyseal dysplasia. Our findings suggest that genetic evaluation of isolated severe myopia should include analysis for the presence of COL9A1 pathogenic variants.

References:

Grants: The Morris Kahn Family Foundation.

Conflict of Interest: None declared.

EP03.017 Spectrum of pathogenic variants in the rhodopsin gene in Slovenian patients with retinal dystrophies

Tjaša Krašovec ¹, Nina Kobal¹, Borut Peterlin², Marija Volk², Maja Šuštar¹, Marko Hawlina¹, Ana Fakin¹

¹Eye Hospital, University Medical Centre Ljubljana, Ljubljana, Slovenia; ²Clinical Institute of Medical Genetics, University Medical Centre Ljubljana, Ljubljana, Slovenia

Background/Objectives: Pathogenic variants in the rhodopsin gene (RHO) cause various forms of retinitis pigmentosa (RP), which is a progressive retinal degeneration, and less commonly congenital stationary blindness. The aim of our study is to review the spectrum of pathogenic variants in RHO in Slovenian patients with retinal dystrophies.

Methods: Study included 40 patients from 11 unrelated families harbouring pathogenic variants in RHO. Patients were identified from database of Slovenian patients with genetic eye diseases at the Eye Hospital, UMC Ljubljana. Phenotypes were classified into four categories according to fundus autofluorescence (FAF): night blindness without degeneration (normal FAF), classic RP (cRP) (hyperautofluorescent ring), sector RP (sRP) (peripheral hyperautofluorescent border extending ≤ 2 quadrants) and pericentral RP (pRP) (pericentral hypoautfluorescent area).

Results: RHO patients represented 11% (40/362) of Slovenian RP cohort (4,5%; 11/247 families) and 41% (40/97 patients) of dominant RP. Pathogenic variants included p.Gly90Asp (4 unrelated families), p.Val87Asp (4 unrelated families), p.Pro347Leu (2 unrelated families) and a novel variant p.Ser297Arg (1 unrelated family). The last two variants were associated only with cRP, whereas p.Val87Asp caused either cRP, sRP or pRP; and all four phenotypes were observed in the p.Gly90Asp cohort. Of the variants only p.Pro347Leu is frequent in other cohorts.

Conclusion: RHO variants represent a frequent cause of RP in Slovenia with specific spectrum of variants. The p.Gly90Asp and p.Val87Asp were the most frequent and displayed significant phenotypic diversity, not usually associated with RHO.

References: /.

Grants: /.

Conflict of Interest: None declared.

EP03.019 ACTG1: a spectrum ranging from non-syndromic hearing impairment to polymalfomative fetal presentations

Margaux Serey-Gaut ^1;2, Isabelle Rouillon³, Natalie Loundon³, Genevieve Lina Granade⁴, Souad Gherbi Halem², Anne-Marie Guerrot⁵, YLINE CAPRI⁶, Arnaud Molin⁷, Christine Poncet Wallet⁸, Ghizlène Lahlou⁹, Tania Attie-Bitach¹⁰, Luke Mansard¹¹, Anne-Françoise Roux¹¹, Abeke-Ralyath Balogoun^1;12, Laurence Jonard¹², Sandrine MARLIN^2;13

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; ³Hôpital Necker, AP-HP.CUP, Service d’ORL, Paris, France; ⁴Hospices Civils de Lyon, Service d’ORL, Lyon, France; ⁵CHU de Rouen, Service de génétique, Unité de génétique clinique, Rouen, France; ⁶Hôpital Robert Debré, AP-HP.NUP, Service de Génétique Clinique, Paris, France; ⁷Hôpital Clémenceau, Service de Génétique Clinique, Caen, France; ⁸Hôpital Rothschild, Centre de Réglage des Implants Cochléaires, Paris, France; ⁹Hôpital de la Pitié-Salpêtrière, AP-HP Sorbonne université, Service d’ORL et de chirurgie cervico-faciale, Paris, France; ¹⁰Hôpital Necker, AP-HP.CUP, Service d’Histologie - Embryologie - Cytogénétique, UF Médecine préimplantatoire, prénatale, périnatale, pathologie fœtale et placentaire, Fédération de Génétique et de médecine génomique, Paris, France; ¹¹CHU de Montpellier, Laboratoire de Génétique Moléculaire, LBMR Pathologies neurosensorielles rares, Montpellier, France; ¹²Hôpital Necker, AP-HP.CUP, Service Médecine Génomique des maladies rares, UF Développement et Morphogenèse, Fédération de Génétique et de médecine génomique, Paris, France; ¹³Institut Imagine, Unité INSERM UMR1163, Paris, France

Background/Objectives: Pathogenic variants of ACTG1 have been reported for 2 distinct phenotypes: Autosomal dominant isolated deafness DFNA20/26 and Baraitser-Winter syndrome 2, that associates intellectual deficiency, ocular malformations, dysmorphism, epilepsy and cerebral malformations. Surprisingly, hearing impairment is seldom associated to Baraitser-Winter syndrome 2. There is a high prevalence of DFNA20/26 patients identified through gene panel sequencing presenting with isolated sensorineural hearing impairment of dominant transmission. DFNA20/26 usually presents as non-syndromic, progressive, postlingual, hearing impairment with an onset between the first and third decade. The objective is to better characterize the phenotypes associated with ACTG1 variants.

Methods: This is a retrospective study on a French cohort of 35 patients and 2 fetuses.

Results: Most of the patients have a typical presentation of DFNA20/26. 3 patients present with developmental delay and a recognizable dysmorphism with flat face and arched eyebrows. 4 patients present with auditory neuropathy. In the 2 fetal cases we found corpus callosum and cerebral anomalies, associated to cardiac and skeletal malformations for 1 of them.

Conclusion: ACTG1-associated phenotype is broader than currently described. We have identified extra-auditory symptoms and a recognizable dysmorphism in a number of patients.

References:

Grants:

Conflict of Interest: None declared.

EP03.022 Mutational analysis and genotype-phenotype correlation of 22 families with GUCY2D variants

Cristina Rodilla ¹, Almudena Avila Fernández^1;2, Inmaculada Martin Merida^1;2, Rosa Riveiro Alvarez^1;2, María José Trujillo Tiebas^1;2, Berta Almoguera Castillo^1;2, Ionut Florin Iancu^1;2, Cristina Villaverde¹, Ascensión Giménez¹, Miguel Ángel López Martínez¹, Fiona Blanco-Kelly^1;2, Ester Carreño Salas^2;3, Belén Jiménez Rolando^2;3, Blanca García Sandoval^2;3, Pablo Mínguez^1;2;4, Marta Corton^1;2, Carmen Ayuso^1;2

¹Health Research Institute - Fundación Jiménez Díaz (IIS-FJD), Genetics Department, Madrid, Spain; ²Carlos III Health Institute (ISCIII), Center for Biomedical Network Research on Rare Diseases(CIBERER), Madrid, Spain; ³Health Research Institute - Fundación Jiménez Díaz (IIS-FJD), Ophtalmology, Madrid, Spain; ⁴Health Research Institute - Fundación Jiménez Díaz (IIS-FJD), Bioinformatics Department, Madrid, Spain

Background/Objectives: The GUCY2D gene, located in chromosome 17, encodes for the retinal-specific guanylate cyclase, which is expressed in photoreceptors. Pathogenic GUCY2D variants are associated with an ample range of inherited retinal dystrophies (IRD). The aim of this study is to describe the mutational and phenotypic landscape of patients with variants in this gene.

Methods: A total of 22 probands characterized with GUCY2D variants were selected from a cohort of more than 4500 families with IRD. Variants were identified by different NGS technologies and/or Sanger sequencing. Clinical data were obtained from questionaries and health records. A detailed ophthalmic evaluation was performed in 5 cases.

Results: Among the studied probands, 14 of them carry a dominant pathogenic variant that is associated with cone-rod dystrophy (CRD) and 8 have a recessive hereditary pattern, mostly associated with a phenotype of Leber congenital amaurosis (LCA). A total of 17 different variants were identified, including 2 novel variants. The variants showing a dominant pattern are exclusively missense, located in the exons 13-14 of GUCY2D. Meanwhile, the recessive-related variants are missense and truncating, distributed throughout the gene. LCA patients, especially those carrying biallelic truncating variants, show a significantly earlier age of onset and loss of visual acuity than CRD cases.

Conclusion: Our study supports the genotype-phenotype model established for GUCY2D variants, and broadens the knowledge of the gene’s mutational diversity.

References:

Grants: This work was supported by Spanish Health Institute Carlos III (PI19/0321), Conchita Rábago Foundation, Spanish National Organization of the Blind (ONCE), Community of Madrid (RAREGenomics, B2017/BMD-3721).

Conflict of Interest: None declared.

EP03.023 Genomic landscape of dominant retinitis pigmentosa in a cohort of 333 Spanish families

Lidia Fernández-Caballero ^1;2, Inmaculada Martin Merida^1;2, Fiona Blanco-Kelly^1;2, Irene Perea-Romero^1;2, Olga Zurita^1;2, Blanca García Sandoval³, Almudena Avila Fernández^1;2, Pablo Mínguez^1;2, Marta Corton^1;2, Carmen Ayuso^1;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; ³Fundación Jiménez Díaz University Hospital (FJD), Department of Ophthalmology, Madrid, Spain

Background/Objectives: Inherited Retinal Dystrophies (IRD) are a group of rare diseases whose prevalence is 1:3000−4000 people worldwide. Currently, the reported diagnostic rate of IRD is 50%−60%. The aim of this work is to update an overview of the molecular characterization rate and gene frequency of autosomal dominant retinitis pigmentosa (adRP) in Spain.

Methods: A cohort of 372 families with a suspected diagnosis of adRP was selected from our database, of which 39 families were excluded because they are deceased, related patients or not collaborative. So, 333 families were studied through classic genotyping methods or targeted next-generation sequencing (NGS). The last up-date of adRP data was reported on 2018 with 258 unrelated adRP families.

Results: Overall, 70% of our adRP cohort has been characterized with an increase of diagnosis rate of ~+10% from 2018. Furthermore, 7.5% of this cohort has been reclassified to other types of inheritance (autosomal recessive or X-linked). Additionally, we identified adRP-causing mutations in 4 not previously reported genes in this cohort (GUCA1A/RP1L1/RAX2/FZD4). Although gene frequency data have changed slightly, mutations in RHO/PRPF31/PRPH2/RP1 continue being the most frequent.

Conclusion: Implementation of NGS has increased the characterization rate. However, there are 30% unsolved cases, which have not been studied by NGS yet or could be caused by undiscovered variants/genes, or genomic rearrangements difficult to identify through this technology. Thus, it is essential to study new molecular mechanisms that explain this disease.

References: https://doi.org/10.1167/iovs.18-23854, https://doi.org/10.1186/gm155.

Grants: CIBERER, ISCIII (PI16/00425, PI19/00321), IIS-FJD_Biobank, CAM (S2017/BMD-3721), University Chair UAM-IIS-FJD Genomic Medicine.

Conflict of Interest: None declared.

EP03.024 Clinical and genetic study of Waardenburg syndrome type 1 in Tunisian patients

Sana Skouri^1;2, Ahlem Achour ^1;2, Malek Nouira², faouzi maazoul¹, Valérie Benoit³, Besbes Ghazi⁴, ridha m’rad^1;5, Mediha Trabelsi^1;2

¹Charles Nicolle Hospital, Department of Congenital and Hereditary Diseases, Tunis, Tunisia; ²University of Tunis El Manar, Faculty of Medicine of Tunis, Human Genetics Laboratory, Tunis, Tunisia; ³Institute of Pathology and Genetics, Department of Molecular and Cell Biology, Gosselies, Belgium; ⁴La Rabta Hospital, Department of OtoRhinoLaryngology, Tunis, Tunisia; ⁵University of Tunis El Manar, Faculty of Medicine of Tunis, Human Genetics Laboratory, Tunis, Tunisia

Background/Objectives: Waardenburg syndrome type 1 (WS1) is a rare autosomal dominant disorder associated with PAX3 gene mutations. It is clinically characterized by sensorineuronal hearing loss and pigmentary anomalies of eyes, skin and hair. Dysmorphic features involve dystopia canthorum, synophrys, and prominent nasal root.

This study aims to present the clinical and genetic characteristics of WS1 in Tunisian patients.

Methods: We included eleven patients with clinical diagnosis of WS1. The entire coding region of PAX3 has been sequenced in six individuals. Multiplex Ligation-dependent Probe Amplification (MLPA) was performed for two patients with normal PAX3 sequencing.

Results: We report eleven patients from eight unrelated families. An affected first-degree relative was identified in 3/8 families. Dysmorphic features involved dystopia canthrom (11/11), prominent nasal root (8/11), synophrys (4/11) and alae nasi hypoplasia (3/11). Clinical findings also included sensorineural deafness (8/11), hypochromic irises (7/11), hair hypopigmentation (9/11) and abnormal skin pigmentation (4/11). We noted an intra-familial phenotype heterogeneity in our patients.

PAX3 sequencing in six individuals revealed three novel pathogenic variants in four of them: c.942delC; c.933_936dupTTAC and c.164delTCCGCCACA. MLPA, performed for two patients with normal sequencing, showed a heterozygous deletion of exons 5 to 9 in one of them (1).

Conclusion: These results highlight the clinical variability associated with WS1. The incomplete penetrance of PAX3 variants, suggested by familial cases study, is an important parameter to consider for genetic counseling.

References: (1) Trabelsi M. et al 2017.

Grants:

Conflict of Interest: None declared.

EP03.025 Phenotype of patients harbouring p.Cys870Ter in USH2A exon 13 amenable for exon skipping therapy

Katja Čadonič ¹, Marko Hawlina¹, Ana Fakin¹

¹Eye Hospital, University Medical Centre Ljubljana, Ljubljana, Slovenia

Background/Objectives: The purpose of our study was to determine the long-term natural history of disease in patients carrying a c.2610C>A; p.(Cys870Ter) variant in USH2A exon 13, who would be amenable to exon skipping therapy.

Methods: Study included 8 patients (3 male) from the Slovenian USH2A cohort, harbouring p.(Cys870Ter) in USH2A (2 homozygous). Median age at first exam was 42 years (range, 25-56) and the median age at last exam was 53 years (range, 25-65); with a median follow up of 10 years (range, 0-23). Phenotypic analysis included age at onset, visual acuity (VA, decimal Snellen), Goldmann perimetry (target II/4), colour vision (Ishihara), fundus autofluorescence imaging (FAF) and optical coherence tomography (OCT). Right eye was taken for analysis.

Results: Median age at onset was median 20 years (range, 8-35 years). At the first and last exam, the median VA were 0,6 (0,16-1,0) and 0,15 (range, 0,005-1); the median number of recognized Ishihara plates were 9/15 and 0/15; and the median visual field diameters were 22° (5°-114°) and 18° (5°-22°), respectively. Hyperautofluorescent ring on FAF delineating preserved photoreceptors on OCT was present in 75% at the first and 63% at the last exam. Kaplan Maier analysis showed that 50 % patients reach legal blindness based on visual acuity on the better eye (VA ≤ 0,1) at the age of 57 (95%CI 52-63) years.

Conclusion: Long term follow-up data of patients with variants in exon 13 of USH2A will be useful in conducting clinical trials that aim to slow down disease progression.

References: None.

Grants: ARRS J3-1750.

Conflict of Interest: None declared.

EP03.026 Pathogenic variant frequencies in autosomal dominant non-syndromic hearing loss genes

Ivanna Fedorenko ^1;2;3, Martin Koenighofer⁴, Christian Schöfer², Thomas Parzefall⁴, Klemens Frei⁴, Trevor Lucas²

¹Medical University of Graz, Graz, Austria; ²Medical University of Vienna, Division of Cell and Developmental Biology, Vienna, Austria; ³University School of Clinical Genetics, Salzburger Landeskliniken and Paracelsus Medical University, Salzburg, Austria; ⁴Medical University of Vienna, Department of Otorhinolaryngology, Head and Neck Surgery, Vienna, Austria

Background/Objectives: Autosomal dominant non-syndromic hearing loss (ADNSHL) is an extremely heterogeneous sensorineural disorder, both genetically and clinically. However, the global prevalence of ADNSHL is yet unknown, as most pathogenic gene variants have a very rare allele frequency. To ascertain whether database searches could be utilized as predictors of pathogenicity, recently, nine genes (REST, COL11A1, PTPRQ, PDE1C, MYO3A, TRRAP, PLS1, SLC12A2 and LMX1A) associated with ADNSHL were identified and analysed in peer-reviewed publications by the developed algorithm.

Methods: Initially, the clinical significance of variants was analysed in public sources such as the Human Gene Mutation Database and ClinVar while predictions of pathogenicity were calculated in silico. Current global and population specific allele frequencies were estimated with high quality sequencing reads in the Genome Aggregation Database (gnomAD). Minor allele frequency of ≤0.00002 defined by PM2_Moderate criteria was estimated from American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) guideline scores adapted for ADNSHL interpretation.

Results: Only MYO3A, TRRAP and PDE1C gene variants were represented in gnomAD and met the PM2_Moderate criteria according to ACMG/AMP guidelines. The remaining variants were not found in any genetic variation database.

Conclusion: In this study, the algorithm may be applied to interpret the pathogenicity of variants and lead to a more precise estimation of global disease frequencies.

References: Oza, A.M., et al., Expert specification of the ACMG/AMP variant interpretation guidelines for genetic hearing loss. 2018. 39(11): p. 1593-1613.

Pandey, S., Advances in Genetic Diagnosis and Treatment of Hearing Loss - A Thirst for Revolution. 2015: IntechOpen.

Grants: Medical University of Graz.

Conflict of Interest: None declared.

EP03.028 Genetic background of adult Cochlear Implantat candidates in Czech Republic

Radka Kremlíková Pourová ¹, Pavel Votypka¹, Marcela Malíková¹, Michaela Zelinová¹, Zdenek Fík², Zdeněk Čada², Jan Bouček²

¹Charles University, 2nd Faculty of Medicine, Department of Biology and Medical Genetics, Prague, Czech Republic; ²University Hospital Motol, Department of Otorhinolaryngology Head and Neck Surgery of the 1st Faculty of Medicine, Prague, Czech Republic

Background/Objectives: The role of genetics in pediatric cochlear implantation (CI) has already been repeatedly investigated, but the same knowledge in adults is still rather vague. While congenital or early onset hearing impairment show mostly autosomal recessive (AR) trait (with GJB2 gene comprising up to 50%), postlingual demonstrate mostly autosomal dominant (AD) inheritance.

Our aim is to map genetic background of patients seeking cochlear implantation in adulthood.

Methods: We performed genetic testing in adult 210 CI users with age of onset from birth to 60 years, 55% having positive family history, mostly of an AD condition.

First we investigated all patients via Sanger sequencing and MLPA of GJB2 gene, second MLPA of STRC gene deletion and sequencing of mtDNA regions m.1237 - m.1892 and m.7001 – m.7982, followed by homemade NGS panel of 174 hearing loss related genes in negatives.

Results: Mutations in GJB2 gene were identified in 46 (22%), STRC deletion in 4 (2,4%) and mtDNA in 6 (3,6%).

From 154 NGS-tested patients 78 (50%) were negative, 52 (34%) VUS (ACMG class 3) and 24 (16%) ACMG class 4/5. Surprisingly only in one third (8 patients) AD diseases were identified. The most common findings were AR Usher syndrome (thrice USH2A, once USH1C) and Pendred syndrome (thrice SLC26A4), followed by AD Stickler syndrome (twice COL11A1, once COL2A1) and nonsyndromic DFNA20 (twice ACTG1 mutation).

Conclusion: We identified genetic background in 80 (38%) investigated subjects.

References:

Grants: NV19-06-00189, IP 9782, IP 6024, ZD-ZDOVA2-001.

Conflict of Interest: Radka Kremlíková Pourová Department of Biology and Medical Genetics, Czech Ministry of Health, Pavel Votypka: None declared, Marcela Malíková: None declared, Michaela Zelinová: None declared, Zdenek Fík: None declared, Zdeněk Čada: None declared, Jan Bouček: None declared.

EP03.029 Genomic study of non-syndromic hearing loss in unaffected individuals: frequency of pathogenic and likely pathogenic variants in a Brazilian cohort of 2097 genomes

Caio Robledo Quaio ^1;2, Antonio Victor Campos Coelho¹, Livia Maria Silva Moura^1;3, Rafael Lucas Muniz Guedes^1;3, Kelin Chen¹, Jose Ricardo Magliocco Ceroni¹, Renata Moldenhauer Minillo¹, Marcel Pinheiro Caraciolo^1;3, Rodrigo de Souza Reis^1;3, Murilo Cervato^1;3, Tatiana Ferreira de Almeida¹, Joao Bosco de Oliveira Filho¹

¹Laboratório Clínico, Hospital Israelita Albert Einstein, São Paulo, Brazil; ²Instituto da Criança (Children’s Hospital), Hospital das Clínicas HCFMUSP, Faculdade de Medicina da Universidade de São Paulo, São Paulo, Brazil; ³VarsOmics, Sociedade Beneficente Israelita Brasileira Albert Einstein, São Paulo, Brazil

Background/Objectives: Hearing loss is a common sensory deficit in humans and represents an important clinical and social burden. We assessed pathogenic and likely pathogenic variants associated with non-syndromic hearing loss (NSHL) in a cohort of unaffected Brazilian individuals referred to genome sequencing.

Methods: We used samples of 2097 patients from participating centers of the Brazilian Rare Genomes Project to sequence the whole genome and search for both sequence variants and copy-number variants in genes associated with NSHL.

Results: We identified 222 heterozygotes (10.59%) for relevant sequence variants, 54 heterozygotes (2.58%) for copy-number variants and four homozygotes (0.19%) for sequence variants. An important fraction of these individuals (n = 104) presented alterations associated with autosomal dominant forms of NSHL. Using data from the heterozygous individuals for recessive forms and the Hardy–Weinberg equation, we estimated the population frequency of affected individuals with autosomal recessive NSHL to be 1:2,222.

Conclusion: Genome sequencing identified relevant frequencies of molecular alterations associated with NSHL in a cohort of unaffected individuals and showed to be a valuable method to study the distinct molecular mechanisms underlying this heterogeneous group of conditions.

References:

Grants: This study was funded by PROADI-SUS, Brazil.

Conflict of Interest: Caio Robledo Quaio Hospital Israelita Albert Einstein, Antonio Victor Campos Coelho Hospital Israelita Albert Einstein, Livia Maria Silva Moura Hospital Israelita Albert Einstein, Rafael Lucas Muniz Guedes Hospital Israelita Albert Einstein, Kelin Chen Hospital Israelita Albert Einstein, Jose Ricardo Magliocco Ceroni Hospital Israelita Albert Einstein, Renata Moldenhauer Minillo Hospital Israelita Albert Einstein, Marcel Pinheiro Caraciolo Hospital Israelita Albert Einstein, Rodrigo de Souza Reis Hospital Israelita Albert Einstein, Murilo Cervato Hospital Israelita Albert Einstein, Tatiana Ferreira de Almeida Hospital Israelita Albert Einstein, Joao Bosco de Oliveira Filho Hospital Israelita Albert Einstein.

EP03.030 Unraveling the genetic basis of early-onset inherited retinal disease in a Saudi Arabian cohort reveals a novel RIMS2-related family

Basamat Almoallem ^1;2, Marta del Pozo-Valero², Laila Jeddawi³, Kristof Van Schil⁴, Toon Rosseel², Sarah De Jaegere², Bart Leroy^2;5;6, Khalid Emara³, Frauke Coppieters², Miriam Bauwens², Elfride De Baere²

¹Department of Ophthalmology, King Saud Medical City, College of Medicine, King Saud University, Riyadh, Saudi Arabia; ²Center for Medical Genetics, Ghent University and Ghent University Hospital, Ghent, Belgium; ³Pediatric Ophthalmology Division, Dhahran Eye Specialist Hospital, Dhahran, Saudi Arabia; ⁴Center for Medical Genetics Antwerp, Universiteit Antwerpen, Antwerpen, Belgium; ⁵Department of Ophthalmology, Ghent University Hospital, Ghent, Belgium; ⁶Division of Ophthalmology, The Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania, United States

Background/Objectives: In this study, we aimed to uncover the molecular causes of Leber Congenital Amaurosis (LCA) and early-onset retinal dystrophy (EORD) in 15 families of Saudi Arabian descent, using an integrated approach consisting of autozygosity mapping and targeted resequencing or whole exome sequencing (WES).

Methods: A total of 18 patients (7 females and 11 males ranging between 2-9 years old) from 15 Saudi consanguineous families underwent ophthalmological examinations to establish a clinical diagnosis. They underwent autozygosity mapping, targeted gene testing combined or not with whole exome sequencing (WES). Variants were validated, classified according to ACMG/ACGS guidelines, and subjected to segregation analysis if family members were available.

Results: Patients displayed decreased best corrected visual acuity, photophobia, amaurotic pupils, congenital nystagmus, and oculo-digital sign. Likely pathogenic variants were found in 13/15 studied families in genes previously implicated in LCA/EORD, including 6 novel variants and a putative founder RPGRIP1 variant (c.1107del;p.(Glu370Asnfs*5)) for the Saudi Arabia population. Interestingly, a novel homozygous RIMS2 splice variant c.1751+1G>T was identified in an EORD patient without any signs of systemic involvement or neurodevelopmental symptoms. All variants were found in runs of homozygosity (ROH), apart from two heterozygous GUCY2D variants.

Conclusion: Our approach uncovered 13 distinct likely pathogenic variants in 13/15 studied families (86%), demonstrating the power of autozygosity-guided WES in a genetically heterogeneous consanguineous cohort with LCA/EORD. Finally, we report a biallelic RIMS2 variant in a seemingly non-syndromic patient and corroborate the previous role of RIMS2 in EORD pathogenesis.

References:

Grants: FAME Postdoctoral Fellow, FWO 1802220N.

Conflict of Interest: None declared.

EP03.031 An update on genetic background of autosomal dominant hearing loss

Monika Ołdak ¹, Dominika Ozieblo¹, Marcin Leja¹, Anna Sarosiak¹, Henryk Skarzynski¹

¹Institute of Physiology and Pathology of Hearing, Warsaw, Poland

Background/Objectives: Autosomal dominant hearing loss (ADHL) is the second most common form of inherited HL. It affects mainly high frequencies and progresses over time. Autosomal‐dominant genes are responsible for about 20% of cases of hereditary non‐syndromic deafness, with 51 different genes identified to date.

Methods: In this study, 105 families with ADHL underwent targeted next-generation sequencing (NGS) using a cochlea-specific HL multi-gene panel (237 genes). Prior to NGS, environmental HL risk factors and DFNB1 locus (GJB2 and GJB6) related hearing impairment had been excluded in all probands. Presence of the selected probably pathogenic variants and their segregation with HL within the family were confirmed by Sanger sequencing.

Results: Genetic cause of ADHL was identified in 43,8% (46/105) of the examined families. Among the 46 identified HL variants only 26% (12/46) have been previously reported and the remaining 74% are novel (34/46). We identified missense variants (27/46; 58,7%), splice site variant (9/46; 19,5%), stop-gain variants (5/46; 10,9%) and frameshift variants (5/46; 10,9%). Among the most common causative genes were MYO6 (n = 8), TBC1D24 (n = 5), KCNQ4 (n = 4), GSDME (n = 4), POU4F3 (n = 4) and WFS1 (n = 4). Pathogenic variants causative of HL in the SLC44A4, NLRP3, LMX1A, FGFR3, CD164, GRHL2, TMC1, COCH, ATP2B2 and CEACAM16 genes were detected in single families.

Conclusion: Considering frequent identification of novel genetic variants it is necessary to perform thorough clinical examination and variant segregation analysis with ADHL in all available family members. In the largest families without genetic diagnosis of HL linkage analysis and whole genome sequencing will be performed.

References:

Grants: NCN grant 2016/22/E/NZ5/00470.

Conflict of Interest: None declared.

EP03.032 Evaluating a causal relationship between Complement Factor I protein level and advanced age-related macular degeneration using Mendelian Randomisation

Amy Jones ¹, Stuart MacGregor², Xikun Han³, James Francis¹, Claire Harris^1;4, David Kavanagh^4;5, Andrew Lotery^6;7, Nadia Waheed^1;8

¹Gyroscope Therapeutics Ltd, London, United Kingdom; ²QIMR Berghofer Medical Research Institute, Statistical Genetics, Brisbane, Australia; ³QIMR Berghofer Medical Research Institute, Statistical Genetics, Brisbane, Australia; ⁴Clinical & Translational Research Institute, Faculty of Medical Sciences, Newcastle University, Newcastle, United Kingdom; ⁵National Renal Complement Therapeutics Centre, Royal Victoria Infirmary, Newcastle upon Tyne, United Kingdom; ⁶University of Southampton, Clinical Neurosciences, Clinical and Experimental Sciences, Faculty of Medicine, Southampton, United Kingdom; ⁷University Hospital Southampton NHS Foundation Trust, Southampton Eye Unit, Southampton, United Kingdom; ⁸Tufts University School of Medicine, Department of Ophthalmology, Boston, United States

Background/Objectives: Advanced age-related macular degeneration (AAMD) risk correlates with rare Complement Factor I (CFI) genetic variants associated with low CFI protein levels(1), but the relationship is unclear.

Methods: Two-sample inverse variance weighted Mendelian Randomisation (MR) was used to evaluate evidence for relationship between CFI and AAMD risk(2), comparing genetically predicted CFI levels in AAMD and Geographic Atrophy (GA) patients and controls.

We derived genetic instruments for systemic CFI level in healthy INTERVAL study participants(3). To evaluate a genetic causal odds ratio (OR) for effect of CFI on AAMD risk, results were combined from an AAMD genome-wide association study(4) with CFI levels from SCOPE and SIGHT AAMD patients.

Results: Common CFI variant rs7439493 strongly associated with low CFI, explaining 4.8% phenotypic variance. Using rs7439493, MR estimates for AAMD odds increase per standard deviation (SD) CFI decrease were 1.47 (95% confidence interval (CI) 1.30–1.65, P = 2.1 × 10⁻¹⁰). MR using rare CFI variant p.Gly119Arg indicated a 1SD decrease in CFI led to increased AAMD odds of 1.79 (95% CI 1.46–2.19, P = 1.9 ×⁻⁸). P.Gly119Arg explained 1.7% phenotypic variance. For benchmarking, results from a CFI-specific immunoassay on 24 p.Gly119Arg positive GA patients indicated each 1 SD (3.5 µg/mL) reduction in CFI associated with OR 1.67 of AAMD (95% CI 1.40–2.00, P = 1.85 × 10⁻⁸).

Conclusion: Concordance in MR calculations provide genetic evidence for a potentially causal role of low CFI increasing AAMD risk.

References: 1. Kavanagh D, et al. Hum Mol Genet. 2015;24(13):3861-3870.

2. Lawlor DA, et al. Stat Med. 2008;15(27):1133-1163.

3. Sun BB, et al. Nature. 2018;558(7708):73-79.

4. Fritsche et al. Nat Genet. 2016;48(2):134-143.

Grants: Gyroscope Therapeutics.

Conflict of Interest: Amy Jones Gyroscope Therapeutics Ltd, Gyroscope Therapeutics stock options, Stuart MacGregor Australian National Health and Medical Research Council: Program, CRE, Fellowship, Gyroscope Therapeutics Ltd, Xikun Han: None declared, James Francis Gyroscope Therapeutics Ltd, Gyroscope Therapeutics stock options, Claire Harris Gyroscope Therapeutics Ltd, Ra Pharmaceuticals, Gyroscope Therapeutics stock options, Consultancy income from Q32 Bio Inc -Payment to Institution.

Consultancy income from Chinook Therapeutics -Payment to Institution.

Consultancy income from Biocryst Pharmaceuticals- Payment to Institution, Royalty income from commercialized factor I ELISA; Hycult Biotech, David Kavanagh Gyroscope Therapeutics Ltd funding source, Alexion pharmaceuticals-Personal.

Novartis-Personal, Gyroscope Therapeutics, Gyroscope Therapeutics-Personal.

Alexion pharmaceuticals- Personal & Institutional.

Novartis-Personal.

Apellis pharmaceuticals-Personal.

Sarepta Therapeutics- Personal, Andrew Lotery Gyroscope Therapeutics Ltd, Gyroscope Therapeutics Ltd, Nadia Waheed Gyroscope Therapeutics Ltd, Carl Zeiss Meditec.

Topcon.

Regeneron.

Heidelberg.

Nidek.

Optovue, Ocudyne.

Gyroscope Therapeutics stock options, Apellis.

Nidek.

Boehringer Ingelheim.

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

EP04.001 Mendelian randomization of eosinophils and other cell types in relation to lung function and disease

Anna Guyatt¹, Catherine John ¹, Alexander T Williams¹, Nick Shrine¹, Nicola F Reeve¹, Ian Sayers^2;3, Ian P. Hall^2;3, Louise Wain^1;4, Nuala A Sheehan¹, Frank Dudbridge¹, Martin D Tobin^1;4

¹University of Leicester, Department of Health Sciences, Leicester, United Kingdom; ²University of Nottingham, Division of Respiratory Medicine, Nottingham, United Kingdom; ³National Institute for Health Research, Nottingham Biomedical Research Centre, Nottingham, United Kingdom; ⁴National Institute for Health Research, Leicester Biomedical Research Centre, Leicester, United Kingdom

Background/Objectives: Eosinophils are involved in airway inflammation in respiratory disease. Interleukin-5 (IL5) partly controls eosinophil production and survival; and response to anti-IL5 agents in asthma is correlated with baseline eosinophil counts. However, it is not clearly understood whether eosinophil levels are causally related to COPD and other respiratory phenotypes. We investigated causality between eosinophils and: lung function, acute exacerbations of COPD (AECOPD), asthma-COPD overlap (ACO), moderate-to-severe asthma, and respiratory infections.

Methods: We performed Mendelian randomization using 151 variants from genome-wide association studies of blood eosinophils in UK Biobank/INTERVAL, and respiratory traits in UK Biobank/SpiroMeta, using methods relying on different assumptions for validity. We performed multivariable analyses using eight cell types for traits where there was possible evidence of causation by eosinophils.

Results: Causal estimates derived from individual variants were highly heterogeneous. This may arise from pleiotropy. The average effect of raising eosinophils was to increase risk of ACO (weighted median OR per SD eosinophils 1.44 [95%CI 1.19,1.74]), and moderate-severe asthma (weighted median OR 1.50 [95%CI 1.23,1.83]), and to reduce FEV₁/FVC and FEV₁ (weighted median estimator, SD FEV₁/FVC: −0.054 [95%CI −0.078,−0.029]. Effects on lung function were more prominent in individuals with asthma.

Conclusion: Broad consistency between methods suggests eosinophils have a causal effect, though of uncertain magnitude. However, given the heterogeneity in results from individual instrumental variables, it is possible that these variants impair respiratory health via pleiotropic mechanisms, rather than by raising eosinophils. Our results suggest anti-IL5 agents may be useful in management of other respiratory conditions, including people with both asthma and COPD.

References:

Grants:

Conflict of Interest: Anna Guyatt: None declared, Catherine John: None declared, Alexander T Williams: None declared, Nick Shrine: None declared, Nicola F Reeve: None declared, Ian Sayers: None declared, Ian P. Hall Funded research collaborations with GSK, Boehringer Ingelheim and Orion, Louise Wain Funding from GSK for collaborative research projects outside of the submitted work, Nuala A Sheehan: None declared, Frank Dudbridge: None declared, Martin D Tobin Funding from GSK for collaborative research projects outside the submitted work.

EP04.002 Relationship between ADAM19, FAM13A, IREB2 genes common variants and COPD susceptibility and severity

Merve Yumrukuz Senel¹, Serkan Kabacam², Pelin Simsek-Kiper², Gulen Eda Utine², Mehmet Alikaşifoğlu ¹

¹Hacettepe University Faculty of Medicine, Medical Genetics, Ankara, Turkey; ²Hacettepe University Faculty of Medicine, Pediatric Genetics, Ankara, Turkey

Background/Objectives: Chronic obstructive pulmonary disease (COPD) is a heterogenous disease characterized by persistent progressive airway limitation caused by both environmental factors and genetic predisposition. In this study, we aimed to investigate relationship between ADAM19, FAM13A, IREB2 genes previously detected common variants and COPD susceptibility and severity.

Methods: The clinical data of 110 patients having persistent airway limitation according to COPD definition of GOLD were collected. Patients were screened for ADAM19, FAM13A, IREB2 genes common variants using BigDye terminator on an ABI Prism 3500 genetic analyzer.

Results: There was a statistically significant relationship between IREB2 rs2568494 GA genotype and COPD disease. The number of patients with FAM13A rs2869967 TC genotype were lower compared to the patients without TC genotype besides; respiratory insufficiency risk was 3.758 fold increase, mMRC □ 2 risk was 2.359 fold increase. GOLD B+D rate was higher in the patients with FAM13A TC variant (74.4%) compared to the patients without FAM13A TC variant (55.2%). FEV1 measures were observed to be lower in the patients with ADAM19 rs1422795 AG genotype.

Conclusion: Our results suggest that IREB2 variants may be associated with COPD. In addition, FAM13A and ADAM19 variants were not related with the disease susceptibility but with the severity. ADAM19, FAM13A, IREB2 genes may be contributor of COPD pathophysiology. Furthermore, associations in different pathways investigated in our study are so important to identify new pathways reflecting COPD heterogeneity.

References: 1. GOLD. Global Strategy for the Diagnosis, Management and Prevention of COPD: 2020 Report. www.goldcopd.org.

Grants: This study was supported by Hacettepe University SRPC Unit (2019-17521).

Conflict of Interest: None declared.

EP04.003 Complex compound inheritance in a four-generation ACDMPV family

Esra Yildiz Bolukbasi ¹, Justyna Karolak², Tomasz Gambin³, Przemyslaw Szafranski¹, Admire Matsika⁴, Sam McManus⁵, Hamish S. Scott^6;7;8, Peer Arts⁶, Thuong Ha^6;9, Christopher P. Barnett^10;11, Jonathan Rodgers^12;13, Pawel Stankiewicz¹

¹Baylor College of Medicine, Molecular and Human Genetics, Houston, United States; ²Poznan University of Medical Sciences, Chair and Department of Genetics and Pharmaceutical Microbiology, Poznan, Poland; ³Warsaw University of Technology, Institute of Computer Science, Warsaw, Poland; ⁴Mater Health, Anatomical Pathology Department, South Brisbane, QLD, Australia; ⁵Mater Hospital Brisbane, Mater Pathology, South Brisbane, QLD, Australia; ⁶An alliance between SA Pathology and the University of South Australia, Department of Genetics and Molecular Pathology, Centre for Cancer Biology, Adelaide, Australia; ⁷University of South Australia, UniSA Clinical and Health Sciences, Adelaide, Australia; ⁸University of Adelaide, Adelaide Medical School, Adelaide, Australia; ⁹An alliance between SA Pathology and the University of South Australia, ACRF Genomics Facility, Centre for Cancer Biology, Adelaide, Australia; ¹⁰University of Adelaide, Adelaide Medical School, Adelaide, SA, Australia; ¹¹Women’s and Children’s Hospital, Paediatric and Reproductive Genetics Unit, South Australian Clinical Genetics Service, North Adelaide, Australia; ¹²Royal Brisbane and Women’s Hospital, Genetic Health Queensland, Brisbane, QLD, Australia; ¹³The University of Queensland, School of Medicine, Brisbane, QLD, Australia

Background/Objectives: Lethal lung developmental disorders (LLDDs) are a rarely diagnosed group of pediatric anomalies presenting with severe progressive respiratory failure and persistent pulmonary arterial hypertension (PAH). They have been histopathologically classified as Alveolar capillary dysplasia with misalignment of the pulmonary veins (ACDMPV), Acinar dysplasia (AcDys), Congenital alveolar dysplasia (CAD), and other unspecified primary pulmonary hypoplasias (PHs). LLDDs are caused by heterozygous alterations involving FOXF1 (ACDMPV), TBX4, FGF10, or homozygous variants involving FGFR2 (AcDys, CAD, and PHs). However, wide variable expressivity observed in carriers of FOXF1 (pLI 0.96), TBX4 (pLI 0.50), or FGF10 (pLI 0.94) heterozygous coding variants suggests that they alone can be insufficient to manifest clinically.

Methods: Using exome and genome sequencing, and functional reporter assay, we have studied a four-generation family with ACDMPV and PAH.

Results: We have identified a frameshift variant c.881_902dup (p.Gly302Profs*46) in FOXF1 in five individuals with ACDMPV (N = 1), PAH (N = 2), or unaffected (N = 2). Interestingly, in the proband’s mother and aunt with PAH, we have found a non-coding rs560517434-A variant within the critical interval of FOXF1 lung-specific enhancer in trans to the FOXF1 coding variant that increased its promoter activity in a reporter assay 10-fold.

Conclusion: Supporting the recent data on complex compound inheritance of LLDD, our results indicate the non-coding variant in trans to the heterozygous coding mutation might have acted as a hypermorph, rescuing the lethal phenotype in the proband’s mother and aunt.

References: PMID:31686214, 30639323, 35075769.

Grants: NIH-NHLBI R01HL137203.

Conflict of Interest: Esra Yildiz Bolukbasi: None declared, Justyna Karolak: None declared, Tomasz Gambin: None declared, Przemyslaw Szafranski: None declared, Admire Matsika: None declared, Sam McManus: None declared, Hamish S. Scott: None declared, Peer Arts: None declared, Thuong Ha: None declared, Christopher P. Barnett: None declared, Jonathan Rodgers: None declared, Pawel Stankiewicz NIH-NHLBI R01HL137203.

EP04.004 Autosomal recessive renal tubular dysgenesis – case report

Monika Pokorna ¹, Monika Koudová¹, Alice Baxova², Katerina Hirschfeldova²

¹Gennet, Prague, Czech Republic; ²Institute of Biology and Medical Genetics, Charles University in Prague, Prague, Czech Republic

Background/Objectives: Autosomal recessive renal tubular dysgenesis (ARRTD, ORPHA:97369) is a rare disease characterised by absent or poor development of proximal renal tubules due to biallelic pathogenic variants in genes encoding proteins of Renin-Angiotensin-Aldosteron system. We present case of anhydramnios in two consecutive pregnancies of unrelated partners. First pregnancy was terminated for early anhydramnios and Potter sequence. Proband was born from the second pregnancy. Prenatal ultrasound revealed anhydramnios. Postnatally, severe hypotension, anuria and abnormal ossification of membranous bones were present.

Methods: Molecular genetic testing was performed by trio exome sequencing of proband and parents (FocusedExome, Agilent). Confirmation of the variants was performed by Sanger sequencing. Cosegregation analysis for assessing germline variant pathogenicity was performed on the DNA isolated from cultivated amniocytes of the fetus from the first pregnancy.

Results: NGS analysis revealed proband´s compound heterozygous status: nonsense pathogenic variant c.1486C>T p.(Arg496Ter) in the ACE gene inherited from heterozygous father and two variants in the ACE gene: in-frame deletion c.41_67del p.(Leu14_Leu22del) and missense variant c.3490G>A p.(Gly1164Arg) inherited from heterozygous mother. Both maternal variants are classified as variants of unknown significance by ACMG. The same genotype was confirmed by cosegregation analysis in the fetus from the first pregnancy.

Conclusion: Based on the molecular properties of in-frame deletions of leucine residues in signal peptide, typical phenotype and results of cosegregation analysis we proposed this in-frame deletion to be likely pathogenic. Identification of disease-causing variants allow us to offer PGT-M or targeted prenatal diagnosis.

References:

Grants:

Conflict of Interest: None declared.

EP04.005 Sex-dependent influence of GSTM1 and GSTT1 polymorphism on asthma control in children observed at the age of 8 to 10 years

Mirela Mačkić-Đurović ¹, Amina Aščerić², Izeta Aganović-Mušinović¹, Selma Dizdar¹

¹Faculty of Medicine, University of Sarajevo, Sarajevo, Bosnia and Herzegovina; ²Faculty of Natural Sciences and Mathematics, Sarajevo, Bosnia and Herzegovina

Background/Objectives: The aim of this study was to examine the association between sex and glutathione S transferase gene polymorphisms in children with asthma, aged 8-10 years. Some studies demonstrate a close association between the and clinical asthma expression. GST gene has a number of polymorphisms, including GSTM1 and GSTT1, which are thought to have a direct impact on the pathophysiology of asthma.

Methods: Blood samples were taken from 50 kids (33 boys and 17 girls born in the term) who, due to respiratory difficulties. Multiplex PCR was used to determine the presence or absence of GSTT1 and GSTM1 genes in the presence of the control β-globin gene.

Results: GSTT1 was detected in 36% (18) of boys and 14% (7) of girls, while GSTM1 was observed in 4% (2) of boys and 2% (1) of girls. GSTT1 and GSTM1 were observed in 22% (11) of boys and 14% (7) of girls. A positive correlation between allergies and GSTT1 was found as strong in boys and medium in girls. GSTM1 and allergies were negatively correlated in males, while the same polymorphism was positively correlated with asthma in females.

Conclusion: GSTT1 polymorphism appears to play a role in asthma development in children, regardless of their sex. However, GSTM1 seems to have a protective effect against allergy development in males, while it potentially increases the risk of childhood asthma in females.

References: Su X, Ren Y, Li M, Kong L, Kang J. Association of glutathione S-transferase M1 and T1 genotypes with asthma: A meta-analysis. Medicine (Baltimore). 2020;99(34):e21732.

Grants: Non.

Conflict of Interest: Mirela Mačkić-Đurović full time, Amina Aščerić full time, Izeta Aganović-Mušinović full time, Selma Dizdar full time.

EP04.007 Genetic analyses in Polish patients with lethal lung developmental disorders

Katarzyna Bzdęga ¹, Anna Kutkowska-Kaźmierczak², Izabela Plaskota², Marta Smyk², Magdalena Niemiec², Artur Barczyk², Małgorzata Nowak-Plęs³, Witold Błaż⁴, Beata Łoniewska⁵, Andrzej Marszałek⁶, Gail Deutsch⁷, Tomasz Szczapa⁸, Pawel Stankiewicz⁹, Justyna Karolak¹

¹Chair and Department of Genetics and Pharmaceutical Microbiology, Poznan University of Medical Sciences, Poznan, Poland; ²Department of Medical Genetics, Institute of Mother and Child, Warsaw, Poland; ³Pro-Familia Hospital, Rzeszow, Poland; ⁴Department of Neonatology and Neonatal Intensive Care Unit, St Jadwiga Provincial Clinical Hospital No. 2, University of Rzeszow, Rzeszow, Poland; ⁵Department of Neonatal Diseases, Pomeranian Medical University in Szczecin, Szczecin, Poland; ⁶Department of Tumour Pathology and Prophylaxis, Poznan University of Medical Sciences and Pathology Department Greater Poland Cancer Centre, Poznan, Poland; ⁷Department of Laboratory Medicine and Pathology, University of Washington School of Medicine and Seattle Children’s Hospital, Seattle, United States; ⁸Department of Newborn’s Infectious Diseases, Neonatal Biophysical Monitoring and Cardiopulmonary Therapies Research Unit, Chair of Neonatology, Poznan University of Medical Sciences, Poznan, Poland; ⁹Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, United States

Background/Objectives: Lethal lung developmental disorders (LLDDs) are rare anomalies characterized by severe respiratory failure in neonates, with lack of treatment. LLDDs include alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV), acinar dysplasia (AcDys), congenital alveolar dysplasia (CAD), and other primary pulmonary hypoplasias (PHs). They are caused by heterozygous alterations involving FOXF1 (80-90% of ACDMPV) and TBX4 or FGF10 (65% of AcDys, CAD, and PHs). The aim of our study was to identify LLDD-causative variants in Polish patients. Recently, we provided evidence for a complex compound inheritance of LLDDs.

Methods: DNA samples from two deceased neonates with clinically and histopathologically diagnosed LLDDs and from one fetus with suspected hydronephrosis were analyzed using arrayCGH and/or Sanger sequencing of TBX4, FGF10, and FOXF1.

Results: Our analyses revealed a heterozygous variant c.16G>A (p.Gly6Ser) in TBX4 (MAF < 0.001) in one patient, inherited from her apparently healthy father; further studies, including in trans non-coding variants that may have influenced the disease phenotype are pending. Prenatal testing in the mother of the fetus with hydronephrosis revealed a de novo heterozygous CNV deletion on 16q24.1, involving FOXF1 and its upstream lung-specific enhancer (chr16:86,053,209-86,705,830; hg19), enabling ACDMPV diagnosis.

Conclusion: Complementary clinical, histopathological, and genetic studies are needed for efficient diagnosis of LLDDs and to further elucidate their reported complex compound inheritance.

References: PMIDs:19500772, 30639323, 31686214, 35075769.

Grants: National Science Centre in Poland 2019/35/D/NZ5/02896.

Conflict of Interest: None declared.

EP04.009 Diagnostic yield and recognized barriers of a pediatric endogenetics clinic

Amir Peleg ¹, Ilana koren¹

¹Carmel Medical Center, Haifa, Israel

Background/Objectives: Background: The advent of molecular genetic technologies paved a path for the diagnosis of many pediatrics endocrine disorders. Joint evaluation by endocrinologists and medical genetics specialists can potentially increase diagnostic effectiveness by ensuring the exclusion of non-genetic mimics, by rationally selecting appropriate genetic diagnostic tools, by shorten the workup process and guide therapy. There is paucity data regarding the potential Diagnostic benefit of an integrated endogenetics clinic.

Objective: Evaluation of the diagnostic’s yield of an integrated endogenetics clinic.

Methods: A retrospective review of medical records of all patients who attended the clinic between 2017 and 2021.

Results: 210 patients were evaluated. The diagnostic’s yield was 20% compared to less than 10% of non-multidisciplinary genetic clinic.

Conclusion: Given the frequent futile use of diagnostic modalities, referral of non-genetic mimics among endogenetic disorders and the complexity of clinical genomic data analysis, a multi-disciplinary endogenetics clinic seems justified. Further research is needed to assess endogenetics clinic effect on larger scale data.

References:

Grants:

Conflict of Interest: None declared.

EP04.010 Diagnostic utility of NGS panel testing including non-coding and mitochondrial DNA variants in patients with monogenic diabetes

Alicia Scocchia¹, Johanna Känsäkoski ², Kimberly Gall¹, Julie Hathaway¹, Archie Taylor¹, Johanna Huusko², Manuel Bernal², Pernilla von Nandelstadh², Johanna Tommiska², Inka Saarinen², Matias Rantanen², Jennifer Schleit¹, Massimiliano Gentile², Pertteli Salmenperä², Jussi Paananen², Samuel Myllykangas², Juha Koskenvuo²

¹Blueprint Genetics Inc, Seattle, United States; ²Blueprint Genetics, Espoo, Finland

Background/Objectives: Identifying the molecular etiology for diabetes can guide treatment, allow early screening and supportive therapy for associated features, and inform familial recurrence. Most panel testing historically performed has not included non-coding regions or mitochondrial genes. We retrospectively assessed the diagnostic utility of NGS panels containing both nuclear and mitochondrial genes, including selected non-coding variants, associated with monogenic diabetes.

Methods: Clinical reports of 507 patients with suspected monogenic diabetes who underwent panel testing at a CLIA laboratory were examined (MODY Panel, n = 387; Comprehensive Monogenic Diabetes Panel; n = 120). Testing included sequence and copy number variant (CNV) analyses of NGS data from a validated clinical exome assay, including established non-coding variants. Mitochondrial genome was included for 199 patients. Molecular diagnosis was defined as the identification of pathogenic or likely pathogenic variant(s) consistent with the patient’s phenotype and known associated disease inheritance.

Results: A molecular diagnosis was established in 24.9% (126/507) of patients in 11 genes. Most molecular diagnoses were identified in GCK (n = 78, 61.9%), HNF1A (n = 22, 17.5%), and HNF1B (n = 7, 5.6%). Diagnostic CNVs were reported in ten patients. A diagnostic non-coding variant in INS was identified in one patient. The mitochondrial MT-TL1 m.3243A>G variant was identified in six patients.

Conclusion: Nearly 25% of patients in this cohort received a molecular diagnosis, including a non-coding variant in one patient and mitochondrial variant in six patients, demonstrating the diagnostic utility of panel testing with concurrent analysis of both nuclear and mitochondrial genes including selected non-coding variants for individuals with suspected monogenic diabetes.

References:

Grants:

Conflict of Interest: Alicia Scocchia Significant; Blueprint Genetics Inc, Johanna Känsäkoski Significant; Blueprint Genetics, Kimberly Gall Significant; Blueprint Genetics Inc, Julie Hathaway Significant; Blueprint Genetics Inc, Archie Taylor Significant; Blueprint Genetics Inc, Johanna Huusko Significant; Blueprint Genetics, Manuel Bernal Significant; Blueprint Genetics, Pernilla von Nandelstadh Significant; Blueprint Genetics, Johanna Tommiska Significant; Blueprint Genetics, Inka Saarinen Significant; Blueprint Genetics, Matias Rantanen Significant; Blueprint Genetics, Jennifer Schleit Significant; Blueprint Genetics Inc, Massimiliano Gentile Significant; Blueprint Genetics, Pertteli Salmenperä Significant; Blueprint Genetics, Jussi Paananen Significant; Blueprint Genetics, Samuel Myllykangas Significant; Blueprint Genetics, Juha Koskenvuo Significant; Blueprint Genetics.

EP04.011 TTC12 loss-of-function mutations cause primary ciliary dyskinesia and unveil distinct dynein assembly mechanisms in motile cilia versus flagella

Lucie THOMAS ¹, Khaled Bouhouche², Marjorie Whitfield³, Guillaume Thouvenin⁴, Andre Coste⁵, Bruno Louis⁶, Claire Szymanski⁷, Emilie Bequignon⁵, Jean-François Papon⁸, Manon Castelli², Michel Lemullois², Xavier Dhalluin⁹, Valérie Drouin-Garraud¹⁰, Guy Montantin⁴, Sylvie Tissier⁴, Philippe Duquesnoy¹, Bruno Copin⁴, Florence Dastot-Le Moal⁴, Sandrine Couvet¹, Anne-Laure Barbotin¹¹, Catherine Faucon¹², Isabelle Honore¹³, Bernard Maitre¹⁴, Nicole Beydon⁴, Aline Tamalet⁴, Nathalie Rives¹⁵, France Koll², Estelle Escudier¹⁶, Anne-Marie Tassin², Aminata Touré³, Valérie Mitchell¹¹, Serge AMSELEM¹⁶, Marie Legendre¹⁶

¹Inserm U933, PARIS, France; ²Institute for Integrative Biology of the Cell, Gif‐sur‐Yvette, France; ³IAB - Institute for Advanced Biosciences. Inserm U1209, Grenoble, France; ⁴APHP - Hôpital Armand-Trousseau, PARIS, France; ⁵Assistance Publique-Hôpitaux de Paris (AP-HP), Hôpital Henri Mondor et Centre Hospitalier Intercommunal de Créteil, Creteil, France; ⁶INSERM U955, Creteil, France; ⁷CHU Lille, Service d’Oto-Rhino-Laryngologie et de Chirurgie cervico-faciale, Lille, France; ⁸AP-HP Service d’Oto-Rhino-Laryngologie et de Chirurgie cervico-faciale, Le Kremlin Bicetre, France; ⁹CHU Lille, Services de Pneumologie et Oncologie Thoracique et d’endoscopie respiratoire, Lille, France; ¹⁰11 CHU-Hôpitaux de Rouen, Service de Génétique Médicale, Rouen, France; ¹¹CHU Lille, Institut de Biologie de la Reproduction-Spermiologie-Cecos, Lille, France; ¹²Centre Hospitalier Intercommunal de Créteil, Laboratoire de microscopie électronique, Service d’Anatomopathologie, Creteil, France; ¹³AP-HP, Hôpital Cochin, Service de Pneumologie et Oncologie Thoracique, Paris, France; ¹⁴AP-HP, Hôpital Henri Mondor et Centre Hospitalier Intercommunal de Créteil, service de Pneumologie, Creteil, France; ¹⁵Rouen University Hospital, Department of Reproductive Biology, Rouen, France; ¹⁶Assistance Publique-Hôpitaux de Paris (AP-HP), Département de Génétique Médicale, PARIS, France

Background/Objectives: Cilia and flagella are evolutionarily conserved organelles whose motility relies on the outer and inner dynein arm complexes (ODAs/IDAs). Defects in ODAs/IDAs result in primary ciliary dyskinesia (PCD), a disease characterized by recurrent airway infections and male infertility. To date PCD mutations in assembly factors cause a combined ODA/IDA defect, affecting both cilia and flagella.

Methods: We identified four loss-of-function mutations in TTC12, which encodes a cytoplasmic protein, in four independent families in which affected individuals displayed a peculiar PCD phenotype characterized by the absence of ODAs and IDAs in sperm flagella, contrasting with the sole absence of IDAs in respiratory cilia.

We analysed both primary cells from individuals carrying TTC12 mutations and human differentiated airway cells invalidated for TTC12 by a CRISPR-Cas9 approach, as well as TTC12 depletion in the ciliated model, Paramecium tetraurelia.

Results: Our results revealed an IDA defect restricted to a subset of single-headed IDAs different in flagella and cilia, while TTC12 depletion in Paramecium tetraurelia recapitulated the sperm phenotype.

Conclusion: Overall, our study, which identifies TTC12 as a new gene involved in PCD, unveils distinct dynein assembly mechanisms in human motile cilia versus flagella.

References:

Grants: Legs Poix and RaDiCo.

Conflict of Interest: None declared.

EP04.012 Clonal culture of renal cells from urine as a model to study somatic mutagenesis in pre-cancerous kidneys

Gianfranco Distefano¹, Isaline Rowe², Alessandro Larcher², Umberto Capitanio², Andrea Salonia², Alessandra Boletta¹, Irene Franco ¹

¹San Raffaele Hospital, Division of Genetics and Cell Biology, Milan, Italy; ²San Raffaele Hospital, Urological Research Institute, Milan, Italy

Background/Objectives: By performing WGS of single somatic genomes from the kidney of 6 healthy donors, we have identified a specific kidney cell subset that is prone to somatic mutagenesis during aging (Franco et al. 2019). This subset expresses markers of kidney tubule damage-repair, such as KIM1 and VCAM1. Here, we aim to better elucidate the mechanisms underlying the excess of somatic mutations in these cells.

Methods: To overcome the limitations connected with low availability of somatic genomes from normal kidneys, we developed a protocol for in vitro expansion of kidney epithelial cell clones, obtained from the urine, a non-invasive, easy-to-collect biopsy. Concomitant whole genome- and transcriptional marker-analyses of clonal populations allow the study of mutation landscapes in specific kidney cell types.

Results: An average of 2 clones per urine sample (range 0-5) expanded enough for WGS and transcriptional marker-analysis (n = 28 urine samples from 12 donors, aged 24–63 years). Both KIM1+VCAM1+ and KIM1-VCAM1- kidney tubule epithelial cells could be cultured. Collected DNA was of suitable quality for WGS. Furthermore, 1 in 7 clones expanded sufficiently for functional experiments aimed to dissect the molecular mechanisms leading to enhanced mutagenesis in the KIM1+VCAM1+ subset.

Conclusion: Kidney cell culture from the urine is a suitable method to profile somatic mutations in single pre-cancer genomes from human kidneys and dissect molecular mechanisms of mutagenesis.

References: Franco, I. et al., Genome Biol, 2019, 20(1): 285.

Grants: MSCA individual fellowship SoMuKT-896832.

Conflict of Interest: None declared.

EP04.013 Molecular diagnosis of cystic kidney disease with NGS panels covering difficult-to-sequence regions

Kimberly Gall¹, Manuel Bernal ², Julie Hathaway¹, Alicia Scocchia¹, Johanna Huusko², Inka Saarinen², Matias Rantanen², Jennifer Schleit¹, Tiia Kangas-Kontio², Tuuli Pietilä², Pertteli Salmenperä², Massimiliano Gentile², Jussi Paananen², Samuel Myllykangas², Juha Koskenvuo²

¹Blueprint Genetics Inc, Seattle, United States; ²Blueprint Genetics, Espoo, Finland

Background/Objectives: Inherited cystic kidney disease (CKD) is a heterogeneous group of conditions commonly leading to end stage kidney disease. Identifying the molecular etiology of CKD can have implications for clinical management. However, genetic testing using NGS is complicated by segmental duplication in several genes. A strategy that addresses these challenging regions is needed to maximize diagnostic potential. Here, we assessed the diagnostic yield of NGS panels in a cohort of CKD patients.

Methods: We examined test results from 1007 patients tested for CKD at Blueprint Genetics. Testing was done with the Polycystic Kidney Disease Panel (up to 12 genes) or the Cystic Kidney Disease Panel (up to 41 genes). The panels included 4 genes challenged by regions of segmental duplication. Copy number analysis was done bioinformatically using two different pipelines, including a proprietary pipeline for the detection of small CNVs.

Results: A genetic diagnosis was established in 557 patients (55%). Diagnostic variants were identified in 17 genes. The most frequently implicated genes were PKD1 (364 patients, 65%), PKD2 (75 patients, 13%), PKHD1 (40 patients, 7%) and HNF1B (37 patients, 7%). Of patients with a diagnostic PKD1 variant, 77% (280/364) were located within the pseudogene regions. Diagnostic CNVs were reported in 9% (49/557) of all diagnosed patients.

Conclusion: These results demonstrate the clinical utility of genetic testing for CKD using NGS panels. Most diagnoses are due to variants in PKD1 emphasizing the importance of including tailored approaches for challenging genomic regions. Given the frequency of CNVs, including high-resolution CNV detection significantly improves the diagnostic potential.

References:

Grants:

Conflict of Interest: None declared.

EP04.014 Fatal case of congenital nephrotic syndrome in a newborn with two bialeal mutations in the NPHS1 gene

Vita Antsupova ¹, Iryna Lastivka², Ludmila Khlunovska², Anastasiya Babintseva², Larysa Sheiko³, ljudmila Brisevac³, Iana Ushko¹, Iryna Malieieva¹, Oleksiy Godovanets², Olha Polodienko⁴, Volodymyr Davidiuk⁵, Olexiy Shapovalov⁴

¹Bohomolets National Medical University, Kyiv, Ukraine; ²Bukovinian State Medical University, Chernivtsi, Ukraine; ³Shupyk National Healthcare University of Ukraine, Kyiv, Ukraine; ⁴Children’s Consultative and Diagnostic Center named after academician Reznik, Odessa, Ukraine; ⁵Pirogov National Memorial Medical University, Vinnytsya, Ukraine

Background/Objectives: Congenital nephrotic syndrome (СNS) is an autosomal recessive kidney disease characterized by massive proteinuria, edema, and hypoproteinemia. Most cases of СNS are caused by mutations in the NPHS1 gene that encodes nephrine. Physical examination, family history and laboratory tests allow to suspect the disease.

Methods: clinical-genealogical, molecular-genetic, paraclinical, instrumental.

Results: A 28-day-old boy showed an increase in the size of the abdomen, a decrease in appetite and a decrease in diuresis. The child was born prematurely, with low body weight, from the first pregnancy against the background of chronic pyelonephritis, the risk of miscarriage and polyhydramnios. Prenatal ultrasound screening revealed an increase in the right kidney of the fetus, signs of intrauterine infection. Objectively on examination: the child’s condition is severe due to edema, hypovolemia, decreased diuresis. Symptom complex of nephrotic syndrome: proteinuria, hypoproteinemia, arterial hypotension. Ultrasound examination of the kidneys revealed diffuse changes in the parenchyma. The child is suspected of congenital nephrotic syndrome of the Finnish type, a molecular genetic study was recommended. The boy was found to be homozygous for two pathological alleles of the NPHS1 gene (c.2053G>T (p.Gly685Cys та с.2746G>A(p.Ala916Thr). Symptomatic treatment of the child was ineffective, the child died. The father and mother are heterozygous carriers of both mutations in the NPHS1 gene.

Conclusion: Taking into account the genotypes of the parents, as well as the fact that recessive mutations in NPHS1 are associated with a severe course of the disease, recommendations are given for planning subsequent pregnancies using reproductive technologies and prenatal diagnosis.

References:

Grants:

Conflict of Interest: None declared.

EP04.015 Characterization of molecular diagnostic findings in an unselected cohort with suspected congenital hypothyroidism or resistance to thyroid hormone

Christele du Souich ¹, Alicia Scocchia², Kimberly Gall², Julie Hathaway², Archie Taylor², Johanna Huusko¹, Manuel Bernal¹, Inka Saarinen¹, Jennifer Schleit², Jussi Paananen¹, Samuel Myllykangas¹, Juha Koskenvuo¹

¹Blueprint Genetics, Espoo, Finland; ²Blueprint Genetics, Seattle, United States

Background/Objectives: Congenital hypothyroidism (CH) and resistance to thyroid hormone syndrome (RTH) are conditions impacting production/use of thyroid hormones. The etiology of monogenic CH is diverse and identifying a precise diagnosis and beginning early treatment can prevent significant neurologic and motor damage. We assess the diagnostic utility of panel tests for individuals with suspected CH or RTH and provide an overview of the yet uncharacterized molecular diagnostic findings identified in this population.

Methods: A retrospective review of 117 individuals who underwent panel testing for CH or RTH at Blueprint Genetics was performed. Testing included both sequencing (NGS) and copy number variant (CNV). The target region included the coding exons of up to 22 genes and up to 16 non-coding variants in these genes.

Results: A molecular diagnosis was established in 19.7% of patients. Molecular diagnoses were identified in seven genes associated with thyroid dysgenesis, dyshormonogenesis, or RTH: THRB, TG, NKX2-1, DUOX2, PAX8, TSHB, and TSHR. Diagnostic variants were not identified in the SLC5A5 or TPO genes. No P/LP CNVs were found. Diagnostic yield for CH was significantly higher among individuals tested in their first 2 years where most P/LP variants associated were presumed truncating variants.

Conclusion: We describe the molecular findings in an unselected cohort undergoing genetic testing for suspected CH or RTH. A molecular diagnosis was found in 19.7% of individuals with a higher diagnostic yield in individuals tested at <2 years. This study reinforces the importance of genetic testing in this patient population.

References:

Grants:

Conflict of Interest: Christele du Souich Full time employee of BlueprintGenetics, Alicia Scocchia Salaried employee of Blueprint Genetics, Kimberly Gall Salaried employee of Blueprint Genetics, Julie Hathaway Salaried employee of Blueprint Genetics, Archie Taylor Salaried employee of Blueprint Genetics, Johanna Huusko Salaried employee of Blueprint Genetics, Manuel Bernal Salaried employee of Blueprint Genetics, Inka Saarinen Salaried employee of Blueprint Genetics, Jennifer Schleit Salaried employee of Blueprint Genetics, Jussi Paananen Salaried employee of Blueprint Genetics, Samuel Myllykangas Salaried employee of Blueprint Genetics, Juha Koskenvuo Salaried employee of Blueprint Genetics.

EP04.016 Molecular diagnosis of classic form of 21-hydroxylase deficiency in a Romanian pediatric group

Sorina Schipor ¹, Andrei Muresan¹, Ioana Nedelcu¹, Camelia Procopiuc¹, ELENA EMANUELA BRAHA¹, Aura Madalina Boboc¹, Andreea Brehar¹, Oana-Monica Popa¹, Susana Vladoiu¹, Dana Manda¹, Andra Caragheorgheopol¹, Iuliana Gherlan^1;2

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

Background/Objectives: 21-Hydroxylase deficiency (21-OHD) is an autosomal recessive disorder caused by CYP21A2 gene mutations and is the most common form of congenital adrenal hyperplasia. Here we present the results of genetic analysis of CYP21A2 gene in a Romanian selected group of paediatric patients with clinical signs of classic 21-OHD.

Methods: All patients were recruited in a tertiary endocrinology centre. Based on biochemical and clinical data we identified 10 patients with classic 21-OHD. The age at diagnosis varied between 5 days and 3 years. CYP21A2 gene molecular analysis was done according to EMQN guidelines¹ including TaqI PCR-RFLP, Sanger sequencing and multiplex ligation-dependent probe amplification (MLPA) to identify gene mutations and large fragment deletions/conversions. Then, the genotypes were analysed in correlation to patients’ clinical presentation (genotype-phenotype correlations).

Results: Genetic analysis confirmed classic form of 21-OHD in all 10 patients. Large deletions/conversions were detected in majority of alleles (30.77 %), followed by IVS2-13A/C>G microconversion (15.38%) and I172N (15.38%), R357W (7.69%), Q318X (3.85%) and P31L (3.85%). We also identified a plethora of polymorphisms, synonymous modifications or missense mutations with predicted benign consequences. Genotypes of all 10 patients were consistent with the suggested clinical phenotype (7 salt-wasting and 3 simple-virilising forms).

Conclusion: The molecular analysis strategy combining MLPA, PCR-RFLP and Sanger sequencing is able to identify the complex mutation spectrum of CYP21A2 gene. In our study large deletions/conversions were dominant in classic 21-OHD patients, and the genotype-phenotype correlation is high.

References: 1.Baumgartner-Parzer S, et al. Eur J Hum Genet. 2020 Oct;28(10):1341-1367.

Grants: None.

Conflict of Interest: None declared.

EP04.018 Next generation sequencing (NGS) for the identification of mutations in genes, associated with various degrees of chronic kidney disease and hyperuricemia

Radosveta Bozhilova ¹, Olga Beltcheva¹, Daniela Pencheva¹, Kunka Kamenarova¹, Galia Zlatanova-Rashkova², Stephka Tsocheva², Ventzislav Shurliev³, Boryana Deliyska³, Maria Gaydarova², Radka Kaneva¹

¹Medical University - Sofia, Molecular Medicine Center, Department of Medical Chemistry and Biochemistr, Sofia, Bulgaria; ²SBAL Pediatric Diseases, Nephrology and Hemodialysis Clinic, Medical University -Sofia, Department of Pediatrics, Sofia, Bulgaria; ³UMHAT “Tsarica Yoanna-ISUL”, Department of Nephrology, Sofia, Bulgaria

Background/Objectives: The aim of this study was to apply next generation sequencing (NGS) for genetic testing of hyperuricemia/gout patients with chronic kidney disease.

Methods: After obtaining informed consent, we collected samples from 5 patients. We used TSO gene panel and MiSeq platform (Illumina) for targeted sequencing of 4 813 genes. For variant confirmation and segregation analysis we applied Sanger sequencing. The pathogenicity of each nucleotide change was evaluated based on the ACMG criteria.

Results: Pathogenic or potentially pathogenic missense variants were found in several genes involved in maintaining normal kidney structure and function - COL4A3, COL4A4, UMOD, GATM and SLC14A2, coding for the α3 and α4 chains of type 4 collagen, uromodulin, the mitochondrial L-arginine: glycine amidinotransferase and UT2 transporter, respectively.

Two of the substitutions, in COL4A3 and UMOD, were novel with unknown effect. The UMOD variant, p.Arg222Cys (rs1313544461), was identified in an individual with prominent family history - multiple affected members in three generations. The variant segregated with the disease, is absent in GnomAD and was judged pathogenic by multiple prediction tools. Based on the ACMG criteria p.Arg222Cys was assumed the disease-causing mutation in this family.

Conclusion: The present study is a first attempt to identify the genetic cause of hyperuricemia associated with impaired kidney function in Bulgaria. Identifying the molecular basis of the disease will guide the choice of therapy in each case and will contribute to our understanding of the biological processes governing the normal renal function.

References: None.

Grants: D-116/24.05.2020, D01-285/17.12.2019, D01-395/18.12.2020, D01-302/17.12.2021.

Conflict of Interest: None declared.

EP04.020 A unique case of severe liver disease associated with the recurrent c.187C>T, p.(Arg63Trp) variant in HNF4A

Catarina Macedo ¹, André Travessa¹, Patricia Costa Reis², Ana Fernandes³, João Campagnolo⁴, Mafalda Bourbon⁵, Gisela Gaspar⁵, Margarida Vaz⁵, Ana Isabel Lopes³, Ana Berta Sousa¹

¹Hospital Santa Maria, Serviço de Genética Médica, Departamento de Pediatria, Lisboa, Portugal; ²Hospital Santa Maria, Unidade de Nefrologia e Transplantação Renal Pediátrica, Departamento de Pediatria, Lisboa, Portugal; ³Hospital Santa Maria, Unidade de Gastrenterologia Pediátrica, Departamento de Pediatria, Lisboa, Portugal; ⁴Centro Hospitalar Universitário de Lisboa Central - Hospital de São José, Serviço de Ortopedia Infantil, Hospital de Dona Estefânia, Lisboa, Portugal; ⁵National Health Institute Dr. Ricardo Jorge, Unidade I&D, Grupo de Investigação Cardiovascular, Departamento de Promoção da Saúde e Doenças Não Transmissíveis, Lisboa, Portugal

Background/Objectives: The recurrent c.187C>T, p.(Arg63Trp) variant in HFN4A is associated with a multisystem metabolic disease characterized by congenital hyperinsulinism, diabetes, and renal Fanconi syndrome. Of the 15 cases reported in the literature, only 6 presented with liver involvement, which was mild and transient.

Methods: We report a 16-year-old boy born to nonconsanguineous parents and presenting with lower limb deformities and short stature secondary to rickets, Fanconi syndrome, chronic liver disease with portal hypertension, esophageal varices requiring endoscopic band ligation, and early-onset diabetes. His mother had a similar phenotype, except for liver disease.

He was born preterm and hypotonia, thrombocytopenia, and cholestatic jaundice with abundant cytoplasmic glycogen on liver biopsy warranted extensive metabolic workup, but no cause was identified.

The clinical picture suggested the diagnosis of Fanconi renotubular syndrome 4 (FRS4) with maturity-onset diabetes of the young (MODY) (MIM#616026).

Results: Sanger sequencing of MODY genes identified the heterozygous variant c.187C>T, p.(Arg63Trp) in HNF4A, confirming the diagnosis. The variant was shown to be maternally inherited.

Conclusion: This FRS4 with MODY patient has the most severe form of liver disease reported so far, showing that, contrary to previous belief, FRS4 with MODY can be associated with liver disease that is not mild or transient. Thus, we suggest that liver function should be monitored in these patients.

The patient’s mother has normal liver tests, emphasizing the variable expressivity of this disorder.

References:

Grants:

Conflict of Interest: Catarina Macedo Medical Genetics Resident, André Travessa Medical Doctor, Patricia Costa Reis Medical Doctor, Ana Fernandes Medical Doctor, João Campagnolo Medical Doctor, Mafalda Bourbon PhD.

PhD, Investigadora Auxiliar/Coordenadora da Unidade de Investigação e Desenvolvimento, Gisela Gaspar INSA Lisboa, Margarida Vaz INSA Lisboa, Ana Isabel Lopes Medical doctor, Ana Berta Sousa Medical Doctor.

EP04.021 Report of 4 patients with pulmonary hypertension and c.622G>T variant in NFU1 gene: is there a founder effect?

Amaia Lasa-Aranzasti ¹, Irene Valenzuela¹, Paula Fernández-Álvarez¹, Anna Maria Cueto Gonzalez¹, Laura Carmen Trujillano Lidon¹, Elena Garcia Arumi¹, Eduardo Tizzano¹

¹Vall d’Hebron University Hospital, Department of Clinical and Molecular Genetics, Barcelona, Spain

Background/Objectives: Multiple mitochondrial dysfunctions syndrome 1 is A recessive disorder due to pathogenic variants in NFU1 gene. Failure to thrive, pulmonary hypertensión and neurological regression are the most common features of this disease.

Methods: Here we report four patients from three unrelated families with homozygous variants in NFU1 gene. In patients 1 and 2 the diagnosis was uncovered by whole-exome sequencing, and in patients 2 and 3 by direct Sanger sequencing because of clinical suspicion.

Results: The main clinical characteristics of our patients include failure to thrive (4/4), pulmonary hypertension (4/4), neurological regression (4/4) and glycine levels (3/3). All patients died in the first 6 months of life. None of the families had a history of consanguinity, but all had a known Basque Country background. Whole exome sequencing in patients 1 and 2 identified a homozygous c.622G>T(p.G208C) variant. Sanger sequencing of c.622G>T variant confirmed the diagnosis of suspect in patient 3 (sibling) and 4.

Conclusion: In infants with pulmonary hypertension, failure to thrive, neurological regression and increased glycine from the Basque country, Multiple mitochondrial dysfunctions syndrome 1 due to NFU1 c.622G>T variant should be considered. The Basque origin of all the patients with a common haplotype suggests a founder effect in this population. Screening in Basque Country population could be performd to estimate an overall carrier rate of the mutation.

References: Navarro-Sastre, Aleix et al. “A fatal mitochondrial disease is associated with defective NFU1 function in the maturation of a subset of mitochondrial Fe-S proteins.” American journal of human genetics vol. 89,5 (2011): 656-67.

Grants:

Conflict of Interest: None declared.

EP04.023 Prediction of intrahepatic cholestasis of pregnancy using a combination of clinical features and plasma cell-free RNA

Jinghua Sun ^1;2, Songchang Chen^3;4, Xuanyou Zhou⁴, Tingyu Yang^1;2, Weihui Shi⁴, Zhongzhen Liu², Lanlan Zhang⁴, Qing Zhou², Zunmin Wan^1;2, Lin Wang², Yiyao Chen⁴, Fang Chen², Hefeng Huang^3;4, Wen-Jing Wang², Chenming Xu^3;4

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

Background/Objectives: Intrahepatic cholestasis of pregnancy (ICP) is a pregnancy-specific liver disease, characterized by skin pruritus and elevated total bile acids (TBA). It can induce preterm birth, fetal distress, and stillbirth. Typically, ICP was diagnosed in the third trimester of pregnancy. This study aimed at constructing a predictive model utilizing clinical features and plasma cell-free RNA (cfRNA) to predict ICP in early pregnancy.

Methods: Detailed clinical information and plasma cfRNA were collected from 149 pregnant women (ICP, N = 43, control, N = 106) between 13 and 25 gestational weeks with a bile acid measurement <10 µmol/L. A predictive model was constructed and validated in an independent cohort of 54 pregnant women (ICP, N = 11, control, N = 43).

Results: The liver function indicators, including TBA (P < 0.001), alanine aminotransferase (P < 0.001), and γ-glutamyl transpeptidase (P < 0.001) were elevated in women subsequently diagnosed as ICP. Consistently, liver-specific signatures in plasma cfRNA were significantly higher in pregnancies with ICP, and gene set enrichment analysis found that pathways related to bile acid transport and liver function were enriched in up-regulated genes. The integrated predictive model was more accurate, with an area under the receiver operating characteristic curve (AUROC) of 0.87 compared with the models derived for clinical features (AUROC, 0.75) and plasma cfRNA (AUROC, 0.80).

Conclusion: Altogether, the combination of clinical information and plasma cfRNA can identify liver-specific signatures in early pregnancy and effectively predict subsequent ICP. These will be benefit in ICP detection and prevention.

References: none.

Grants: none.

Conflict of Interest: Jinghua Sun: None declared, Songchang Chen National Natural Science Foundation of China (No.81901495) the research grant from the National Key R&D Program of China (2018YFC1004900) the Shanghai “Rising Stars of Medical Talent” Youth Development Program Clinical Laboratory Practitioners Program (201972).

Shanghai Municipal Commission of Health and family planning (202140110), Xuanyou Zhou: None declared, Tingyu Yang: None declared, Weihui Shi: None declared, Zhongzhen Liu: None declared, Lanlan Zhang: None declared, Qing Zhou: None declared, Zunmin Wan: None declared, Lin Wang: None declared, Yiyao Chen: None declared, Fang Chen the research grant from the National Key R&D Program of China (2018YFC1004900), Hefeng Huang Shanghai Municipal Health Commission (GW-10.1-XK07), Wen-Jing Wang the research grant from the National Key R&D Program of China (2018YFC1004900) the Science, Technology and Innovation Commission of Shenzhen Municipality under grant (No.JCYJ20180703093402288), Chenming Xu National Natural Science Foundation of China (Nos.81771638 and 81971344) the Shanghai Municipal Commission of Science and Technology Program (No.21Y21901002).

EP05 Skeletal, Connective Tissue, Ectodermal and Skin Disorders

EP05.001 Pathognomonic oral findings in three rare syndromes caused by KCNK4, MIA, and GZF1 gene mutations

Nehal Hassib ¹, Inas Sayed^1;2, rasha elhossini³, mennat mehrez¹, ahmad abdelazeem¹, usama hilal¹, mohamed abdelhameed⁴

¹national research centre, orodental genetics, cairo, Egypt; ²national research centre, cairo, Egypt; ³national research centre, clinical genetics, cairo, Egypt; ⁴national research centre, medical molecular genetics, cairo, Egypt

Background/Objectives: Orodental findings as amelogenesis imperfecta (AI), dentinogenesis imperfecta (DI) and gingival fibromatosis (GF) could be characteristic pathognomonic and diagnostic features in rare syndromes.

Methods: Three patients were recruited from the Centre of Excellence clinics, National Research Centre, Cairo, Egypt. The first patient was 7 years old male with negative parental consanguinity. The clinical examination revealed hypertrichosis and gingival fibromatosis. The second patient was 6 years old male with positive parental consanguinity. Clinically, the patient had an extreme short stature and opalescent teeth. The third patient was 13 years old female with positive parental consanguinity. The patient complained of short stature and amlogenesis imperfecta. Preliminary diagnosis went to Hypertrichosis/Gingival fibromatosis (OMIM# 135400) and Goldblatt syndrome (OMIM# 184260) for patient 1 and patient 2, respectively. The diagnosis remained unknown for patient 3.

Results: Targeted sequence for TRIP11 gene in Patient 2 was performed and was negative. Blood samples were sent for WES that revealed KCNK4, MIA, and GZF1 mutations, respectively. The diagnosis of patient 1 was Facial dysmorphism, hypertrichosis, epilepsy, intellectual/developmental delay, and gingival overgrowth syndrome (FHEIG) (OMIM# 618381), patient 2 was odontochdrondysplasia type 2 without hearing loss or diabetes (OMIM# 619269), patient 3 was joint laxity, short stature and myopia (OMIM# 617662).

Conclusion: Orodental geneticists play an important role in cooperation with the multidisciplinary team to reach the accurate diagnosis and the proper genetic counseling. Orodental findings as tooth structure defects could be pathognomonic to some rare syndromes. Moreover, a dental management plan was designated according to the confirmed diagnosis.

References:

Grants: STDF project 33458 fund.

Conflict of Interest: Nehal Hassib STDF project 33458, Inas Sayed: None declared, rasha elhossini STDF project 33458 fund, mennat mehrez: None declared, ahmad abdelazeem: None declared, usama hilal: None declared, mohamed abdelhameed STDF project 33458 fund.

EP05.002 First Interim Analysis of the International X-Linked Hypophosphataemia (XLH) Registry: Family history and genetic findings

Outi Mäkitie¹, Jonathan Liu², Angela Williams², Sue Wood²

¹Helsinki University Hospital, Children’s Hospital, Helsinki, Finland; ²Kyowa Kirin International Marlow Office, Marlow, Buckinghamshire, United Kingdom

Background/Objectives: X-Linked Hypophosphataemia (XLH) is a rare, progressive, hereditary phosphate-wasting disorder characterised by excessive fibroblast growth factor 23 activity, caused by changes in the PHEX gene. The International XLH Registry is an ongoing 10-year data collection programme. Here, we describe baseline family history and genetic data from the first interim analysis.

Methods: The International XLH Registry (NCT03193476), initiated August 2017, aims to recruit 1,200 children and adults with XLH. This analysis describes results of patients eligible as of 29/03/2021. Data are presented on XLH family history and genetic findings.

Results: Overall, 579 subjects (64.6% female) were included in this analysis. Data on XLH family history were collected for 466 (80.5%) subjects; 220 (47.2%) recorded their biological mother affected with XLH, 71 (15.2%) recorded their biological father affected. The 311 subjects reporting siblings had a total of 309 affected siblings (mean = 0.7; SD = 0.78). Information on genetic evaluation was available for 495 subjects; of them 309 (62.4%) had undergone genetic testing. Two subjects’ ages were not reported; the proportion of subjects with recorded genetic testing results was higher in children (239/330, 72.4%) vs adults (68/163, 41.7%). Of these subjects, the majority had a confirmed PHEX variant (88.7% children; 91.2% adults). Non-PHEX variants were reported in the paediatric population only: FGF23 variants in 3 children, SLC4A3 in 1 child, and “other” in 7.

Conclusion: The International XLH Registry provides real-world, genetic findings and family history in people affected by XLH.

The authors acknowledge the contribution of all members of the XLH Registry Steering Committee.

References:

Grants: N/A

Conflict of Interest: Outi Mäkitie Kyowa Kirin, Alexion, Merck, Kyowa Kirin, BridgeBio, Jonathan Liu Full time employee of Kyowa Kirin International., Angela Williams Full time employee of Kyowa Kirin International., Sue Wood Full time employee of Kyowa Kirin Internationa.

EP05.003 A family with Ehlers-Danlos syndrome and novel mutation in COL5A1 gene

Maria Sredkova-Ruskova ¹, Tsvetina Veleva¹, Trayan Delchev¹, Daniela Avdjieva-Tzavella¹

¹University Pediatrics Hospital, Medical University, Department of Clinical Genetics, Sofia, Bulgaria

Background/Objectives: Classic Ehlers-Danlos syndrome (cEDS) is a connective tissue disorder, mainly caused by heterozygous COL5A1 or COL5A2 variants encoding type V collagen. The diagnosis is established with the minimal clinical diagnostic criteria and molecular testing. Herein, we report a family with four affected persons in three generations with identified variant of uncertain significance in COL5A1 gene.

Case report: The index patient, a female at age of 12y, presented with mitral valve prolapse, photophoby, chronic fatigue, chronic pain in knees and ankles, epicanthus, mild scoliosis, arachnodactyly, skin hyperextensibility and joint hypermobility. Family history revealed affected father, brother and grandfather. Father at age of 47y presented with progressive and painful contractual arachnodactyly with Heberden’s nodes, foot pain, easy fatigue, shortness of breath, hypertrophic obstructive cardiomyopathy and discal hernias. Brother at age of 24y presented with contractual arachnodactyly and pectus excavatum. Grandfather was expired and history of contractual arachnodactyly was given.

Methods: As the index patient fulfilled the criteria for cEDS with skin hyperextensibility and joint hypermobility and minor criteria of epicanthus, complications of joint hypermobility and family history we performed next generation sequencing (NGS) connective tissue disorders panel.

Results: A novel variant of uncertain significance, c.260_262del (p.Thr87del), was identified in COL5A1 gene in the index patient. Family testing of the father and the sibling was performed and the same variant was identified in both family members.

Conclusion: The clinical manifestations range in severity, and families with mild to severe expression have been described. Genotype-phenotype correlations remain to be determined.

References:

Grants:

Conflict of Interest: None declared.

EP05.006 Bleeding assessment in 195 patients with osteogenesis imperfecta

Koert Gooijer ¹, Gabriëla Heidsieck¹, Arjan Harsevoort¹, Daniëlle Bout¹, Guus Janus^1;2, Anton Franken^1;3

¹Isala, Expert Center for adults with Osteogenesis Imperfecta, Zwolle, Netherlands; ²Isala Zwolle, department of orthopedic surgery, Zwolle, Netherlands; ³Isala, Department of internal medicine, endocrinology, Zwolle, Netherlands

Background/Objectives: Osteogenesis Imperfecta (OI) is commonly defined as “brittle bones” disease, but there are also more characteristics like blue sclerae, hearing loss, dental problems, ligamentous laxity and a short stature. Easy bruising is also a very common feature and there are multiple case reports on haemorrhagic events in OI. Large population studies on bleeding tendency in OI are very sparse, while other connective tissue disorders with easy bruising have much more relevant research. This paper reviews the clinical aspects of bleeding and bruising in OI based on the self-bleeding assessment tool (BAT) questionnaire among a large cohort of OI patients. The aim of this study is to make a first translation to clinical consequences of bleeding due to surgery, tooth extraction, menstrual and obstetrical bleeding and to present therapeutic considerations relevant to bleeding in OI.

Methods: This explorative study was conducted at the national expert center for adults with OI in the Netherlands. The self-BAT was digitally distributed among 354 adults with different clinically confirmed types of OI.

Results: 195/354 patients with OI types 1,3 and 4 were included. Self-BAT scores were increased in 37-44%.

Conclusion: Bleeding tendency seems to be a relevant feature in OI patients. This study should be a wakeup call for all clinicians who treat OI patients to consider assessment of bleeding tendency and take the right interventions to reduce haemorrhagic symptoms and improve quality of life.

References: n/a.

Grants: None.

Conflict of Interest: None declared.

EP05.008 Likely pathogenic and known variants in EDA, EDAR and NECTIN4 in Egyptian families with different forms of Ectodermal dysplasia

Inas Sayed ¹, Ghada El-Kamah², Hoda Radwan³, Eman Rabie^3;4, Suher Zada⁴, Mostafa Mostafa¹, Nehal Hassib¹, mennat mehrez¹, Khalda Amr³

¹Human Genetics and Genome Research Institute, National Research Centre, Oro-dental Genetics, Cairo, Egypt; ²Human Genetics and Genome Research Institute, National Research Centre, Clinical Genetics Department, Cairo, Egypt; ³Human Genetics and Genome Research Institute, National Research Centre, Medical Molecular Genetics, Cairo, Egypt; ⁴School of Sciences and Engineering, The American University in Cairo (AUC), Biology, Cairo, Egypt

Background/Objectives: Ectodermal dysplasia (ED) is a heterogenous nosologic group of disorders characterized by primary defect in at least two of the ectodermal derived tissues, namely hair, nails, teeth and sweat glands. The most common features are teeth agenesis, absent or reduced sweating (anhydrosis or hypohidrosis), hypotrichosis and dysplastic nails. The oral and dental findings include tooth agenesis, microdontia, abnormal shaped teeth and decreased salivary flow. More than 163 ED phenotypes have been reported with different patterns of inheritance, however the X-linked hypohidrotic ED (OMIM#305100) caused by EDA gene represents the most frequent type. Only 75 ED phenotypes were linked to 77 genes which makes molecular diagnosis challenging and drives the search for potential disease-causing variants.

Methods: Fifteen ED Patients from 12 Egyptian families were subjected to oro-dental and general clinical examination focusing on the skin, and other ectodermal elements. Whole exome sequencing (WES) was performed for the probands. Familial segregation was confirmed using Sanger sequencing.

Results: We identified eight pathogenic and likely pathogenic variants in three genes (EDA, EDAR and NECTIN4) including five novel mutations. We report five variants of unknown significance (VUS) in four other genes (NFKB2, RSPO4, BTD and KRT14).

Conclusion: WES provides a valuable tool in identifying potentially disease-causing variants in a phenotypically diverse group of ED. The detection of four VUS requires further functional studies for evaluation. Recognizing the genetic causes of the condition help in genetic counselling and in possible finding of new treatments.

References:

Grants: Science and Technology Development Fund (STDF) grant no: 33494.

Conflict of Interest: Inas Sayed STDF 33494, Ghada El-Kamah: None declared, Hoda Radwan: None declared, Eman Rabie: None declared, Suher Zada: None declared, Mostafa Mostafa: None declared, Nehal Hassib: None declared, mennat mehrez: None declared, Khalda Amr: None declared.

EP05.009 A novel homozygous mutation of TCIRG1 gene in a case of infantile malignant osteopetrosis

Tsvetina Veleva ¹, Daniela Avdjieva-Tzavella¹, Maria Sredkova-Ruskova¹, Trayan Delchev¹

¹University pediatric hospital, Clinical genetics, Sofia, Bulgaria

Background/Objectives: Osteopetrosis is a genetically heterogeneous group of skeletal disorders, which is result of differentiation or functional defects of osteoclasts. That leads to bone thickening, abnormal bone marrow cavity formation and impaired bone remodeling. The most severe form of osteopetrosis is infantile malignant osteopetrosis. It is a rare genetic disorder, the incidence is 1:250000, most often a result of mutation in TCIRG1 gene. The reported patient is a 1-year old boy presented with visual impairments, bone marrow failure, hepatosplenomegaly, hypocalcemia and supportive X-ray changes of increased bone density and bone-to-bone appearance. There is history for parental consanguinity.

Methods: Upon physical examination he was found to have impaired visual milestones, abnormal craniofacial appearance, short limbs, hepatosplenomegaly, inguinal and umbilical hernias. Laboratory results shows anemia, hypocalcemia, hypophosphatemia, hyperparathyroidism, high alkaline phosphatase, low 25(OH)D. There is MRT data for hydrocephalus. The molecular genetic analysis was performed by NGS (panel, that includes the associated with osteopetrosis genes).

Results: The clinical manifestation, laboratory tests and x ray changes make the diagnosis osteopetrosis highly likely. It was confirmed by genetic testing, that revealed a novel homozygous pathogenic variant, c.205C>T (p.Gln69*) in TCIRG1. The couple of parents are carriers of the mutation.

Conclusion: Early diagnosis is important to direct the appropriate treatment to prevent the disease progression before irreversible sequelae occur. The surveillance and treatment are managed by multidisciplinary team. The only treatment of infantile malignant osteopetrosis is haemopoetic stem cell transplantation. Our patient was referred to specialized center for treatment of osteopetrosis.

References:

Grants:

Conflict of Interest: None declared.

EP05.010 Identification of rare novel TSPEAR variants in autosomal recessive ectodermal dysplasia using whole exome sequencing

Eman Rabie ^1;2, Inas Sayed³, Ghada El-Kamah⁴, Suher Zada², Hoda Radwan¹, Nehal Hassib³, Mostafa Mostafa⁵, Khalda Amr¹

¹National Research Centre, Medical Molecular Genetics Department, Human Genetics & Genome Research Division (HGGR), Giza, Egypt; ²The American University in Cairo (AUC), Biotechnology program, School of Sciences and Engineering, Cairo, Egypt; ³National Research Centre, Orodental Genetics Department, Human Genetics & Genome Research Division (HGGR), Giza, Egypt; ⁴National Research Centre, Clinical Genetics Department, Human Genetics & Genome Research Division (HGGR), Giza, Egypt; ³National Research Centre, Orodental Genetics Department, Human Genetics & Genome Research Division (HGGR), Giza, Egypt

Background/Objectives: Ectodermal dysplasia (ED) is a group of heterogenous inherited disorders due to the dysfunction of ectodermal developmental processes with subsequent defects in at least two of four ectodermal derivatives, these are hair, teeth, nails and sweat glands. ED can be classified into five clusters according to the disrupted developmental pathway. The disease-causing gene remains unknown in approximately 50% of ED. We used whole exome sequencing (WES) to identify disease causing variants in a cohort of phenotypically variable ED patients whose underlying molecular pathology could not be characterized through targeted NGS panel containing EDA, EDAR, EDARADD&WNT10A genes.

Methods: DNA was extracted from blood samples of nine ED patients from eight consanguineous families. WES was performed, followed by Sanger sequencing for segregation of variants of interest. For novel missense variants, changes in protein structure were predicted in silico.

Results: Five novel TSPEAR variants disrupting functional domains were identified. A frameshift homozygous variant was identified in one patient with severe clinical picture. An in-frame deletion was identified in seven patients with variable clinical presentations: in five patients in homozygous form, and in a compound heterozygous form with a missense variant in two patients. One patient had two compound heterozygous missense variants.

Conclusion: We expanded the clinical and molecular spectrum of TSPEAR variants which might propose an additional ED cluster since TSPEAR is known to function via Notch signalling pathway.

References: Wright et al., 2019 (PMID: 30703280).

Bowles et al., 2021 (PMID: 34042254).

Grants: The American University in Cairo research grant#R36 and Science and Technology Development Fund grant#33494.

Conflict of Interest: None declared.

EP05.012 Positive association between a common polymorphism within the GPR126 gene and idiopathic scoliosis in Bulgarian patients

Svetla Nikolova ¹, Milka Dikova², Alexandre Loukanov³

¹Sofia University, Faculty of Medicine, Department of Biology, Medical Genetics and Microbiology, Sofia, Bulgaria; ²Medical University-Sofia, Faculty of Medicine, Department of Pediatrics, Sofia, Bulgaria; ³National Institute of Technology, Gunma College, Department of Materials Engineering, Gunma, Japan

Background/Objectives: Several genome wide association studies suggested that polymorphic variants of GPR126 gene could take part in the pathogenesis of idiopathic scoliosis. The present case-control study investigated the association between a common polymorphism, GPR126 (rs6570507, A/G), and idiopathic scoliosis in Bulgarian patients.

Methods: The association study was performed on 127 patients and 254 controls after obtaining written informed consent. The mean Cobb angle was 53.8 ± 21.2°. The mean age of patients was 11.2 ± 2.9 years. The cases were divided into subgroups based on disease onset, sex, family history, and curve progression. The genotyping was carried out by TaqMan Real-Time PCR method. The statistical analysis was performed by Pearson’s chi-squared test and Fisher’s exact test with p-value less than 0.05 as statistically significant.

Results: The frequencies of the variant G allele and the GG genotype in the total group of patients and in the subgroup of patients with Cobb angle above 40° were significantly higher than those in the controls (p < 0.05). In addition, this case-control study revealed statistically significant association between GPR126*rs6570507 and primary scoliosis in females, adolescents, and sporadic cases.

Conclusion: The results confirmed previously reported associations between a common variant of GPR126 gene and idiopathic scoliosis in Caucasian and Asian populations and suggested that the molecular marker rs6570507 is an independent predisposing and modifying factor of idiopathic scoliosis in different subgroups of Bulgarian patients.

References: Kou et al. Sci Rep. 2018;8(1):11575.

Grants: MEXT/JSPS KAKENHI №T20K05260 and Jikoshunyu Kyoinhaibun-keihi №T5452.

Conflict of Interest: Svetla Nikolova: None declared, Milka Dikova: None declared, Alexandre Loukanov MEXT/JSPS KAKENHI №T20K05260 and Jikoshunyu Kyoinhaibun-keihi №T5452.

EP05.013 Targeted next-generation sequencing contributes to genetic diagnosis of osteogenesis imperfecta in Bulgarian patients

Darina Kachakova ¹, Kunka Kamenarova¹, Kalina Mihova¹, Ivanka Dimova^1;2, Radka Kaneva¹

¹Laboratory of Genomic Diagnostics, Molecular Medicine Center, Department of Medical Chemistry and Biochemistry, Medical Faculty, Medical University of Sofia, Sofia, Bulgaria; ²Medical Faculty, Medical University of Sofia, Department of Medical Genetics, Sofia, Bulgaria

Background/Objectives: Osteogenesis imperfecta (OI) is a systemic connective tissue disease characterized by low bone mass, bone fragility, and deficient growth. The clinical phenotype is highly variable and there is also genetic heterogeneity. Clinical exome sequencing (CES) help genetic diagnosis, counseling, and treatment of OI patients.

Methods: Ten patients with OI were directed for targeted next-generation sequencing (NGS) in Molecular Medicine Center in the period 2019–2021 year. NGS was performed on MiSeq platform. Direct sequencing by Sanger was used in order to confirm estimated pathogenic variants.

Results: Genetic cause of the disease was estimated in 5 from 10 analyzed patients. Pathogenic/probably pathogenic variants in COL1A1 were found in 4 patients and a nonsense variant in COL5A1 in one patient. The variants that we have detected in COL1A1 are one missense, one nonsense, and 2 frameshift mutations. In 1 from 5 of undiagnosed patients, we found heterozygous missense pathogenic variant in BMP1. In an OI patient girl with deafness genetic cause for the disease was not found but two missense variants in MYO7A and COCH were observed. These genes are associated with deafness and probably they could contribute to hearing loss in the patient. In another patient with not estimated genetic cause for OI we found a heterozygous pathogenic variant in SLC26A2 gene, mutations in which are associated with autosomal recessive chondrodysplasias.

Conclusion: Targeted NGS contributes substantially to genetic diagnosis of OI and allowed detection of disease-causing mutation in 50% of the patients.

References: OMIM.

Grants: D01-285/17.12.2019, D01-395/18.12.2020, D01-302/17.12.2021.

Conflict of Interest: None declared.

EP05.014 A previously unreported de novo FBN1 missense variant associated with a severe phenotype of neonatal Marfan syndrome

Jens Skaerbaek¹, Sara Markholt ², Pernille Axél Gregersen^2;3, Kim Munk⁴, Brian Nauheimer Andersen³, Jesper Padkær Petersen⁵, Jesper Vandborg Bjerre⁶, Kasper Jacobsen Kyng⁵

¹Vejle Hospital, Lillebælt Hospital, Department of Clinical Genetics, Vejle, Denmark; ²Aarhus University Hospital, Department of Clinical Genetics, Aarhus, Denmark; ³Aarhus University Hospital, Centre for Rare Diseases, Department of Paediatrics, Aarhus, Denmark; ⁴Aarhus University Hospital, Department of Cardiology, Aarhus, Denmark; ⁵Aarhus University Hospital, Neonatal Intensive Care Unit and Perinatal Research Unit, Department of Paediatrics, Aarhus, Denmark; ⁶Aarhus University Hospital, Department of Paediatrics, Aarhus, Denmark

Background/Objectives: Neonatal Marfan syndrome (nMFS) is characterized by a severe neonatal or infantile phenotype. We present a patient with nMFS and lethal cardiovascular phenotype.

Methods: Fetal ultrasound performed at GA 41+3 showed pronounced tricuspid regurgitation with a dilated right atrium, atrial flutter with 2:1 conduction and atrial rate of 280. Incipient circulatory overload led to acute caesarean section. The patient was flaccid at birth and needed respiratory assistance. ECG showed sinus rhythm of 120 bpm, birth weight 3800 g, length 60 cm and head circumference 33 cm. Persistent pulmonary hypertension of the newborn and worsening cardiorespiratory distress was evident the first 24 hours. Other phenotypic features involving enlarged hands and feet with arachnodactyly, elbow and knee flexion contractures, inverted thumbs, disproportionately long forearms and lower legs, long narrow face, micrognathia, premature craniosynostosis of sutura metopica and coronalis, deep set eyes, hypoplastic ear cartilage and loose skin were striking.

Echocardiography showed that all cardiac structures were enlarged with prolapse, annular dilatation and regurgitation of both atrioventricular valves, and aortic dilatation with regurgitation. Also, a non-restrictive PFO/ASD was noted. Following medical supportive therapy and palliative care, the boy deceased from congestive heart failure at 4 months of age.

Results: Genetic analysis revealed heterozygosity for a de novo FBN1 variant: c.3284G>C (p.Cys1095Ser).

Conclusion: The clinical and genetic findings and considerations in this case can be helpful for the prenatal and clinical diagnosis, management and genetic counseling in patients with a similar clinical picture and/or the same variant in FBN1.

References:

Grants:

Conflict of Interest: None declared.

EP05.015 Expanding the phenotypic spectrum of B3GAT3-associated disorders

Cecilia Bracco ¹, Patrizia Dentelli^1;2, Giovanni Botta³, Andrea Sciarrone⁴, Enrico Grosso¹, Barbara Pasini^1;2

¹Medical Genetics Unit, AOU Città della Salute e della Scienza di Torino, Torino, Italy; ²Department of Medical Sciences, University of Turin, Torino, Italy; ³Pathology Unit, AOU Città della Salute e della Scienza di Torino, Torino, Italy; ⁴Department of Obstetrics and Gynecology, AOU Città della Salute e della Scienza di Torino, Torino, Italy

Background/Objectives: First trimester ultrasound on a foetus of a 33-years-old lady at her third pregnancy (previous ones ended in spontaneous abortions) identified increased NT (3.60 mm, >99th centile). A chromosomal analysis was performed, which showed a normal male karyotype (46,XY).

Ultrasound in the 17th week of gestation showed an abnormal flection with lateral deviation of both hands and bilateral talipes equinovarus, movements of all limbs were normal. A further ultrasound in the 19th week of gestation showed short ribs with abnormal curvature, abnormal conformation of the column, reduced fetal movements, amniotic fluid at the upper limit. An array-CGH-analysis (60K, AgilentTechnologies) did not show any pathogenic CNV.

Because of the prognosis, the pregnancy was interrupted. In addition to the ultrasound findings, fetal autopsy showed facial dysmorphisms (flat face, anteverted nares, long philtrum, macroglossia, short neck), arachnodactyly, platyspondyly of the thoracic vertebrae and reduced ossification of cervical vertebrae.

Methods: SureSelect-Agilent Custom Constitutional Panel 17Mb encompassing 5219 genes. Virtual panel “skeletal disorders” (342 genes).

Results: The analysis led to the identification of a homozygous likely pathogenic variant (c.668G>A, p.(Gly223Asp)) in the B3GAT3 gene (Larsen-like Syndrome). Both parents were confirmed to be carrier of the same missense variant and it was found that they had ancestors in a small village located north-west of the Italian city of Turin. This might therefore be a founder mutation of the valleys nearby Turin.

Conclusion: The identified variant might be a founder mutation of the valleys nearby Turin. This case expands the knowledge of the B3GAT3 phenotypic spectrum to the prenatal manifestations.

References:

Grants:

Conflict of Interest: None declared.

EP05.016 Application of aCGH technique in Ehlers-Danlos syndrome diagnostics

Anna Junkiert-Czarnecka ¹, Ewelina Łazarczyk¹, Maria Pilarska-Deltow¹, Anna Repczyńska¹, Aneta Bąk¹, Marta Heise¹, Olga Haus¹

¹Collegium Medicum in Bydgoszcz, Nicolaus Copernicus University in Toruń, Bydgoszcz, Department of Clinical Genetics, Faculty of Medicine, Bydgoszcz, Poland

Background/Objectives: Ehlers-Danlos syndrome (EDS) is a heterogeneous group of heritable connective tissue disorders. The 2017 International Classification of EDS recognized 13 subtypes caused by pathogenic variants in 19 different genes, encoding different types of collagen as well as protein involved in collagen metabolism or functioning. For all types of EDS, the genetic background was determined, except for the hypermobile type (hEDS).

Aim of our study was an evaluation by aCGH (Array Comparative Genomic Hybridization) a large alterations in genome as a potential background of hEDS.

Methods: The study group included 43 hEDS patients, 33 women and 10 men, negative for NGS-EDS panel (Illumina).

aCGH was performed using Agilent SurePrint G3 Unrestricted CGH 8 × 60K microarrays (Agilent Technologies) which provide an average resolution of 120 kb. These microarrays contain approximately 60000 probes. Results were analyzed using CytoGenomics software.

Results: In tested patients no large deletions or duplications were detected. In 42 only benign variants were found, in 2 of them any alterations in genome were observed.

Conclusion: Application of different molecular methods still did not give us an answer to the question about genetic background of hEDS. Our investigation shows that also large genome changes are not the basis of this connective tissue disorder.

References: 1. Malfait F, Francomano C, Byers P, et al.: The 2017 international classification of the Ehlers-Danlos syndromes. Am J Med Genet C Semin Med Genet. 2017;175(1):8-26.

Grants: This investigation was supported by the CM UMK grant MN-10/WL/2020.

Conflict of Interest: None declared.

EP05.018 Male-pattern hair loss: Integration of GWAS and single-cell RNASeq data to identify pathobiologically relevant hair follicle cell types

Nicole Engelmann¹, Sabrina Henne¹, Stefanie Heilmann-Heimbach ¹

¹University of Bonn, School of Medicine & University Hospital Bonn, Institute of Human Genetics, Bonn, Germany

Background/Objectives: Male-pattern hair loss (MPHL) is a prevalent and highly heritable form of hair loss. GWAS have identified >350 genomic risk loci and have implicated numerous candidate genes and pathways. However little is known about the cell types and hair-cycle stages in which these genes and pathways exert their pathobiological effects.

Methods: We used (i) a statistical model that relies on the assumption that genes with critical functions in pathogenic cell types are likely to be located within disease-associated loci (Hu et al.,2011) together with (ii) published GWAS-data (Yap et al., 2018), and (iii) a comprehensive single-cell RNASeq dataset of the murine hair follicle (HF) during hair growth and rest (Joost et al.,2020) to identify MPHL-relevant HF-cell types across hair-cycle stages.

Results: Our analyses revealed a role of different HF-cell types across and in specific hair-cycle stages. While e.g. dermal fibroblasts seemed to be of pathogenic relevance across hair-cycle stages, sebaceous gland cells and endothelial cells seemed to specifically contribute to MPHL-pathogenesis during growth or rest, respectively. Pathway-based analyses of the most specifically expressed genes in associated cell types suggest a similar picture on the molecular level, where e.g. androgen-signalling plays a role across cell types and hair-cycle stages whereas ErbB- or EDA-signalling are only active in specific cell types and hair-cycle stages.

Conclusion: Our data provide novel insight into MPHL-relevant HF-cell types and cellular processes and constitute an important basis for systematic functional follow-up of GWAS findings in relevant cell types.

References: Hu et al. (2011)-PMID:21963258.

Joost et al. (2020)-PMID:32109378.

Yap et al. (2018)-PMID:30573740.

Grants:

Conflict of Interest: Nicole Engelmann: None declared, Sabrina Henne: None declared, Stefanie Heilmann-Heimbach SHH receives a salary from the Life & Brain GmbH.

EP05.019 Biallelic copy number variations in both upstream & downstream enhancers of SHOX gene causes mesomelia and clubfoot without short stature

Bengisu Güner Yılmaz ¹, Saygın Abalı², Azad Akberzade³, Beril Ay⁴, Sait Tümer⁵, Özlem Akgün Doğan^1;6, Gen Nishimura⁷, Yasemin Alanay^1;6

¹Acibadem University, Pediatric Genetics, Department of Pediatrics, Istanbul, Turkey; ²Acibadem University, Pediatric Endocrinology, Department of Pediatrics, Istanbul, Turkey; ³Sağlam Ailə Medical Center, Bakı, Azerbaijan; ⁴Acıbadem University School of Medicine, Istanbul, Turkey; ⁵Acıbadem Labmed, Istanbul, Turkey; ⁶Rare Diseases and Orphan Drugs Application and Research Center, Acibadem University, Istanbul, Turkey; ⁷Saitama Medical University Hospital, Moroyama, Japan

Background/Objectives: SHOX-related disorders include idiopathic X-linked short stature (MIM#300582), Leri-Weill dyschondrosteosis (LWD)(MIM#127300), and Langer mesomelic dysplasia (LMD) (MIM#249700). Duplications and deletions in the upstream and downstream conserved non-coding regulatory elements (CNEs) of SHOX gene are also reported.

Methods: A 7-year-old girl, born at term with bilateral clubfeet was examined. Birth measurements were unremarkable. Mesomelic shortness of forearms and legs, Madelung deformity, ulnar deviation and cubitus valgus were noted. Her height was at −1.45 SDS. First cousin parents had proportionate average height.

Results: Xrays showed mesomelic dysplasia. Whole genome sequencing (WGS) was negative. Reevaluation of WGS for SHOX regulatory regions revealed four copies of GAIN: two upstream and two downstream. Parents were carriers (Table). The unaffected mother’s X-ray showed mild Madelung deformity.

Conclusion: This is the first report of biallelic inheritance of both upstream and downstream duplicated CNEs of SHOX gene. Biallelic CNVs in regulatory regions of SHOX gene may be the cause of mesomelia resembling mild Langer Mesomelic Dysplasia without short stature.

References: 1. Spurna Z, Capkova P, Srovnal J, et al. Clinical impact of variants in non-coding regions of SHOX - Current knowledge Gene. 2022;818:146238.

2. Bunyan DJ, Baffico M, Capone L, et al. Duplications upstream and downstream of SHOX identified as novel causes of Leri-Weill dyschondrosteosis or idiopathic short stature. Am J Med Genet A. 2016;170A(4):949-957.

Grants: No grants.

	UPSTREAM	DOWNSTREAM
PATIENT	arr[GRCh37] Xp22.33(168552_451049)x4	arr[GRCh37] Xp22.33(614734_802868)x4
MOTHER	arr[GRCh37] Xp22.33(168552_451049)x3	arr[GRCh37] Xp22.33(629999_802868)x3
FATHER	arr[GRCh37] Xp22.33 or Yp11.32(168552_451049 or 118552_401049)x3	arr[GRCh37] Xp22.33 or Yp11.32(614734_802868 or 564734_752868)x3

Conflict of Interest: None declared.

EP05.020 An aberrant IRAK1 gene generates a hypersensitive and hyperactive IRAK1 protein in the synovial fibroblast, when in presence of Escherichia coli Group D lipopolysaccharides effectuates an amplified expression of IL-1, IL-6 and TNFα, resulting in seropositive rheumatoid arthritis

Lane Scheiber II¹

¹Osteoporosis & Arthritis Center, Richmond, United States

Background/Objectives: Rheumatoid arthritis is postulated to be the result of a combination of genetic and environmental influences; the mechanistic factors which remain undefined. To determine cause of RA.

Methods: Root cause analysis of genetic/cellular processes associated with seropositive rheumatoid arthritis (SPRA) demonstrates: (1) Proliferation of synovial fibroblasts and expression of inflammatory cytokines cultivates synovitis. (2) Synovial fibroblasts mount Toll-like receptor 4 (TLR4) on their surface. (3) Escherichia coli Group D (EcGD) has been strongly associated with SPRA. (4) TLR4 triggers due to EcGD lipopolysaccharides. (5) TLR4 utilizes crucial transducer Interlukin-1 Receptor Associated Kinase-1 (IRAK1) protein to generate IL-2, Il-6 and TNFα, cytokines responsible for RA. (6) IRAK1 gene located on x-chromosome, with SPRA demonstrating a preponderance for women, and variable penetrance likely facilitated by random switching on/off the x-chromosome gene.

Results: Examination of systemic cellular mechanisms and genetic factors associated with SPRA demonstrate an overactive variant of the pivotal IRAK1 protein is the root cause of SPRA.

Conclusion: An anomalous IRAK1 gene results in generation of hypersensitive and hyperactive IRAK1 protein in fibroblasts, which when the TLR4 is triggered by serum lipopolysaccharides generated by presence of EcGD, amplifies expression of IL-1, IL-6 and TNFα, which results in synovial hypertrophy; left untreated, manifests into erosive arthritis. Blocking the hyperactive IRAK1 protein, specifically in synovial fibroblast cells, with targeted therapy, would lead to optimal management of SPRA. In women, whom develop SPRA, deactivating the aberrant IRAK1 gene in synovial fibroblasts and actuating IRAK1 demonstrating normal function, would be highly curative for SPRA.

References: to provide.

Grants: not applicable.

Conflict of Interest: None declared.

EP05.022 Ethnospecific markers of osteoarthritis in women from the Republic of Bashkortostan, Russia

Anton Tyurin ¹, Rita Khusainova¹

¹Bashkir State Medical University, Ufa, Russian Federation

Background/Objectives: Osteoarthritis (OA) is a common joint disease, with at least 30% genetic determination (Sophie C Warner, 2017). By 2020, more than 50 target genes have been identified (Ioanna Tachmazidou, 2019), but the results require validation on different ethnic group (Louise N Reynard, 2013).

Methods: DNA samples from 417 women (51.67 ± 11.5 y.o.) with OA and 161 healthy women from Ufa (Republic of Bashkortostan, Russia) were analyzed for the associations of polymorphic variants in 3′ UTR regions of COL1A1, COL11A1, ADAMTS5, MMP1, MMP13, SOX9, FGFR1, FGFRL1 and incidence of OA using kompetitive allele-specific PCR. Ethnic composition was presented as follows: 144 Russian (Slavic group of the Indo-European language family), 159 Tatar (Turkic branch of the Altai language family), 114 mixed and representatives of small ethnic groups.

Results: Identified associations are presented in Table 1. All associations remained statistically significant after Benjamini-Hochberg correction (p*).

Table 1. Associations of the miRNA target sites loci in different ethnical groups

SNP	Gene, loci	Ethnicity	Allele	p	p*	OR; 95% CI
rs6854081	FGF2 (4q28.1)	Tatar	G	0.0001	0.0002	OR = 4.78;(1.89–12.02)
rs1061237	COL1A1 (1p21.1)	Russian	С	0.017	0.034	OR = 1.77;(1.07–2.94)
rs229069	ADAMTS5 (21q21.3)	Mixed	Т	0.0002	0.0004	OR = 2.25;(1.30–3.89)
rs73611720	GDF5 (20q11.22)	Mixed	Т	0.004	0.008	OR = 3.02;(1.38–6.60)

Conclusion: Ethnospecific markers of OA development in the FGF2 (rs6854081), COL1A1 (rs1061237), ADAMTS5 (rs229069) and GDF5 (rs73611720) genes were identified in women from the Republic of Bashkortostan.

References:

Grants: This work was financially supported by a grant from the Republic of Bashkortostan for young scientists SEC-GMU-2021.

Conflict of Interest: None declared.

EP05.023 Congenital defects in a patient carrying a novel homozygous AEBP1 variant could expand the phenotype of Ehlers-Danlos syndrome classical-like type 2

Niccolò Di Giosaffatte¹, Alessandro Ferraris ¹, Federica Gaudioso¹, Valentina Lodato¹, Emanuele Savino¹, Giulia Parise¹, Filippo Camerota², Claudia Celletti², Simone Bargiacchi¹, Luigi Laino¹, Irene Bottillo¹, Paola Grammatico¹

¹Laboratory of Medical Genetics; San Camillo-Forlanini Hospital, Department of Experimental Medicine, Sapienza University of Rome, Rome, Italy; ²Umberto I University Hospital of Rome, Physical Medicine and Rehabilitation Division, Rome, Italy

Background/Objectives: In 2018 a new clinical subtype, caused by biallelic variants in the AEBP1 gene, was added to the current nosological classification of the Ehlers Danlos Syndromes (EDS). This new phenotype, provisionally termed EDS classical-like type 2 (clEDS2), has been not yet fully characterized, as only seven cases have been reported to date. Here we describe a patient, homozygous for a novel AEBP1 pathogenic variant, whose phenotype is reminiscent of classical EDS but presenting also with previously unreported clinical features.

Methods: Besides the EDS typical features, this patient presented with multiple congenital defects including cleft palate, agenesis of multiple phalanxes of the left foot, and partial agenesis of the right pectoral muscle. Cytogenomic array and targeted exome sequencing, according to the clinical features, were performed on DNA isolated from a blood sample. Full clinical examination and medical history were obtained and compared to the previously reported cases of AEBP1-related EDS.

Results: An homozygous novel AEBP1 variant, c.2123_2124delTG (p.Val708AlafsTer5), was detected by exome sequencing, segregating from heterozygous parents.

Conclusion: Our case recapitulates most clinical features previously reported in clEDS2, especially those reminiscent of classical EDS. Conversely, the additional congenital defects in this patient might be novel manifestations, expanding the phenotype of AEBP1 biallelic mutations. Although a different concomitant etiology for cleft palate and foot phalanges agenesis cannot be formally excluded, the connection of AEBP1 protein with TGF-beta and WNT pathways, for instance through the interaction with frizzled receptors, may suggest an intriguing common underlying etio-pathological mechanism.

References:

Grants:

Conflict of Interest: None declared.

EP05.024 Steroid sulfatase deficiency in Tunisian patients: awareness of STS pseudogene technical trap

hamza CHOUK ¹, Sarra Saad², Rima Gammoudi², Lobna Boussofara², sarra dimassi³, ALI SAAD³, Mohamed Denguezli², Dorra H’mida³

¹Higher Institut of Biotechnology of Monastir, Department of Cytogenetics, Molecular Genetics and Human Reproduction Biology, Sousse, Tunisia; ²Faculty of Medicine of Sousse, Department of Dermatology, Sousse, Tunisia; ³Faculty of Medicine of Sousse, Department of Cytogenetics, Molecular Genetics and Human Reproduction Biology, Sousse, Tunisia

Background/Objectives: X-linked recessive ichthyosis (XLI) is a genetic disorder that affects the skin, caused by a deficiency of the steroid sulphatase enzyme encoded by the STS gene (OMIM # 300747). Our work aims to study the clinical and genetic characteristics of 8 Tunisian patients with XLI.

Methods: We collected eight patients with XLI, all males, from three unrelated Tunisian families from central Tunisia. Genetic diagnosis was conducted through Sanger Sequencing, haplotype analysis of STR markers, MLPA analysis, FISH and CGH techniques.

Results: Our 8 patients presented with collodion baby at birth that evolved into a thick, polygonal, dirty, dark scaly ichthyosis with a generalized distribution. Direct sequencing revealed the same 13 bp deletion in all patients. However, their mothers were not carriers of this variant and no common haplotype was shared between affected patients around STS gene. Sequence alignment with reference human genome revealed an unprocessed pseudogene of the STS gene located on the Y chromosome, on which the 13 bp deletion was actually located. STS MLPA analysis revealed a deletion of the entire STS gene for the 3 families, confirmed by FISH and CGH array techniques.

Conclusion: Geneticists must be aware of the presence of STS pseudogenes that can lead to misdiagnosis. Pseudogenes sequence similarities with the gene of interest must be taken into account when designing primers for sequencing to avoid mistaken results.

References: Ballabio, A. et al. Isolation and characterization of a steroid sulfatase cDNA clone: Genomic deletions in patients with X-chromosome-linked ichthyosis.

Grants:

Conflict of Interest: None declared.

EP05.025 Early onset end-stage renal disease and retinal dystrophy in a cranioectodermal dysplasia patient with WDR35 variants

Joanna Walczak-Sztulpa ¹, Anna Wawrocka¹, Weronika Sikora², Marta Pawlak³, Ewelina Bukowska-Olech¹, Bartłomiej Kopaczewski⁴, Heleen Arts^5;6, Agnieszka Urzykowska⁷, Anna Gotz-Wieckowska³, Ryszard Grenda⁷, Anna Latos-Bieleńska¹, Renata Glazar⁸

¹Poznan University of Medical Sciences, Department of Medical Genetics, Poznan, Poland; ²Poznan University of Medical Sciences, Students’ Research Group of Medical Genetics, Poznan, Poland; ³Poznan University of Medical Sciences, Department of Ophthalmology, Poznan, Poland; ⁴Poznan University of Medical Sciences, Department of Neurosurgery, Karol Jonscher Clinical Hospital, Poznan, Poland; ⁵Dalhousie University, Department of Pathology and Laboratory Medicine, Nova Scotia, Canada; ⁶IWK Health Centre, Clinical Genomics Laboratory, Nova Scotia, Canada; ⁷The Children’s Memorial Health Institute, Department of Nephrology, Kidney Transplantation and Hypertension, Warsaw, Poland; ⁸Centers for Medical Genetics GENESIS, Poznan, Poland

Background/Objectives: Cranioectodermal dysplasia (CED), is a clinically and genetically heterogenous disorder characterized by skeletal, craniofacial, and ectodermal abnormalities. CED belongs to a group of disorders known as ciliopathies and is associated with defective cilia function and structure. To date six genes have been associated with this syndrome (WDR35, IFT122, IFT140, IFT144, IFT52, and IFT43). Here we describe on a 4-year-old male CED patient whose features include dolichocephaly, multi suture craniosynostosis, facial dysmorphism, narrow thorax, limb shortening, and brachydactyly. The patient presented early-onset chronic kidney disease and early onset of retinal degeneration. He developed the end-stage of renal disease at the age of 11 months and has been transplanted at the age of 2 years and 5 months. Retinal dystrophy has been diagnosed at the age of 3.5 years.

Methods: The NGS custom designed SureSelect (Agilent Technologies) panel consists of 61 genes and 11 single nucleotide variants (SNVs) known to be associated with craniosynostosis has been performed in the patient.

Results: Targeted NGS detected two heterozygous variants p.(Gly303Arg) [c.907A>G] in exon 9 and p.(Leu641*) [c.1922T>G; rs199952377] in exon 18 in the WDR35 gene. The presence of both variants was confirmed by Sanger sequencing and segregation analysis revealed that the mother and father are each carriers of p.(Gly303Arg) or p.(Leu641*), respectively.

Conclusion: CED patients with variants in the WDR35 gene should be monitored regularly for kidney function, and standard ophthalmologic evaluation including ERG and fundoscopy should be performed to detect early signs of retinal degeneration.

References: Tan, W., Lin, A., & Keppler-Noreuil, K. (2021). Cranioectodermal Dysplasia. Definitions.

Grants:

Conflict of Interest: None declared.

EP05.027 Expanding the spectrum of PAX9 mutations associated with selective tooth agenesis type 3

Federico Rondot ¹, Patrizia Dentelli¹, Cecilia Bracco², Barbara Pasini^1;2

¹Department of Medical Sciences, University of Turin, Torino, Italy; ²Medical Genetics Unit, AOU Città della Salute e della Scienza di Torino, Torino, Italy

Background/Objectives: We present here a 10-years old girl with oligondontia and abnormal dental shape with positive family history (mother, maternal uncle and grandfather). She presented with eruption of 4 definitive elements (two peg shaped maxillary central incisors and two firsts inferior molars). Moreover, she showed a delay in the replacement of the inferior deciduous incisors. Given the suspicion of oligodontia, an Orthopantomagram was performed, which showed only 10 more still unerupted definitive elements, confirming the diagnosis. Given the positive family history, a clinical exome was performed in duo with the mother.

Methods: SureSelect-Agilent Custom Constitutional Panel 17Mb encompassing 5219 genes.

Virtual panel “oligodontia” (31 genes).

Results: The analysis led to the identification of a heterozygous likely pathogenic variant in the PAX9 gene (c.771+1G>A, p.?) in both mother and daughter. The variant, never reported, absent in the main population and mutation databases, alters the canonical donor splicing site. Skipping of exon 4 would lead to a frameshift. However in absence of functional data, the use of an alternative upstream or downstream splice site cannot be excluded.

Conclusion: PAX9 is associated with non-syndromic selective tooth agenesis type 3 (OMIM # 604625). This condition is inherited in an autosomal dominant way and affected people mostly show lack of permanent molars and possibly of second premolars and central incisors, which may also show shape anomalies. This phenotype is highly overlapping with that of our family.

Further segregation analysis in the family and functional studies are needed in order to expand the pathogenic mechanisms underlying PAX9-associated oligodontia.

References:

Grants:

Conflict of Interest: None declared.

EP05.028 Rare association of Cone-rod dystrophy in a patient with Neurofibromatosis type 1

Cristian Marinau ^1;2, Filip Mirodot^2;3, Dan Bembea⁴, Larisa Niulas^2;3, Cristian Sava^2;3, Claudia Jurca³

¹University of Oradea, Faculty of Medicine and Pharmacy, Oradea, Romania; ²“Dr Gavril Curteanu” Municipal Hospital, Oradea, Romania; ¹University of Oradea, Faculty of Medicine and Pharmacy, Oradea, Romania; ⁴Iuliu Hațieganu University of Medicine and Pharmacy, Cluj-Napoca, Romania

Background/Objectives: Neurofibromatosis type 1 (NF1; MIM 613113) is a autosomal dominant disease, characterised by the presence of cafe-au-lait macules, Lisch nodules and neurofibromas. Cone-rod dystrophy (CORD; MIM 602225) is characterized by decreasing visual acuity, loss of color vision, decreasing peripheral vision and nyctalopia leading to blindness.

Methods: The authors present a case of a 16 year old girl diagnosed with NF1 associating Cone-rod dystrophy.

Results: The patient is in our genetics department record from the first year of life. Family history is positive for NF1 with one affected parent, the father. At the latest follow-up she presents with multiple café-au-lait macules, bilateral axillary freckles, emerging neurofibromas, strabismus and astigmatism. Molecular diagnosis confirmed the diagnosis of NF1 identifying the pathogenic variant c.3871-2A>T on NF1 gene (17q11.2) and the c.119G>A varinat on CRX gene (19q13.33) classified as likely pathogenic based on ACMG guidelines.

Conclusion: NF1 associating CORD has been described only in a few cases in literature. Although positive diagnostic for NF1 is based on clinical signs, molecular analysis can confirm the diagnosis and provide additional information.

References:

Kylstra JA, Aylsworth AS. Cone-rod retinal dystrophy in a patient with neurofibromatosis type 1. Can J Ophthalmol. 1993 Apr;28(2):79-80. PMID: 8508343.

Zobor D, Kaufmann DH, Weckerle P, Sauer A, Wissinger B, Wilhelm H, Kohl S. Cone-rod dystrophy associated with amelogenesis imperfecta in a child with neurofibromatosis type 1. Ophthalmic Genet. 2012 Mar;33(1):34-8. https://doi.org/10.3109/13816810.2011.592178. Epub 2011 Jul 5. PMID: 21728811.

Grants: None.

Conflict of Interest: None declared.

EP05.029 9 new cases of spondylometaphyseal dysplasia with corner fractures: enhancement of the phenotypic spectrum of FN1 gene mutations

Caroline Michot ¹, Pauline Marzin¹, Genevieve Baujat¹, Eugénie Koumakis², Anne Dieux-Coeslier³, Massimiliano Rossi⁴, Coralie Haudry⁵, Anne-Laure Tourre⁵, Julie Steffann⁵, Sophie Rondeau⁵, Valérie Cormier-Daire¹

¹Reference Center for Skeletal Dysplasias, INSERM UMR1163, Paris Descartes - Sorbonne Paris Cité University, Imagine Institute, Necker-Enfants Malades Hospital, Paris, France; ²Reference Center for Calcium and Phosphate Metabolism Diseases, Paris Descartes University, Cochin Hospital, Rheumatology Department, Paris, France; ³CHU Lille, Clinic of Genetics « Guy Fontaine », Lille, France; ⁴Reference Center for Developmental Anomalies and Competence Center for Skeletal Dysplasias, Hospices Civils de Lyon, Genetics Department, Bron, France; ⁵Department of Genomic Medicine of Rare Diseases, Paris Descartes - Sorbonne Paris Cité University, Necker-Enfants Malades Hospital, Paris, France

Background/Objectives: Spondylometaphyseal dysplasia with corner fractures (SMDCF, MIM 184255) is an autosomal dominant skeletal dysplasia, characterized by irregular metaphyses, coxa vara, scoliosis and secondary ossification sites (“corner fractures”). SMDCF is linked to mutations in FN1, encoding fibronectin, an extracellular matrix glycoprotein. FN1 heterozygous mutations are also involved in glomerulopathy with fibronectin deposits (GFD). GFD mutations are located in C-terminal part of fibronectin type-III domain.

We aim to describe the SMDCF natural history.

Methods: We recruited 9 cases from 5 families: 2 females and 7 males; 6 children (3 to 14 years) and 3 adults.

Results: All families harbor FN1 heterozygous mutations: 4 in fibronectin type-I domain; 1 in N-terminal part of fibronectin type-III domain.

The 3 adult men measure 121 to 160 cm. 5 children displayed IUGR. 4 children grow between −3 and −5,5 SD (1 GH deficiency). 2 children grow at -1,5/-2 SD, one under GH therapy. Joint hyperlaxity is noted in 2 families; genu valgum is frequent. 3 cases have scoliosis; lumbar hyperlordosis is almost systematic.

Metaphyseal dysplasia and coxa vara are constant. 8 cases had hip surgeries. 4 children display corner fractures, which are no longer visible in adults. Vertebrae are: tall in adulthood, ovoid in 3 children. A child has basilar impression with posterior fossa cyst.

No patient has impaired renal function.

Conclusion: We confirm a correlation between mutation localization and clinical presentation. The basilar impression case indicates systematic spine assessment. Joint laxity assessment is recommended. We underline endocrinological management, even if the relevance of GH treatment requires further investigations.

References:

Grants:

Conflict of Interest: None declared.

EP05.030 Epidermolysis Bullosa: Literature Review and Three Case Presentation

Laura Popa ^1;2, Nicoleta Andreescu^1;2, Cristian Zimbru², Adela Chirita-Emandi^1;2, Simona Farcas², Doriana Chilom², Maria Puiu^1;2

¹Emergency Hospital for Children Louis Turcanu, Clinical Genetics, Timișoara, Romania; ²Victor Babes University of Medicine and Pharmacy, Medical Genetics, Timișoara, Romania

Background/Objectives: Epidermolysis bullosa (EB) is a genodermatosis in which clinical manifestations varies in severity and character according to subtype. There are three main EB categories: simplex, junctional, and dystrophic. EB can be inherited in recessive (AR) or dominant (AD) manner, AR form being the most severe.

Methods: Here we present three cases of EB, two cases of dystrophic EB (DEB) and one case of simplex EB (SEB), genetically analysed by NGS sequencing. All three patients present bullous lesions and skin exfoliation, diagnosed by dermatologist.

Results: Following sequencing, we determined three different mutations. One of the patients with DEB has been identified with a missense homozygous mutation (c.425A>G) in COL7A1 gene, also known as the most common variant among DEB patients. For the other patient with DEB, two different heterozygote mutations have been identified, both in COL7A1 gene (frameshift c.5960del, missense heterozygote c.425A>G). The frameshift c.5960del mutation is less common; therefore, there are few information available for this mutation. The sequencing analysis for the SEB patient revealed a missense heterozygote mutation (c.1400T>C) in KRT5 gene. The two DEB variants have been associated with AR inheritance, while SEB variant has been associated with AD inheritance.

Conclusion: The first conclusion reached is that the same EB type with similar manifestations can be determined by different mutations. The second conclusion is that the mutation information and knowing the inheritance pattern, can help us predict the evolution of the disease, create the basis for an accurate genetic counselling and increase the prevalence of prenatal diagnosis among families with risk.

References:

Grants:

Conflict of Interest: None declared.

EP05.031 Two sibs with overgrowth, macrocephaly, intellectual disability and homozygous novel pathogenic FIBP variant, Thauvin-Robinet Faivre Syndrome

Esra Kilic¹

¹Univerctiy of Health Sciences, Ankara City Hospital, Pediatric Genetics, Ankara, Turkey

Background/Objectives: Thauvin-Robinet Faivre Syndrome is a newly defined rare autosomal recessive overgrowth sydrome characterized with intellectual disability, facial dysmorphism, macrocephaly and variable congenital malformations. It is caused by homozygous FIBP mutations. FIBP gene locates on 11q13.1 and codes fibroblast growth factor intracelluar binding protein. To date only four patient has been reported with this disorder. Here we report 2 siblings born from consanginous parents.

Methods: Case 1, 14 years old boy with tall stature, macrocephaly, moderate intellectual disability. He has downslanting palpebral fissures, widely spaced/deep set eyes, thick lips, scoliosis, athrophic-dysplastic right kidney, large hands and feet, 4/5 mild motor deficit in right arm and leg. His metabolic workup chromosome analysis, 5q35 FISH analysis, CGG repeat on FMR1 gene, eye examination and echocardiography were normal. Microarray analysis was also normal. Case 2, 3 years old girl with tall stature, macrocephaly, developmental delay, round face, widely spaced eyes, epichantic folds, flat mid face, thick lips. Her methabolic workup, chromosome analysis and eye examination were normal.

Results: Whole exome sequencing analyses of case1 reveaed homozygous pathogenic c.412-3_415 dupCAGTTTG, (p. Asp139Alafs*Ter3) variant. Sanger sequencing of FIBP gene revealed, homozygous pathogenic c.412-3_415 dupCAGTTTG variant in in both sibs. This pathogenic variant was also confirmed heterozygous state in parents with direct sequencing.

Conclusion: Here we report a rare autosomal recessive overgrowth sydrome in two affected siblings. Reporting two new cases with pathogenic FIBP mutation will be support and expand the clinical spectrum of Thauvin-Robinet Faivre syndrome.

References: Thauvin‐Robinet, et al.2016. Clinical Genetics, 89(5).

Grants:

Conflict of Interest: None declared.

EP05.032 A long-term surveillance of a 32 years old patient with Osteogenesis imperfecta type III

Filip Mirodot ¹, Anamaria Orosz-Cosmescu¹, Cristian Marinau¹, Claudia Jurca², Marius Bembea²

¹University of Oradea, Faculty of Medicine and Pharmacy, Romania, Medical, Oradea, Romania; ²University of Oradea, Faculty of Medicine and Pharmacy, Romania, Preclinical, Oradea, Romania

Background/Objectives: Osteogenesis imperfecta (OI) is a group of rare disorders. It is caused by mutations in the COL1A1 and COL1A2 genes that codify the alpha 1 and alpha 2 chains of type 1 collagen. The incidence of OI is 1:10,000. The main important signs include spontaneous fracture or fracture from minor trauma, joint hypermobility, skin fragility, easy bruising.

Methods: The authors tracked the physical, mental and social integration of a 32-year-old patient diagnosed with severe OI from birth.

Results: Phenotypically the patient presents relative macrocephaly frontal bossing, triangular face, blue sclera; He has had numerous spontaneous fractures (over 100) since birth, leading to extremely severe disharmonious dwarfism, with marked shortening and curving of the limbs, pectus deformity and binding him to a wheelchair. People with OI are usually lonely, self-conscious, sensitive, suspicious of the opinions of others, depressive, with poorer quality of life (QOL); our patient proved to be the opposite: he is cheerful, optimistic, passionate about history and politics, surrounded by friends, lives life to the fullest; he has a Master`s degree in environmentalism; currently working as a customer service operator.

Conclusion: People with OI can live a happy and satisfying life. These people need a number of specific support measures provided by an adapted lifelong support system delivered through specialized genetic disease centers.

References: Palomo T, Vilaça T, Castro ML. Osteogenesis imperfecta: diagnosis and treatment. Curr Opin Endocrin Diabetes Obes 2017 24(6):381-388.

Grants: None.

Conflict of Interest: None declared.

EP05.033 Autophagy polymorphisms are associated to hip fracture outcome

Carlos Gutiérrez-Cerrajero ^1;2;3, Antonio Cerdán-Morala¹, Carmen Dacasa-Pérez^2;4, Helena Fidalgo^2;4, María Agustina Hierro-Estévez^2;4, Carmen Pablos^2;4, Alfonso González-Ramírez^2;4, Ana-Belén Herrero^1;2;3, Juan Francisco Blanco-Blanco^2;4, Rogelio González-Sarmiento^1;2;3

¹Molecular Medicine Unit, Department of Medicine, University of Salamanca, Salamanca, Spain; ²Biomedical Research Institute of Salamanca (IBSAL), Salamanca, Spain; ³Institute of Molecular and Cellular Biology of Cancer (IBMCC), Salamanca, Spain; ⁴University Hospital of Salamanca-USAL, Department of Traumatology, Salamanca, Spain

Background/Objectives: Hip fracture in the older population is associated to high morbidity and mortality dependent on, among others, factors like genetics (1). Autophagy is a process involved in bone metabolism linked to aging-related diseases. Thus, we analysed the relationship of single nucleotide polymorphisms (SNPs) in autophagy related genes (ATGs) with the clinical variables and outcome of hip fracture in older patients.

Methods: 87 patients from the University Hospital of Salamanca, presenting osteoporotic hip fracture and aged 80 years or older, were included in the study after signing informed consent in compliance with the Declaration of Helsinki. Biodemographic, clinical and functional variables were recorded. Genomic DNA was extracted from peripheral blood by standard phenol/chloroform protocol and SNP genotyping was carried out using TaqMan probe-mediated qPCR. Four SNPs were analysed: rs3759601 (ATG2B), rs2245214 (ATG5), rs1864183 (ATG10) and rs2241880 (ATG16L1). Statistical analysis was carried out using Chi-square, Student-t and ANOVA tests with statistical significance considered at p-values < 0.05.

Results: rs3759601 heterozygotes showed lower 90-day survival, while rs2245214 heterozygotes, rs1864183 heterozygotes and A/A rs2241880 carriers showed higher 90-day survival. Both rs2245214 and rs1864183 heterozygotes showed better functional status and rs1864183 heterozygotes showed lower incidence of comorbidities.

Conclusion: Our results point to a relationship between autophagy and the outcome of hip fracture. This highlights an understudied area that could help assess the prognosis of these patients.

References: 1. B. Abrahamsen, T. Van Staa, R. Ariely, M. Olson, C. Cooper, Osteoporos. Int. 20, 1633–1650 (2009).

Grants: This project was funded by FIS-FEDER PI20/01569.

Conflict of Interest: None declared.

EP05.034 Differential expression of TLR7 and miRNA-146a genes in peripheral blood and skin samples of patients with systemic sclerosis

Vesna Spasovski ¹, Misa Vreca¹, Marina Andjelkovic¹, Maja Stojiljkovic¹, Anita Skakic¹, Kristel Klassen¹, Ana Zekovic², Nemanja Damjanov², Sonja Pavlovic¹

¹Institute of Molecular Genetics and Genetic Engineering, Laboratory for Molecular Biomedicine, Belgrade, Serbia; ²Institute of Rheumatology, Clinical Center of Serbia, Belgrade, Serbia

Background/Objectives: Progressive fibrosis of the skin and internal organs is one of the hallmarks of systemic sclerosis (SSc). Accumulative evidence demonstrates that Toll-like receptors (TLRs) may represent the link between immune activation and tissue fibrosis. Releasing of endogenous TLR ligands and their binding to TLR receptors complexed to autoantibodies might be one of the mechanisms that initiate fibrotic events. It arose recently that ‘fine-tuning’ of the TLR/NF-kB signaling pathway is taken place through down-regulation of IRAK1 gene via miR-146a.

Methods: The expression of TLR7 and miRNA-146a genes in PBMNC of 50 SSc patients and 13 healthy individuals using RT-qPCR technique was performed. Comparative analysis of these genes in affected and unaffected skin areas of the five SSc patient was performed in addition.

Results: In skin tissue samples the expression of TLR7 gene was 56% lower in affected (mRSS score>10) compared to unaffected tissue sample. When peripheral blood samples were examined, we found that patients with severe skin involvement (mRSS score>10) showed 26% lower TLR7 expression compared to patients with mild skin involvement (mRSS score≤10).

In addition, 19% lower level of miR-146a expression was detected in affected compared to unaffected skin sample. In peripheral blood samples, 37% lower expression of miR-146a was detected in patients with severe skin involvement compared to patients with mild skin involvement.

Conclusion: Synchronized expression TLR7 and miR-146a genes in skin tissue samples and blood samples suggest that both of these molecules should be further investigated as noninvasive biomarkers for skin involvement in SSc patients.

References:

Grants: MESTD, RS (III41004 and 451-03-68/2022-14/200042).

Conflict of Interest: None declared.

EP05.035 Phenotype diversity associated with TP63 mutations

ariane schmetz¹, Xing Xiong ², nicole cesarato², F. Buket Basmanav², Petra Gierthmuehlen³, Jörg Schaper⁴, Daniel Schlieper⁵, Maria Wehner², Holger Thiele⁶, Jorge Frank⁷, Regina C. Betz², Silke Redler^1;4

¹Institute of Human Genetics, Düsseldorf, Germany; ²Institute of Human Genetics, Bonn, Germany; ³Department of Prosthodontics, Düsseldorf, Germany; ⁴Center of Rare Disorders, Düsseldorf, Germany; ⁵Interdisciplinary Centre for Palliative Medicine, Düsseldorf, Germany; ⁶Department of Dermatology, Göttingen, Germany; ⁷Cologne Center for Genomics (CCG), Cologen, Germany

Background/Objectives: We saw a 41-year old South Indian patient (index) and his 10-year old son. They reported a history of diverse cutaneous and extracutaneous symptoms affecting the ectodermal appendages. Our aim was to find the underlying genetic cause of this autosomal dominant ectodermal dysplasia associated with hypotrichosis, hyperpigmentation, hypohidrosis and syndactyly for our patients.

Methods: Detailed physical examination was done for both affected family members. Whole-exome sequencing (WES) was performed in both patients. Results were confirmed by Sanger sequencing in the patients and the unaffected parents of the index.

Results: WES identified the heterozygous variant c.1922C>T; p.(Ala641Val) in exon 14 of TP63 (NM_003722.5) in father and son. The variant could not be detected in the unaffected wife/mother. The parents of the index both did not carry the variant, which indicates a de novo pathogenic variant.

Conclusion: Since the first description of a TP63 variant, the broad spectrum of molecular defects within this gene became increasingly clear. Each patient with a TP63 mutation reported displayed a plethora of distinct cutaneous and extra-cutaneous symptoms, reflecting the clinical heterogeneity of these disorders. The symptoms of the here described family do not fit into any of the TP63-related entities. The findings of our patients and critical review of the literature point to a phenotype diversity associated with TP63 mutations. It seems therefore reasonable to critically question the old clinical classification and to overcome the historical terminology. We propose the use of the term TP63-associated disorder for the future.

References:

Grants:

Conflict of Interest: None declared.

EP05.037 A novel frameshift variant of EDAR gene in a patient diagnosed with hypohidrotic ectodermal dysplasia

Mert Coşkun ¹, Aslı Toylu¹, Alp Peker¹, Özden Altıok Clark¹, banu nur², ercan mihci²

¹Akdeniz University School of Medicine, Department of Medical Genetics, Antalya, Turkey; ²Akdeniz University School of Medicine, Pediatric Genetics, Antalya, Turkey

Background/Objectives: Hypohidrotic ectodermal dysplasia (HED) is a hereditary disease characterized by the triad of hypohidrosis, anodontia/hypodontia and hypotrichosis signs. In this study we present a patient with clinical diagnosis of HED. The patient had periorbital hyperpigmentation, low-set ears, short philtrum in addition to the characteristic clinical findings of the disease. His mother and father were second cousins. We aimed to analyse DNA sequence of the reported candidate genes for HED disease in order to confirm the diagnosis of the patient.

Methods: Genomic DNA was extracted from the patient’s peripheral blood sample. Exons and exon-intron transition regions of the HED associated genes (EDA, EDAR, EDARADD, EDA2R, TRAF6, IKBKG and WNT10A) were investigated by next generation sequencing. Variants were analyzed using Ion Reporter Software (ThermoFisher Scientific Inc.) and SEQ (Genomize Inc.) programs. Variant information servers dbSNP, ClinVar, Ensembl and ACMG criteria were used for evaluations.

Results: In the eighth exon of the EDAR gene, c.677_678dup (p.Lys227GlyfsTer8) frameshift variant was detected in homozygous state. It was predicted as a “pathogenic” change due to premature termination of protein translation. The variant was not reported in population databases (gnomAD, ExAC) and disease-specific databases (ClinVar, OMIM).

Conclusion: The detected frameshift variant in our study is presumed a candidate variant that could be attributed to HED, considering predicted functional effects and its homozygosity. To better elucidate genotype-phenotype association, variant segregation status in the patient’s family and larger population data will be examined.

References: https://doi.org/10.1111/ijd.14048, https://doi.org/10.1007/s13353-015-0307-4.

Grants: Akdeniz University Scientific Research Projects Coordination Unit, Project Number: TSA-2021-5594.

Conflict of Interest: None declared.

EP05.038 Melnick-Needles syndrome: a male with severe and perinatally lethal phenotype

NIKOLAOS MARINAKIS ¹, Anastasia Konstantinidou², Danai Veltra¹, Christalena Sofocleous^1;3, Faidon-Nikolaos Tilemis³, Konstantina Kosma¹, Jan Traeger-Synodinos¹

¹National and Kapodistrian University of Athens, Laboratory of Medical Genetics, Medical School, Athens, Greece; ²National and Kapodistrian University of Athens, 1st Department of Pathology, Medical School, Athens, Greece; ³National and Kapodistrian University of Athens, Research University Institute for the Study and Prevention of Genetic and Malignant Disease of Childhood, Athens, Greece

Background/Objectives: Melnick-Needles syndrome (MNS) is a rare X-linked skeletal dysplasia, with severe lethal phenotype in males and less than 70 cases in the literature. MNS presents with extreme clinical heterogeneity whereby phenotypes in female carriers range from severe to very mild. A pathogenic FLNA variant was detected in a male fetus and is presented in the context of X-Linked Otopalatodigital Spectrum Disorders.

Methods: A 45 years-old female was referred for clinical evaluation and counselling, after 4 miscarriages, a successful IVF and delivery, and 2 therapeutic abortions due to severe anomalies (IUGR, hydrops, narrow chest etc). Specialised post-mortem examination on one of the aborted foetuses with a 46,XY karyotype, indicated renal dysplasia, cleft palate, lungs hypoplasia, constructures, short neck and multiple congenital anomalies. Subsequent genetic studies on DNA extracted from frozen tissues included Whole Exome Sequencing (WES) and targeted Sanger sequencing.

Results: The NM_001110556.2:c.3562G>A missense variant detected in hemizygosity is expected to cause a p.(Ala1188Thr) substitution which based on ACMG guidelines is classified as pathogenic (PM1, PM2, PP3, PP5). Segregation analysis with Sanger sequencing verified findings and indicated that the mother is a manifesting carrier, with very mild characteristics (hoarse voice, short stature) and the female born to IVF is normal.

Conclusion: To the best of our knowledge, this is only the fifth time a male with MNS was genetically diagnosed, and the second record of this specific variant. NGS supports marked improvement in diagnosis of cases with unusual severe phenotypes even before complete clinical presentation.

References: PMID: 34008892, 29575627.

Grants:

Conflict of Interest: None declared.

EP05.039 Familial and bilateral Poland Syndrome with hepatic hemangioma

Mustafa Gunes ^1;2, Sena Cetin^1;2, Filiz Ozen², Elif Yilmaz Gulec^1;2

¹Istanbul Medeniyet University Medical School, Department of Medical Genetics, Istanbul, Turkey; ²Istanbul Goztepe Prof. Dr. Suleyman Yalcin City Hospital, Department of Medical Genetics, Istanbul, Turkey

Background/Objectives: Poland Syndrome is a rare congenital disease known as hypoplasia or absence of pectoralis major muscle and upper limb abnormalities. Most cases are unilateral and sporadic, rare familial cases show reduced penetrance. Etiology of this disorder is still unknown, however intrauterine vascular defects have been suspected. Up-to-date only one case with a hemangioma has been reported (1). We present a familial Poland Syndrome in three generations with bilateral involvement and hepatic hemangioma in the proband.

Methods: A 27-year-old male patient was referred with the complaint of inability to raise his arms. The patient who has abnormal physical examination findings was evaluated with imaging and whole exome sequencing.

Results: In physical examination, limited abduction was observed in both shoulders and bilateral pectoralis major muscles could not be palpated. Family pedigree was compatible with an autosomal dominant inheritance with variable expression. In the proband, bilateral absence of the pectoralis major muscle was confirmed with ultrasound. No abnormalities in other muscles and thoracic structures were detected with tomography, however coincidentally, hepatic hemangioma measuring 34x30 mm was revealed with abdominal MRI. No candidate gene was found in whole exome sequencing.

Conclusion: Our case is unique with autosomal dominant inheritance pattern of Poland syndrome and with the presence of additional vascular defect, a hepatic hemangioma, probably a genetic vascular disorder without known locus.

References: 1. Riyaz N, Riyaz A. Poland syndrome (anomaly) with congenital hemangioma: a new association. Indian J Dermatol Venereol Leprol. 2006 May-Jun;72(3):222-3. https://doi.org/10.4103/0378-6323.25785. PMID: 16766839.

Grants:

Conflict of Interest: None declared.

EP05.040 Novel DTDST mutation in a Moroccan patient with diastrophic dysplasia

Maria Mansouri ¹, Meriem El Qabli¹, abdmajid moussaoui¹, hassan akallakh¹, Fatima Zahrae Bouzid¹, Loubna SOUFIAN¹, Nisrine Aboussair^1;2

¹Mohammed VI University Hospital, department of genetics,Clinical Research Center, Marrakech, Morocco; ²Cadi Ayyad University, School of Medicine, Marrakech, Morocco

Background/Objectives: Diastrophic dysplasia is a rare disorder marked by short stature with short extremities and joint malformations leading to multiple joint contractures, it’s a rare syndrome with a prevalence estimated at 1-1.3/100,000. A hitchhiker thumbs, cleft palate and cystic ear swelling in the neonatal period may be suggestive signs of this syndrome. Diastrophic dysplasia is caused by mutations in the SLC26A2 (DTDST), which encodes a sulfate transporter that is predominantly expressed in the cartilage.

Objective: Confirm the clinic diagnostic of Diastrophic dysplasia in our patient.

Methods: We studied the DNA from the family by a direct sequencing analysis of PCR amplified DNA from the proband and their parents.

Results: We report through this work a undescribed SLC26A2 mutation, in a girl with diastrophic dysplasia.

Conclusion: Interest of genetic consultation and dysmorphology expertise in the orientation of the diagnosis for certain constitutional bone diseases.

References: Superti-Furga A, Neumann L, Riebel T, Eich G, Steinmann B, Spranger J, Kunze J (1999) Recessively inherited multiple epiphyseal dysplasia with normal stature, club foot, and double layered patella caused by a DTDST mutation. J Med Genet 36:621–624.

Superti-Furga A, Unger S, the Nosology group of the international skeletal dysplasia society (2007) Nosology and classification of genetic skeletal disorders: 2006 revision. Am J Med Genet A 143:1–18.

Mégarbané A, Haddad FA, Haddad-Zebouni S, Achram M, Eich G, Le Merrer M, Superti-Furga A (1999) Homozygosity for a novel DTDST mutation in a child with a ‘broad bone-platyspondylic’ variant of diastrophic dysplasia. Clin Genet 56:71–76.

Grants: No grants.

Conflict of Interest: None declared.

EP05.042 A large family with short stature and genu varum: expanding the phenotype associated with ACAN variants

André Travessa ¹, Patrícia Dias¹, Belinda Campos-Xavier², Catarina Ferreira², Andrea Superti-Furga², Ana Berta Sousa¹

¹Serviço de Genética Médica, Departamento de Pediatria, Centro Hospitalar Universitário Lisboa Norte, Lisbon, Portugal; ²Division of Genetic Medicine, Lausanne University Hospital and University of Lausanne, Lausanne, Switzerland

Background/Objectives: Since 2010, heterozygous ACAN variants have been found in patients with short stature (SS), advanced bone age, early-onset osteoarthritis and/or osteochondritis dissecans.

Here, we report a multigenerational family with 12 patients with SS, in whom we found an ACAN variant. Some of them also presented genu varum, thus expanding the phenotype associated with ACAN variants.

Methods: Two 20-year-old twin sisters were referred at the age of 2 years due to growth delay and a family history of SS. Pregnancy was unremarkable but height at birth was <5th centile. They evolved with mildly disproportionate SS (136,2 and 138,7cm at adult age) and macrocephaly, and went on to develop lumbar hyperlordosis and genu varum that required surgical correction.

The family history was remarkable for several relatives with SS and lumbar hyperlordosis (son, sister, nephew, father, paternal grandfather, aunt, uncle, three first cousins, and a second cousin). Some also had genu varum (paternal grandfather and aunt), and others had degenerative joint disease (two first cousins).

Results: Whole-exome sequencing performed in the probands’ second cousin documented the presence of a heterozygous pathogenic variant in ACAN: c.1180C>T, p.(Arg394Ter). This variant was shown to be present in six affected relatives, including the twin sisters.

Conclusion: This case highlights the intrafamilial variability of ACAN-related SS and expands the phenotype to include genu varum. In the literature, the variant c.1180C>T, p.(Arg394Ter) was found in one patient with SS, advanced bone age, and osteochondritis dissecans, reinforcing that genotype-phenotype correlations in ACAN-related SS and very limited.

References: None.

Grants: None.

Conflict of Interest: André Travessa Medical Geneticist, Patrícia Dias Medical Geneticist, Belinda Campos-Xavier Laboratory Geneticist, Catarina Ferreira Laboratory Geneticist, Andrea Superti-Furga Medical Geneticist, Ana Berta Sousa Medical Geneticist.

EP05.043 Ectodermal-dysplasia-like phenotype associated with null variant in LRP6 gene

Franziska Roessler ¹, Rami Abou Jamra¹, Vincent Strehlow¹

¹University of Leipzig Medical Center, Institute of Human Genetics, Leipzig, Germany

Background/Objectives: Heterozygous pathogenic variants in the LRP6 gene are a confirmed cause of selective tooth agenesis 7 (OMIM # 616724). Recently, an 11-year-old male with oligodontia and sparse scalp hair carrying a truncating LRP6 variant was reported¹.

Methods: In our genetic counseling unit, we saw a 19-year-old woman with microcephaly, short stature, sparse scalp hair, pale skin, severe myopia and impressive gestalt. Her teeth were normal in quantity and morphology. We performed trio-exome-sequencing.

Results: We identified a heterozygous de novo variant c.4361dup, p.(Ser1455Lysfs*7) in LRP6. Loss of function is a known mechanism of tooth agenesis 7. Nonetheless, truncating LRP6 variants are listed in gnomAD (pLI 0.7 and o/e 0.22). Distribution of pathogenic and gnomAD-variants in LRP6 does not point to a genotype-phenotype-correlation. There is evidence for an incomplete penetrance. However, our patient’s characteristics show hardly any overlap with the established LRP6-associated phenotype.

Conclusion: Although our patient does not show the typical symptoms of tooth agenesis 7, we still consider the null variant in LRP6 to be a candidate explaining her phenotype. The variant occurred de novo and an overlap of the pathomechanisms of an ectodermal-dysplasia-like phenotype and oligodontia seems plausible. Also, there is a recent report of a patient with a null variant in LRP6 and sparse hair. However, larger cohorts are needed to clarify if there is a broader phenotypic spectrum including ectodermal dysplasia.

References: 1.Yu, M. et al. Lrp6 Dynamic Expression in Tooth Development and Mutations in Oligodontia. J. Dent. Res. 100, 415–422 (2021).

Grants:

Conflict of Interest: Franziska Roessler Institute of Human Genetics, University of Leipzig Medical Center, Rami Abou Jamra Institute of Human Genetics, University of Leipzig Medical Center, Principal investigator of “Genetics of rare diseases based on Next Generation Sequencing”, Vincent Strehlow Institute of Human Genetics, University of Leipzig Medical Center.

EP05.044 Genetic variants in epidemolysis bullosa patients in Lithuania

Zivile Zemeckiene^1;2, Rimvydas Jonikas ¹, Kristina Aleknavičienė^1;2, Marius Šukys^1;2, Inga Poceviciene¹, Rasa Traberg^1;2, Rasa Ugenskiene^1;2

¹Hospital of Lithuanian University of Health Sciences Kaunas Clinics, Kaunas, Lithuania; ²Lithuanian University of Health Sciences, Kaunas, Lithuania

Background/Objectives: Epidermolysis bullosa (EB) is a group of genetic skin diseases that cause fragile, blistering skin. Genetic testing of these diseases is important to better management and future predictions.

Methods: Whole exome sequencing and skin disease virtual gene panel analysis was performed for seven patients who were referred to clinical geneticist due to clinical diagnosis of epidermolysis bullosa in The Hospital of Lithuanian University of Health Sciences in 2021. All patients developed symptoms soon after birth or in early infancy.

Results: Likely pathogenic variants in KRT5 (NM_000424.4) gene were found for two patients with no family history of epidermolysis bullosa, one patient with EB dystrophica had novel variant c.1423G>T and other with EB simplex – known variant c.74C>T. Two other EB simplex patients with negative family history had known heterozygous pathogenic c.927+1G>A and likely pathogenic c.1162C>G variants in KRT14 (NM_000526.5) gene. Three patients had heterozygous variants in COL7A1 (NM_000094.3) gene. First EB dystrophica patient had known likely pathogenic variant c.6119G>A. Another patient with EB dystrophica was heterozygote for two likely pathogenic variants – one known maternal c.933C>G, and other novel paternal variant –c.6449G>A. As parents are healthy, we suspect autosomal recessive inheritance. Third patient with undefined EB type had novel VUS-leaning pathogenic variant in COL7A1 c.7886G>T and likely pathogenic variant in FLG gene, his family had similar symptoms in four generations.

Conclusion: Eigth different pathogenic/likely pathogenic variants in three different genes were determined in seven patients. Three of the variants were novel.

References:

Grants:

Conflict of Interest: None declared.

EP05.045 High prevalence of scoliosis in Koolen-de Vries syndrome: An international observational retrospective cohort study

Arianne Bouman ¹, Romy Bouwmeester², David Koolen¹, Joyce Geelen², Willemijn Margriet Klein³, Leo van Vlimmeren⁴, Marinus de Kleuver⁵, Pauline Burger^6;7;8;9, Jean Louis Mandel^6;7;8;9;10

¹Radboud university medical center, Human Genetics, Nijmegen, Netherlands; ²Radboud university medical center, Pediatrics, Nijmegen, Netherlands; ³Radboud university medical center, Medical Imaging, Nijmegen, Netherlands; ⁴Radboud university medical center, Rehabilitation, Pediatric Physical Therapy, Nijmegen, Netherlands; ⁵Radboud university medical center, Orthopedic Surgery, Nijmegen, Netherlands; ⁶Institute of Genetics and Molecular and Cellular Biology (IGBMC), Neurogenetics and Translational Medicine, Illkirch, France; ⁷Institut National de la Santé et de la Recherche Médicale, Illkirch, France; ⁸Centre National de la Recherche Scientifique, Illkirch, France; ⁹University of Strasbourg, Strasbourg, France; ¹⁰University of Strasbourg Institute for Advanced Studies (USIAS), Strasbourg, France

Background/Objectives: The Koolen-de Vries syndrome (KdVS, OMIM #610443), a rare neurodevelopmental disorder secondary to 17q21.31 microdeletion or mutation in KANSL1-gene, is associated with scoliosis. We describe the prevalence, clinical and radiological characteristics of scoliosis in KdVS.

Methods: In this international retrospective cohort study, 54 participants with KdVS were included. Mean age of participants was 13.6 years (SD 8.4). We retrospectively analyzed participants’ spine radiographs, MRI’s and corresponding radiology reports for scoliosis and additional anomalies. Presence of scoliosis-related clinical conditions were assessed in medical records and patient surveys.

Results: Scoliosis was present in 30/54 participants (56%). Prevalence increased with age, from 36% at age 10 to 67% at age 18. Mean age at diagnosis was 10.6 years. During follow-up, the number of coronal curves increased and the curve magnitude progressed. At the time of inclusion, most curves (60%) were below 30°. Participants with scoliosis received less often physiotherapy (p 0.002). Bracing therapy was received in 7/24 participants (29%), and surgical spinal fusion in 3/30 participants (10%).

In KdVS, we found that scoliosis was radiologically associated with hyperkyphosis (47%) and hyperlordosis (50%). A majority of the patients had (mild) hypotonia (83%) and all patients (100%) were able to walk.

Conclusion: Prevalence of scoliosis in KdVS is high: 56%. Scoliosis in KdVS cannot be included in one of the existing scoliosis’ categories, therefore we label it as scoliosis due to neurodevelopmental disorder. We advise routine coronal and sagittal full-standing spine X-ray screening before the start of the growth spurt and at the age of 18 years.

References:

Grants:

Conflict of Interest: Arianne Bouman full-time, Romy Bouwmeester full-time Radboudumc, David Koolen full-time Radboudumc, Joyce Geelen full-time Radboudumc, Willemijn Margriet Klein full-time Radboudumc, Leo van Vlimmeren full-time Radboudumc, Marinus de Kleuver full-time Radboudumc, Pauline Burger: None declared, Jean Louis Mandel: None declared.

EP05.048 Craniosynostosis as an additional feature of rare genetic syndromes

Ewelina Bukowska-Olech ¹, Dawid Larysz², Aleksander Jamsheer¹

¹Poznan University of Medical Sciences, Poznan, Poland; ²University of Warmia and Mazury in Olsztyn, Olsztyn, Poland

Background/Objectives: Craniosynostosis (CS) represents a highly heterogeneous genetic condition whose genetic background has not been fully revealed yet. The abnormality occurs either as an isolated form or syndromic, an element of hundreds of different inborn syndromes. Consequently, CS may often constitute a challenging diagnostic issue.

Methods: gDNA samples were subjected to whole-exome sequencing. The coding and flanking intronic regions were enriched using a custom-designed in-solution exome enrichment (TWIST bioscience, San Francisco, USA) and sequenced using the Illumina NovaSeq system (Illumina, San Diego, USA).

Results: We revealed seven heterozygous variants in the seven patients in the following genes – ARID1A (linked to Coffin-Siris type 2 syndrome), KMT2A (linked to Wiedemann-Steiner syndrome), KMT2D (linked to Kabuki type 1 syndrome), MN1 (linked to MN1 C-terminal truncation syndrome; MCTT syndrome, and CEBALID syndrome), NSD1 (linked to Sotos type 1 syndrome), and FAM111A (linked to Gracile bone dysplasia, and Kenny-Caffey syndrome type 2).

Conclusion: We have shown that CS may be an additional feature of different, not associated earlier with it, syndromes. We have also pinpointed the possible underestimated co-occurrence of CS and intellectual disability, suggesting it may be overlooked when intellectual disability constitutes a primary clinical complaint.

References: Morriss-Kay GM, Wilkie AO. Growth of the normal skull vault and its alteration in craniosynostosis: insights from human genetics and experimental studies. J Anat 2005;207:637–53.

Lattanzi W, Barba M, Di Pietro L, Boyadjiev SA. Genetic advances in craniosynostosis. Am J Med Genet Part A Published Online First: 2017. https://doi.org/10.1002/ajmg.a.38159.

Grants: None.

Conflict of Interest: None declared.

EP05.049 The role of rare variants in male-pattern hair loss: Analysis of whole-exome sequencing data in the UK Biobank

Sabrina Henne ¹, Sugirthan Sivalingam^2;3;4, Lara Hochfeld¹, Carlo Maj², Oleg Borisov², Andreas Buness^2;3;4, Markus M Nöthen¹, Peter Krawitz², Stefanie Heilmann-Heimbach¹

¹Institute of Human Genetics, University of Bonn, School of Medicine & University Hospital Bonn, Bonn, Germany; ²Institute for Genomic Statistics and Bioinformatics, University of Bonn, Bonn, Germany; ³Department of Medical Biometry, Informatics and Epidemiology, University of Bonn, Bonn, Germany; ⁴Core Unit for Bioinformatics Data Analysis, University of Bonn, Bonn, Germany

Background/Objectives: Male-pattern hair loss (MPHL) is a highly heritable and prevalent form of hair loss. Genome-wide association studies (GWAS) have identified more than 350 genomic risk loci, providing insights into the contribution of common genetic variants (MAF>1%) to MPHL etiology. However, systematic studies on the relevance of rare variants (MAF < 1%) in MPHL development are lacking. Using a tranche of 200,629 exomes from the UK Biobank, we tested for a contribution of rare coding variants to MPHL etiology.

Methods: 2,632,801 nonsynonymous variants (MAF < 1%) in protein-coding genes were tested for association with MPHL using SKAT-O gene-based and GWAS-style single-variant analyses in a case-control and extreme phenotypes setting. Association testing was followed by data interpretation, integration with MPHL GWAS data, and testing for enrichment in biological pathways.

Results: Both our single-variant and gene-based analyses identified significant associations of MPHL and rare variants in the X-chromosomal HEPH and EDA2R genes. Nominally significant associations were identified for an additional 17,892 single variants and 3,119 genes. This included associations in previously implicated candidate genes (e.g. WNT10A) and novel candidate genes at and beyond known GWAS loci (e.g. LAMA5, HOX13). Notably, nominally significantly associated genes were enriched for genes causative for monogenic trichoses as well as for genes located within topologically associated domains of MPHL-GWAS loci.

Conclusion: Our data point to a contribution of rare coding variants to MPHL etiology, broaden the MPHL-associated allelic spectrum at and beyond known risk loci, and suggest a biological association between monogenic trichoses and the common MPHL phenotype.

References:

Grants:

Conflict of Interest: Sabrina Henne: None declared, Sugirthan Sivalingam: None declared, Lara Hochfeld: None declared, Carlo Maj: None declared, Oleg Borisov: None declared, Andreas Buness: None declared, Markus M Nöthen Professor Markus M. Nöthen receives a salary from the Life & Brain GmbH., Peter Krawitz: None declared, Stefanie Heilmann-Heimbach Stefanie Heilmann-Heimbach receives a salary from the Life & Brain GmbH.

EP06 Cardiovascular Disorders

EP06.001 Aspirin reverses Fibronectin-mediated inhibitory effect on trophoblast invasion through Akt and ERK signaling in preeclampsia

Meitsz Su^1;2

¹National Cheng Kung University Hospital, Obs/Gyn, Tainan, Taiwan; ²National Cheng Kung University Hospital, Tainan, Taiwan

Background/Objectives: Preeclampsia is a severe gestational hypertensive disorder that represents a major threat to mortality and morbidity in maternal and fetal health worldwide. The pathoetiology of preeclampsia is believed to have defects of trophoblast differentiation and trophoblast invasion in placenta. Recent clinical trials showed that 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 pathoetiology in preeclampsia is unclear.

Methods: FN knockdown (si-FN) and exogenous treatment of recombinant FN protein in HTR-8/SV neo cells.

Results: In our presenting study, FN was upregulated in the placenta of preeclamptic patients and aspirin suppressed trophoblast FN expression in a dose-dependent way. FN was shown to inhibit trophoblast migration and invasion abilities without affecting cell proliferation. Moreover, FN activated signaling pathway of ERK and Akt. Aspirin was demonstrated to not only suppressing FN expression but also reversing FN-mediated inhibitory effect on cell motility and signaling pathway.

Conclusion: In conclusion, FN inhibited trophoblast invasion and could be a potential biomarker for preeclampsia. Aspirin may exert its prevention effect from preeclampsia development through suppressing FN expression and alleviating FN-mediated inhibitory effect on trophoblast invasion.

References:

Grants: National Cheng Kung University Hospital, Tainan, Taiwan (NCKUH-11102045); Ministry of Health and Welfare, Tainan, Taiwan (TNHPA11001).

Conflict of Interest: Meitsz Su National Cheng Kung University Hospital, Tainan, Taiwan (NCKUH-11102045); Ministry of Health and Welfare, Tainan, Taiwan (TNHPA11001).

EP06.002 Biology of telomeres in patients with coronary heart disease without acute cardiovascular events

Maxim Asanov ¹, Nadezhda Deeva¹, Anastasia Ponasenko¹

¹Research Institute for Complex Issues of Cardiovascular Diseases, Kemerovo, Russian Federation

Background/Objectives: Cardiovascular disease is the leading cause of death worldwide. Decrease or loss of function of myocardial cells or blood vessels is the cause of coronary heart disease. Telomeres are repetitive DNA sequences located at the ends of chromosomes that maintain genetic stability. The biology of telomeres is associated with several human disorders and diseases, especially cardiovascular diseases.

Methods: The study included 30 patients with ischemic disease and 30 healthy people. The standard phenol-chloroform extraction method was used to isolate the DNA. RNA isolation was carried out by the standard method using TRIzol. There was qPCR for measuring relative telomere lenght (RTL) and hTERT gene expression.

Results: The results of the study showed that the RTL of patients before surgery is 2 times shorter compared to this indicator of healthy people living in the same area (p < 0.01). The area under the ROC curve is 0.667 ± 0.045 (p = 0.01). Through a systematic analysis, it was found that the OCT in patients with coronary artery disease was significantly shorter than in the control group, while inversely correlated with the severity of coronary artery disease. The level of hTERT expression in both groups was extremely low, slightly higher in the control group (p > 0.05).

Conclusion: Presumably, inflammatory processes and oxidative stress, complementing each other, are the causes of irreparable damage to telomeres, accelerating the aging process and leading to irreversible consequences in atherogenesis. More research is needed to elucidate the biology of telomeres in atherogenesis.

References:

Grants: Program of Basic Research of Siberian Branch of the Russian Academy of Sciences (0419-2022-0001).

Conflict of Interest: None declared.

EP06.004 Combined exosomal and plasma non-coding RNA signature associated with urinary albumin excretion in hypertension

Ana Ortega ¹, Olga Martinez-Arroyo¹, Angela L Riffo-Campos², Javier Perez-Hernandez³, Ana Flores-Chova¹, Josep Redon¹, Raquel Cortés¹

¹Biomedical Research Institute Hospital Clinico - INCLIVA, Cardiometabolic and Renal Risk Research Group, Valencia, Spain; ²Universidad de La Frontera, Vicerrectoria Académica, Temuco, Chile; ³Université de Paris, Institut Co-chin, CNRS, INSERM, T-cell tolerance, Biomarkers and Therapies in Type 1 Diabetes Team, Paris, France

Background/Objectives: Non-coding RNA (ncRNA), released into circulation or packaged into exosomes, play important roles in many biological processes in the kidney. The purpose of the present study is to identify a common ncRNA signature from exosomes, urine and plasma associated with early renal damage and its related molecular pathways by constructing a RNA-based transcriptional network.

Methods: This is an observational case-control study which included twenty-one patients with essential hypertension (n = 21) and twenty-two without persistent elevated urinary albuminuria (UAE) (≥30 mg/g urinary creatinine). Three individual libraries (plasma and urinary exosomes and total plasma) were prepared from each hypertensive patient for ncRNA sequencing analysis. Next, a RNA-based transcriptional regulatory network was constructed.

Results: The three RNA biotypes with the greatest number of differentially expressed transcripts were long-ncRNA (lncRNA), microRNA (miRNA) and piwi-interacting RNA (piRNA). We identified a common 24 ncRNA molecular signature related to hypertension-associated albuminuria, of which lncRNA was the most representative. In addition, the transcriptional regulatory network analysis showed five lncRNA (LINC02614, BAALC-AS1, FAM230B, LOC100505824 and LINC01484), and the miR-301a-3p to play a significant role in network organization and to target critical pathways regulating filtration barrier integrity, tubule reabsorption and systemic endothelial dysfunction.

Conclusion: Our study found a combined ncRNA signature associated with albuminuria, independently of biofluid origin identifies a handful of potential targets involved in filtration barrier, tubule reabsorption and endothelial function that may be utilized to treating hypertension-associated albuminuria and cardiovascular damage progression.

References:

Grants: Health Institute Carlos III” [PI12/02615; PI19/01796]. European Regional Development Fund (ERDF).

Conflict of Interest: None declared.

EP06.005 Expression of innate immune response genes in the native heart valves obtained from patients with infective endocarditis

Maxim Sinitsky¹, Anna Sinitskaya ¹, Maxim Asanov¹, Yana Kazachek¹, Alexey Evtushenko¹, Anastasia Ponasenko¹

¹Research Institute for Complex Issues of Cardiovascular Diseases, Kemerovo, Russian Federation

Background/Objectives: Invective endocarditis (IE) is an inflammatory disease characterized by a dysfunction of heart valves and associated with high level of in-hospital mortality. Innate immune response playing the important role in the IE pathophysiology is genetically determined [1]. This study aimed to access the expression of TLR1, TLR2, TLR4 and TLR6 genes in the native heart valves obtained from IE patients.

Methods: The expression of TLR1, TLR2, TLR4 and TLR6 genes has been investigated in biopsies of native heart valves obtained from 26 patients with IE and from 12 patients who underwent surgical correction of non-infectious heart defects (control) using RT-qPCR with TaqMan probes. ACTB, GAPDH and B2M were used as a reference gene. The expression level was calculated by ΔCt method.

Results: We discovered no differences in the mRNA level of TLR2 in the native heart valves obtained from IE patients and from patients involved in the control group (fold-change was 0.97). At the same time, the expression of TLR4, TLR1 and TLR6 was significantly decreased in IE patients (fold-change was 0.65, 0.56 and 0.38, respectively) compared to the control.

Conclusion: A decreased activity of TLR-receptors leads to atypical forms of inflammation due to a failure of the innate immune response and an increased susceptibility of valve tissue to bacterial invasion.

References: 1. Weinstock M, Grimm I, Dreier J, et al. Genetic variants in genes of the inflammatory response in association with infective endocarditis. PLoS One. 2014; 9(10):e110151.

Grants: The fundamental research project of Research Institute for Complex Issues of Cardiovascular Diseases No. 0419-2022-0001.

Conflict of Interest: None declared.

EP06.006 Diagnostic yield of genetic testing in an unselected cohort of patients with congenital heart disease

Julie Hathaway¹, Marcos Cicerchia², Johanna Tommiska², Saija Ahonen ², Eija H. Seppala², Kimberly Gall¹, Alicia Scocchia¹, Inka Saarinen², Matias Rantanen², Jennifer Schleit¹, Tiia Kangas-Kontio², Massimiliano Gentile², Pertteli Salmenperä², Jussi Paananen², Samuel Myllykangas², Juha Koskenvuo²

¹Blueprint Genetics, Seattle, United States; ²Blueprint Genetics, Espoo, Finland

Background/Objectives: Chromosomal microarray analysis is typically the first genetic test offered to patients with congenital heart disease (CHD), particularly in syndromic cases where copy number variants (CNVs) and aneuploidies are more common. Few studies describe the genetic findings in CHD cohorts referred for Next Generation Sequencing (NGS) panel testing. We present the diagnostic yield of >200 patients with CHD receiving NGS panel testing to inform the utility of this test in these patients.

Methods: We undertook a retrospective review of 204 consecutive CHD patients who underwent testing for the Congenital Structural Heart Disease panel. Patients were considered syndromic if their requisition described ≥1 extracardiac anomaly. The identification of a pathogenic (P) or likely pathogenic (LP) variant(s) (with a modified ACMG/AMP classification scheme) was considered diagnostic. Chi-square analysis and Fisher’s exact test determined statistical significance (P value < 0.05).

Results: In this cohort, 14.2% of patients (n = 29) had a diagnostic test result. P/LP CNVs were identified in 3.4% (n = 7) of patients; two of these were intragenic. The diagnostic yield was significantly higher in patients with a syndromic presentation (n = 17, 20.7%) compared to an isolated presentation (n = 10, 9.5%) (P = 0.015). There was no significant difference in diagnostic yield by age group at the time of testing (P = 0.8737).

Conclusion: One in seven patients in this cohort received a diagnostic test result. CNVs were common; more than one quarter of these were intragenic. This work demonstrates that: high resolution CNV detection capabilities are important and NGS panel testing should be considered for patients with CHD.

References:

Grants:

Conflict of Interest: Julie Hathaway Blueprint Genetics, Marcos Cicerchia Blueprint Genetics, Johanna Tommiska Blueprint Genetics, Saija Ahonen Blueprint Genetics, Eija H. Seppala Blueprint Genetics, Kimberly Gall Blueprint Genetics, Alicia Scocchia Blueprint Genetics, Inka Saarinen Blueprint Genetics, Matias Rantanen Blueprint Genetics, Jennifer Schleit Blueprint Genetics, Tiia Kangas-Kontio Blueprint Genetics, Massimiliano Gentile Blueprint Genetics, Pertteli Salmenperä Blueprint Genetics, Jussi Paananen Blueprint Genetics, Samuel Myllykangas Blueprint Genetics, Juha Koskenvuo Blueprint Genetics.

EP06.008 Cardiomyopathies (CMs): from clinical characterization to Whole Exome Sequencing (WES) analysis in Italian patients

Beatrice Alessandrini ¹, Stefania Lenarduzzi², Matteo Dal Ferro³, Alessia Paldino^1;3, Gianfranco Sinagra^1;3, Paolo Gasparini^1;2, Giorgia Girotto^1;2

¹University of Trieste, Department of Medicine, Surgery and Health Sciences, Trieste, Italy; ²Institute for Maternal and Child Health – I.R.C.C.S. “Burlo Garofolo”, Medical Genetics, Trieste, Italy; ³Azienda Sanitaria Universitaria Giuliano Isontina (ASUGI), Cardiothoracovascular Department of Trieste, Trieste, Italy

Background/Objectives: CMs are functional and structural abnormalities of the heart muscle characterised by a high clinical and genetic variability with more than 70 genes so far described. The main aim is to define the genetic cause of CMs in patients carefully clinical selected.

Methods: 200 Italian cases (68 familial and 132 sporadic) were analysed by WES through the Illumina NextSeq 550 platform focused on an in-silico panel of 66 genes related to cardiac diseases and distinguished into the three categories of CMs (structural, channelopathies (CAPs) and aortopathies (AORTO)).

Results: Among the enrolled patients, 87.5% were clinically classified as CMs, 7% as CAPs, and 5.5% as AORTO. WES allowed the molecular characterization of 21.5% patients with the highest detection rate in familial cases (34% vs 15% sporadic ones). TTN, MYH7, MYBPC3, PKP2 and FLNC are the most mutated genes explaining 70% of the CMs cases. Three main findings are the identification of 1) two novel gross deletions in the PKP2 gene confirmed by MLPA and SNPs array 2) a novel missense variant in the KCNH2 gene in a family affected by Long-QT Syndrome and 3) a non-canonical splicing variant in the ACVRL1 gene in a woman affected by Rendu-Osler-Weber Syndrome.

Conclusion: This study highlighted the importance of a multidisciplinary approach for the characterization of CMs patients. The advent of next-generation sequencing technologies facilitated the molecular diagnosis of the CMs and shed light on the complexity of this class of diseases, providing important information for clinical management and recurrence risk estimation.

References: NA

Grants: NA

Conflict of Interest: None declared.

EP06.009 Genetic findings in a family with hereditary spherocytosis, haemolytic anaemia and pulmonary hypertension

Memoona Shaukat ^1;2;3, Mareike lankeit⁴, Ding Cao^1;2;3, Nicola Benjamin^2;3, Ekkehard Grünig^2;3, Michal Zawada¹, Katrin Hinderhofer¹, Christina Eichstaedt^1;2;3

¹Institute of Human Genetics, Laboratory for Molecular Genetic Diagnostics, Heidelberg, Germany; ²Thoraxklinik-Heidelberg gGmbH, Zentrum für Pulmonale Hypertonie, Heidelberg, Germany; ³Deutsches Zentrum für Lungenforschung, Translational Lung Research Center Heidelberg, Heidelbrg, Germany; ⁴Campus Virchow Klinikum (CVK), Charité - University Medicine, Department of Internal Medicine and Cardiology, Berlin, Germany

Background/Objectives: Genetic defects are known in up to 18 genes to cause pulmonary arterial hypertension (PAH). However, in many cases the genetic background is unclear. The objective of this study was to identify the genetic background of a family with several cases of hereditary spherocytosis (HS), cholelithiasis and pulmonary hypertension (PH).

Methods: Whole exome sequencing for four family members was performed. We included the index patient who manifests both PH and HS, daughter with spherocytosis only, healthy son and healthy wife of index patient. Variants in identified genes were sought in the database Online Mendelian Inheritance in Man to investigate the genotype-phenotype relationship.

Results: No pathogenic variant in any PAH gene could be identified in the index patient. Instead, a missense variant, c.2204C>T p. (Ala735Val), in a gene causative of HS, Solute Carrier Family 4 Member 1 (SLC4A1), was identified in the index patient and his daughter. The variant was absent in the healthy members of the family. It was barely present in controls (0.003%), classified inconclusive by in silico prediction programs, but showed co-segregation with the disease in two family members. No further variant could be identified in the other genes responsible for HS (ANK1, SPTA1, SPTB, EBP42).

Conclusion: The identified variant in the SLC4A1 gene might be causative for HS, hemolytic anemia and cholelithiasis as identified in the index patient and his daughter. All PH patients had undergone splenectomy prior to the onset of the disease. Therefore, PH in this family has been classified as group V, PH due to hematological disorders.

References:

Grants:

Conflict of Interest: None declared.

EP06.010 Hereditary Hemorrhagic Telangiectasia: role of MGP as modifier gene

Claudia Cantarini ¹, Anna Sbalchiero¹, Giuseppe Spinozzi², Fabio Pagella^2;3, Elina Matti², Elisabetta Buscarini⁴, Guido Manfredi⁴, Carla Olivieri¹

¹University of Pavia - Unit of General Biology and Medical Genetics, Molecular Medicine, Pavia, Italy; ²Fondazione IRCCS Policlinico San Matteo, UOC of Otorhinolaryngology, Pavia, Italy; ³University of Pavia, Surgical Sciences, Pavia, Italy; ⁴ASST Ospedale Maggiore di Crema, Crema, Italy

Background/Objectives: Hereditary Hemorrhagic Telangiectasia (HHT) is a rare autosomal dominant disease. Genes associated are involved in the TGF-β/BMPs signaling pathway; arteriovenous malformations in lungs (pAVMs) are a clinical sign of HHT. Matrix Gla Protein (MGP) encodes for an extracellular protein, which inhibits BMP2 and BMP4, regulating ALK1 and VEGF expression. It also plays a protective role in pAVMs development in HHT-mouse model. The study aims to search for MGP variants in HHT patients.

Methods: In 40 pAVMs-HHT patients and 11 non-pAVMs HHT relatives, the whole MGP gene was sequenced by Sanger. The variants’ allele frequency was compared to GnomAD database controls, using Chi-Squared Test.

Results: Eight variants were observed, one in 5′UTR, three in 3′UTR, one in the coding region and three in deep intronic regions. They are present in heterozygous and homozygous state in pAVMs patients, while they are present only in heterozygous state in non-pAVMs patients. The haplotype reconstruction on chromosome 12 was possible for 12 families. Chi-Squared Test produced significative p values.

Conclusion: These preliminary results, together with evidence from literature, suggest a role of MGP in affecting HHT-pAVMs. This can be related to the regulation of HHT-related pathways involving BMP4, ALK1 and VEGF by MGP. Haplotype reconstruction suggests a common pattern for the totality of HHT-patients. The statistically significant differences in allele frequencies between patients and controls encourage the hypothesis of MGP-involvement in HHT phenotypic modulation.

References:

Grants: Italian Ministry of Education, University and Research to the DMM-University of Pavia “Dipartimenti di Eccellenza (2018-2022)”.

Conflict of Interest: None declared.

EP06.011 Ventricular fibrillation during acute myocardial infarction could be associated with a novel missense variant in the Fibroblastic Growth Factor (EGF) gene

Luis Mariñas Pardo¹, Ricardo Pan-Lizcano ¹, Lucía Núñez Fernández¹, Pablo Piñón², Xacobe Flores², Guillermo Aldama², Ramón Calvino-Santos², José Manuel Vázquez Rodríguez³, Manuel Hermida-Prieto¹

¹Instituto de Investigación Biomédica de A Coruña (INIBIC)-CHUAC-UDC, Cardiology Research Group, A Coruña, Spain; ²Complexo Hospitalario Universitario de A Coruna (CHUAC)-INIBIC, Department of Cardiology, A Coruña, Spain; ³Complexo Hospitalario Universitario de A Coruña (CHUAC)-CIBERCV, Instituto de Investigación Biomédica de A Coruña (INIBIC), Universidad de A Coruña (UDC), Department of Cardiology, A Coruña, Spain

Background/Objectives: Ventricular fibrillation (VF) during acute myocardial infarction (AMI) is considered the most serious cardiac rhythm disturbance, causing collapse and cardiac arrest. However, the understanding of the genetic contribution to VF is very limited. Variants in genes related to ion homeostasis may increase the risk of developing such cardiac events.

Methods: Rare genetic variants in 244 genes encoded proteins were analysed by NGS in 46 patients with FV during AMI. Visualization of variants was performed using IGV and the bioinformatic analysis of its possible effect was performed with MutationTester, SNAP2, SIFT2, Polyphen and PhD-SNP.

Results: The variant c.1752A>C in EGF gene was identified in a 50 years old male, leading to the aminoacidic substitution p.Lys584Phe.

It encodes a member of the EGF superfamily, that after processing leads to the 53-amino acid EGF peptide. Previous studies have shown that EGF is involved in regulation of various epithelial ion channels that govern Na⁺, K⁺, Cl⁻ or Mg²⁺ homeostasis. Its receptor (EGFR) has also a role in maintaining contractile homeostasis under physiologic conditions in the adult heart.

EGF acts as an autocrine/paracrine magnesiotropic hormone that stimulates Mg²⁺ reabsorption in the kidney. Mutations in this gene are associated to a renal reabsorption defect, leading to hypomagnesaemia with normocalciuria and normocalcaemia.

Conclusion: c.1752A>C in EGF gene may have a pathological role in the development of VF during AMI due to a constitutive ionic disbalance.

References:

Grants: Instituto de Salud Carlos III (PI18/01737)-FEDER.

Conflict of Interest: None declared.

EP06.013 Complex arrythmogenic right ventricular cardiomyopathy – case presentation and genetic mutation screening by NGS

Tsenka Boneva ^1;1, Kiril Karamfilov¹, Raya Ivanova², Galina Zlatancheva¹, Monika Shumkova¹, Teodora Yaneva¹, Dobrin Vassilev³, Kristina Stoyanova¹, Ivanka Dimova⁴

¹UMHAT Alexandrovska, Cardiology, Sofia, Bulgaria; ¹UMHAT Alexandrovska, Cardiology, Sofia, Bulgaria; ³SBALK „Medica COR“, Department of Cardiology, Rousse, Bulgaria; ⁴Medical University Sofia, Department of Medical genetics, Sofia, Bulgaria

Background/Objectives: Arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C) is characterized by progressive fat or fibrofatty replacement of the right ventricular (RV) myocardium. The cause of ARVD/C is not yet clear, but recent studies suggest it is most often related to genetic mutations. Left ventricular (LV) or biventricular involvement are increasingly identified in ARVD/C patients. The genetic background of cardiomyopathies could shed light on the mechanism of their development, clinical presentations and new treatment options.

Methods: We describe here a patient affected by arrythmogenic right ventricular cardiomyopathy with left ventricular involvement, with the presence of coronary artery anomalies. There were aneurisms of coronary arteries and left circumflex artery (LCx) arises from right coronary sinus. Physiological, imaging and invasive study was performed in details. In addition, genetic analysis by next generation sequencing (NGS), using panel of genes for hereditary cardiomyopathy, was done.

Results: We detected a missense mutation in MYBPC3 gene (c.1316G>A, p.Gly439Asp), classified as variant of unknown significance. We discuss myosin-related mutations in different cardiomyopathies with dosage-dependent effects of MYBPC3 on myosin that occur across the cardiac cycle.

Conclusion: The association of MYBPC3 mutation with ARVD/C is worthy to be investigated, since it could have an important impact on the application of new treatment, using specific myosin targeted agents.

References: 1. Vimalanathan, A. K., Ehler, E. & Gehmlich, K. Genetics of and pathogenic mechanisms in arrhythmogenic right ventricular cardiomyopathy. Biophys. Rev. 10, 973–982 (2018).

2. Hoorntje, E. T. et al. Arrhythmogenic cardiomyopathy: Pathology, genetics, and concepts in pathogenesis. Cardiovasc. Res. 113, 1521–1531 (2017).

Grants:

Conflict of Interest: None declared.

EP06.014 No effect of CRP rs1800947, TNF-α rs1800629 and IL6 rs1800795 polymorphisms on plasma levels of inflammatory markers after treatment with PCSK9 inhibitors

Tina Levstek ¹, Nik Podkrajšek¹, Inge Sotlar¹, Andreja Rehberger Likozar²

¹Institute of Biochemistry and Molecular Genetics, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia; ²Department of Vascular Diseases, University Medical Centre Ljubljana, Ljubljana, Slovenia

Background/Objectives: Inflammation plays a key role in the pathogenesis of atherosclerosis. However, the role of genetic variability on inflammation after treatment with proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitors remains to be elucidated. For the first time, we examined the influence of polymorphisms in CRP, TNF-α, and IL6 genes on plasma levels of hsCRP, TNF-α, and IL6 at baseline and after treatment with PCSK9 inhibitors.

Methods: A total of 69 patients with stable coronary artery disease after a premature myocardial infarction were included in the study. All patients had extremely elevated lipoprotein(a) levels and received a PCSK9 inhibitor. Genotyping for CRP rs1800947, TNF-α rs1800629, and IL6 rs1800795 was performed.

Results: Our results showed no significant association between single nucleotide polymorphisms in CRP, TNF-α, and IL6 and plasma levels of hsCRP, TNF-α, and IL6, respectively. Consistent with previous studies, no significant change in levels of inflammatory biomarkers was observed after 6 months of treatment with PCSK9 inhibitors. Moreover, genetic variability in selected genes was not significantly associated with the change in plasma levels of corresponding inflammatory markers.

Conclusion: Genetic variability did not affect plasma levels of inflammatory markers, which could be due to background therapy with statins or extremely elevated lipoprotein(a) levels, because lipoprotein(a) itself contributes to inflammation. Further studies are needed to clarify which factors contribute most to the modulation of inflammation in high-risk patients.

References: Ruscica et al., Atherosclerosis, 2019.

Grants: The study was funded by the Slovenian Research Agency (P1-0170, P3-0308). T.L. was granted a scholarship from the University Foundation of ing. Lenarčič Milan.

Conflict of Interest: None declared.

EP06.015 Circulating microRNAs as biomarkers for pulmonary arterial hypertension

Mauro Lago-Docampo¹, Jesús Eduardo Martín-Salazar ¹, Ainhoa Iglesias-López¹, Isabel Blanco², Adolfo Baloira Villar³, Joan Albert Barbera², Diana Valverde Pérez¹

¹CINBIO, Universidade de Vigo, Vigo, Spain; ²Hospital Clínic de Barcelona, Barcelona, Spain; ³Complexo Hospitalario Universitario de Pontevedra, Universidad de Vigo, Pontevedra, Spain

Background/Objectives: Pulmonary Arterial Hypertension (PAH) is a rare disease where the thickening of the precapillary pulmonary arteries ends up inducing right heart failure. The prognosis of PAH patients depends on multiple factors, being the time of diagnosis a critical one. Currently, diagnosis is complicated and usually delayed until performing right-heart catheterization.

Methods: We perform small RNA sequencing in plasma of idiopathic PAH patients and controls. We used classification models to analyse the potential of the microRNAs, that we found differentially expressed, as PAH predictors. Also, we use miRBase to predict the targets for the dysregulated miRNAs we detected. Finally, we performed functional assays based on qPCR and western blotting to confirm our results.

Results: We were able to find 29 differentially expressed microRNAs and validate 7 of them in a nationwide cohort (let-7a-5p, let-7b-5p, let-7c-5p, let-7f-5p, miR-9-5p, miR-31-5p, miR-3168). In our cohort, we obtained a model with an AUC of 0.738. Also, we identified miR-3168 as a novel upregulated miRNA in PAH patients. We demonstrate that it targets the Bone Morphogenetic Protein Receptor type 2 (BMPR2), as validated at mRNA and protein levels. Preliminary results show that miR-3168 overexpression increases resistance to apoptosis and enhanced angiogenesis.

Conclusion: We found novel downregulated and upregulated microRNAs in idiopathic PAH patients. We were able to develop a 3-microRNA signature for diagnosis and functionally characterized in vitro the effect of miR-3168 as a possible modulator of the disease.

References:

Grants:

Conflict of Interest: None declared.

EP06.016 22q11.2 microdeletion is the most common genomic abnormality in Serbian newborns with critical congenital heart disease and could be rapidly detected by Multiplex ligation probe amplification analysis

Aleksandra Miletic¹, Goran Cuturilo ^1;2, Jelena Ruml Stojanovic¹, Danijela Drakulic³, Marija Mijovic¹, Brankica Bosankic¹, Hristina Petrovic¹, Milena Stevanovic^3;4;5

¹University Children’s Hospital, Belgrade, Department of Clinical Genetics, Belgrade, Serbia; ²Faculty of Medicine, University of Belgrade, Belgrade, Serbia; ³Institute of Molecular Genetics and Genetic Engineering, University of Belgrade, Belgrade, Serbia; ⁴Faculty of Biology, University of Belgrade, Belgrade, Serbia; ⁵Serbian Academy of Sciences and Arts, Belgrade, Serbia

Background/Objectives: Genetic tests may facilitate rapid and effective diagnostics but unfortunately their high costs usually limit their application in all patients (1). We aimed to investigate the utility of rapid, cost effective and high sensitive Multiplex ligation probe amplification analysis (MLPA) for detection copy number variants (CNV) in newborns with critical CHD, admitted to the Neonatal Intensive Care Unit (NICU).

Methods: Study included 100 consecutive newborns admitted to the NICU, University Children’s Hospital in Belgrade from August 2014 to September 2019. Patients with viable trisomies (21, 18 and 13) were excluded. All participants were tested by MLPA analysis using SALSA MLPA P250-B2 Di George and SALSA MLPA P311-B1 Congenital Heart Disease probemixes (MRC Holland, The Netherland).

Results: Pathogenic CNVs were identified in ten (10%) patients. Nine of them had 22q11.2 deletion detected by both kits while one patient had 3p25 deletion detected by P311 kit.

Conclusion: Genetic evaluation of all newborns with critical CHD admitted to the NICU by rapid and inexpensive MLPA analysis using combination P250 and P311 SALSA probemixes could contribute to high detection rate of pathogenic variants.

References: 1. Monteiro RA, Freitas ML, Vianna GS et al. (2017) Major contribution of genomic copy number variation in syndromic congenital herat disease: the use of MLPA as the first genetic test. Mol Syndromol 8: 227-23.

Grants: This work was supported by the Ministry of Education, Science and Technological Development of the Republic of Serbia (Agreement number 451-03-68/2020-14/200042) and the Serbian Academy of Sciences and Arts (MIKRONEURO_no. 01-2021).

Conflict of Interest: None declared.

EP06.017 Is the phenotype for ARVC caused by TMEM43 (p.S358L) becoming more severe in women over time?

Ashley Baker¹, Stephen Duffett², Fiona Curtis³, Emily Bonnell³, Susan Stuckless⁴, Sean Connors², Fred Paulin², Lesley Turner³, Terry-Lynn Young⁵, Kathy Hodgkinson ⁴

¹Memorial University of Newfoundland, St. John’s, Canada; ²Memorial University of Newfoundland, Division Of Cardiology, St. John’s, Canada; ³Eastern Health, Provincial Medical Genetics Program, St. John’s, Canada; ⁴Memorial University of Newfoundland, Unit of Clinical Epidemiology, St. John’s, Canada; ⁵Memorial University of Newfoundland, Faculty of Medicine, St. John’s, Canada

Background/Objectives: Autosomal dominant arrhythmogenic right ventricular cardiomyopathy (ARVC) due to TMEM43 p.S358L is a sex influenced cause of sudden cardiac death (median age 40 yrs. in men, 67yrs. in women) and heart failure. We have demonstrated that the implantable cardioverter-defibrillator (ICD) significantly improves survival and described the natural history and clinical course using 27 large multiplex families. Anecdotal evidence suggests that women are presenting with an ICD discharge ≥240 beats per minute (previously shown to be a death equivalent) earlier than family history would suggest. We wished to investigate temporal changes in severity using our dataset spanning 100 years.

Methods: We compared 76 affected women (obligate carriers and/or positive for p.S358L TMEM43) in each of two birth cohorts (DOB 1960–1999 and DOB 1920–1959). Using SPSS v 22 we performed Kaplan Meier survival analysis, and cox proportional regression to the end point of death or an ICD firing of ≥240 beats per minute.

Results:

Cohort	N	# Events	Survival time KM		Test of equality KM			Cox PR
			Mean	Std. Error	Log Rank	Breslow	Tarone Ware
1920-1959	76	32	75.3	2.26	p = 0.06	p = 0.03	p = 0.04	p = 0.06
1960-1999	76	10	52.7	1.08

Conclusion: There is a trend toward earlier presentation in the younger cohort. We will re-analyse, adjusting for life expectancy differences between 1920 and 1999. These results suggest that non- genetic influences may be affecting the younger cohort, thus women may not be as protected as previously thought. Historic family data is powerful as it allows temporal changes to be seen.

References:

Grants:

Conflict of Interest: None declared.

EP06.018 Aortic disease and cardiomyopathy in patient with a novel variant in the DNMT3A gene causing Tatton-Brown-Rahman syndrome

Dovile Jancauskaite ^1;2, Ruta Masiuliene^2;3, Egle Sadauskiene^2;4, Jurate Barysiene^2;4, Violeta Mikstiene¹, Egle Preiksaitiene¹

¹Department of Human and Medical Genetics, Institute of Biomedical Sciences, Faculty of Medicine, Vilnius University, Vilnius, Lithuania; ²Centre of Cardiology and Angiology, Vilnius University Hospital Santaros klinikos, Vilnius, Lithuania; ³Faculty of Medicine, Vilnius University, Vilnius, Lithuania; ⁴Clinic of Cardiac and Vascular Diseases, Institute of Clinical Medicine, Faculty of Medicine, Vilnius University, Vilnius, Lithuania

Background/Objectives: Tatton-Brown-Rahman syndrome (TBRS) is an autosomal dominant overgrowth syndrome caused by heterozygous and usually de novo pathogenic variant in DNMT3A. This syndrome was first described in 2014, since then an increasingly wider spectrum of neurological, skeletal and cardiological clinical features has been found in adults.

Methods: We present a 34-year-old male who was referred to a cardiologist due to episodes of weakness, dizziness, and a cold sweat. The patient had psychomotor retardation, tall stature, obesity, kyphoscoliosis, coarse facial features, synophrys and corrected pectus excavatum in childhood. Cardiovascular evaluation revealed left ventricular (LV) dilatation with non-compaction, aortic root dilatation, mitral prolapse with moderate regurgitation and short episodes of ventricular tachycardia. During follow-up, aortic dilatation (from 5.0 to 6.0 cm) and mitral valve regurgitation (from moderate to severe) progressed, indicating the need for surgical correction.

Results: Genetic analysis revealed a likely pathogenic heterozygous variant of the DNMT3A gene NM_022552.4:c.2324C>A, NP_072046.2:p.(Ser775Tyr). This variant has not been described in literature before. Segregation analysis in the family showed that c.2324C>A variant was inherited from the 58-year-old mother. Additionally, the genetic alteration was identified to the 39-year-old brother of the proband. Both affected relatives have LV dilatation, mild to moderate mitral valve regurgitation. Furthermore, the older brother was diagnosed with aortic root dilatation (4.5 cm).

Conclusion: Clinical examination of the affected individuals in the family showed variable expressivity and incomplete penetrance of specific clinical features of TBRS, including the aortic disease and cardiomyopathy, which might be major complications of the syndrome and require specific therapeutic management.

References:

Grants: None.

Conflict of Interest: None declared.

EP06.019 Exploiting the Whole Exome Sequencing for the identification of new candidate genes associated with Brugada Syndrome

Giulia Molli¹, Valeria Kirpichnikova², Anna Santoni¹, Matteo Di Giacobbe³, Giovanni Battista Pipitone³, Andrea Villatore², Stefania Merella³, Silvia Bonfiglio⁴, Giovanni Peretto⁵, Simone Sala⁵, Sara Benedetti³, Chiara Di Resta ^1;2

¹IRCCS San Raffaele Hospital, Genomic Unit for the diagnosis of Human Pathologies, Milan, Italy; ²Vita-Salute San Raffaele University, Milan, Italy; ³IRCCS San Raffaele Hospital, UOC Clinical Genomics, Milan, Italy; ⁴IRCCS San Raffaele, Center for Omics Science, Milan, Italy; ⁵IRCCS San Raffaele Hospital, Department of Cardiac Electrophysiology and Arrhythmology, Milan, Italy

Background/Objectives: Brugada Syndrome (BS) is an inherited arrhythmogenic disease with risk of sudden cardiac death in young asymptomatic adults. SCN5A is the only known causative gene, though 22 genes are associated with BS susceptibility; 70% patients remain genetically undiagnosed. To identify new candidate genes, we performed Whole Exome Sequencing (WES) of 172 patients, both sporadic and familiar cases. To investigate the oligogenic hypothesis, 22 SCN5A-positive patients were enrolled too.

Methods: WES was performed on Illumina Platforms (mean coverage: 180X, 97% target region >20X). Reads were analyzed using Dragen Bio IT Platform, coding and splice regions variants (MAF≤0.01%) prioritized and classified according to ACMG guidelines, with support of eVai-EnGenome software. Burden Test extrapolated genes with higher mutation burden, suggesting a possible pathogenic role.

Results: Most prioritized variants were missense (88%) located in new candidate genes belonging to ion channels (25%), sarcomeric (55%), desmosomal (8%) and nuclear proteins (5%), consistent with disease phenotype. In particular, burden test confirmed the role of SCN5A and highlighted also possible association of three genes encoding cytoskeletal and channel proteins.

Conclusion: Our data identified new candidate genes which should be further investigated and confirmed in a larger cohort, evaluating also possible copy number variations. Genotype-phenotype correlations will be performed to better stratify patients, taking into account also the putative oligogenic inheritance of the disease, evaluating the possible role of multiple variants in the clinical phenotype.

References:

Grants: Funded by Italian Ministry of Health (GR-2016-02362316).

Conflict of Interest: None declared.

EP06.020 DNA methylation patterns in NSTEMI and unstable angina pectoris patients

Walter Pulverer ¹, Silvia Schoenthaler¹, Tanja Zeller^2;3, Mahir Karakas^2;3, Vita Rovite⁴, Janis Klovins⁴, Andreas Weinhaeusel¹, Christa Nöhammer¹

¹AIT Austrian Institute of Technology GmbH, Molecular Diagnostics, Vienna, Austria; ²University Medical Center Eppendorf, Clinic for Intensive Care Medicine, Hamburg, Germany; ³German Center for Cardiovascular Research (DZHK), Hambug, Germany; ⁴Latvian Biomedical Research and Study Centre, Riga, Latvia

Background/Objectives: Acute coronary syndrome (ACS) contributes significantly to mortality accounting for nearly one third of all deaths worldwide. ACS can be divided into ST-segment elevation myocardial infarction (STEMI), non-ST-segment elevation infarction (NSTEMI) and unstable angina pectoris (uAP). There is a need for accurate diagnostic tools for the early identification and monitoring of CAD, also with respect for the differentiation of NSTEMI and uAP. The role of DNA methylation has attracted attention in the field of CAD diagnostics.

Methods: A genome-wide DNA methylation discovery was performed (Illumina EPIC BeadChips; n = 23 NSTEMI, n = 25 uAP, n = 24 NCCP). Technical and biological validation on 89 selected methylation marks was done using a qPCR approach based on methylation sensitive restriction enzymes.

Results: A DNA methylation pattern differentiating NSTEMI, uAP and NCCP was identified. 89 single CpGs with p < 0.05 and a delta-β of >0.15 were selected for further validation. Validation of selected CpGs confirmed differentiation of NSTEMI and uAP from controls with high accuracy (AUC: 0.83; sensitivity: 0.84; specificity: 0.68). Clear differentiation of NSTEMI and uAP was hampered by biological similarity of theses two groups (AUC-value: 0.71; sensitivity: 0.72; specificity: 0.6).

Conclusion: A unique epigenetic profile is present in the investigated sample cohort. However, the epigenetic profile between NSTEMI and uAP is quite similar and the observed DNA methylation differences between the two groups are low. Nonetheless, we were able to confirm a set of 14 CpGs between NSTEMI/uAP and NCCP, as well as 7 CpGs, which may facilitate the differentiation of NSTEMI vs. uAP.

References:

Grants:

Conflict of Interest: None declared.

EP06.021 Validation of new gene variant classification methods: a field-test in diagnostic cardiogenetics

Mohamed Alimohamed¹, Helga Westers¹, Yvonne J Vos¹, Joeri Van Der Velde¹, Rolf Sijmons¹, Paul van der Zwaag¹, B. Sikkema-Raddatz¹, Jan Jongbloed ¹

¹University of Groningen, University Medical Center Groningen, Dept. of Genetics, Groningen, Netherlands

Background/Objectives: In the molecular genetic diagnostics of Mendelian disorders, solutions are needed for the major challenge of dealing with the large number of variants of uncertain significance (VUS) identified using next-generation sequencing (NGS). Recently, promising approaches to calculate case excess scores (CE) and etiological fractions (EF) and gnomAD-derived constraint metrics scores have been reported that estimate the likelihood that rare variants in specific genes or regions are pathogenic. Our objective was to study the usability of these scores into diagnostic variant interpretation, using our clinical cardiomyopathy cohort.

Methods: Patients (N = 2002) referred for clinical genetic diagnostics underwent NGS testing of 55-61 genes associated with cardiomyopathies. Previously classified likely pathogenic (LP) and pathogenic (P) variants were used to validate the use of data from CE, EF and gnomAD constraint analyses for (re)classification of associated variant types in specific cardiomyopathy subtype-related genes. Next, we applied these constraint data to interpret and (re)classify 1229 variants (identified in 812 patients) previously classified as VUS.

Results: Using constraint scores in variant interpretation and classification, the classifications of (L)P’s was corroborated in 94% (354/378) of cases. Moreover, 23 unique VUSs were reclassified to LP, increasing the diagnostic yield with 1.2%. In addition, 106 unique VUSs (5.3% of patients) were prioritized for co-segregation or functional analyses.

Conclusion: Our analysis confirms that the use of constraint metrics data can improve cardiogenetic variant interpretation and classification. We therefore recommend the inclusion of constraint scores in variant interpretation protocols and to also apply these in other cohorts and disorders.

References:

Grants:

Conflict of Interest: None declared.

EP06.022 A case report of a truncating variant altering the extreme C-terminal region of desmoplakin (DSP) suggests its crusial functional role

Eleni-Malena Pantou ¹, POLYXENI GOURZI¹, Alexandros Tsoutsinos², Dimitrios Degiannis¹, Aris Anastasakis³

¹Onassis Cardiac Surgery Center, Molecular Immunopathology and Histocompatibility Unit, Division of Genetics, Kallithea-Athens, Greece; ²Onassis Cardiac Surgery Center, Department of Pediatric Cardiology, Kallithea-Athens, Greece; ³Onassis Cardiac Surgery Center, Unit of Inherited and Rare Cardiovascular Diseases, Kallithea-Athens, Greece

Background/Objectives: Homozygous DSP truncating mutations located in the C-terminal region of DSP are known to mainly cause Carvajal syndrome, an autosomal recessive syndromic form of arrhythmogenic cardiomyopathy with an extra-cardiac cutaneous phenotype.

Methods: The index case and her relatives underwent full cardiological assessment. Genetic analysis of the index case was performed using SOPHiA Extended Cardio solution and Sanger sequencing was used to screen members of the family for the presence of the reported mutation.

Results: We describe a female proband with a severely manifesting arrhythmogenic left ventricular cardiomyopathy and a syncopal episode at the age of 10, who was found homozygous for the novel DSP mutation: NM_004415.4:c.8586delC, p.(Ser2863Hisfs*20) at the extreme C-terminal region of the protein, just 8 amino acids upstream the stop codon. She did not have any of the typical dermatological symptoms that characterize Carvajal syndrome. Her brother had died suddenly during exercise and was post mortem found homozygous for the same variant, while their parents were found heterozygous. When interviewed they stated as region of origin the same geographic area of Greece but they were not aware of any common ancestor. Detailed clinical examination revealed that the mother displayed a mild arrhythmic profile, while the father was asymptomatic.

Conclusion: These observations pinpoint to a significant functional role of the extreme C-terminal tail of the protein.

References:

Grants: GR i CARDIAC NET: Greek National Network of Precision Medicine in Cardiology and the Prevention of Sudden Death in the Young.

Conflict of Interest: None declared.

EP06.026 Molecular genetics profile of inherited cardiomyopathies in Bahrain

CRISTINA SKRYPNYK ¹, Neale Kalis², Leena Sulaibeekh², Mary Jozeph Lynch², Adel Khalifa², Noureddine Ben Khalaf³, Sfoug AlShammary⁴

¹Al Jawhara Center, Arabian Gulf University, Molecular Medicine, Manama, Bahrain; ²Bahrain Defense Force Royal Medical Services Military Hospital, Cardiac Center, Manama, Bahrain; ³College of Graduate Studies, Arabian Gulf University, Life Sciences Department, Health Biotechnology Program, Manama, Bahrain; ⁴Arabian Gulf University, Regenerative Medicine Center, Manama, Bahrain

Background/Objectives: Cardiac diseases are a priority for the health system and the cardiomyopathies are known for their impact into morbidity and mortality. This study, the first in Bahrain, aims to investigate the inherited cardiomyopathies molecular genetics profile and identify a phenotype-genotype correlation.

Methods: 58 patients diagnosed with cardiomyopathies were recruited from Cardiac Center, Bahrain Defense Force Hospital. Genomic DNA was extracted from peripheral blood and saliva samples. Gene sequencing and deletion/duplication analysis was performed using a next generation sequencing (NGS) panel of 106 genes linked with inherited cardiomyopathies. The candidate disease-causing variants were verified by Sanger sequencing, MLPA-seq, array CGH. PolyPhen-2 was used to analyze mutation effect and to predict the effect of the variants of unknown significance (VOUS).

Results: Pathogenic variants were identified in 17/58 patients (29.3%). TNNT2, MYBPC3, MYH7, CACNA1C, PTPN11, MYBPC3, MYH7 and FHL1 genes mutations were identified in 14/17 hypertrophic cardiomyopathy. ALMS1 and TTN genes mutations were found in 3/17 patients with dilated cardiomyopathy. Syndromic cardiomyopathies (Alstrom Syndrome, Noonan Syndrome, Emery Dreyfuss dystrophy) were identified in 4/17 patients. The cardiology management was immediately adjusted for 6/17 patients (4/6 for ICD, 2/6 for medication). 131 VOUS in 52/106 genes were identified in 41/58 patients and 89/131 VOUS were assessed by bioinformatics algorithms as possible damaging.

Conclusion: Genetic results provided a certitude causal diagnosis in 29.3% of the patients, establishing a genotype-phenotype correlation, offering a better estimated prognosis, an accurate genetic counselling and providing therapeutic guidance for the treating cardiologists.

References: Invitae Cardiomyopathy Comprehensive panel https://www.invitae.com.

Grants: AGU G-E016-Pi-11/18.

Conflict of Interest: CRISTINA SKRYPNYK Al Jawhara Center, Arabian Gulf University, Research grant G-E016-PI-11/18 PI, Neale Kalis Cardiac Center, Bahrain Defense Force Royal Medical Services Military Hospital, Research grant G-E016-PI-11/18 collaborator, Leena Sulaibeekh Cardiac Center, Bahrain Defense Force Royal Medical Services Military Hospital, Research grant G-E016-PI-11/18 collaborator, Mary Jozeph Lynch Cardiac Center, Bahrain Defense Force Royal Medical Services Military Hospital, Research grant G-E016-PI-11/18 collaborator, Adel Khalifa Cardiac Center, Bahrain Defense Force Royal Medical Services Military Hospital, Research grant G-E016-PI-11/18 collaborator, Noureddine Ben Khalaf Life Sciences Department, Health Biotechnology Program, College of Graduate Studies, Arabian Gulf University, Research grant G-E016-PI-11/18 collaborator, Sfoug AlShammary Regenerative Medicine Center, Arabian Gulf University, Research grant G-E016-PI-11/18 collaborator.

EP06.028 Ryanopathy in two Iranian families with premature coronary artery disease and sudden cardiac death

Maryam Asadnezhad ¹, Sepideh Mehvari¹, fatemeh ghodratpour¹, Sara Saki², Kaveh Hosseini³, Saeed Sadeghian³, Reza Malekzadeh², Hossein Najmabadi¹, Kimia Kahrizi¹

¹Genetics Research Center, University of Social Welfare and Rehabilitation Sciences, Tehran, Iran; ²Digestive Disease Research Institute, Tehran University of Medical Sciences, Tehran, Iran; ³Department of Cardiology, Tehran Heart Center, Tehran University of Medical Sciences, Tehran, Iran

Background/Objectives: Coronary artery disease (CAD), assigns 32% of all deaths to itself with an estimated 17.9 million deaths annually. Approximately 50% of all CAD-related deaths are attributed to sudden cardiac death (SCD) and the proportion of all SCDs resulting from CAD are about 80%. Four etiologies exist for SCD, comprising CAD, cardiomyopathies, cardiac channelopathies, and aortopathy. Ryanodine receptors (RyRs), of which, three members are known, are calcium (Ca2+) release channels located on the endoplasmic/sarcoplasmic reticulum (ER/SR). RyR2 is the major (Ca2+) channel in cardiomyocytes, in which mutations can lead to cardiac arrhythmia and SCD. Due to several evidence on the contribution of rare variants to CAD, we aimed at identifying the potential rare disease-causing variants in Iranian patients with premature CAD and a positive family history for the disease.

Methods: Whole exome sequencing (WES) was performed on the proband of each family followed by Sanger sequencing for confirmation of the results and co-segregation study.

Results: We identified two heterozygous missense variants, p.Arg3260Trp and p.Thr1730Met, in RYR2 gene (OMIM:*180902) in two unrelated families with history of CAD and SCD. Both variants have been recently reported in ClinVar database with ventricular tachycardia, catecholaminergic polymorphic, 1 (CPVT1) and/or cardiomyopathy, classified as VUS.

Conclusion: There are some forms of CAD with familial segregation consistent with being a single gene disorder. In a cohort of premature CAD Iranian patients, RYR2 variants have been identified in two families, one suffered from SCD. Identifying asymptomatic individuals prior to disease onset can help to disease prevention and management.

References:

Grants: D/206/1851 to Kimia Kahrizi.

Conflict of Interest: None declared.

EP06.029 Heterozygous ABCG8 variant as the cause of premature coronary artery disease in an Iranian family

Sepideh Mehvari ¹, nahid karimian fathi¹, fatemeh ghodratpour¹, Reza Najafipour¹, Kaveh Hosseini², Saeed Sadeghian², Reza Malekzadeh³, Hossein Najmabadi¹, Kimia Kahrizi¹

¹Genetics Research Center, University of Social Welfare and Rehabilitation Sciences, Tehran, Iran; ²Department of Cardiology, Tehran Heart Center, Tehran University of Medical Sciences, Tehran, Iran; ³Digestive Disease Research Institute, Tehran University of Medical Sciences, Tehran, Iran

Background/Objectives: Coronary artery disease (CAD) is the major cardiovascular disease with an estimated 17.9 million deaths annually. One of the major pathways with a significant influence on CAD is lipid metabolism. Sitosterolemia 1 [STSL1;MIM:#210250], characterized by elevated cholesterol and plant sterols (xenosterols) in plasma, is associated with premature coronary atherosclerosis that results from mutations in a member of ABC transporters, known as ABCG8. Sterolins, comprised of ABCG8 and ABCG5, form an obligate heterodimer that pumps xenosterols/cholesterol out of enterocytes and hepatocytes back into the intestinal lumen and bile canaliculi. This function is severely affected upon ABCG5/8 mutations. In our study, we aimed at identifying the potential rare disease-causing variants in Iranian patients who show familial clustering of premature CAD (PCAD).

Methods: Whole-exome sequencing was performed on the proband of each family followed by Sanger sequencing for confirmation of the results and co-segregation analysis.

Results: We identified a likely pathogenic missense variant [c.G562C(p.V188L)] in ABCG8 gene (MIM:*605460) in an Iranian family affected by PCAD with autosomal dominant pattern of inheritance. There was only one affected offspring in the family with previously angiographically proven PCAD. However, subsequent segregation analysis showed the carrier genotype in four out of five apparently healthy offspring, for whom coronary CT angiography confirmed the genetic results.

Conclusion: Although the rare STSL1 is caused by homozygous/compound heterozygous mutations in ABCG8 gene, recent research found that ABCG8 variants are more common than previously assumed. Furthermore, even heterozygous carriers have altered sterol profiles and are at higher risk of cardiovascular disease.

References:

Grants: D/206/1851 to Kimia Kahrizi.

Conflict of Interest: None declared.

EP06.030 Whole Exome Sequencing (WES) in unravelling complex cases of bicuspid aortic valve

Ginevra Morgante¹, Maria Teresa Bonati², Flavio Faletra ², Anna Morgan², Elisa Rubinato², Agnese Feresin¹, Agnese Luglio¹, Beatrice Spedicati¹, Giorgia Girotto^1;2, Paolo Gasparini^1;2

¹Università degli Studi di Trieste, Scuola di Specializzazione in Genetica Medica, Trieste, Italy; ²IRCCS Burlo Garofolo, Trieste, Italy

Background/Objectives: Congenital Heart Defects (CHD) are among the most frequently observed malformations and, to date, several genes have been described (both in syndromic and non-syndromic conditions). Here we present the cases of two patients assessed by genetic counseling for bicuspid aortic valve (BAV) and diagnosed through WES.

Methods: Both patients (P1 and P2) have received a deep clinical characterization through cardiological and genetic counselling and their DNA has been analyzed through WES. After standard WES filtering procedures, pathogenic variants within genes likely associated to CHD have been selected.

Results: P1 and P2 present with similar clinical features. In particular, P1, a boy aged 4 y.o., was diagnosed with BAV at birth. He showed later onset of language delay, facial dysmorphisms and short stature, while P2 is an 18 y.o. girl with BAV, cardiomyopathy and arrhythmias, facial dysmorphisms, joint pain and disproportionate short stature. WES analyses highlighted two variants within TAB2 gene (NM_001292034.3): P1 carries a novel pathogenic truncating variant c.712C>T,p.Gln238 predicted as deleterious by all the in silico predictor tools, while P2 carries a truncating known mutation c.823C>T,p.Gln275* (NM_015093.4). This genotype-phenotype correlation is in agreement with recent literature studies showing the involvement of this gene in syndromic CHD.

Conclusion: Although TAB2 has been mostly described for non-syndromic CHD, our results, together with recent literature studies, highlighted its role for syndromic CHD in a precise phenotypic spectrum (i.e. developmental delay, facial dysmorphisms, short stature). In conclusion, we believe the importance of a deep evaluation in CHD patients together with a TAB2 genetic characterization.

References: PMID:2561515;PMID:26493165;PMID: 21128281.

Grants:

Conflict of Interest: None declared.

EP06.031 Genomic diagnosis usefulness in clinical approach and genetic counseling in vascular Ehlers-Danlos Syndrome (vEDS). A case report

Jesica Ramirez ¹, Nicolás García², Pablo Bernasconi³, Eliel Ramirez³, Nicolas Renna³

¹Central Hospital from Mendoza, Genetic and Oncology Department, Mendoza, Argentina; ²Central Hospital from Mendoza, Vascular Surgery Department, Mendoza, Argentina; ³Spanish Hospital from Mendoza, Cardiology Department, Mendoza, Argentina

Background/Objectives: Vascular Ehlers-Danlos syndrome (vEDS) is a genetic disease with dominant autosomal inheritance caused by alterations in COL3A1 gen. Clinical criterias are useful tools to identify affected patients in which mortalitity associated with vascular complications is elevated. The aim of this work is to report the case of male patient, 38 years old, with genomic certification of vEDS. Patient is second son from nonconsanguineous parents. He has been studied by Cardiology Unit for thoraco-abdominal aneurism that required prothesic replacement. Among his clinical background chronical hypertension, vertebral cerebellar artery dissection, bruising and dissection of right kidney artery has been referred. The anatomo-pathologic analysis post splenectomy reveled compatible features with muscular fibrodysplasia on splenic artery. Genealogy study supported the clinical suspect because his father had thoracic aneurysm corrected by surgery. On the other hand his mother died after recurrent strokes and his brother died at 32 years by sudden death.

Methods: Considering mayor and minor criterias COL3A1 gen was analyzed by multipanel sequencing (NGS) using ILLUMINA platform.

Results: Genetic testing reported one germline pathogenic, missense variant (c.709G>A/p.Gly237Arg) in COL3A1 gen. According Varsome and Clinvar COL3A1 is a gen with very low proportion of benign missense variants.

Conclusion: Genomic diagnosis allowed to identify the pathology and offer guidelines to prevent complications. Cosegregation variant studies discarded vEDS in offsprings. In families identified on the basis of clinical complications, penetrance of the vEDS phenotype appears to be close to 100% in adults with a missense or exon-skipping alteration; the age at which the pathogenic variant becomes penetrant may vary.

References:

Grants:

Conflict of Interest: None declared.

EP06.032 Periodontal pathogens in atherosclerosis. A metagenomic and in-vitro analysis

Talia Marroquin¹, Sandra Guauque-Olarte ², Juan Camilo Hernandez^3;4, Lizdany Florez^5;6

¹Universidad Coperativa De Colombia, Dentistry, Pasto, Colombia; ²University Cooperativa De Colombia - Medellín, Dentistry, Medellín, Colombia; ³University Cooperativa De Colombia - Medellín, Medicin, Medellín, Colombia; ⁴University Cooperativa De Colombia - Medellín, Medicine, Medellín, Colombia; ⁵Universidad de Antioquia, Medicin, Medellín, Colombia; ⁶Universidad de Antioquia, Medicine, Medellín, Colombia

Background/Objectives: It has been stablished that periodontitis is a contributing factor for atherosclerosis, being transitory bacteremia one of the mechanisms involved (1). The aim of this study is to describe the bacteria diversity and detect common species between peripheral blood and dental plaque of Colombian patients affected by both diseases.

Methods: A Metagenomic analysis of peripheral blood and dental plaque samples from 12 adults with atherosclerosis was performed. After DNA extraction (QIAamp® DNA Mini-Kit), the sequencing libraries were obtained using the Illumina HiSeq-2500 platform. Bioinformatic analysis was performed in the platform QIIME 2 (Version 2020.11.1).

Results: The metagenomic and bioinformatic analysis of peripheral blood and dental plaque revealed one bacteria specie (Prevotella melaninogenica), six genus, eleven families, and five phyla common for both sample types. Some of the genus present in blood are associated to periodontal disease such as Prevotella, Pseudomonas, and Streptococcus. The richness was greater in plaque samples (mean:47. SD = 5.24) than in blood samples (mean: 4.6. SD = 5.8).

Conclusion: The findings of this study confirm the presence of bacteria associated to periodontal disease (2) in peripheral blood of Colombian patients affected by periodontitis and atherosclerosis. This shows a possible contributing role of periodontal pathogens in atherosclerosis probably by mean of transitory bacteremia.

References: 1. Aarabi G, Heydecke G, Seedorf U. Roles of Oral Infections in the Pathomechanism of Atherosclerosis. Int J Mol Sci. 2018 Jul 6;19(7).

2. Solbiati J, Frias-Lopez J. Metatranscriptome of the Oral Microbiome in Health and Disease. J Dent Res. 2018;97(5):492-500. https://doi.org/10.1177/0022034518761644.

Grants: SGO received funding by CONADI and TYM by Minciencias.

Conflict of Interest: None declared.

EP06.034 Two novel mutations, two different clinical phenotypes associated with SCN5A; Brugada and Long QT syndromes

Alp Peker ¹, Aslı Toylu¹, Mert Coşkun¹, Gokcen Karamik², Feyza Altunbaş¹, Duygu Gamze Aracı¹, nuray ozturk², banu nur², Aytül Belgi Yıldırım³, Filiz Ekici⁴, Özden Altıok Clark¹, ercan mihci²

¹Akdeniz University School of Medicine, Medical Genetics, Antalya, Turkey; ²Akdeniz University School of Medicine, Pediatric Genetics, Antalya, Turkey; ³Akdeniz University School of Medicine, Cardiology, Antalya, Turkey; ⁴Akdeniz University School of Medicine, Pediatric Cardiology, Antalya, Turkey

Background/Objectives: SCN5A gene encodes the alpha subunit of myocardial expressed sodium channel protein, Nav1.5. Generally, loss-of-function mutations of SCN5A are expected to exhibit Brugada syndrome phenotype, meanwhile gain-of-function mutations portray Long QT syndrome. Here we present two patients with novel mutations in SCN5A, who display characteristics of different clinical entities, Brugada and Long QT syndromes.

Methods: Patient-1 (aged 13) was led to cardiac stress test that indicated long QT syndrome, then to our genetics clinic for further analysis. Patient-2 (aged 39), prediagnosed with Brugada syndrome in Ajmaline provocation test, was forwarded to genetic evaluation. Next generation sequencing method for target genes of cardiac arrhythmias was performed on patients’ genomic DNA. Data were analysed via Ion Reporter Software (ThermoFisher Scientific Inc.) and SEQ (Genomize) programs.

Results: In Patient-1, a heterozygous missense variant, c.1575C>A (p.Ser525Arg), was identified. This variant’s position is not conserved, however is a hot-spot region for nucleotide alterations.

In Patient-2, also a heterozygous missense variant, c.2548G>C (p.Val850Leu) was detected which is located in a highly conserved region.

Both alterations weren’t found in population studies (gnomAD); were unidentified in disease specific databases (ClinVar, ClinGen, OMIM) and predicted to have damaging effect on protein function, therefore they were classified as likely pathogenic variants.

Conclusion: As nucleotide changes in SCN5A gene might dysregulate the protein’s function, in-vitro studies are crucial for further understanding their effect on channel activity in terms of increase/decrease. Novel mutations in SCN5A gene and the clinical phenomena they resemble might elucidate how structural changes affect the phenotypical result.

References: https://doi.org/10.1016/j.jacep.2018.03.006, https://doi.org/10.1038/ncomms1717.

Grants: None.

Conflict of Interest: None declared.

EP06.035 A polygenic risk score improves the prediction of cardiovascular risk associated with obstructive sleep-apnoea

Christian Thorball ¹, Mathieu Berger², Geoffroy Solelhac², Adrien Waeber², Claire Redin¹, Pedro Marques-Vidal³, Peter Vollenweider³, Jacques Fellay^1;4;4, Raphael Heinzer²

¹Lausanne University Hospital, Precision Medicine Unit, Lausanne, Switzerland; ²Lausanne University Hospital, Center for Investigation and Research in Sleep (CIRS), Lausanne, Switzerland; ³Lausanne University Hospital, Department of Medicine, Internal Medicine, Lausanne, Switzerland; ⁴EPFL School of Life Sciences, Lausanne, Switzerland

Background/Objectives: Obstructive sleep apnoea (OSA) is a prevalent disease characterized by repetitive episodes of partial or complete airway obstruction during sleep. It is associated with an increased risk of cardiovascular disease (CVD) and its severity is based on the apnoea-hypopnoea index (AHI). However, AHI is only partially associated with CVD events. Therefore, we sought to assess the potential benefit of adding a coronary artery disease (CAD) polygenic risk score (PRS) to classical predictors of CVD risk in individuals with moderate or severe OSA.

Methods: We applied the CAD PRS developed by Inouye et al. (2018) and classified the OSA status in 1558 participants of the CoLaus-HypnoLaus population-based cohort, following full night polysomnography. We assessed the association between OSA, clinical factors, PRS and CVD events using multivariate Cox proportional hazard regressions.

Results: A total of 442 study participants (67% men; average 64 years old) had moderate or severe OSA (AHI ≥ 15/h). Of those, 77 experienced a CVD event during a mean follow-up of 7 years. A 10% increase in the CAD PRS was significantly associated with CVD events in a model adjusted for all cofounding factors (HR = 1.22, 95% CI (1.02–1.5), P = 0.03). Adding the PRS to the clinical risk factors significantly improved CVD event prediction (P = 0.03).

Conclusion: A CAD PRS is significantly associated with the occurrence of CVD in individuals with moderate or severe OSA. The addition of a CAD PRS has the potential to improve risk stratification of OSA patients, thereby enabling more personalized treatment management.

References:

Grants:

Conflict of Interest: None declared.

EP06.036 Development and validation of a new cardiac screening test

Skevi Kyriakou ¹, Achilleas Achilleos¹, Michaella Georgiadou¹, Charalambos Loizides¹, Christos Lemesios¹, Michalis Nicolaou¹, Chrisovalando Soteriou¹, Charalambos Kkoufou¹, Louiza Constantinou¹, Krystallo Christou¹, Antonia Matsentidou¹, Michalis Spyrou¹, Stelia Pissaridou¹, Demetra Panayiotou¹, Kyriakos Tsangaras¹, Elena Kypri¹, Marios Ioannides¹, George Koumbaris¹, Philippos Patsalis¹

¹NIPD Genetics, Nicosia, Cyprus

Background/Objectives: Cardiovascular diseases are the leading cause of illness and death worldwide. There are many types of congenital cardiovascular diseases, ranging from simple to complex diseases with severe life-threatening symptoms. Overlapping symptoms can make it challenging to identify the underlying cardiovascular condition. We have developed and validated an NGS-based targeted cardiac screening test for the detection of multiple genetic conditions with complex phenotypes.

Methods: The test was developed using hybrid-capture enrichment followed by next generation sequencing. Custom target capture sequences (TACS) were designed to capture all coding exons and flanking regions of 292 disease associated genes. A blind validation study was performed on samples with unknown variant status and samples that were found to carry a genetic disorder previously identified by an independent lab to determine the sensitivity and specificity of single nucleotide variant (SNV), INDEL and copy number variant (CNV) detection.

Results: SNVs and INDELS were detected at sensitivity of 100% (CI: 79.4–100%) and specificity of 100% (CI: 99.8-100%). The algorithm was designed to detect CNVs at high-resolution with high sensitivity and specificity when applied to single or few exon CNV. All variants were confirmed by an orthogonal method.

Conclusion: We have developed and validated a cardiac screening test of a great diagnostic and prognostic importance. Clear diagnosis can lead to a notable increase in diagnostic success rate, effective treatment, and management for many cardiac genetic defects.

References:

Grants:

Conflict of Interest: Skevi Kyriakou NIPD Genetics, Achilleas Achilleos NIPD Genetics, Michaella Georgiadou NIPD Genetics, Charalambos Loizides NIPD Genetics, Christos Lemesios NIPD Genetics, Michalis Nicolaou NIPD Genetics, Chrisovalando Soteriou NIPD Genetics, Charalambos Kkoufou NIPD Genetics, Louiza Constantinou NIPD Genetics, Krystallo Christou NIPD Genetics, Antonia Matsentidou NIPD Genetics, Michalis Spyrou NIPD Genetics, Stelia Pissaridou NIPD Genetics, Demetra Panayiotou NIPD Genetics, Kyriakos Tsangaras NIPD Genetics, Elena Kypri NIPD Genetics, Marios Ioannides NIPD Genetics, George Koumbaris NIPD Genetics, Philippos Patsalis NIPD Genetics.

EP06.037 The challenge of interpreting GLA VUS: a case of late-onset Fabry disease

Clara Herrero-Forte ¹, Victoria Cañadas-Godoy², Raluca Oancea-Ionescu¹, Carmen Cotarelo-Pérez¹, M. Alejandra Restrepo-Córdoba³, María Fenollar-Cortés¹

¹Hospital Clínico San Carlos, Unidad de Genética Clínica. Servicio de Análisis Clínicos. Instituto de Medicina del Laboratorio. IdISSC., Madrid, Spain; ²Hospital Clínico San Carlos, Consulta de Cardiopatías Familiares/Unidad de Arritmias. Servicio de Cardiología. Instituto Cardiovascular., Madrid, Spain; ³Hospital Clínico San Carlos, Unidad de Insuficiencia Cardiaca y Cardiopatías Familiares. Servicio de Cardiología. Instututo Cardiovascular, Madrid, Spain

Background/Objectives: Fabry disease is an X-linked recessive inborn metabolic disorder caused by alterations in the GLA gene and with heterogeneous phenotypes, ranging from severe early forms diagnosed in childhood to atypical late-onset forms.

We report the case of a 71-year-old woman with hypertrophic cardiomyopathy (HCM) detected at the age of 26, who was finally diagnosed of an atypical form of Fabry disease many years later.

Methods: NGS analysis using virtual panel of 18 genes. Sanger sequencing for segregation studies. Biochemical study by determining α-galactosidase and lyso-GL-3 levels.

Results: We identified the heterozygous variant c.207C>A in the GLA gene. It was previously reported in a hemizygous male with late-onset Fabry disease¹. In our patient, the variant was initially classified as a variant of uncertain significance (VUS) according to the ACMG/AMP guidelines². A segregation and biochemical study were carried out on 3 clinically unaffected relatives (table).

Case	Age	Phenotype	Genotype	α-Galactosidase	lyso-GL-3
Index	71	HCM	+/-	Normal	↑
Daughter	45	-	-/-	Normal	Normal
Son	43	-	-/-	Normal	Normal
Brother	68	Septal hypertrophy secondary to hypertension	-/-	-	Normal

An endomyocardial biopsy showed typical intracelular inclusions, confirming the diagnosis and allowing reclassification of the variant as likely pathogenic. Enzymatic replacement therapy was initiated.

Conclusion: The interpretation of VUS in recessive X-linked diseases as Fabry, poses a diagnostic challenge that requires a multidisciplinary approach and additional individual and family clinical and genetic studies³.

References: ¹Umeda. Hum Genome Var. 2015;2:15044.

²Richards. Genet Med. 2015;17(5):405-24.

³Germain. Clin Genet. 2021. https://doi.org/10.1111/cge.14102.

Grants:

Conflict of Interest: None declared.

EP06.038 Massive parallel sequencing in postmortem genetic analyses of sudden unexplained deaths in the young reveals genetic predispositions for cardiac diseases

Ainur Akilzhanova ^1;2, Madina Zhalbinova^1;2, Ayaulym Chamoieva^1;2, Aidana Gabdulkayum¹, Gulbanu Akilzhanova³, Saule Rakhimova¹, Diana Samatkyzy¹, Ulykbek Kairov¹, Ulan Kozhamkulov¹, Kenes Akilzhanov³, Tatyana Polyakova^4;5, Kulzhami Ospanova^5;6, Tolkin Zhakupova^5;6, Makhabbat Bekbossynova⁷, Dos Sarbassov^1;8

¹Nazarbayev University, National Laboratory Astana, Nur-Sultan, Kazakhstan; ²Eurasian National University named after L.N. Gumilyov, Nur-Sultan, Kazakhstan; ³Medical University Semey, Pavlodar Branch, Pavlodar, Kazakhstan; ⁴Medical University Astana, Nur-Sultan, Kazakhstan; ⁵Center for Forensic medicine, Nur-Sultan, Kazakhstan; ⁴Medical University Astana, Nur-Sultan, Kazakhstan; ⁷National Research Cardiac Surgery Center, Nur-Sultan, Kazakhstan; ⁸Nazarbayev University, School of Science and Humanities, Nur-Sultan, Kazakhstan

Background/Objectives: Cases of sudden cardiac death (SCD) in young and apparently healthy individuals represent a devastating event in affected families. Coronary artery disease accounts for the majority of SCD in the older population whereas cardiomyopathies and arrhythmogenic abnormalities predominate in younger SCD victims (<40 years) with a significant genetic component. Purpose of this study was to define the portion of underlying genetic heart diseases among young Kazakhstani unexplained SCD victims revealed by massive next-generation sequencing (NGS).

Methods: We included 12 forensic cases of unexplained SCD victims aged less than 40 years. DNA was analyzed by NGS panel of 174 candidate genes with known associations to cardiomyopathies, arrhythmias, aortopathies, and more.

Results: We identified within 1 year 12 cases of SCD among 183 forensic cases. The data were evaluated bioinformatically and detected sequence variants were assessed using common databases and applying in silico prediction tools. Evidence for a genetic disposition was found in 9 of 12 (75%) cases, with likely pathogenic effect in 4 and variants of uncertain significance in 7 of SCD cases.

Conclusion: The study provides strong evidence that molecular genetics improves the post mortem diagnosis of fatal genetic heart diseases among SCD victims and should be integrated in forensic and pathological routine practice. NGS has nonetheless brought new challenges to molecular autopsy, especially regarding the clinical interpretation of the large number of variants of unknown significance detected in each individual.

References:

Grants: Study was supported by a program targeted funding from the Ministry Education and Science, Republic of Kazakhstan (BR10965164).

Conflict of Interest: None declared.

EP06.039 Characterization and dating of a novel variant in the MYH7 gene exclusive of the Balearic Islands that is associated to Hypertrophic Cardiomyopathy

Jéssica Hernández Rodríguez ¹, Guido Antonioutti², Fiama Caimi Martinez², Jorge Alvarez-Rubio³, Paula Morlanes-García⁴, Jaume Pons-Llinares⁵, Elena Fortuny-Frau³, Blanca Rodriguez-Picon⁶, Emilia Amengual-Cladera¹, Teresa Carrion-Mera⁷, Victor Asensio-Landa⁷, Rosa Martorell⁷, Antonia Obrador⁷, Fernando Santos⁷, Maria Vidal⁷, Iciar Martínez⁷, Laura Torres⁷, DAMIAN HEINE SUÑER⁷, Tomás Ripoll-Vera³

¹Health Research Institute of the Balearic Islands (IdISBa), Genomics of Health research group, Palma, Spain; ²Hospital Universitario Son Llàtzer, Cardiology, Palma, Spain; ³Hospital Universitario Son Llàtzer/Health Research Institute of the Balearic Islands (IdISBa), Cardiology, Palma, Spain; ⁴Hospital Clínico Universitario Lozano Blesa, Cardiology, Zaragoza, Spain; ⁵Hospital Universitari Son Espases, Cardiology, Palma, Spain; ⁶Hospital Mateu Orfila, Cardiology, Menorca, Spain; ⁷Hospital Universitari Son Espases/Health Research Institute of the Balearic Islands (IdISBa), Unit of Molecular Diagnostics and Clinical Genetics, Palma, Spain

Background/Objectives:.

Methods:.

Results: Hypertrophic cardiomyopathy (HCM) is a genetic disease characterized by increased left ventricle (LV) wall thickness caused by mutations in sarcomeric genes. We performed an observational and genetic study by NGS sequencing of patients with HCM and found a novel variant in the MYH7 gene (NM_000257.4:c.1955G>A) which putatively causes a p.Arg652Lys missense protein change. This previously non-described variant was found in twelve families with HCM. Out of 8 families that were clinically characterized 64% of carriers developed HCM. The LV hypertrophy was asymmetric septal in 75% of cases, with LV outflow tract obstruction in 28%. The incidence of a composite of serious adverse cardiovascular events (sudden death, aborted sudden death, appropriate implantable cardiac defibrillator discharge, an embolic event, or admission for heart failure) was observed in five (20%) patients. This p.Arg652Lys variant was classified as likely pathogenic (LP) and associated with the development of HCM for the following reasons: 1) It is found in patients with HCM, but not in controls, 2) There is evident segregation with HCM, and 3) It is located in an active site of the protein where a variant in the same amino acid has already been clearly established as pathogenic (p.Arg652Gly). Interestingly, the exclusive presence of the variant in our region could correspond to a founder effect in the Balearic Islands, Spain, which we have further investigated. IBD/coalescent-based allele dating analysis reveals that the origin of this allele is 96 generations away which would correspond to 1900-2400 years ago.

Conclusion:.

References:

Grants:

Conflict of Interest: None declared.

EP06.040 DNA libraries preparation optimization for sequencing of long DNA fragments using Nanopore sequencing for patients with heart disorders

Diana Samatkyzy ¹, Aidana Gabdulkayum¹, Saule Rakhimova¹, Ulykbek Kairov², Ulan Kozhamkulov¹, Makhabbat Bekbossynova³, Ainur Akilzhanova¹

¹Laboratory of Genomic and Personalized Medicine, Center for Life Sciences, National Laboratory Astana, Nazarbayev University, Nur-sultan, Kazakhstan; ²Laboratory of Bioinformatics and Computational Systems Biology, Center for Life Sciences, National Laboratory Astana, Nazarbayev University, Nur-sultan, Kazakhstan; ³National Research Cardiac Surgery Center, Nur-sultan, Kazakhstan

Background/Objectives: Heart failure (HF) is characterized by the failure of the heart’s function and may be a result of cardiomyopathy, which brings to cardiovascular death and disability. We conducted the whole genome sequencing of patients with HF, by using the method of Nanopore sequencing (Oxford Nanopore), which allowed us to read longer DNA fragments. Nanopore long-read sequencing technology greatly expands the capacity of long-range single-molecule DNA-modification detection as well. Sequenced long DNA fragments allowed evaluating new genetic alterations as well as complex structural variants, repetitive regions and DNA-modification associated with HF in Kazakhstani population.

Methods: Genomic DNA was isolated from 68 frozen blood samples of patients with heart failure, and cardiomyopathy by using the Gentra Puregene Blood (QIAGEN). Then, NanoDrop 2000 and Qubit 2.0 assessed samples quantitatively and qualitatively. Optimizing the original fragmentation protocol obtained Long-fragments of gDNA. We used 3 µg gDNA at a concentration of 100 ng/µl using g-TUBE (Covaris) in 49 µl volume. LabChip GX Touch II (PerkinElmer) analyzed fragmentation results. DNA libraries were prepared using Ligation-sequencing kit (SQK-LSK109) and were sequenced on PromethION 48 (Oxford Nanopore) with loading three times after each 24 h.

Results: Attaining whole genome sequencing with approximate coverage of 30× and N50 between 20 and 23kb.

Conclusion: Optimization methods allowed obtaining long DNA fragment reads. Currently, bioinformatics analysis of obtained sequenced data is performing.

References:

Grants: Study was supported by a program targeted funding from the Ministry Education and Science, Republic of Kazakhstan (BR10965164).

Conflict of Interest: None declared.

EP06.041 Evaluation of subclinical myocardial damage in Cornelia de Lange syndrome

LAURA TRUJILLANO^1;2, Ariadna Ayerza Casas^3;4, Beatriz Puisac², Gonzalo Gonzalez Garcia⁵, Angela Ascaso², Ana Latorre-Pellicer², Maria Arnedo³, Cristina Lucia Campos³, Marta Gil-Salvador³, Frank J. Kaiser^6;7, Feliciano Ramos ^2;8, Juan Pie², Gloria Bueno Lozano⁹

¹Department of Clinical and Molecular Genetics. Vall d’Hebron Barcelona Hospital Campus., Barcelona, Spain; ²Unit of Clinical Genetics and Functional Genomics, Department of Pharmacology-Physiology-Legal Medicine, School of Medicine, Universidad de Zaragoza, CIBERER-GCV02 and IIS-Aragon, Zaragoza, Spain, Zaragoza, Spain; ³Unit of Clinical Genetics and Functional Genomics, Department of Pharmacology-Physiology-Legal Medicine, School of Medicine, Universidad de Zaragoza, CIBERER-GCV02 and IIS-Aragon, Zaragoza, Spain., Zaragoza, Spain; ⁴Department of Pediatrics, Hospital Universitario Miguel Servet, Zaragoza, Spain., Zaragoza, Spain; ⁵Department of Pediatrics, Hospital Clínico Universitario Lozano Blesa, Zaragoza, Spain., Zaragoza, Spain; ⁶Institute for Human Genetics, University Hospital Essen, University of Duisburg-Essen, 45147 Essen, Germany, Essen, Germany; ⁷Center for Rare Diseases (Essener Zentrum für Seltene Erkrankungen, EZSE), University Hospital Essen, Essen, Germany, Essen, Germany; ⁸Department of Pediatrics, Hospital Clínico Universitario “Lozano Blesa”, CIBERER-GCV02 and IIS-Aragon, Zaragoza, Spain, Zaragoza, Spain; ⁹Department of Pediatrics, Hospital Clínico Universitario Lozano Blesa, Zaragoza, Spain, Zaragoza, Spain

Background/Objectives: Cornelia de Lange Syndrome (CdLS) is a rare multisystemic congenital developmental disorder. Among others, cardiovascular defects represent a significant cause of morbility and mortality in patients with CdLS. The aim of this study is to evaluate a possible cardiac dysfunction in individuals with CdLS without diagnosed congenital heart disease (CHD) using speckle tracking echocardiography.

Methods: This is a case control study including 20 individuals with CdLS without CHD and 20 healthy controls selected of same ages and gender. All individuals with CdLS were submitted to molecular analysis (16 individuals carrying pathogenic variants in NIPBL, two in SMC1A, one in RAD21 and one in HDAC8). Cardiac function was evaluated using conventional echocardiography, tissue doppler imaging (TDI), two-dimensional speckle tracking and biochemical analyses.

Results: The analytical markers of cardiovascular risk and cardiac function showed no alterations. However the left ventricular global longitudinal strain (GLS) was altered (>−15.9%) in 55% of patients. All values obtained by speckle tracking (strain, strain rate and velocity) showed a downward trend correlated with age (p < 0.05). Likewise, the ejection fraction, shortening and TAPSE, although preserved, also decreased with age (p < 0.05).

Conclusion: The results of this study suggest that patients with CdLS may develop subclinical myocardial dysfunction, which can be detected by speckle tracking even before the appearance of clinical symptoms and the alteration of other echocardiographic or analytical parameters. Taking this into account, cardiological follow up is suggested even in the absence of CHD in individuals with CdLS.

References:

Grants:

Conflict of Interest: None declared.

EP06.042 Novel variants in ALPK3 and GATA3 in a pedigree with primary cardiomyopathy and multiple congenital abnormalities: concurrence of two monogenic diseases in one family

Roman Myasnikov¹, Anna Bukaeva ^1;2;3, Olga Kulikova¹, Anna Petukhova^1;2;3, Evgenia Zotova^1;2;3, Alexey Meshkov¹

¹National Medical Research Center for Therapy and Preventive Medicine, Moscow, Russian Federation; ²Federal State Budgetary Institution “National Medical Research Center of Endocrinology” of the Ministry of Health of Russia, Moscow, Russian Federation; ³Moscow Institute of Physics and Technology, Dolgoprudny, Russian Federation

Background/Objectives: Prominent phenotypic heterogeneity of primary cardiomyopathies makes precise genetic diagnosis difficult, especially in case of presentation of extracardiac phenotypic abnormalities that may indicate either a syndromic cardiomyopathy or an independent disease. We report a case of successful genetic investigation of familial noncompaction cardiomyopathy complicated with diverse dysmorphologic features.

Methods: The proband is 40-year-old man with noncompaction cardiomyopathy, heart failure, arrhythmic complications and short stature. His sister has hypertrophic cardiomyopathy (HCM), myocardial hypertrabeculation, compensated heart failure, sensorineural deafness and congenital abnormalities of genitourinary system. The siblings’ father has HCM. Besides, the proband’s eldest daughter demonstrates developmental delay. We performed clinical examination and whole-exome sequencing for all available family members (three affected and six unaffected, a total of nine persons). Variants with gnomAD frequency <0.01% were selected for clinical interpretation.

Results: In affected siblings and their father, we found a c.4411-2A>C variant in canonical acceptor splice site in the ALPK3 gene. Recently, heterozygous truncating variants in ALPK3 were identified as the cause of adult-onset HCM. Based on sequence properties, we suggest that our finding causes premature truncation of ALPK3 product. None of unaffected persons harbored the c.4411-2A>C variant. Additionally, in the proband’s sister we found a p.Trp329Gly missense in GATA3, the gene responsible for the rare syndrome “hypoparathyroidism, sensorineural deafness, and renal dysplasia”. Both findings in ALPK3 and GATA3 are previously unreported.

Conclusion: The accurate genetic diagnosis is the way to optimize the follow-up scheme and to improve our understanding of complex clinical cases.

References:

Grants: The Ministry of Science and Higher Education of Russia (agreement №075-15-2020-899).

Conflict of Interest: None declared.

EP06.043 Mitomycin C induced genotoxic stress in endothelial cells is associated with inflammatory response

Maxim Sinitsky ¹, Anna Sinitskaya¹, Daria Shishkova¹, Maxim Asanov¹, Anastasia Ponasenko¹

¹Research Institute for Complex Issues of Cardiovascular Diseases, Kemerovo, Russian Federation

Background/Objectives: Genotoxic stress triggers endothelial disfunction and atherosclerosis [1], but mechanism of this process is poorly investigated. Inflammation plays the important role in atherogenesis. This study aimed to access the expression of inflammatory markers in endotheliocytes exposed to mitomycin C (MMC).

Methods: Primary human coronary- (HCAEC) and internal thoracic artery endothelial cells (HITAEC) exposed to 500 ng/ml MMC (experimental group) and 0.9% NaCl (control) was used in this research. Gene expression was evaluated by RT-qPCR after 6-h exposure to MMC with MMC followed by 24-h incubation in the mutagen-free cell growth media. The level of cytokine release in endotheliocytes was studied by dot blotting.

Results: We discovered that HCAEC exposed to MMC are characterized by increased mRNA level of IL-8, MCP-1, IP-10; decreased expression of TIMP-2 and no differences in the expression of MIF, MIP-1b, PDGFB compared to the control. In HITAEC, increased mRNA level of IL-8, IP-10; decreased expression of MIF, TIMP-2 and no differences in the expression of MCP-1, MIP-1b, PDGFB was shown. At the proteome level MIF, IL-8, MCP-1, IP-10, PDGFB were upregulated both in HCAEC and HITAEC, and there are no differences in MIP-1b release. TIMP-2 was upregulated in HCAEC but not in HITAEC.

Conclusion: Genotoxic stress in endotheliocytes induces by MMC leads to differential inflammatory response that can trigger endothelial dysfunction.

References: Sinitsky M.Y. et al. The gene expression profile in endothelial cells exposed to mitomycin C. Biochemistry (Moscow), Supplement Series B: Biomedical Chemistry, 15(3):255-261, 2021.

Grants: Grant of Russian Science Foundation №27-75-10052, https://rscf.ru/project/21-75-10052/.

Conflict of Interest: None declared.

EP06.044 Non-desmosomal genes in Arrhythmogenic Cardiomyopathy: genetic variants rating

Marco Cason ¹, Maria Bueno Marinas¹, Rudy Celeghin¹, Riccardo Bariani¹, Monica De Gaspari¹, Stefania Rizzo¹, Gaetano Thiene¹, Martina Perazzolo Marra¹, Domenico Corrado¹, Cristina Basso¹, Barbara Bauce¹, Kalliopi Pilichou¹

¹University of Padua, Department of Cardiac, Thoracic, Vascular Sciences and Public Health, Padova, Italy

Background/Objectives: Arrhythmogenic Cardiomyopathy (AC) is an inherited disorder of the myocardium with a highly heterogeneous clinical presentation including life-threatening arrhythmias at risk of sudden death. Genetic testing impacts greatly in reaching AC diagnosis, but gene-disease associations has yet to be determined for the increasing number of genes included in clinical panels.

Methods: Genetic variant re-appraisal was undertaken in most relevant non-desmosomal disease genes, based on current adjudication guidance, identified in 320 unrelated Italian AC patients who did not carry pathogenic/likely pathogenic (P/LP) variants in desmosome-coding genes and reported literature data.

Results: In our cohort, 28 rare genetic variants in non-desmosomal genes were identified in 30 patients, of which 17 FLNC (Filamin C), 7 DES (Desmin), 2 TMEM43 (Transmembrane protein 43), and 2 CDH2 (Cadherin-2). No P/LP variants were found in PLN (Phospholamban) and TJP1 (Tight junction protein-1) genes. Gene-based burden analysis, including P/LP variants reported in literature, showed significant enrichment only for TMEM43 (3.52-fold), DES (9.55-fold), PLN (117.8-fold) and FLNC (93.22-fold). Evolutionary conservation analysis made evident a positive selection pressure (Ka/Ks ratio >1) for CDH2 and TJP1, indicating that missense variants impact less the protein structure. Genotype-phenotype correlation highlighted 71% and 89% of left-dominant AC in FLNC and DES carriers, respectively.

Conclusion: Genes lacking robust clinical and genetic evidences impact greatly the number of variants-of-unknown-significance detected and should be removed from clinical AC-targeted genetic panels since the findings cannot drive clinical decision-making. About two thirds of non- desmosomal P/LP variants occur in FLNC leading to fully-penetrant left-dominant AC.

References:

Grants:

Conflict of Interest: None declared.

EP07 Metabolic and Mitochondrial Disorders

EP07.001 Nuclear DNA changes in patients with suspected mitochondrial disorder

Kristina Grigalionienė ¹, Birutė Burnytė¹, Algirdas Utkus¹, Laima Ambrozaitytė¹

¹Vilnius University, Faculty of Medicine, Institute of Biomedical Sciences, Department of Human and Medical Genetics, Vilnius, Lithuania

Background/Objectives: Mitochondrial diseases represent genetically and phenotypically diverse group of disorders, characterized by dysfunctional mitochondria. Pathogenic variants have been identified in more than 350 genes, but at least 40% of patients remain undiagnosed (Stenton, 2020).

Methods: Next-generation sequencing (NGS) methods were applied for 24 patients in a group of 80 unrelated individuals with suspected mitochondrial disease after exclusion of mtDNA mutations by whole mtDNA (Sanger) sequencing. Gene panels for mitochondrial and other neuromuscular disorders were performed for 8 patients, whole exome sequencing was performed for 16 patients.

Results: In the study four patients (16.7%) were found to have pathogenic variants in POLG, SURF1, PNPLA8, RRM2B, and BTD genes. SURF1 gene is involved in the biogenesis of the cytochrome c oxidase complex. POLG and RRM2B genes are involved in mtDNA synthesis, pathogenic variants lead to qualitative or quantitative changes in mtDNA. The protein encoded by PNPLA8 gene is essential for maintaining efficient bioenergetic function through tailoring mitochondrial membrane lipid metabolism. Pathogenic variants in the genes CACNA1A, DDX3X, TPP1, YARS and ANO5, leading to other neuromuscular diseases, were identified in six patients (25.0%). Two patients were diagnosed with two diseases caused by pathogenic variants in nuclear (RRM2B and BTD) or in both mitochondrial (MT-ND4) and nuclear (ANO5) genes.

Conclusion: The application of NGS allowed to confirm rare mitochondrial or neuromuscular disorders emphasizing the importance of clinical genetic-based research in improving the care of these patients. However, the genetic causes of other patients remain unknown and challenging to investigate the molecular mechanisms as well as disease progression.

References:

Grants:

Conflict of Interest: None declared.

EP07.006 Medium chain acyl co-A dehydrogenase (MCAD) deficiency due to an exon 8 duplication in ACADM

aviva eliyahu ¹, orna Staretz-Chacham², Ben Pode-Shakked³, Dina Marek-Yagel⁴, haike reznik wolf¹, elon pras¹, Yair Anikster³, shlomo almashano⁵

¹Sheba Medical Center, Israel, The Danek Gertner Institute of Human Genetics, Tel Hashomer, Israel; ²Soroka Medical Center, Israel, Metabolic Clinic, Beer Sheva, Israel; ³Sheba Medical Center, Israel, Edmond and Lily Safra Children’s Hospital, Tel Hashomer, Israel; ⁴Sheba Medical Center, Israel, Metabolic Laboratory, Tel Hashomer, Israel; ⁵ministry of health, national newborn screening, Tel Hashomer, Israel

Background/Objectives: Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is the most common disorder of fatty acid β-oxidation, caused by bi-allelic variants in the ACADM gene. Targeted sequencing of ACADM establishes a molecular diagnosis for most- but not all- patients.

We sought to establish the molecular basis of MCAD deficiency (MCADD) in a cohort of patients with a biochemical working diagnosis of MCADD, for whom sequencing of ACADM failed to detect bi-allelic pathogenic variants.

Methods: sequencing of the ACADM gene could not establish a bi-allelic pathogenic variant in fourteen of the 63 newborns detected by the Israeli Newborn Screening (NBS) Program with suspected MCADD. We pursued multiplex ligation-dependent probe amplification (MLPA) of ACADM for these fourteen patients.

Results: The fourteen patients, 22% of all detected MCADD by NBS, were found to harbor a common duplication of exon 8 of the ACADM gene. The exon 8 duplication was in either homozygous form or compound heterozygous with a previously recognized mutation by sequencing. All patients share a North-African Jewish (Tunisian, Moroccan) descent. Exon 8 duplication was not detected in 110 chromosomes of matched ethnic background controls.

Conclusion: A likely pathogenic exon 8 duplication in the ACADM gene leading to MCAD deficiency consists a founder mutation in the North-African Jewish population. Due to the beneficial effect of early diagnosis and intervention towards a positive outcome and better prognosis, we suggest that in patients with a biochemical working diagnosis of MCADD for whom sequencing of ACADM fails to establish a molecular diagnosis, MLPA should be considered.

References:

Grants:

Conflict of Interest: None declared.

EP07.008 A novel heterozygous mutation of CYP17A1 gene in a child with micropenis and isolated 17,20-lyase deficiency

Rossella Ferrante ¹, Lucrezia Pilenzi¹, Maria Alessandra Saltarelli², Francesca Di Marcello², Daniela David², Silvia Valentinuzzi¹, Claudia Rossi¹, Stefano Tumini², Liborio Stuppia¹

¹Center for Advanced Studies and Technology (CAST), University “G. d’Annunzio” of Chieti-Pescara, 66100 Chieti, Italy, CHIETI, Italy; ²Department of Maternal and Child Health, UOSD Regional Center of Pediatric Diabetology, Chieti Hospital, Chieti, Italy

Background/Objectives: Disorders of sexual development (DSD) are a heterogeneous group of congenital conditions associated with atypical development of chromosomal, gonadal or anatomical sex. These alterations can be associated with numerous hormonal, genetic and often physical alterations. The micropenis consists in the presence of a stretched penile length of less than 2–2.5 SD for age, can represent a clinical sign of various forms of DSDs.

Methods: In the present study we report the case of a 5-year-old child with isolated micropenis, a typical feature of 46,XY DSD, showing low basal and post hCG stimulation testosterone levels. Molecular analysis using a NGS panel of 50 genes involved in DSDs was performed. Then a serum steroid profiling for the child was also determined by Liquid Chromatography coupled to Tandem Mass Spectrometry (LC–MS/MS) analysis.

Results: NGS analysis evidenced the presence of an heterozygous mutation in the CYP17A1 gene. Segregation analysis in the unaffected parents show the presence of the same mutation in the mother of the child, who however presented no features of the disease. Biochemical analysis of the child revealed low levels of testosterone, progesterone and dehydroepiandrostenedione.

Conclusion: These results suggest that in some cases even heterozygous mutations in recessive genes involved in adrenal steroidogenesis can influence the patient’s phenotype. The presence of mutation in the mother, who does not have the disease, does not contrast with the results obtained, since low testosterone levels in female subjects do not necessarily correlate with a pathological phenotype.

References: 1. Hiort et al,2014-Nat Rev Endocrinol.

Grants:

Conflict of Interest: None declared.

EP07.009 Ethylmalonic encephalopathy masquerading as meningococcemia

Ari Horton ^1;2;3;4, Kai Mun Hong⁵, Dinusha Pandithan⁶, Meredith Allen⁷, Caroline Killick⁷, Stacy Goergen⁸, Amanda Springer¹, Dean Phelan⁵, Melanie Marty⁵, Rebecca Halligan⁶, Joy Lee⁶, James Pitt⁵, Belinda Chong⁵, John Christodoulou^5;9, Sebastian Lunke^5;10, Zornitza Stark^5;10, Michael C. Fahey^1;4;11

¹Monash Medical Centre, Monash Genetics, Monash Health, Clayton, Australia; ²Monash Medical Centre, Monash Heart and Monash Children’s Hospital, Monash Health, Clayton, Australia; ³Monash University Clayton Campus, Monash Cardiovascular Research Centre, Clayton, Australia; ⁴Monash University Clayton Campus, Department of Paediatrics, Clayton, Australia; ⁵Victorian Clinical Genetics Services, Parkville, Australia; ⁶The Royal Children’s Hospital Melbourne, Department of Metabolic Medicine, Parkville, Australia; ⁷Monash Children’s Hospital, Department of Paediatric Intensive Care, Clayton, Australia; ⁸Monash Medical Centre, Monash Health Imaging, Monash Health, Clayton, Australia; ⁹University of Melbourne, Department of Paediatrics, Parkville, Australia; ¹⁰University of Melbourne, Parkville, Australia; ¹¹Monash Children’s Hospital, Department of Neurology, Clayton, Australia

Background/Objectives: Ethylmalonic encephalopathy (MIM #602473) is a rare autosomal recessive metabolic condition caused by biallelic variants in ETHE1 (MIM #608451), characterized by global developmental delay, infantile hypotonia, seizures and microvascular damage. The microvascular changes result in a pattern of relapsing spontaneous diffuse petechiae and purpura, positional acrocyanosis and pedal edema, hemorrhagic suffusions of mucous membranes and chronic diarrhea. This case provides a timely reminder to consider rare genetic diagnoses when atypical features of more common conditions are present, with an early referral to ensure prompt biochemical and genomic diagnosis. Here we describe an instructive case in which ethylmalonic encephalopathy masqueraded as meningococcal septicemia and shock.

Methods: Ultra-rapid whole genome testing (time to result 60 hours) and prompt biochemical analysis facilitated accurate diagnosis and counselling with rapid implementation of precision treatment for the metabolic crisis related to this condition.

Results: WGS identified a homozygous frameshift variant in exon 2 of ETHE1 (MIM #608451), c.131_132del; p.(Glu44Valfs*62).

Conclusion: This case provides a timely reminder to consider rare genetic diagnoses when atypical features of more common conditions are present, with an early referral to ensure prompt biochemical and genomic diagnosis.

References: Nil

Grants: Nil

Conflict of Interest: None declared.

EP07.010 Glutaric aciduria type 1: Variant classification and distribution in the GCDH gene

Isabelle Rinke ¹, Alexandra Tibelius², Christine Fischer², Anna Jauch², Katrin Hinderhofer²

¹Institute of Human Genetics, Heidelberg, Germany; ²Institute of Human Genetics, Heidelberg, Germany

Background/Objectives: Glutaric aciduria type I (GA-1) is a rare autosomal-recessive inherited dysfunction of the enzyme glutaryl-CoA dehydrogenase (GCD) catalyzing lysine, hydroxylysine and tryptophan break down. The disease is caused by pathogenic variants in the GCDH gene and leads to an irreversible movement disorder as main symptom when untreated. The molecular genetic basis of GA-1 is poorly investigated. Therefore, the main objective of this project was to provide an extensive collection and classification of variants in combination with reported phenotypes in GA-1 in the openly accessible Leiden Open (source) Variation Data base (LOVD). Further investigations on this up-to-date largest GA-1-patient-cohort were conducted and will soon be admitted to publication.

Methods: An intensive literature research was conducted on PubMed. Variants were classified according to adjusted ACMG guidelines.

Results: The preexisting GCDH-section of LOVD was complemented by 96 new publications, 342 new patient data and 71 new variants. 227 of the overall 229 listed variants were classified. Finally, more than 220 variants and phenotypes from more than 500 mostly published patients are listed in the LOVD database so far. Almost one third of these variants are classified as VUS and the remaining as (likely) pathogenic. Most variants were missense variants and found towards the middle and 3’ end of the gene localized at functional domains on protein level without clear hot spots.

Conclusion: Internationally uniform criteria for variant classification, such as the ACMG criteria, are important tools. Nevertheless, they aren’t easily applicable and need to be adjusted in case of rare diseases.

References: https://databases.lovd.nl/shared/genes/GCDH.

Grants: No.

Conflict of Interest: Isabelle Rinke Voluntary unpaid curator of GCDH-section of Leiden Open (Source) Variation Database (LOVD), Alexandra Tibelius Voluntary unpaid curator of GCDH-section of Leiden Open (Source) Variation Database (LOVD), Christine Fischer: None declared, Anna Jauch: None declared, Katrin Hinderhofer Voluntary unpaid curator of GCDH-section of Leiden Open (Source) Variation Database (LOVD).

EP07.011 Mutation analysis of the PAH gene in phenylketonuria patients from West Ukraine

Liliia Chorna ¹, Halyna Makukh¹, Yelyzaveta Poliakova¹, Marta Tyrkus¹, Hayane Akopyan¹

¹State Institution «Institute of Hereditary Pathology of National Academy of Medical Sciences of Ukraine», Lviv, Ukraine

Background/Objectives: More than 1280 pathogenic variants have been described in the international database of patients and genotypes causing hyperphenylalaninemia (HPA)/PKU (BIOPKUdb, accessed on 26 August 2021). The occurrence of different mutant alleles varies among ethnic groups and geographic regions worldwide. We report the spectrum and frequency of PAH gene mutations in 160 subjects with PAH deficiency from Western Ukraine.

Methods: DNA was isolated from peripheral blood by treatment with proteinase K with subsequent salting-out protocol and target PAH gene mutations testing was performed by RFLP-PCR. The majority of the patients were diagnosed by newborn screening.

Results: Mutation analysis revealed that 62.5% of all variant alleles carry five mutations. The most prevalent mutation c.1222C>T (p.R408W) was detected on 185 alleles (57.8%) with a very high degree of homozygosity (35%). Mutation p.R408W presented on one or two alleles in 81% patients (129/160). The other 16% were accounted for c.1066-11G>A (8%), c.473G>A (4%), c.1241A>G (2.7%) and c.754C>T (1.2%). Using the direct automated DNA sequencing it has been found 5 different mutations: c.722G>A and c.838G>A in compound heterozygous state, one case each of c.727C>T and c.116_118del, and rare pathogenic variant c.581T>C in homozygous state. The additional testing is required to identify the rest of 37.5% alleles.

Conclusion: The results of study have showed that the most common mutation in PKU patients from West Ukraine is c.1222C>T accounted for 58% of mutated chromosomes. The mutational spectrum is corresponded to that observed for the East European population.

References:

Grants:

Conflict of Interest: None declared.

EP07.013 Novel variants in COQ7 gene cause infantile fatal multysistemic mitochondrial disorder

Francesca Montanari^1;2, Mina Grippa^1;2, Giulia Lanzoni¹, Giulia Severi ¹, Irene Ambrosetti^1;2, Sara Taormina^1;2, Veronica Di Pisa³, Duccio Maria Cordelli^2;3, Maria Cristina Mondardini⁴, Tommaso Pippucci¹, Federica Isidori¹, Mariantonietta Capristo⁵, Valerio Carelli^5;6, Marco seri^1;2, Caterina Garone^1;2;3

¹IRCCS Azienda Ospedaliero-Universitaria di Bologna, U.O. Genetica Medica, Bologna, Italy; ²Alma Mater Studiorum - Università di Bologna, Dipartimento di Scienze Mediche e Chirurgiche, Bologna, Italy; ³IRCCS Istituto delle Scienze Neurologiche di Bologna, UOC Neuropsichiatria dell’età Pediatrica, Bologna, Italy; ⁴IRCCS Azienda Ospedaliero-Universitaria di Bologna, U.O.C Anestesiologia e Rianimazione Pediatrica, Bologna, Italy; ⁵IRCCS Istituto delle Scienze Neurologiche di Bologna, Programma di Neurogenetica, Bologna, Italy; ⁶University of Bologna, Department of Biomedical and Neuromotor Sciences (DIBINEM), Bologna, Italy

Background/Objectives: Coenzyme Q10 is a major electron transporter in mitochondrial chain, thus essential for energy production. Primary COQ10 deficiency is a heterogeneous disease caused by biallelic variants in genes involved in the CoQ10 biosynthesis pathway: 10 “COQ genes” have been identified for being responsible of this disease, including most rarely involved COQ7. We report a case of Primary COQ10 deficiency due to COQ7 gene mutation.

Methods: A multidisciplinary protocol including biochemical and genetic analyses of patient’s biological samples was implemented to diagnose a complex metabolic case with suspected mitochondrial dysfunction.

Results: Patient presented a prenatal onset of increased nuchal translucency, cardiac hypertrophy, increased bowel wall echogenicity. At birth, meconium ileus required corrective ileal steno-atresia surgery; in the following months, intestinal resections have been needed due to recurrent intestinal occlusions. Clinical course was complicated by left ventricular non-compaction cardiomyopathy, ascending aorta dilation, arterial hypertension, renal dysfunction, diffuse skin desquamation, axial hypotonia, neurodevelopmental delay, and growth retardation. Brain MRI showed thalamic abnormalities. Mitochondrial respiratory chain activity revealed an increase in citrate synthase (234.7 nmol/mg/min). Riboflavin and high-dose oral CoQ10 supplementation ameliorated psychomotor development and skin. Patient suddenly died at 16 months. Exome sequencing revealed compound heterozygosis rare variants in COQ7 gene, c.613_617delGCCGGinsCAT (p.Ala205HisfsTer48) and c.403A>G (p.Met135Val), whose pathogenicity was predicted by several bioinformatic tools and confirmed by functional studies.

Conclusion: Our case expands the clinical spectrum of manifestation of primary COQ10 deficiency due to COQ7 gene, and highlights the essential role of multidisciplinary and combined approaches for a timely diagnosis.

References:

Grants:

Conflict of Interest: None declared.

EP07.014 A novel gross deletion in SLC25A15 gene not detected by regular WES analysis

Laura Gort ¹, Laura Pacheco¹, Maria Unceta², Arantzazu Arza², Raquel Bernado³, Javier Adolfo De las Heras⁴, Antonia Ribes¹, Judit Garcia-Villoria¹

¹Section of Inborn Errors of Metabolism - IBC. Department of Biochemical and Molecular Genetics. Hospital Clínic de Barcelona. IDIBAPS, CIBERER, Barcelona, Spain; ²Biochemistry Laboratory. Metabolopathies. Hospital Universitario Cruces, Barakaldo, Spain; ³Department of Neuropediatrics. Hospital Universitario de Navarra, Pamplona, Spain; ⁴Department of Metabolic Pediatrics. Hospital Universitario Cruces, Barakaldo, Spain

Background/Objectives: Hyperornithinemia-hyperammonemia-homocitrullinuria syndrome (HHHS) is an autosomal recessive disorder of the urea cycle. The main clinical symptoms are hypotonia, developmental delay, mental regression and motor dysfunction. In the acute phase hyperammonemia accompanied by vomiting, ataxia, lethargy and confusion are characteristics of the disease. We detected a Pakistani 3 year-old girl with mental and motor retardation, spastic paraplegia and vomiting. Parents were consanguineous. Urine and plasma amino acids were suggestive of HHHS. We wanted to confirm molecular diagnostic.

Methods: We performed WES analysis using Nextera DNA Exome (Illumina). We also analysed the candidate genes individually in the generated bam files. We also performed PCR analysis and Sanger sequencing of introns 1 and 2 and exon 2 of SLC25A15 gene.

Results: To identify the genetic defect in our patient, we performed WES but no variants were found in SLC25A15 gene or in any other urea cycle genes. Due to the HHHS suggestive biochemical and clinical phenotype, we checked SLC25A15 gene in the bam files and we observed a possible deletion involving exon 2. Oligonucleotides designed to delimitate and characterize the deletion gave rise to the detection of a novel homozygous gross indel. The deletion was c.-69-1221_55+1683delinsAGA, which deleted 3028bp and inserted 3bp instead. Her parents were both carriers for the mutation. This variant classified as pathogenic could be considered the disease causing mutation.

Conclusion: When clinical and biochemical phenotype is very suggestive of a disease, it is crucial to further investigate the candidate genes, even though WES results are negative.

References:

Grants:

Conflict of Interest: None declared.

EP07.015 Adult-onset CblC deficiency: a challenging diagnosis involving different adult clinical specialists

Silvia Kalantari ¹, Brigida Brezzi², Valeria Bracciamà¹, Antonella Barreca³, Paolo Nozza⁴, Tiziana Vaisitti¹, Antonio Amoroso^1;5, Silvia Deaglio^1;5, Marco Manganaro², Francesco Porta⁶, Marco Spada⁶

¹University of Turin, Department of Medical Sciences, Turin, Italy; ²Azienda Ospedaliera “SS. Antonio e Biagio e Cesare Arrigo”, Nephrology and Dialysis Unit, Alessandria, Italy; ³Città della Salute e della Scienza University Hospital, Anatomia e Istologia Patologica, Turin, Italy; ⁴Azienda Ospedaliera “SS. Antonio e Biagio e Cesare Arrigo”, S.C. Anatomia e Istologia Patologica, Alessandria, Italy; ⁵Città della Salute e della Scienza University Hospital, Immunogenetics and Biology of Transplantation, Turin, Italy; ⁶Città della Salute e della Scienza University Hospital, Department of Pediatrics, Turin, Italy

Background/Objectives: Methylmalonic aciduria and homocystinuria, CblC type (OMIM #277400) is the most common disorder of cobalamin intracellular metabolism, an autosomal recessive disease, whose biochemical hallmarks are hyperhomocysteinemia, methylmalonic aciduria and low plasma methionine. Despite being a well-recognized disease for pediatricians, there is scarce awareness of its adult presentation.

Methods: The PubMed database was searched for adult-onset CblC cases. All cases with first symptom at onset ≥18 years old were included.

Results: Available clinical, biochemical and molecular data from 22 reports on cases and case series were collected, resulting in 45 adult-onset CblC cases, including the description of a patient followed in our center. We describe the onset of the disease in adulthood, encompassing neurological, psychiatric, renal, ophthalmic and thromboembolic symptoms. From a molecular point of view adult patients are usually compound heterozygous carriers of a truncating and a non-truncating variant in the MMACHC gene.

Conclusion: Adult onset CblC disease is a rare disorder whose diagnosis can be delayed due to poor awareness regarding its presenting insidious symptoms and biochemical hallmarks. To avoid misdiagnosis, we suggest that adult onset CblC deficiency is acknowledged as a separate entity from pediatric late onset cases. To further aid diagnosis, it is important that genes belonging to the intracellular cobalamin pathway are included within gene panels routinely tested for atypical hemolytic uremic syndrome and chronic kidney disorders.

References: Kalantari et al., Adult-onset CblC deficiency: a challenging diagnosis involving different adult clinical specialists. Orphanet J Rare Dis. 2022 Feb 2;17(1):33. https://doi.org/10.1186/s13023-022-02179-y.

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

Conflict of Interest: None declared.

EP07.016 A challenging metabolic acidosis management in a young patient with Trasnsalodase deficiency, T1DM, and pRTA

Hajar AlAkeel ¹, Wafaa Eyaid², Abdullah Alzaben³

¹King Abdullah Specialized Children’s Hospital., Genetics and Precision Medicine., Riyadh., Saudi Arabia; ¹King Abdullah Specialized Children’s Hospital., Genetics and Precision Medicine., Riyadh., Saudi Arabia; ³King Abdullah Specialized Children’s Hospital., Endocrinology., Riyadh., Saudi Arabia

Background/Objectives: We report a rare case of a 14 year old girl with Eyaid syndrome - TransAldolase deficiency1,2 (OMIM 606003) based on both clinical and molecular finding of homozygous pathogenic variant in TALDO1 gene, c.793del p.(Gln265Argfs*56).

She developed type 1 diabetes at around the age of nine and was found to have a baseline non-ionic gap metabolic acidosis that was persistent despite adequate management of her diabetes. Extensive work up for possible renal causes -giving that they are part of her primary syndrome1,3– revealed proximal renal tubular acidosis evident by increased urinary excretion of amino acids and glucose, phosphate and normal renal ultrasound1,2 she also had developmental delay and progressive liver failure resulting in liver cirrhosis, portal hypertension and esophageal varices.

Methods: Case report.

Results: In one ER visit; she presented with one day history of mild abdominal pain, vomiting, diarrhea and lethargy, labs showed metabolic acidosis, her VBG was: pH 6.93, HCO3 3.3, K 3.8, N 136.

Conclusion: Her metabolic acidosis could be related to her underlying pRTA, missed insulin and sodium bicarbonate dosage, the acute illness itself (viral gastroenteritis); Making the diagnosis and management challenging to identify which of them is the major contributor to her acidosis, and what would be the best course of action in regards to when to stop insulin infusion and when to start sodium bicarbonate for which we will highlight in this case report.

References: Clinical, biochemical, and molecular overview of transaldolase deficiency, Clinical utility of anion gap in deciphering acid–base disorders.

Grants:

Conflict of Interest: None declared.

EP07.017 Phenotypic and genotypic spectrum of MTRFR-related disorders

Catarina Olimpio ¹, Rita Horvath²

¹East Anglian Medical Genetics Service, Cambridge University Hospitals NHS Foundation Trust, Cambridge, United Kingdom; ²Department of Clinical Neurosciences, University of Cambridge, Cambridge, United Kingdom

Background/Objectives: Biallelic loss of function variants in MTRFR (previously known as C12orf65) have been reported to cause a variable clinical spectrum which includes combined oxidative phosphorylation deficiency 7 and spastic paraplegia 55 (*613541). MTRFR is a nuclear gene involved in mitochondrial translation. It contains a functional RF-1 domain with a conserved GGQ motif.

Methods: A retrospective review of all reported patients with biallelic MTRFR pathogenic variants was undertaken, focusing on phenotype and possible genotype-phenotype correlations.

Results: 31 patients and 13 pathogenic variants in MTRFR are reported in the literature. Clinical presentations were varied but the core triad of optic atrophy, peripheral neuropathy and spastic paraparesis was present in most patients. Other common features included intellectual disability, reduced visual acuity, ophthalmoplegia, nystagmus and ataxia. Ophthalmological abnormalities were the most common initial presentation. Approximately half the patients had normal early milestones, with regression after the first year of life. Variants within the RF-1 domain, and in particular those disturbing the GGQ motif, appeared to produce a more severe phenotype with bulbar dysfunction, respiratory insufficiency and brain MRI abnormalities but this was not the case in all reported patients.

Conclusion: It has been suggested that the length of the truncated protein is inversely proportional to the severity of the clinical phenotype. However, this is not consistent in all reported patients, with three pairs of patients sharing the same variants with divergent phenotype severity. Further work is ongoing recruiting patients with MTRFR-related disorders to further understand the natural history of this condition and clarify genotype-phenotype correlations.

References:

Grants:

Conflict of Interest: None declared.

EP07.018 Identifying the molecular basis of Glycogen-storage-disease III in a family with negative exome testing

Dr. Michal Barzily Rokni ¹, Lina Berkun¹, Batel Terespolsky¹, Omer Murik¹, Adi Ben Yehuda¹, Pinhas Renbaum¹, Reeval Segel¹, Stanley Korman², ephrat lahad¹

¹Shaare Zedek Medical Center, Medical Genetics Institute, Jerusalem, Israel; ²Shaare Zedek Medical Center, Wilf Children’s Hospital, Jerusalem, Israel

Background/Objectives: Glycogen storage disease type III (GSD-III) is characterized by liver, cardiac, and skeletal muscle involvement, and is caused by mutations in the AGL gene alone. We studied a highly consanguineous Palestinian family of two boys affected with GSD-III. WES testing was performed on DNA samples from the two boys and their parents. No pathogenic variants were found, but a homozygous deep intronic variant of uncertain significance (VOUS) AGL:c.1735+130A>G, in intron 13 was identified in both probands. Our objective was to discover the genetic basis for the disease in this family.

Methods: cDNA sequencing and qRT-PCR methods were used to evaluate alternative splicing and gene expression. Long-range PCR and NGS methods were used to identify structural variants.

Results: cDNA sequencing of exons surrounding the VOUS variant, showed no exon skipping in the probands and their parents. But, expression analysis using qRT- PCR showed lack of expression of exons 28-30, at the end of the gene, in the two probands. Long-range PCR of genomic DNA revealed a homozygous deletion of 5.5Kb. This deletion causes a frameshift in the AGL gene with deleterious effect. Their parents were found to be heterozygous.

Conclusion: Traditional Exome sequencing mainly concentrates on the detection of SNVs and small indels. Larger deletions may be missed by these methods. The development of long-read sequencing can help the detection of small CNVs. These methods helped to identify the disease causing variant in this family which will enable the parents to proceed to PGT/pre-natal diagnosis in the future.

References:

Grants:

Conflict of Interest: None declared.

EP07.020 GM1-Gangliosidosis in four Romanian patients: clinical picture and mutation report

CRISTINA COLDEA ¹, Dragos Serban², Felicia Galos³, Eliza Cinteza⁴, Anna Caciotti⁵, Gabriel Smarandache²

¹Clinical Emergency Hospital for Children “Grigore Alexandrescu”, Pediatrics, Bucharest, Romania; ²Clinical Universitary Emergency Hospital, Surgery, Bucharest, Romania; ³Clinical Emergency Hospital for Children “MS Curie”, Pediatrics, Bucharest, Romania; ⁴Clinical Emergency Hospital for Children “MS Curie”, Pediatric Cardiology, Bucharest, Romania; ⁵Diagnostic Laboratory of the Nervous System and Metabolic Disorders, Molecular and Cellular Biology, Florence, Italy

Background/Objectives: GM1 gangliosidosis (GM1) is an autosomal recessive lysosomal storage disease caused by deficiency of acid beta-galactosidase activity, due to mutations in the GLB1 gene. This work reports clinical and molecular characteristics of four Romanian patients with GM1.

Methods: All patients presented type I (infantile) form. A full biochemical and imagistic workout was performed. Molecular diagnosis was confirmed by low enzyme activity of beta-galactosidase measurement in leukocytes, using artificial 4-methylumbeliferyl beta-galactosidase. Mutations were identified by PCR amplification and direct sequencing of the entire coding region, plus exon / intron boundaries of the GLB1 gene, or for the specific exon, only¹.

Results: Four infants, three males and one female, aged between 8 and 13 months, coming from three different families were analyzed. Natural history and presentation were similar: onset in early infancy, coarse facial features, skin infiltration, abdominal distention due to visceromegaly, hydrocele (males), respiratory distress. Developmental regression, seizures, generalized hypotonia, cortical and optic atrophy were hallmarks of this disease. Hypertrophic cardiomyopathy was associated, with variable severity. Fatality was 100%, occuring between 10 and 18 months of age. Βeta-galactosidase activity ranged from 4 to 14 % of the inferior limit. A single mutation in homozygous state was identified in all patients: c.176G>A (p.Arg59His).

Conclusion: This is the first report of GM1-gangliosidosis cases from Romania. Similar phenotypic traits were found in all four patients, as they proved homozygous for c.176G>A (p.Arg59His) mutation. Poor outcome was associated with this genetic defect.

References: ¹Silva et al. Hum Mutat. 1999;13 (5):401-409.

Grants: None.

Conflict of Interest: CRISTINA COLDEA: None declared, Dragos Serban: None declared, Felicia Galos: None declared, Eliza Cinteza: None declared, Anna Caciotti For research purpose genetic analysis, pro bono, Gabriel Smarandache: None declared.

EP07.021 Three cases of GLB1-related disorders of the same genotype in patients in Ukraine

Nataliia Samonenko ^1;2, Nataliia Trofimova³, Nataliia Mytsyk², Nataliia Olhovich², Nataliia Pichkur², Olena Okhotnikova⁴, Nataliia Gorovenko⁵

¹Shupyk National Healthcare University of Ukraine, Pediatrics 1, Kyiv, Ukraine; ²National Specialise Children Hospital, Kyiv, Ukraine; ²National Specialise Children Hospital, Kyiv, Ukraine; ¹Shupyk National Healthcare University of Ukraine, Pediatrics 1, Kyiv, Ukraine; ⁵Institute of Genetic and Regenerative Medicine, Kyiv, Ukraine

Background/Objectives: GLB1-related disorders comprise two phenotypically distinct lysosomal storage disorders: GM1 gangliosidosis and mucopolysaccharidosis type IVB (MPS IVB). GM1 is characterized by severe degenerative lesions of the central nervous system, regression of development, convulsive syndrome, ataxia. While MPS IVB is characterized by lesions of the skeletal system without cognitive lesions: joint contractures, short stature, scoliosis, lesions of the heart valves and retina. Mutations in the gene cause a deficiency of the lysosomal enzyme galactosidase. And the disease is divided purely clinically.

Methods: Herein we present three patients 9–15 years with the same phenotype and genotype.

Results: All children presented the onset of the disease at the age of 3 years with pronunciation disorders and gradual regression of mental development. From the age of 7 they began to develop an atactic syndrome, muscle weakness, stiffness and contractures of the joints, scoliosis, hip dysplasia. At the age of 9 they had seizures. At the age of 8, they were diagnosed with myopia. No patient had hepatolienal syndrome.

Galactosidase activity is increase. Molecular genetic work-up heterozygous c.841C>T (p.His281Tyr) and c.602G>A (p.Arg201His) gene GLB1.

Conclusion: According to the existing phenotype, it is difficult to classify patients as GM1 or MPS IVB. In terms of manifestations, they occupy an intermediate position between these two diseases. Mutation c.841C>T is common in patients with GM1. Mutation c.602G>A is rare. The presence of the same clinical manifestations in these patients by the same genotype gives us additional data on the gene-phenotypic correlation of gene GLB1 damage.

References:

Grants:

Conflict of Interest: None declared.

EP07.022 Molecular diagnosis of MCAD in the Macedonian neonates with elevated medium-chain acylcarnitines identified through MS/MS-based newborn screening

Violeta Anastasovska ¹, Mirjana Kocova², Nikolina Zdraveska³, Tine Tesovnik⁴, Marusa Debeljak⁴, Jernej Kovac⁴

¹University Clinic for Pediatrics, Ss. Cyril and Methodius University in Skopje, Faculty of Medicine, Department of Neonatal screening, Skopje, Macedonia; ²Ss. Cyril and Methodius University in Skopje, Faculty of Medicine, Skopje, Macedonia; ³University Clinic for Pediatrics, Ss. Cyril and Methodius University in Skopje, Faculty of Medicine, Department of Neonatology, Skopje, Macedonia; ⁴University Children’s Hospital, University Medical Centre Ljubljana, Clinical Institute for Special Laboratory Diagnostics, Ljubljana, Slovenia

Background/Objectives: Medium Cchain acyl-CoA dehydrogenase (MCAD) deficiency is an autosomal recessive disorder of fatty acid oxidation, with potential fatal outcome. It is diagnosed by acylcarnitine analysis on newborn screening blood spot cards by tandem mass spectrometry. Early diagnosis of MCAD and presymptomatic treatment can potentially reduce morbidity and mortality.

Methods: A total of 38,578 newborns were screened for inborn errors of metabolism during May 2014 - Jan 2022, using the LC/MS/MS method. Eight newborns showed elevations of medium-chain acylcarnitines with predominance of octanoylcarnitine, and C8/C10 ratio as well. Molecular ACADM gene analysis was performed by whole exome sequencing using NovaSeq 6000 (Illumina), and confirmed by Sanger sequencing. Sequencing data were analysed using the bcbio_nextgen bioinformatics pipeline (version 1.2.7; GRCh37 genome).

Results: Molecular analysis of the ACADM gene was performed in eight patients with positive metabolic screening for MCAD deficiency. Two different ACADM mutations were obtained in a total of 15/16 (93.75%) alleles of the patients. The common c.958A>G mutation (p.Lys329Glu) was detected in 12/16 (75%) alleles, while 3/16 (18.75%) alleles had c.244dupT pathogenic variant (p.Trp81fs). Five of the patients were homozygous for c.985A>G variant, and one was homozygous for c.244dupT. One of the patients was compound heterozygote (c.985A>G/c.244dupT) while another was a heterozygote for c.958A>G without second mutant allele detection.

Conclusion: The sensitivity of medium-chain acylcarnitines as screening markers for early detection of MCAD was confirmed through the molecular analysis of the ACADM gene. Early detection and treatment have successfully prevented adverse health outcomes in patients with MCAD.

References:

Grants:

Conflict of Interest: None declared.

EP07.023 A novel homoallelic founder variant of RTN4IP1 in a consanguineous population

Mazhor Aldosary ¹, Hanan AlQudairy¹, Rawan Almas², Albandary Al-Bakheet¹, Maysoon AlSagob¹, Alya Qari², Saif Alshahrani², Dilek Colak³, Moeen Al-Sayed², Mohammed Alowain², Robert Taylor⁴, Namik Kaya²

¹Center for Genomic Medicine, King Faisal Specialist Hospital and Research Centre, Translational Genomics, Riyadh, Saudi Arabia; ²Center for Genomic Medicine, King Faisal Specialist Hospital and Research Centre, Medical Genomics, Riyadh, Saudi Arabia; ³Research Centre, King Faisal Specialist Hospital and Research Centre, Biostatistics, Epidemiology and Scientific Computing Department, Riyadh, Saudi Arabia; ⁴Centre for Mitochondrial Research, Translational and Clinical Research Institute, Newcastle University, Highly Specialised Mitochondrial Diagnostic Laboratory, Newcastle upon Tyne Hospitals NHS Foundation Trust, Newcastle Upon Tyne, United Kingdom

Background/Objectives: RTN4IP1 encodes a mitochondrial ubiquinol oxydoreductase that interacts with reticulon 4. Such interaction is considered to be vital for reticulon-induced inhibition of neurite growth. Inherited deficiencies in RTN4IP1 causes mitochondrial deficiency in human and characterized by early-onset recessive optic neuropathy, atrophy, and encephalopathy (1).

Methods: We performed mtDNA sequencing on the collected DNA samples that revealed no pathogenetic variants. We then performed whole exome sequencing (WES) on the index cases and also utilized genome-wide SNP screening to detect shared runs of homozygosity (ROHs) in the families. Sanger sequencing was used to confirm variants’ segregation in the families. The age of the mutation calculation was done using published protocols (2).

Results: We identified a missense variant (NM:032730; c.G475T, p.Val159Phe) in RTN4IP1. The variant was fully segregated with the phenotype in both families and absent among large ethnically matching controls. The variant was predicted to pathogenic by different classifiers. Amino acid sequence alignment revealed that the mutation site is highly conserved in various species. Immunoblotting experiments revealed a decrease in RTN4IP1 steady-state levels in the index’s fibroblasts. Mutation age calculation predicted that the mutation goes back to 56 generations.

Conclusion: Our study expands phenotypic spectrum and pathological outcome of RTN4IP1 deficiency.

References: 1. Angebault, C., et al., Recessive Mutations in RTN4IP1 Cause Isolated and Syndromic Optic Neuropathies. Am J Hum Genet, 2015. 97(5): p. 754-60.

2. Al-Hassnan, Z.N., et al., ISCA2 mutation causes infantile neurodegenerative mitochondrial disorder. J Med Genet, 2015. 52(3): p. 186-94.

Grants: KACST#11-BIO2221-20.

KSCDR/RAC#2180004.

Conflict of Interest: None declared.

EP07.024 Identification and classification of a childhood form of hereditary hypophosphatasia

Márta Szegedi ¹, Robert Petrovic², Maria Judit Molnár¹

¹Semmelweis University, Institute of Genomic Medicine and Rare Disorders, Budapest, Hungary; ²University Hospital, Institute of Medical Biology, Genetics and Clinical Genetics, Bratislava, Slovakia

Background/Objectives: Hypophosphatasia (HPP) is a rare, multisystemic disorder caused by loss-of-function mutations in the ALPL gene that encodes the tissue-nonspecific alkaline phosphatase(ALP) responsible for calcium-, and phosphate mineralization. Our aim was to associate the suspected childhood HPP with the test results and classify the manifestation.

Methods: An 8-year-old child was investigated. Neurological, electrophysiological, radiological, biochemical and genetic tests were performed. Pyridoxal-5′-phosphate(PLP) in blood and phosphoethanolamine(PEA) in blood and urine were measured by HPLC. ALPL gene was analysed by Sanger sequencing and copy number variants were detected by MLPA. Bone manifestation was classified by the Radiographic Global Impression of Change(RGI-C) and Rickets Severity Score(RSS).

Results: Patient had craniosynostosis reconstruction surgery, dental anomalies, generalized muscular atrophy, spastic tetraparesis, significant flexion contarctures, serious somatomental retardation and movement disability, convulsions. Midline and right hemisphere epileptiform signs occurred by EEG. Brain MRI detected periventricular leukomalatia with pons and mesencephalon atrophy. RGI-C and RSS scoring:1/10. Bone age appropriate. Extremely low serum ALP value (-2SD compared to the age-specific average) was detected. PLP in blood and also PEA in blood and urine increased.

Heterozygous missense mutation c.1374C>A(p.Asp458Glu) was detected, which had not been described. The predictive software classifies it as likely pathogenic.

Conclusion: Hypophosphatasia is confirmed by biochemical and genetic level. As to the clinical history and the phenotype, moderate form of childhood hypophosphatasia (HPPC) was diagnosed.

References: 1. OMIM https://www.omim.org/entry/ 241510.

2. Tournis S et al.J ClinMed.2021 Dec; 10(23):5676.

3. Varsome database https://varsome.com.

Grants: This study was supported by KTIA_13_NAP-A-III/6; KTIA_NAP and with FIKP program.

Conflict of Interest: None declared.

EP07.025 Mutations in PPOX gene in Czech patients with variegate porphyria

Daniela Zahorakova ¹, Karel Medek², Jiri Zeman², Pavel Martasek²

¹General University Hospital in Prague and First Faculty of Medicine, Charles University, Department of Pediatrics and Inherited Metabolic Disorders, Prague, Czech Republic; ¹General University Hospital in Prague and First Faculty of Medicine, Charles University, Department of Pediatrics and Inherited Metabolic Disorders, Prague, Czech Republic

Background/Objectives: Variegate porphyria (PV) is an acute hepatic porphyria resulting from partial deficiency of the protoporphyrinogen oxidase (PPO). This enzyme is encoded by the PPOX gene and catalyzes the seventh step of the haem biosynthesis pathway. PV is an autosomal dominant disorder with low penetrance. Clinical manifestations may be triggered by many nongenetic factors and include acute neurovisceral attacks and/or cutaneous photosensitivity. Biochemical diagnosis of PV is based on an abnormal faecal porphyrin profile (increased faecal coproporphyrin and protoporphyrin and reversal of the ratio of copro III/I) and characteristic fluorometric plasma scan. A rare variant of PV is caused by biallelic PPOX gene mutations and presents with developmental delay, severe skin and neurologic symptoms in early infancy.

Methods: PPOX gene was analyzed in 6 probands with clinical and biochemical diagnosis of PV and 2 probands with suspected severe biallelic variant of PV. All exons and adjacent non-coding regions were analyzed by Sanger sequencing.

Results: Pathogenic mutations were confirmed in all probands and subsequent genetic analysis of 22 family members revealed 14 carriers with mutations.

Conclusion: Identification of asymptomatic family members is very important to reduce the risk of PV symptoms by avoiding triggering factors.

References:

Grants: Supported by grants RVO-VFN 64165 and AZV 17-32727A.

Conflict of Interest: None declared.

EP07.026 Functional RNA analysis of a TAFAZZIN intronic variant in a patient with Barth syndrome

Berta Campos ¹, Laura Carmen Trujillano Lidon¹, Anna Tenes¹, Alejandro Moles-Fernández¹, Marta Codina-Sola¹, Paula Fernández-Álvarez¹, Mar Costa-Roger¹, Orland Diez Gibert¹, Elena Garcia Arumi¹, Eduardo Tizzano¹

¹Vall d’Hebron Barcelona Hospital Campus., Department of Clinical and Molecular Genetics., Barcelona, Spain

Background/Objectives: Barth syndrome is a monogenic X-linked recessive disorder characterized by cardiomyopathy, skeletal myopathy, prepubertal growth delay, neutropenia, and 3-methylglutaconic aciduria. It is caused by cardiolipin deficiency and is associated with pathogenic variants in the TAFAZZIN (TAZ) gene. To date, few patients with a molecular diagnosis have been reported. We describe the characterization of an intronic variant identified by whole-exome sequencing (WES) in a 14-month-old boy who presented with cardiogenic shock.

Methods: Genomic DNA was obtained from skin fibroblasts. We used SpliceAI for in silico splicing predictions. RNA samples from cultured skin fibroblasts and whole blood underwent splicing analysis by Sanger sequencing.

Results: WES analysis identified an unreported variant in hemizygosity in TAFAZZIN: c.109+3G>C in the NM_000116.5 transcript (short), corresponding to the variant c.112G>C, p.(Glu38Gln) in the less expressed NM_001303465.2 transcript (long). The variant was inherited from his mother, in whom presumably it appeared de novo.

SpliceAI indicated the disruption of exon 1 donor site (DS) of the short transcript and the activation of a cryptic DS in the c.109+34 position. Nevertheless, RNA analysis showed the activation of a different cryptic DS located in the c.82 position, causing the deletion of the last 28 nucleotides of exon 1, and leading to a truncated protein p.(Val28SerfsTer3). The full-length short transcript was not detected, and the expression of the long transcript was significantly increased.

Conclusion: Our findings show that discrepancies between in silico predictions and in vitro results may exist, highlighting the requirement of RNA studies to characterize splicing complex variants.

References:

Grants:

Conflict of Interest: None declared.

EP07.027 Deciphering MALSU1 in a consanguineous family with mitochondrial cardiomyopathy

Laura Holthöfer ¹, Mareike Selig², Verena Engelhardt¹, Dewi Hartwich¹, Hanna Müller¹, Clemens Sommer³, Torsten Konrad⁴, Alexandra Marx⁴, Anne Schänzer⁵, Matthias Bauer⁶, Jennifer Winter¹, Susann Schweiger¹

¹Institute of Human Genetics, University Medical Centre of the Johannes Gutenberg University Mainz, Mainz, Germany; ²Institute of Human Genetics, University Medical Centre of the Johannes Gutenberg University Mainz, Mainz, Germany; ³Institute of Neuropathology, University Medical Centre of the Johannes Gutenberg University Mainz, Mainz, Germany; ⁴Department of Cardiology Cardiology II, University Medical Centre of the Johannes Gutenberg University Mainz, Mainz, Germany; ⁵Institute of Neuropathology, University Medical Centre of the Justus Liebig University Gießen, Gießen, Germany; ⁶Institute of Laboratory Diagnostics, Hygiene and Transfusion Medicine of the Medical Centre Ludwigshafen, Ludwigshafen, Germany

Background/Objectives: We describe a consanguineous family with two brothers showing the clinical picture of a mitochondrial cardiomyopathy. The older boy has a history of learning difficulties but was considered as healthy until the age of 17. He then showed signs of a decompensated heart insufficiency and was diagnosed with non-compaction cardiomyopathy (LVNC) and died at the age of 19. The younger brother had been diagnosed with hypertrophic cardiomyopathy (HCM) at the age of 3 years. He also showed learning difficulties. At the age of 16 he showed progressive heart insufficiency. The family´s third child is completely healthy.

Methods:.

Results: A homozygous missense variant in MALSU1 (C7orf30) was detected in genomic DNA of the two patients. Both parents and the healthy brother were heterozygous carriers. MALSU1 encodes an assembly and stability factor of the large subunit of the mitochondrial ribosomes (mt-LSU), suggesting a critical role in mitochondrial translation for MALSU1. Protein modelling suggested substantial destabilization of MALSU1 through this variant. Western blot analysis confirmed significant reduction of protein expression in cells of the homozygous family members. Comparative seahorse analysis of fibroblasts of the two homozygous and the heterozygous brother showed a decrease in mitochondrial activity in the homozygous samples but not in the heterozygous sample. Ongoing Western blot analysis of mitochontrial translated proteins versus cytosolic synthesized proteins will show if the MALSU1 variant influences mitrochondrial protein synthesis.

Conclusion: Mutations in mito-ribosomal proteins are a common cause of mitochondrial protein synthesis deficiencies. Here, we provide further evidence for MALSU1 to likely play a role in mitochondriopathies.

References:

Grants:

Conflict of Interest: None declared.

EP07.028 The c.1274_1277dupTATC variant causing Tay-Sachs disease in Tunisian family: a case report

Manel Guirat ¹, Mariem Ben Said², Ikhlas Ben Ayed^1;2, Fatma Kamoun³, Alaa Ziadi¹, Olfa Jallouli³, Salma Mallouli³, Hassen Kamoun¹, Chahnez Charfi Triki³, Saber Masmoudi²

¹Hedi Chaker hospital, Medical genetic department, Sfax, Tunisia; ²Center of Biotechnology of Sfax, Laboratory of Molecular and Cellular Screening Processes, Sfax, Tunisia; ³Hedi Chaker hospital, Neuropediatric department, Sfax, Tunisia

Background/Objectives: Tay-Sachs disease (TSD) (GM2 gangliosidosis I) is an autosomal recessive lysosomal-storage disorder confined to the central nervous system, resulting from deficiency of β-hexosaminidase A.

Methods: Present case is a 3-year-old girl with normal early developmental milestones until 10 months of age when she developed psychomotor regression. She started paroxysmal tonic deviation at the age of 22 months and she subsequently developed myoclonic jerks and an exaggerated startle reaction to sharp noise. Neurological evaluation showed normal head conference, axial hypotonia and slight spastic tetraplegia. EEG showed interictal bi-temporal spikes, spikes-waves discharges and an episode of increased startle response. Brain MRI showed diffuse white matter and striatum hyperintensity on T2 with swollen appearance and thin corpus callosum. Bilateral cherry-red spots were found with a low level (5%) of HEXA enzyme activity.

Results: The homozygous pathogenic variant in HEXA gene (NM_000520.5):c.1274_1277dupTATC was detected using Human Inherited Disease QIAseq Targeted DNA panel. This variant creates a premature translational stop signal (p.Tyr427Ilefs*5), resulting in a disrupted protein product.

Conclusion: Our patient presented an acute infantile form of TSD supporting the low level of the residual HEXA enzyme activity. The c.1274_1277dupTATC variant has previously reported as the most frequent mutation (80%) in TSD of Ashkenazi Jews (AJ), suggesting the occurrence of a common founder in AJ population or the localization in a hotspot mutation region.

References: 1. https://doi.org/10.1007/s12687-011-0057-x.

2. https://doi.org/10.1007/s00439-003-1072-8.

Grants: Not available.

Conflict of Interest: None declared.

EP07.029 A clinical presentation of patient with ATP6AP1 -CDG and liver transplantation

Natalia Semenova ¹, Olga Shatokhina¹, Elena Kamenec¹, Ainur Aliyeva¹, Natalia Taran², Tatyana Strokova²

¹Research Centre for Medical Genetics, Moscow, Russian Federation; ²Federal Research Centre of Nutrition and Biotechnology, Moscow, Russian Federation

Background/Objectives: Congenital disorder of glycosylation, type IIs (OMIM# 300972) is a rare X-linked recessive complex syndrome characterized by liver dysfunction, recurrent bacterial infections, hypogammaglobulinemia, and defective glycosylation of serum proteins.

Methods: Here we report the 1-years-old male patient of Buryat origin, who presented with hypogammaglobulinemia and liver dysfunction. At the age of 3 months, he was hospitalized with jaundice and hepatosplenomegaly. Laboratory evaluation revealed thrombocytopenia, elevated transaminases, alkaline phosphatase of 4164 IU/L, alpha-fetoprotein of 85602 ME/mL and normal level of gamma-glutamyltransferase. MS/MS analysis of acylcarnitines and amino acids in plasma was normal. At the age of 10 months, the child successfully underwent orthotopic liver transplantation. After transplantation, the use of Tacrolimus led to the development of severe colitis with perforation. It required a change of Tacrolimus to Everolimus.

Results: The whole-exome sequencing identified an ATP6AP1 gene missense variant NM_001183.6:c.938A>G (p.Tyr313Cys) in the hemizygous state, which was previously reported by Jansen et al. in the patient with immunodeficiency type 47.

Conclusion: The previously reported patient demonstrated abnormal N- and O-glycosylation, and as for our patient, isoelectric focusing (IEF) of serum transferrin was performed only after liver transplant and showed a normal IEF pattern. We suggest that a possible explanation for normal glycosylation of transferrin in our patient might be a normalization of the glycosylation profile due to liver transplant, as Mirian et al. reported on the first successful liver transplantation in a patient with congenital disorder of glycosylation, after which, normal glycosylation of transferrin was found.

References:

Grants:

Conflict of Interest: None declared.

EP07.030 Novel mtDNA variants: a key role of muscle biopsy in defining their pathogenicity

Hana Štufková¹, Tereza Danhelovská¹, Katerina Lokvencova¹, Hana Kolarova¹, Silvie Kelifova¹, Tomas Honzik¹, Hana Hansikova¹, Marketa Tesarova ¹

¹Charles University, First Faculty of Medicine, and General University Hospital in Prague, Department of Paediatrics and Inherited Metabolic Disorders, Prague, Czech Republic

Background/Objectives: Pathogenic variants in mitochondrial DNA (mtDNA) are associated with variable clinical symptoms usually with neuromuscular manifestation. The variability of the symptoms may be affected by heteroplasmy level of the mtDNA variant in tissues. We report on two novel (MT-ND1: m.4135>C (p.Tyr277His), MT-TK: m.8315A>C) and one extremely rare (MT-ATP6: m.8719G>A (p.Gly65*) mtDNA variants identified in 3 patients by mitochondrial genome sequencing. To characterize the pathogenicity of the variants, muscle biopsies were necessary.

Methods:.

Results: Patient 1 is 39-years-old man with progressive loss of visually acuity due to mtDNA variant MT-ND1: m.4135>C (p.Tyr277His). In muscle with mutation load 93%, markedly decreased activities of complex (C) I and CI+III were observed and only mildly lower amount of CI holoenzyme. Patient 2 is 39-year-olf woman with fatigue, epilepsia partialis continua, and myoclonus based on variant MT-TK: m.8315A>C. A combine deficiency of CI, CIV, and CV was found in her muscle (heteroplasmy 85%). A 55-year-old man with cataract, hearing loss, and leukoencephalopathy (Patient 3) is carrier of a variant MT-ATP6: m.8719G>A (p.Gly65*). Separation of muscle (heteroplasmy 70%) mitochondria by native electrophoresis revealed reduced amount of CV (<20% of controls) with accumulated sub-complexes: V^*(F1-part with c-ring), free F1-part and a free c-ring.

Conclusion: We described two novel (MT-ND1: m.4135>C (p.Tyr277His), MT-TK: m.8315A>C) and one extremely rare (MT-ATP6: m.8719G>A (p.Gly65*)) pathogenic mtDNA variants and defined their impact on oxidative phosphorylation complexes. Our data underscores the necessity of muscle biopsy especially for characterization of pathogenicity of novel mtDNA variants.

References:

Grants: Supported by grants AZV 17-30965A, NV19-07-00149, RVO VFN64165.

Conflict of Interest: None declared.

EP07.031 The role of exome sequencing in newborn screening

Steven Brenner ^1;2, Aashish Adhikari^1;2, Renata Gallagher², Yaqiong Wang¹, Robert Currier², George Amatuni², Laia Bassaganyas², Flavia Chen², Kunal Kundu^1;3, Mark Kvale², Sean Mooney⁴, Robert Nussbaum^2;5, Savanna Randi⁶, Jeremy Sanford⁶, Joseph Shieh², Rajgopal Srinivasan⁷, Uma Sunderam⁷, Hao Tang⁸, Dedeepya Vaka², Yangyun Zou¹, Barbara Koenig², Neil Risch², Jennifer Puck²

¹University of California, Berkeley, United States; ²University of California, San Francisco, United States; ³University of Maryland, Gaithersberg, United States; ⁴University of Washington, Seattle, United States; ⁵InVitae, San Francisco, United States; ⁶University of California, Santa Cruz, United States; ⁷Tata Consultancy Services, Hyderabad, India; ⁸California Department of Public Health, Richmond, United States

Background/Objectives: Public health newborn screening (NBS) programs provide population-scale ascertainment of rare, treatable conditions that require urgent intervention. Tandem mass spectrometry (MS/MS) is currently used to screen newborns for a panel of rare inborn errors of metabolism (IEMs). The NBSeq project evaluated whole-exome sequencing (WES) as an innovative methodology for NBS.

Methods: We obtained archived residual dried blood spots and data for nearly all IEM cases from the 4.5 million infants born in California between mid-2005 and 2013 and from some infants who screened positive by MS/MS, but were unaffected upon follow-up testing.

Results: WES had an overall sensitivity of 88% and specificity of 98.4%, compared to 99.0% and 99.8%, respectively for MS/MS, although effectiveness varied among individual IEMs.

Conclusion: WES alone was insufficiently sensitive or specific to be a primary screen for most NBS IEMs. However, as a secondary test for infants with abnormal MS/MS screens, WES could reduce false-positive results, facilitate timely case resolution and in some instances even suggest more appropriate or specific diagnosis than that initially obtained. This study represents the largest, to date, sequencing effort of an entire population of IEM-affected cases, allowing unbiased assessment of current capabilities of WES as a tool for population screening.

References: Adhikari AN et al. 2020. The role of exome sequencing in newborn screening for inborn errors of metabolism. Nature Medicine 26:1392-1397. pmid:32778825. https://doi.org/10.1038/s41591-020-0966-5.

Grants: Work supported by NIH U19 HD077627 and a research agreement with Tata Consultancy Services.

Conflict of Interest: Steven Brenner S.E.B. receives support at the University of California Berkeley through a research agreement from TCS., Aashish Adhikari A.A. is currently an employee of Illumina, Inc., Renata Gallagher: None declared, Yaqiong Wang: None declared, Robert Currier: None declared, George Amatuni: None declared, Laia Bassaganyas: None declared, Flavia Chen: None declared, Kunal Kundu K.K. was an employee of Tata Consultancy Services (TCS), Mark Kvale: None declared, Sean Mooney: None declared, Robert Nussbaum R.N. is an employee of Invitae., Savanna Randi: None declared, Jeremy Sanford: None declared, Joseph Shieh: None declared, Rajgopal Srinivasan R.S. is an employee of Tata Consultancy Services (TCS)., Uma Sunderam U.S. is an employee of Tata Consultancy Services (TCS)., Hao Tang: None declared, Dedeepya Vaka: None declared, Yangyun Zou Y.Z. is currently an employee of Yikon Genomics Co., Ltd., Barbara Koenig: None declared, Neil Risch: None declared, Jennifer Puck J.P. is the spouse of R. Nussbaum, an employee of Invitae.

EP07.032 Circulating transcripts in maternal blood for non-invasive prediction of gestational diabetes mellitus

Zunmin Wan ¹, Songchang Chen², Jinghua Sun¹, Xuanyou Zhou³, Zhongzhen Liu¹, xi yang¹, taifu wang¹, Lanlan Zhang³, Qing Zhou¹, Lin Wang¹, Yiyao Chen³, wanqiu peng³, Fang Chen¹, Hefeng Huang², Chenming Xu², Wen-Jing Wang¹

¹BGI-Shenzhen, Shenzhen 518083, China, guangdong, China; ²Obstetrics and Gynecology Hospital, Institute of Reproduction and Development, Fudan University, Shanghai, China, shanghai, China; ³International Peace Maternity and Child Health Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200030, P. R. China, shanghai, China

Background/Objectives: Gestational diabetes mellitus (GDM) is the most prevalent pregnancy disease, which can lead to serious adverse pregnancy outcomes. Early intervention in pregnant women at high risk of GDM can reduce the incidence. Plasma cell-free RNA (cfRNA) comes from various tissues and cells, it can provide a valid marker for risk prediction of GDM.

Methods: We included 54 GDM and 57 matched healthy control pregnant women between 14 and 19 weeks of gestation. Transcripts profiling of plasma cfRNA was performed in whole samples. Then, we divided them into two sets, predictive models were constructed by using multiple machine-learning algorithms in the training set and tested the performance in the validation set.

Results: The altered mRNAs were enriched in acute phase response, leukocyte migration, bone marrow leukocyte activation and response to bacteria pathways, both of which are associated with insulin resistance. This suggests that cfRNA could reflect the mechanism of GDM. Using lasso and random forest (RF) algorithm, five mRNAs and six lnRNAs were screened from all transcripts based on multiple iterations, then we constructed prediction model with the AUC of 0.89 in validation set. And, the significance of the model was further evaluated by permutation tests. Subsequently, we included 9 clinical features that AUC can up to 0.94. Tetraiodothyronine had a good predictive value.

Conclusion: Plasma cfRNA transcripts can characterize GDM noninvasively, and in combination with clinical features can easily and accurately predict the risk of GDM, thereby reducing the incidence of GDM and improving pregnancy outcomes.

References:

Grants:

Conflict of Interest: None declared.

EP07.033 Expanding the phenotype of MSTO1 related mitochondrial cytopathy and questioning the existence of an autosomal dominant transmission

lola lessard ^1;2, Sylvie Gerber^3;4, Cécile Rouzier^5;6, Samira Ait-el-mkadem Saadi^5;6, Roxana Ameli⁷, Stéphane Thobois⁸, Lucie Abouaf⁹, Françoise Bouhour^1;2, Josseline Kaplan^3;4, Audrey Putoux^10;11, Antoine Pégat¹, Jean-Michel Rozet^3;4

¹Service d’Electroneuromyographie et de pathologies neuromusculaires, Hôpital Neurologique Pierre Wertheimer, Hospices Civils de Lyon, Bron, France; ²Institut NeuroMyoGène, PGNM, UMR 5261 CNRS, U1315 Inserm, Lyon, France; ³IHU Imagine - Institut des Maladies Génétiques, Paris, France; ⁴Université Paris Descartes, Laboratoire de Génétique Ophtalmologique, Paris, France; ⁵Service de Génétique, Hôpital l’Archet 2, CHU de Nice, Nice, France; ⁶Université Côte d’Azur, CHU, Inserm, CNRS, IRCAN, Nice, France; ⁷Service de Neuroradiologie, Hôpital Neurologique Pierre Wertheimer, Hospices Civils de Lyon, Bron, France; ⁸Service de Neurologie C - Pathologies du mouvement et pathologies neuromusculaires, Hôpital Neurologique Pierre Wertheimer, Hospices Civils de Lyon, Bron, France; ⁹Cabinet d’Ophtalmologie des Tullistes, Ecully, France; ¹⁰Service de Génétique, Unité de Génétique Clinique, Centre Labellisé Anomalies du Développement, Hospices Civils de Lyon, Bron, France; ¹¹Centre de Recherche en Neurosciences de Lyon, Equipe GENDEV, INSERM U1028, UMR CNRS 5292, Université Claude Bernard Lyon 1, Bron, France

Background/Objectives: MSTO1 is a nuclear gene encoding a mitochondrial distribution and morphology regulator, protein misato homolog 1. The MSTO1 gene-related disease was first described as a dominant mitochondrial cytopathy. Three siblings and their affected mother were reported to carry the c.22G>A, p.Val8Met substitution and suffer from cognitive and/or psychiatric disorders, distal muscle weakness and normal creatine kinase (CK) level. To our knowledge, this is the only reported dominant MSTO1 variant. In contrast, recessive were reported in 27 patients from 21 unrelated families. The MSTO1 recessive phenotype contrasts the dominant one: early-onset proximal muscle weakness, elevated CK level and a variable constellation of symptoms including non-progressive cerebellar atrophy/ataxia, visual impairment, motor developmental delay, cognitive alteration, corticospinal tract involvement, and skeletal abnormalities.

Methods: Studying a multiplex family presenting with early-onset muscle weakness and adult-onset optic neuropathy,.

Results: we identified two novel MSTO1 variants: c.65C>A (p.Ala22Glu) predicted as “probably pathogenic” and absent from public databases, and c.220+5G>C which resulted in exon skipping in patient fibroblasts. The phenotype was consistent with typical recessive MSTO1-gene related disease, but this is the first report of a severe bilateral optic neuropathy as part of the MSTO1-gene related disease phenotype.

Conclusion: Through the subsequent analysis of a cohort of patients with bilateral optic neuropathy, we uncovered that the G>A substitution reported as a dominant MSTO1 mutation was highly frequent and is most probably located in the MSTOP2 pseudogene instead of MSTO1, questioning the dominant inheritance of the MSTO1-gene related disease.

References:

Grants:

Conflict of Interest: None declared.

EP07.034 Genomic risk prediction of type 2 diabetes in a clinical trial setting

Xiao Jiang ¹, Abhishek Nag¹, Katherine Smith¹, Benjamin Challis², Philip Ambery³, Björn Carlsson², Jan Oscarsson³, Dirk Paul¹

¹Centre for Genomics Research, Discovery Sciences, BioPharmaceuticals R&D, AstraZeneca, Cambridge, United Kingdom; ²Research and Early Development, Cardiovascular, Renal and Metabolism, BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden; ³Late-stage Development, Cardiovascular, Renal and Metabolism, BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden

Background/Objectives: Polygenic risk scores (PRS) can capture the aggregate contribution of the physiological effects of known type 2 diabetes (T2D) risk alleles [1]. We tested the predictive performance of PRS for T2D risk in a clinical trial setting.

Methods: We constructed distinct PRS for overall T2D, six pathophysiological pathways in T2D, and diabetic complications (liver dysfunction markers, CAD, and CKD). We estimated the relative risk overall and across PRS percentiles using logistic regression in a post-hoc analysis of 4,872 European T2D patients from AstraZeneca clinical trials, EXSCEL [2] and Cotadutide-T2D-Ph2 [3]. We used 4,987 randomly selected UK Biobank Europeans without diabetes as controls.

Results: The genetic T2D risk for trial participants was 1.98-time higher against controls (P = 3.72 × 10⁻¹⁸⁹). The top quintile of the T2D PRS had an odds ratio (OR) of 6.32 (P = 2.82 × 10⁻¹⁵⁰). Among the T2D PRS subtypes, adiposity showed the highest genetic risk (OR = 1.32, P = 2.29 × 10⁻⁴⁰). T2D patients had a 1.37-fold increased risk for CAD (P = 7.13 × 10^-52) and 1.19-time higher liver transaminase levels (P = 1.99 × 10⁻¹⁷). T2D patients in the top quintile of the T2D Insulin Secretion PRS showed 1.11-time higher transaminase levels (P = 2.61 × 10^-3) against the remaining T2D patients.

Conclusion: Our study demonstrated the predictive performance of PRS for T2D risk in a post-hoc analysis of clinical trials. We will expand on this work by testing the utility of the derived PRS for determining the response to anti-diabetes treatment.

References: 1. Mahajan et al. Nature Genet. 50 (2018): 559-571.

2. Holman et al. N Engl J Med. 377 (2017): 1228-1239.

3. Ambery et al. Lancet. 391 (2018): 2607-2618.

Grants: None.

Conflict of Interest: Xiao Jiang Xiao Jiang is a current full-time employees of AstraZeneca., Abhishek Nag Abhishek Nag is a current full-time employee of AstraZeneca., Katherine Smith Katherine Smith is a current full-time employee of AstraZeneca., Benjamin Challis Benjamin challis is a current full-time employee of AstraZeneca, Philip Ambery Philip Ambery is a current full-time employee of AstraZeneca., Björn Carlsson Björn Carlsson is a current full-time employee of AstraZeneca., Jan Oscarsson Jan Oscarsson is a current full-time employee of AstraZeneca., Dirk Paul Dirk Paul is a current full-time employee of AstraZeneca.

EP07.035 a shared pattern of altered gene expression in mitochondrial respiratory chain deficient human embryos

Kalliopi Chatzovoulou¹, Anne Mayeur², Nicolas Cagnard¹, mohammed zarhrate¹, christine bole¹, Patrick Nitschké¹, fabienne jabot-hanin¹, Agnès Rötig¹, sophie monnot³, Jean-Paul BONNEFONT¹, arnold munnich¹, nelly achour², Julie Steffann ¹

¹Imagine Institute, Paris, France; ²Antoine Beclere Hospital, Clamart, France; ³Necker Hospital, Paris, France

Background/Objectives: Quantitative and qualitative anomalies of the mitochondrial DNA (mtDNA) are reportedly associated with impaired human embryonic development, but the underlying mechanisms remain hitherto unexplained. The goal of the present study was to investigate whether mitochondrial mutations affect gene expression in human blastocyst embryos.

Methods: A total of 42 blastocyst embryos (day-5/6/7) from 27 unrelated couples were collected after a preimplantation genetic testing analysis. Among them, 9 embryos were carrying pathogenic variants in either mtDNA genes or in a nuclear gene encoding a mitochondrial protein (mitochondrial group) and 33 were affected by a non-metabolic genetic disorder (control group). Gene expression profiling was performed on whole blastocyst embryos, by RNA-Sequencing.

Results: Transcriptomic analyses showed a similarly decreased gene expression pattern in embryos from the mitochondrial group, altering a number of differentiation factors and nuclear genes encoding mitochondrial proteins. Expression of oxidative phosphorylation genes was most impacted, cell survival and autophagy were also severely decreased, questioning embryonic viability.

Conclusion: The presence of a pathogenic mitochondrial variant induces changes in gene expression programs of human preimplantation embryos and probably compromises development, cell differentiation and survival of the embryo. While identification of reliable markers of normal embryonic development is urgently needed in assisted reproductive technologies, we suggest considering the under-expressed genes reported here as predictive biomarkers of mitochondrial dysfunction during preimplantation development.

References: None.

Grants: This work was supported by the “Association Française contre les Myopathies” (Grant Number: 20304) and the “Fondation des Maladies Rares” (Grant Number: GenOmics_202003003).

Conflict of Interest: None declared.

EP08 Immunology and Hematopoietic System

EP08.001 Revisiting diagnostic procedures in patients with hereditary red blood cell membranopathies– Description of 9 new genetic variants

Friederike Haeuser ¹, Heidi Rossmann¹, Anke Adenaeuer¹, Berhnhard Laemmle^2;3;4, Claudia Paret⁵, Karl J. Lackner¹, Joerg Faber⁵, Olaf Beck⁵

¹University Medical Center of the Johannes Gutenberg-University, Institute of Clinical Chemistry and Laboratory Medicine, Mainz, Germany; ²University Medical Center of the Johannes Gutenberg University, Center for Thrombosis and Hemostasis (CTH), Mainz, Germany; ³Inselspital, Bern University Hospital, University of Bern, Department of Hematology and Central Hematology Laboratory, Bern, Switzerland; ⁴University College London, Haemostasis Research Unit, London, United Kingdom; ⁵University Medical Center of the Johannes Gutenberg-University, Center for Pediatric and Adolescent Medicine, Department of Pediatric Hematology, Oncology & Hemostaseology, Mainz, Germany

Background/Objectives: Red blood cell membrane defects are a group of hereditary diseases with highly variable severity of symptoms and caused by a set of different variants of genes encoding for erythrocyte membrane and skeleton proteins. Although the most common hereditary hemolytic disease in Caucasians, hereditary spherocytosis (HS) is underdiagnosed, because mild clinical course is common and only the minority of patients have been genetically characterized so far. We aimed to develop a simple strategy to confirm the suspected diagnosis of HS using an NGS (next generation sequencing)-based approach.

Methods: 12 patients with clinical and laboratory signs of HS and one patient with initially inconclusive laboratory results, were genetically (exome sequencing) and phenotypically (standard laboratory tests) characterized. Family members of the index patients were studied by Sanger sequencing.

Results: In all 13 patients, pathogenic variants fulfilling ACMG-criteria were found, limited to only four genes: ANK1, SLC4A1, SPTA1, and SPTB. Four predescribed and nine new variants were detected, typically for HS or pyropoikilocytosis respectively.

Medical history and standard laboratory tests are usually sufficient to establish the diagnosis of HS. More sophisticated methods such as SDS- and native PAGE or ectacytometry can elucidate unclear cases.

Conclusion: The diagnosis of membranopathies was confirmed in all 13 patients by NGS. Panel sequencing of the five most common genes (incl. EPB42) is nowadays a comparatively rapid and inexpensive method to confirm the diagnosis of HS and related disorders. Time-consuming or low-specificity tests can be avoided, and family members can be easily genotyped.

References:

Grants:

Conflict of Interest: Friederike Haeuser: None declared, Heidi Rossmann: None declared, Anke Adenaeuer: None declared, Berhnhard Laemmle Potential conflict outside the topic of this Abstract: Bernhard Lämmle is chairman of the data safety monitoring committees (DMC) for the Baxalta 281102 study (recombinant ADAMTS13 in congenital TTP), the Shire SHP655-201 study (recombinant ADAMTS13 in acquired TTP) and the TAK-755-3002 study (Phase 3b continuation study of recombinant ADAMTS13 in congenital TTP), now all three run by Takeda; he was a member of the Advisory Board of Ablynx, now part of Sanofi, for the development of caplacizumab for the treatment of autoimmune TTP; he received congress travel support and/or lecture fees from Baxter, Ablynx, Alexion, Siemens, Bayer, Roche, and Sanofi., Claudia Paret: None declared, Karl J. Lackner: None declared, Joerg Faber: None declared, Olaf Beck: None declared.

EP08.002 Dominant negative effect of ETV6 germinal mutations as common pathogenic mechanism

Daniele Ammeti ¹, Michela Faleschini¹, Nicole Papa², Caterina Alfano³, Roberta Bottega¹, Giorgia Fontana¹, Valeria Capaci², Melania Eva Zanchetta¹, Federico Pozzani⁴, Claudio Graziano⁵, Francesca Montanari⁵, Valeria Petroni⁶, Paola Giordano⁷, Patrizia Noris⁸, Fiorina Giona⁹, Anna Savoia^1;2

¹Institute for Maternal and Child Health – IRCCS Burlo Garofolo, Trieste, Italy; ²University of Trieste, Department of Medical Sciences, Trieste, Italy; ³Fondazione Ri.MED, Structural Biology and Biophysics Unit, Palermo, Italy; ⁴University of Trieste, Department of Life Sciences, Trieste, Italy; ⁵Policlinico S. Orsola-Malpighi, Medical Genetics Unit, Bologna, Italy; ⁶University Hospital “Ospedali Riuniti”, SOSD Pediatric Oncohematology, Ancona, Italy; ⁷University “A.Moro” of Bari, Interdisciplinary department of Medicine, Pediatric Unit, Bari, Italy; ⁸IRCCS Policlinico San Matteo Foundation, University of Pavia, Department of Internal Medicine, Pavia, Italy; ⁹Sapienza University of Rome, Department of Translational and Precision Medicine, Rome, Italy

Background/Objectives: ETV6-related thrombocytopenia (ETV6-RT) is a rare autosomal dominant platelet disorder characterized by an increased risk to develop haematological malignancies. The disease is caused by mutations in ETV6, a gene encoding a transcription factor that plays a key role in haematopoiesis. Since ETV6-RT has been discovered only in 2015, many aspects of this disease still remain unknown. For this reason, we studied the pathogenic effect of seven germline missense variants identified in thrombocytopenic patients.

Methods: We performed bioinformatic analysis and gene reporter assays on a target promoter of ETV6 to determine the potential effect of the variants. Western Blot and immunofluorescence assays were used to evaluate the pathogenic mechanism.

Results: Analysing thrombocytopenic families by NGS approach, we identified seven different germline missense variants in ETV6 gene, including four novel amino acid substitutions.

Consistent with bioinformatic analysis, we demonstrated the pathogenicity of all but two variants, whose protein products are no longer able to migrate into the nucleus, impairing the ETV6 repressive activity. Moreover, we ascertained that the pathogenic mutations act through a dominant negative effect, which retains the WT-mutant dimers of ETV6 in the cytoplasm consequently affecting its function.

Conclusion: We confirmed the pathogenicity of five out of seven variants and evaluated their pathogenic mechanism. Understanding the mechanism by which mutations exert their effect is important to clarify the ETV6 role during megakaryopoiesis, to identify possible therapeutic approaches to correct platelet biogenesis and to prevent the onset of leukaemia in ETV6-RT patients.

References:

Grants:

Conflict of Interest: None declared.

EP08.003 Autoimmune Polyglandular Syndrome Type 1 with Clinical and Genetic Characteristics

Asli Subasioglu ¹, Roya Gasimli², Derya Sema Yaman Kalender³, Baris Onder Pamuk³

¹Izmir Katip Çelebi University, Faculty of Medicine, Department of Medical Genetics, Izmir, Turkey; ²Ege University Faculty of Medicine, Department of Medical Biology, Izmir, Turkey; ³Izmir Katip Çelebi University Faculty of Medicine, Department of Internal Diseases, Endocrinology Clinic, Izmir, Turkey

Background/Objectives: Autoimmune polyglandular syndrome type 1 is a rare autoimmune disease characterized by polyendocrinopathy and enteropathies [1,2], resulting from a mutation in the autoimmune regulator (AIRE) gene located on chromosome 21q22.3 [3]. AIRE protein which acts as a transcription factor is responsible for immune system functions such as thymic self-representation and immune self-tolerance. Therefore, defects in the protein levels cause enhancement of autoreactive T cells and autoimmunity. Autoimmune polyglandular syndrome type 1 progresses majorly with chronic mucocutaneous candidiasis, hypoparathyroidism and adrenocortical failure [4]. We aimed to report a 41-year-old female patient with APS-1 disorder.

Methods: We performed clinical exome sequencing from the patient’s DNA which was isolated from peripheral blood leukocytes.

Results: As a result of the analysis, it was detected that the patient had a homozygous c.927 C>G mutation in the AIRE gene which is responsible for APS-1.

Conclusion: It was decided to follow up the patient’s clinical course with the light of the literature and genetic results.

References: 1. Ahonen et al. Clinical variation of autoimmune Polyendocrinopathy–candidiasis–ectodermal dystrophy (APECED) in a series of 68 patients. N Engl J Med. 1990;322:1829–36.

2. Neufeld M et al. Two types of autoimmune Addison’s disease associated with different polyglandular autoimmune (PGA) syndromes. Medicine (Baltimore). 1981;60:355–62.

3. Buzi F et al. Autoimmune Polyendocrinopathy-candidiasis-ectodermal dystrophy syndrome: time to review diagnostic criteria? J Clin Endocrinol Metab. 2003; 88:3146–8.

4. Betterle C et al. Autoimmune Polyglandular syndrome type 1. J Clin Endocrinol Metab. 1998;83:1049–55.

Grants:

Conflict of Interest: None declared.

EP08.004 De novo mutation in the C1 inhibitor gene in the female patient with hereditary angioedema

Olga Ukhanova¹, Pavel Budnikov ², Lyudmila Barycheva¹, Sophia Podsvirova³, Vyacheslav Podsvirov³

¹Stavropol State Medical University, Immunology department, Stavropol, Russian Federation; ²Kazan (Volga region) Federal University, Institute of Fundamental Medicine and Biology, Kazan, Russian Federation; ³Stavropol State Medical University, Dermatovenereology department, Stavropol, Russian Federation

Background/Objectives: Hereditary angioedema (HAE) is a rare hereditary disease potentially dangerous for patients due to angioedema of larynx, intestines and other vital organs. However, there are HAE de novo in 25% of cases. We describe the clinical case of the patient with characteristic symptoms of HAE I (registered since 2017) without family history. The symptoms manifested after labor as angioedema and abdominal attacks up to 20 per year (AAS28 – 105 points) and were accompanied by decreased levels of complement component C4 to 0,04 g/l, C1-INH quantitative value to 3,3 mg/dl (N = 15–35 mg/dl), C1-INH functional activity to 24,1% (N = 70–130%). The waiting period for a diagnosis was 4.5 years.

Methods: A DNA study of a 27-year-old patient was carried out for the presence of mutations in the SERPING1 gene (C1NH) (NM_000062) with the method of direct Sanger sequencing of all exons and exon-intron junctions.

Results: A new variant c.308_311dup (p.(Gln104Hisfs*30)) in the heterozygous state was found in exon 3. It had not been described in literature and databases (HGMD, ClinVar, LOVD) yet.

Conclusion: SERPING1 mutations heterogeneity is confirmed by discovering new gene variations, including de novo mutations. The identified DNA sequence variant should be considered as probably pathogenic and confirming the HAE diagnosis by a molecular genetic method. Genetic epidemiology demonstrates at least 25% of unrelated HAE cases. It should be taken into consideration during a patient consultation. For all patients with suspected HAE without family history, prompt genetic consultation or molecular genetic testing is necessary.

References:

Grants:

Conflict of Interest: None declared.

EP08.005 Tumor-specific methylation of p53-responsive oncosuppressive microRNA genes in Diffuse Large B-cell Lymphoma

Elena Voropaeva¹, Mariia Churkina ², Tatyana Pospelova², Anna Gurazheva¹, Vladimir Maksimov¹, Olga Berezina²

¹Institute of Therapy and Preventive Medicine - Branch of the Institute of Cytology and Genetics, Novosibirsk, Russian Federation; ²Novosibirsk State Medical University, Novosibirsk, Russian Federation

Background/Objectives: To identify the frequency and specificity of p53-responsive oncosuppressive MIR-34B/C, MIR-34A, MIR-203 and MIR-129-2 genes methylation in Diffuse Large B-cell Lymphoma (DLBCL).

Methods: Methylation status of the genes in tumor tissue (n = 73) was studied by methyl-specific PCR and methyl-sensitive analysis of high-resolution melting curves. DNA isolated from lymph node biopsies with reactive polyclonal B-cell proliferation (n = 11) was used to control the tumor-specificity of the detected methylation. The quantitative analysis of the combined methylation of the studied genes was carried out with the calculation of the one-sided Fisher exact criterion (p-value) and the frequency of false discoveries (FDR) (q-value) was calculated using the Benjamin-Hochberg procedure.

Results: Aberrant methylation of the promoters of the studied genes can serve as a significant mechanism for reducing the miR-34B/C, miR-34A, miR-203 and miR-129 micro-RNAs expression in the tumor tissue of DLBCL. It is occurs in combination and is tumor-specific. Thus methylation of MIR-129-2, MIR-203, MIR-34A and MIR34B/C in lymphoma samples occurred with a frequency of 67%, 66%, 27% and 62%, respectively, and there was no reactive lymph node tissue. Combined methylation of MIR-203, MIR-129-2 and MIR-34B/C genes (p < 0.013, q < 0.020), as well as pair of MIR-34B/C and MIR-34A genes (p = 0.010, q = 0.029) was detected.

Conclusion: Aberrant methylation of oncosuppressive microRNA genes associated with underlying p53 signaling pathways is a potentially useful molecular biomarker in the diagnosis, prognosis of tumors and the development of a strategy for targeted therapy of DLBCL.

References:

Grants: The research was carried out at the expense of the grant from the Russian Science Foundation No. 22-25-00222.

Conflict of Interest: None declared.

EP08.006 Non-coding RNA profile associated with systemic lupus erythematosus activity, relevance of exosomal fraction

Olga Martinez-Arroyo ¹, Ana Ortega¹, Ana Flores-Chova¹, Carlos Bea², Maria J Forner², Raquel Cortés¹

¹Biomedical Research Institute Hospital Clinico - INCLIVA, Cardiometabolic and Renal Risk Research Group, Valencia, Spain; ²Hospital Clinico, Internal Medicine, Valencia, Spain

Background/Objectives: Systemic lupus erythematosus (SLE) is a chronic autoimmune disease where the exosomes have a regulating role throughout their cargos, especially non-coding RNA (ncRNA). The aim of this study is to identify a global ncRNA profile in plasma and plasma exosomes, associated to SLE activity.

Methods: Plasma samples were obtained from 96 SLE patients and 25 controls. RNA was extracted from both plasma and plasma-exosomes (EXO-P). NcRNAs were identified using SmallRNA sequencing analysis. Results were validated in a higher cohort by qPCR.

Results: MicroRNAs (miRNAs) were the biotype with the highest mapped reads in all groups for both biofluids, followed by piRNAs, lncRNA and Y-RNA. Then, it was observed that plasma presented the greatest diversity of differentially expressed ncRNA biotypes, being the miRNA and lncRNA the most representative. Analysing only miRNAs in SLE, it was observed that they are biofluid-specific, being up-regulated in exosomes and down-regulated in plasma, and only 1.2% were common in both fractions. MiR-144-3p and miR-144-5p were the highest up-regulated in EXO-P versus P in all patient groups (3.92 and 3.03, p < 0.001, respectively). The ROC curve analysis showed the discriminatory power only of the hsa-miR-144-3p in EXO-P for the presence of SLE (AUC = 0.71, p < 0.01).

Conclusion: The results showed a biofluid specificity for the ncRNA profile, being up-regulated in plasma exosomes. MiRNAs are the most representative biotype of ncRNA, and exosomal miR-144-3p could be a potential biomarker of SLE activity.

References:

Grants: Health Institute Carlos III” [PI12/02615; PI19/01796]. European Regional Development Fund (ERDF).

Conflict of Interest: None declared.

EP08.007 The molecular genetic and laboratory features of HBB gene mutations and prevalence of beta-thalassemia in Russia: preliminary results

Alina Khachaturian ¹, Vladimir Nazarov², Sergey lapin², Vladimir Emanuel³

¹I.P. Pavlov First Saint-Petersburg State Medical University, Saint-Petersburg, Russian Federation; ²First Pavlov State Saint-Petersburg Medical University, Laboratory of Autoimmune diagnostics, Center for Molecular Medicine, Saint-Petersburg, Russian Federation; ³First Pavlov State Saint-Petersburg Medical University, Clinical Laboratory Diagnostics Department, Saint-Petersburg, Russian Federation

Background/Objectives: HBB gene encodes the beta-globin that is a subunit of hemoglobin. HBB gene mutations can lead either to the decreased beta-chains synthesis and beta-thalassemia or to the beta-chains conformation changes and hemoglobinopathy. Thalassemia is the most common inherited disease in the world however the diagnostics is still a complicated complex process. Our aim was to characterize the molecular genetic and laboratory features of HBB gene mutations and to evaluate the prevalence of beta-thalassemia in Russia due to a little data about it.

Methods: The first group consisted of 268 patients with supposed inherited anemia in accordance with clinical and laboratory data. Capillary electrophoresis (CE) and Sanger sequencing the HBB gene were performed. Calculation of erythrocyte indexes, CE of hemoglobin and the HBB gene genotyping were carried out consistently to 4918 patients to evaluate the approximate prevalence of beta-thalassemia in Russia.

Results: The first group had 33 electrophoresis positive patients: 69,6% had the elevated HbF, 60,6% – HbA2. There were also detected HbS, Hb Shepherds Bush and the unknown pathological variant. 73% of patients had aberrations with the most frequent HBB:c.25_26delAA. The second group had 11 electrophoresis positive patients out of 32 chosen by indexes. 9 of them had the aberrations in the HBB gene.

Conclusion: It is necessary to evaluate both HbA2 and HbF fractions while screening for beta-thalassemia. The approximate prevalence of beta-thalassemia in Russia is 0,18%. The HBB:c.93-96CT mutation evaluated by NCBI base as mutation of uncertain significance was defined by us as pathogenic due to the detected unknown pathological hemoglobin variant.

References:

Grants:

Conflict of Interest: None declared.

EP08.008 The First report of sextuplicated alpha genes in a heterozygote beta thalassemia revealed by a severe beta thalassemia intermedia phenotype

Ahlem Achour^1;2, Tamara Koopmann¹, Jeroen Knijnenburg ¹, raz amir³, Cornelis Harteveld⁴, Frank Baas¹

¹Leiden University Medical Center (LUMC), LDGA-Clinical genetics, Leiden, Netherlands; ²Hospital Charles Nicolle, Departement of Congenital and Hereditary diseases, Tunis, Tunisia; ³Chapman University, Orange, United States; ⁴Leiden University Medical Center (LUMC), Leiden, Netherlands

Background/Objectives: Co- inheritance of an extra copy of the alpha-globin gene cluster in individuals heterozygous for beta-thalassemia usually results in beta thalassemia intermedia. This study reports two members of an American family having eight alpha-globin genes due to an alpha-cluster segmental triplication combined with a beta+-thalassemia gene variant expressing a severe beta-thalassemia intermedia.

Methods: Standard hematology, HPLC and Sanger sequencing have been performed. FISH analysis, Multiplex Ligation-dependent Probe Amplification, SNP- and fine tiling array analysis of the alpha-globin gene cluster were performed to determine the triplication location and breakpoints.

Results: Two patients became transfusion dependent at age 15 and 10 years respectively. Sequencing the HBB gene has revealed a heterozygote beta+-thalassemia variant (HBB:c.*113A>G) inherited from father. MLPA and array analysis, performed in the probands and the parents, have shown a maternally inherited interstitial triplication of the alpha-globin gene cluster on chromosome 16p13.3 (approx. 900 kb), for which the mother was 25% mosaic. One child inherited eight alpha-genes, without the beta+-thalassemia variant of father. The triplicated segment involves the complete alpha-globin gene cluster and 18 additional protein coding genes. Still the only phenotype expressed is that of a thalassemia intermedia when in association with beta-thalassemia trait.

Conclusion: Our results clearly show that the presence of eight alpha globin genes does not have a discernible phenotype on its own, however, it actively contributes to globin chain unbalance when co-inherited with a beta thalassemia variant.

References: 1-Harteveld, C.L.et al. (2008). Blood Cells, Molecules, & Diseases.

2-Clark B, et al. (2018) Br J Haematol.

Grants: None.

Conflict of Interest: None declared.

EP08.009 PADI4 and PADI2 enhance collagen-initiated inflammatory responses

Akari Suzuki¹

¹RIKEN, Center for Integrative Medical Sciences, Yokohama, Japan

Background/Objectives: Previously, peptidylarginine deiminase type 4 (PADI4) was identified as a susceptibility gene for Rheumatoid arthritis (RA) by genome-wide association studies. Peptidyl citrulline is a target antigen of anti-citrullinated peptide antibodies, and only PADs (translated protein from PADI genes) can provide peptidyl citrulline via modification of protein substrates. Also the distribution of PADI4 and PADI2 has overlap in immune cells. The aim of this study was to investigate the relationship between PADI4 and PADI2 in the progression of RA.

Methods: To clarify the physiological function of PADI4 and PADI2 in RA, we used collagen-induced arthritis (CIA), known as a RA model mouse. We generated PADI4Knockout (KO) and PADI2KO mice, and performed CIA. In PADI4KO mice sera, serum anti-type II collagen (CII) IgM, IgG, and inflammatory cytokine levels were also significantly decreased compared with those in wild-type mice sera. We also examined that the clinical disease score of CIA mice and expression levels of Padi genes in PADI2KO CIA mice.

Results: We demonstrated that the clinical disease score of CIA was significantly decreased in PADI4KO mice. Interestingly, PADI2 expression was complementary induced in CD11b+ cells of PADI4KO mice. Gene expression levels of CIA of Padi2 using wild type mice was not observed significant difference between CIA and control mice, however, the clinical disease score was decreased in PADI2KO CIA mice.

Conclusion: It appears that PADI4 and PADI2 related with collagen-initiated inflammatory responses.

References:

Grants:

Conflict of Interest: Akari Suzuki RIKEN.

EP08.010 De novo gain-of-function variations in LYN lead to an early onset systemic autoinflammatory disorder

Camille Louvrier ^1;2, Elma El Khouri¹, Martine Grall Lerosey³, Pierre Quartier⁴, Anne-Marie Guerrot⁵, Brigitte Bader-Meunier⁴, Julie Chican¹, Malaika Mohammad¹, Eman Assrawi¹, Angela Arenas-Garcia¹, Bruno Copin², William Piterboth², Florence Dastot-Le Moal², Sonia Karabina¹, Serge AMSELEM^1;2, Irina Giurgea^1;2

¹Sorbonne Université, Inserm, Childhood Genetic Disorders, Hôpital Armand-Trousseau, Paris, France; ²Département de Génétique médicale, Assistance Publique–Hôpitaux de Paris (APHP), Hôpital Armand-Trousseau, Paris, France; ³Département de pédiatrie et médecine de l’adolescent, CHU-Hôpitaux de Rouen, Rouen, France; ⁴RAISE reference centre for rare diseases, Unité d’Immunologie-Hématologie et Rhumatologie pédiatrique, and Imagine Institute, Hôpital Necker-Enfants Malades, Assistance Publique Hôpitaux de Paris, Paris, France; ⁵Normandie Univ, UNIROUEN, Inserm U1245 and Rouen University Hospital, Department of Genetics and Reference Center for Developmental Disorders, F 76000, Normandy Center for Genomic and Personalized Medicine, Rouen, France

Background/Objectives: To identify the molecular basis of a severe systemic autoinflammatory disorder (SAID) and define its main phenotypical features. To functionally assess the sequence variations identified in LYN, a gene encoding a non-receptor tyrosine-kinase.

Methods: (i) Targeted next-generation sequencing; (ii) In vitro functional studies of Lyn phosphorylation state and of Lyn-dependent NF-κB activity after expression of recombinant forms of Lyn carrying different sequence variations.

Results: We identified a de novo LYN variation (Tyr508His) in a patient presenting since birth with recurrent fever, chronic urticaria, atopic dermatitis, arthralgia, increased inflammatory biomarkers and elevated plasma cytokine levels. We studied the consequences of the Tyr508His variation and of the two LYN variations reported so far (Tyr508Phe and Tyr508*), on Lyn phosphorylation state, and showed that all three variations prevent phosphorylation of residue 508 and lead to autophosphorylation of Tyr397. Additionally, these three LYN variations activate the NF-κB pathway. These results reflect a gain-of-function (GOF) effect of the variations involving Tyr508 on Lyn activity.

Conclusion: This study, which demonstrates the pathogenicity of the first three LYN variations identified in SAID patients, delineates the phenotypic spectrum of a disease entity characterized by an early onset severe inflammatory disease affecting neonates with no family history of SAID. All three variations affect the same tyrosine residue located in the C-terminus of Lyn, thereby underlining the critical role of this residue in the proper regulation of Lyn activity in humans.

References:

Grants: Agence Nationale de la Recherche (ANR-17-CE17-0021-01), ImmunAID - European Union’s Horizon 2020 research and innovation programme (No 779295), Sorbonne Université EMERGENCE-PhenomAID.

Conflict of Interest: None declared.

EP08.011 Searching for rare genetic variants associated with thrombosis using high-throughput sequencing technology

Radek Vrtel ^1;2, Petr Vrtel¹, Radek Vodicka¹, Ludek Slavik³, Jana Prochazkova³, Jana Ulehlova³, Julia Stellmachova¹

¹University Hospital, Medical Genetics, Olomouc, Czech Republic; ²Palacky University, Olomouc, Czech Republic; ³University Hospital, Department of Hemato-Oncology, Olomouc, Czech Republic

Background/Objectives: We are using high-throughput sequencing (HTS) in search for rare genetic variant in thrombophilia cases and this approach we are implementing to clinical practice. We designed HTS panel for genes (PROS1, PROC, SERPINC1 and PROCR) which encode proteins with important role in anticoagulation system. In addition to FV Leiden and FII Prothrombin, mutations in these genes are an important risk factor for thrombosis. In these genes was recently found more than 800 mutations without mutational hot-spots.

Methods: The selection of patients for HTS testing is primarily based on repeated low levels of a given anticoagulant protein by an adequate functional test while excluding possible exogenous causes. After exclusion was indicated 31 unrelated patients. The Ion Torrent PGM platform and the newer Ion Torrent S5 platform were used. Variant annotation is performed according to the variant description in the ClinVar database, based on low population frequencies, prediction software (PolyPhen, SIFT, etc.), PhyloP score, VarSome, etc.

Results: Overall mutation detection rate was 67.7%. Representation of mutations in responsible genes will be presented in a poster.

Conclusion: This is a pilot project in the Czech Republic, which assesses the impact of rare genetic variants on thrombophilic conditions using the HTS methodology. It is an effective adaptation of the HTS methodology to the sequencing platforms used at our department for searching variants in genes encoding anticoagulant protein in clinical practice.

References:

Grants: Supported by MH CZ – DRO (FNOl, 00098892).

Conflict of Interest: None declared.

EP08.012 Host genomic susceptibility to severe respiratory syncytial virus infection

Ali Saadat ^1;2, Dylan Lawless^1;2, Jacques Fellay^1;2;3

¹École Polytechnique Fédérale de Lausanne, School of Life Sciences, Lausanne, Switzerland; ²Swiss Institute of Bioinformatics, Lausanne, Switzerland; ³Lausanne University Hospital and University of Lausanne, Precision Medicine Unit, Lausanne, Switzerland

Background/Objectives: Infection with respiratory syncytial virus (RSV) is mild in most children, still a fraction suffers from severe disease, which might be due to specific variants in the human genome. We here use exome sequencing and bioinformatics analysis to search for genetic variants predisposing previously healthy children to severe clinical presentation of RSV infection.

Methods: We sequenced the exome of 127 previously healthy children who needed hospitalization and oxygen therapy due to RSV infection. We used GATK, VEP, and Annovar for variant calling and annotation. For variant prioritization, we implemented a Bayesian framework based on ACMG/AMP guidelines. Only variants with odds of pathogenicity >80% were included in downstream analysis. We used random walk with restart (RWR) on STRING networks to search for genes with similar function to known disease-associated nodes (called seeds). RSV-associated seeds were extracted from DisGeNET and OpenTargets. To control for the impact of network structure on the RWR output, we used permutation test, keeping genes with FDR <5%.

Results: We identified 28 deleterious variants in genes of interest, including: [a] three IFIH1 loss-of-function previously shown to decrease interferon response to RNA viruses; [b] a frameshift NOD2 variant resulting in a lack of synergistic response to RSV stimulation; and [c] a stop-gained IFI44L variant known as a risk factor for multisystem inflammatory syndrome in children.

Conclusion: To search for host genetics variants involved in severe RSV infection, we used a combination of ACMG/AMP guidelines and network-based methods. We identified 28 potentially causal variants, many of which in interferon-related genes.

References:

Grants:

Conflict of Interest: None declared.

EP08.013 Methylation of RUNX3 promoter 2 in the whole blood of children with ulcerative colitis

Emilia Dybska ¹, Jan K. Nowak¹, Aleksandra Banaszkiewicz², Anna Szaflarska-Poplawska³, Jaroslaw Kierkus⁴, Jarosław Kwiecień⁵, Urszula Grzybowska-Chlebowczyk⁶, Mariusz Szczepanik¹, Jaroslaw Walkowiak¹

¹Poznan University of Medical Sciences, Department of Pediatric Gastroenterology and Metabolic Diseases, Poznan, Poland; ²Medical University of Warsaw, Department of Pediatric Gastroenterology and Nutrition, Warsaw, Poland; ³Nicolaus Copernicus University in Torun, Collegium Medicum in Bydgoszcz, Department of Pediatric Endoscopy and Gastrointestinal Function Testing, Bydgoszcz, Poland; ⁴The Children’s Memorial Health Institute, Department of Gastroenterology, Hepatology, Feeding Disorders and Pediatrics, Warsaw, Poland; ⁵Medical University of Silesia in Katowice, Faculty of Medical Sciences in Zabrze, Department of Pediatrics, Zabrze, Poland; ⁶Medical University of Silesia in Katowice, Faculty of Medical Sciences, Department of Pediatrics, Katowice, Poland

Background/Objectives: Runt-related transcription factor 3 (RUNX3) regulates Th1/Th2 balance, and therefore the synthesis of cytokines and inflammation. Ulcerative colitis (UC) results from a complex interplay between the environment and immune cells, in which the relationship between gene methylation and disease activity remains unclear. We aimed to analyze RUNX3 promoter 2 (P2) methylation level depending on several clinical factors.

Methods: This cross-sectional study recruited hospitalized children with UC. Clinical characteristics were assessed: age, sex, body mass index (BMI), C-reactive protein (CRP), serum albumin, disease duration, pediatric ulcerative colitis activity index (PUCAI), the Paris classification, and exposure to medications. Whole blood DNA was isolated and RUNX3 P2 methylation level was measured with methylation-sensitive restriction enzymes (OneStep qMethyl).

Results: Sixty-four children were enrolled, at a mean age of 14.5 ± 2.8 years. Half of them were female (51.6%) and the mean BMI was 18.0 ± 3.2 kg/m² (Z-score −0.44 ± 1.14). The mean methylation level of RUNX3 P2 was 54.1 ± 13.3%. The methylation level of RUNX3 P2 did not correlate with age, sex, nutritional status, CRP, albumin, PUCAI or the extent of colitis (Paris E1–E4). RUNX3 P2 methylation did not differ between patients recruited within two months of diagnosis and children who had UC for at least a year. Current or past exposure to biologics, immunosuppressants or steroids was not associated with RUNX3 P2 methylation.

Conclusion: Methylation of RUNX3 promoter 2 in whole blood DNA does not seem to associate with clinical characteristics of UC in children.

References: N/A

Grants: Polish National Science Center 2017/25/B/NZ5/02783, awarded to JW.

Conflict of Interest: None declared.

EP08.014 A novel missense variant in RPS19 gene causing Diamond-Blackfan anemia 1

Ivona Sansovic ¹, Ana-Maria Meašić¹, Mijana Kero¹

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

Background/Objectives: Diamond-Blackfan anemia (DBA) is a rare congenital erythroid aplasia that usually presents in infancy. It is characterized by a normochromic macrocytic anemia, growth retardation, and approximately 30 to 50% patients have congenital malformations, and predisposition to cancer. Haploinsufficiency of gene encoding ribosomal protein S19 (RPS19; MIM 603474) accounts for approximately 25% of DBA patients. These patients (DBA type 1) usually don’t have craniofacial abnormalities.

We report a three-month-old girl of a healthy nonconsanguineous parents, with clinical features of Diamond-Blackfan anemia: low birth weight, pale skin, sleepiness, severe normocytic anemia, congenital heart defects, but without craniofacial dysmorphism. She responded well to steroids.

Methods: Clinical exome sequencing including genes associated with Diamond-Blackfan anemia was performed in family members using Illumina TruSight One Kit.

Results: In the RPS19 gene, one novel, de novo, missense variant p.Arg56Gly was identified. According ACMG classification is pathogenic.

Conclusion: Already reported alternative variant Arg56Gln is also classified pathogenic according ACMG. We assume that these variants at codon 56 may disrupt/weaken interactions between arginine and 18S rRNA. In order to develop better diagnostics and treatment, it is necessary to resolve the molecular basis of the pathogenic variants, especially missense that are challenging to decipher. The novel reported variant will further contribute to that knowledge.

References:

Grants: This work was supported by Scientific Center of Excellence for Reproductive and Regenerative Medicine and by the EU through the European Regional Development Fund, under grant agreement No. KK.01.1.1.01.0008, project „Reproductive and Regenerative Medicine - Exploring New Platforms and Potentials”.

Conflict of Interest: Ivona Sansovic Scientific Center of Excellence for Reproductive and Regenerative Medicine and by the EU through the European Regional Development Fund, under grant agreement No. KK.01.1.1.01.0008, project „Reproductive and Regenerative Medicine - Exploring New Platforms and Potentials”, Ana-Maria Meašić Scientific Center of Excellence for Reproductive and Regenerative Medicine and by the EU through the European Regional Development Fund, under grant agreement No. KK.01.1.1.01.0008, project „Reproductive and Regenerative Medicine - Exploring New Platforms and Potentials”, Mijana Kero Scientific Center of Excellence for Reproductive and Regenerative Medicine and by the EU through the European Regional Development Fund, under grant agreement No. KK.01.1.1.01.0008, project „Reproductive and Regenerative Medicine - Exploring New Platforms and Potentials”.

EP08.015 Next-generation sequencing with a 64-gene panel in Slovenian patients with myelofibrosis

Spela Stangler Herodez^1;2, Danijela Krgović^1;2, Zlatko Roškar³, Nadja Kokalj Vokač ^1;2, Mojca Dreisinger³

¹Univeristy Medical Centre Maribor, Laboratory of Medical Genetics, Maribor, Slovenia; ²University of Maribor, Faculty of Medicine, Maribor, Slovenia; ³Univeristy Medical Centre Maribor, Department of Haematology and Haematologic Oncology, Maribor, Slovenia

Background/Objectives: Myelofibrosis (MF) is a chronic myeloproliferative neoplasm that may present as primary disease (PMF) or as post-polycythemia vera MF (PPV-MF) or post-essential thrombocythemia MF (PET-MF). Over the last decade, many additional secondary mutations have been found to have a prognostic impact on disease course.

Methods: We performed analysis of 80 MF (PMF, N = 62; PET-MF, N = 11; PPV-MF, N = 7) samples with our custom 64-gene NGS panel. he variants were manually checked in the ClinVar and COSMIC databases to determine if they had been previously reported as pathogenic.

Results: Fifty-two patients were tested positive for the JAK2 V617F driver mutation, and 5 were positive for CALR and 2 for MPL driver mutations. Aside from driver mutations an initial total of 190 variants were detected across 75 clinical samples, five patients did not show mutations in any of the gene tested. The majority of cases had mutations in ≥ 1 genes (1 mutation: N = 20, 25%, 2 mutations: N = 23, 28.8%, 3 mutations: N = 15, 18.8%, 4 mutations: N = 10, 12.5%, 5 mutations: N = 5, 6.3%, 6 mutations: N = 2, 2.5%). Ultimately, a total of 40 unique variants (which include known driver mutations) with VAFs above 1% were identified. The most common mutations and those with the worst prognostic impact were found as follows: TET2 (N = 37), SETBP1 (N = 17), ASXL1 (N = 9), EZH2 (N = 11), IDH1 (N = 10), IDH2 (N = 7), SF3B1 (N = 6), SRSF2 (N = 2), TP53 (N = 1), U2AF1 (N = 1) and FLT3 (N = 3).

Conclusions: With the obtained results, we will be able to more appropriately classify patients to prognostic groups and treat them accordingly.

Conflict of Interest: Spela Stangler Herodez Research was supported by the internal research project IRP-2019/02-10 of the University Medical Centre Maribor, Maribor, Slovenia., Danijela Krgović Research was supported by the internal research project IRP-2019/02-10 of the University Medical Centre Maribor, Maribor, Slovenia., Zlatko Roškar Research was supported by the internal research project IRP-2019/02-10 of the University Medical Centre Maribor, Maribor, Slovenia., Nadja Kokalj Vokač Research was supported by the internal research project IRP-2019/02-10 of the University Medical Centre Maribor, Maribor, Slovenia., Mojca Dreisinger Research was supported by the internal research project IRP-2019/02-10 of the University Medical Centre Maribor, Maribor, Slovenia.

EP08.016 A small deletion in FAS causes splicing disturbances in a patient with suspected ALPS

Gunda Petraitytė ¹, Evelina Siavrienė¹, Živilė Maldžienė¹, Violeta Mikštienė¹, Ilma Tavorienė^2;3, Laimonas Griškevičius^2;3, Egle Preiksaitiene¹

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

Background/Objectives: Autoimmune lymphoproliferative syndrome (ALPS; MIM#601859) is a disorder of a programmed cell death. The known cause of this syndrome is heterozygous variants in FAS (MIM#134637) – Fas Cell Surface Death Receptor. The receptor belongs to the TNF–superfamily receptors and plays a central role in apoptosis. The penetrance of ALPS is variable.

Methods: The proband, 20 years of age female, had autoimmune hemolytic anemia, pancytopenia, and accumulation of double-negative T cells. Exome sequencing was applied to analyse the proband’s DNA. The genetic findings were confirmed by Sanger sequencing in the DNA samples of the proband and her healthy mother. This method was also applied to investigate the mRNA structure of the gene FAS in the RNA samples of the proband and the mother, extracted from fibroblasts.

Results: A heterozygous deletion NC_000010.11(NM_000043.6):c.16_30+1del which covers a first nucleotide of a donor splice site in FAS was identified in the proband’s DNA. The variant was inherited from healthy mother. The impact of the splice site variant on mRNA structure was determined by Sanger sequencing which revealed a cryptic splice site formation. In silico, a frameshift and a premature stop codon (NP_000034.1:p.(Thr6Phefs17Ter)) is formed.

Conclusion: The disrupted splicing process and most probably truncated and dysfunctional FAS protein were revealed by the molecular analysis of a small deletion NC_000010.11(NM_000043.6):c.16_30+1del in FAS. The disturbed molecular process presumably causes the phenotype of ALPS of the affected individual.

References:

Grants: This work was funded by the Research Council of Lithuania (No. S-MIP-21-15, ATGC project).

Conflict of Interest: None declared.

EP08.017 Association of rare genetic variants at the ULBP3 locus with alopecia areata

Paula Schwerbrock ¹, Axel Schmidt¹, Rana Aldisi², hanna zieger¹, Nicole Cesarato¹, Stefan Herms¹, swapnil Awasthi³, nadine fricker¹, daisy axt¹, stephan ripke^3;4;5, Stefanie Heilmann-Heimbach¹, Kerstin Ludwig¹, Peter Krawitz², Carlo Maj², Per Hoffmann¹, Regina C. Betz¹, F. Buket Basmanav¹

¹Institute of Human Genetics, University of Bonn, School of Medicine & University Hospital Bonn, Bonn, Germany; ²Institute of Genomic Statistics and Bioinformatics, University of Bonn, Bonn, Germany; ³Department of Psychiatry and Psychotherapy, Charité - Universitätsmedizin, Berlin, Germany; ⁴Analytic and Translational Genetics Unit, Massachusetts General Hospital, Boston MA, United States; ⁵Stanley Center for Psychiatric Research, Broad Institute of MIT and Harvard, Cambridge MA, Germany

Background/Objectives: Alopecia areata (AA) is a common hair loss and autoimmune disorder with an estimated lifetime risk of about 2%. Genome-wide association studies reported 14 susceptibility loci, one of which implicates ULBP3, encoding for a ligand of the NKG2D receptor expressed on natural killer cells and a subset of CD8+ T cells. ULBP3 expression was shown to be highly upregulated in the hair follicle of AA patients. The aim of the current study was to discover whether rare variants in the ULBP3 locus are associated with genetic susceptibility to develop AA.

Methods: The entire coding and non-coding region around ULPB3 (10 kb) was sequenced in 1.000 AA patients and 1.000 controls. The results were analyzed by a rare variant burden analysis pipeline, which implements a gene-based scoring system using functional annotations and allele frequency weighting function.

Results: A very restricted number of rare variants were identified in the coding sequence. The analysis of the entire region including the non-coding sequences revealed a significant association of rare ULBP3 variants (MAF < 0.01) with AA (p = 0.028).

Conclusion: Our study shows the contribution of rare genetic variation at the ULBP3 locus to disease susceptibility in AA. A functional follow-up of some of the identified variants can provide mechanistic insights into the development of AA.

References: Petukhova, L. et al. Genome-wide association study in alopecia areata implicates both innate and adaptive immunity. Nature 466, 113-7 (2010).

Grants: This work was supported by a grant from Deutsche Forschungsgemeinschaft (German Research Foundation), under the auspices of the Germany Excellence Strategy - EXC2151-390873048 (to R.C.B.).

Conflict of Interest: Paula Schwerbrock: None declared, Axel Schmidt: None declared, Rana Aldisi: None declared, hanna zieger: None declared, Nicole Cesarato: None declared, Stefan Herms: None declared, swapnil Awasthi: None declared, nadine fricker Nadine Fricker receives salary from Life & Brain GmbH, daisy axt: None declared, stephan ripke: None declared, Stefanie Heilmann-Heimbach Stefanie Heilmann-Heimbach receives salary from Life & Brain GmbH, Kerstin Ludwig: None declared, Peter Krawitz: None declared, Carlo Maj: None declared, Per Hoffmann Per Hoffmann receives salary from Life & Brain GmbH, Regina C. Betz: None declared, F. Buket Basmanav: None declared.

EP08.018 Sickle cell trait - a case report

Anastasiya Hryshkina ¹, Liliana Gonçalves¹, Marisa Teixeira¹, Rita Cerqueira¹, Carolina Vaz de Macedo², Juliette Dupont³, Joaquim Sá¹

¹CGC Genetics, Unilabs, Porto, Portugal; ²Hospital Garcia de Orta, E P E, Lisboa, Portugal; ³Centro Hospitalar Universitário de Lisboa Norte, E.P.E., Lisboa, Portugal

Background/Objectives: Sickle cell disease is caused by mutations in the gene encoding the haemoglobin subunit β (HBB), and is characterized by chronic haemolytic anaemia, ischemia, severe pain, and necrosis. The disease occurs when HBB gene is mutated in both alleles, and at least one of the mutations is c.20A>T p.(Glu7Val) (HbS). Individuals with this mutation alone carry the sickle cell trait and generally are healthy.

We report a case of a couple with a sickle cell trait and normal hemograma (pregnant has also elevated Hb F), and their fetus, that were studied for the variant c.20A>T p.(Glu7Val) in the HBB gene.

Methods: Sanger sequencing of the exon 1 of the HBB gene and MLPA analysis.

Results: Sanger sequencing revealed the heterozygous mutation c.20A>T p.(Glu7Val) in the father, while both mother and fetus had the c.20A>T p.(Glu7Val) mutation in apparent homozygosity. Those results were confirmed by MLPA: no deletions/duplications on the HBB gene were detected in the fetus, that was proven to be homozygous; while the mother has the c.20A>T p.(Glu7Val) mutation in one allele and deletion of the HBB and HBD genes (known as HPFH-2 Ghanaian) in the other allele.

Conclusion: Heterozygous individuals for the HPFH-2 Ghanaian deletion have normal hematology, with increased levels of Hb F (fetal hemoglobin) and decreased levels of Hb A2, which justifies the phenotype presented by the mother. Homozygous individuals for the HBB c.20A>T p.(Glu7Val) variant have sickle cell disease.

References: PMID: 29542687; 23065522; 28356267; 6196781; 2307466; 33238542.

Grants:

Conflict of Interest: None declared.

EP08.019 JAK2 variants associated with congenital erythrocytosis and Polycythemia vera

Monika Banfi¹, Aleša Kristan ², Irena Preložnik Zupan³, Andrej Vuga⁴, Tanja Kunej⁵, Nataša Debeljak²

¹Biotechnical Faculty, University of Ljubljana, Ljubljana, Slovenia; ²Institute of Biochemistry and Molecular Genetics, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia; ³Clinical Department of Hematology, University Medical Centre Ljubljana, Ljubljana, Slovenia; ⁴Kemomed Ltd., Kemomed Research and Development, Ljubljana, Slovenia; ⁵Department of Animal Science, Biotechnical Faculty, University of Ljubljana, Domžale, Slovenia

Background/Objectives: Erythrocytosis is a condition with an increased erythrocyte mass. The JAK2 gene is involved in the development of acquired type Polycythemia vera (PV), however its role in development of congenital erythrocytosis (CE) is still poorly understood. The aim of our research was to identify JAK2 variants associated with different types of erythrocytosis and to analyse their presence in patients suspected with CE.

Methods: Literature and Ensembl database were reviewed for potentially pathogenic JAK2 variants and their position in the gene. Patients were screened for variants in selected JAK2 gene regions using targeted NGS and confirmed by Sanger sequencing.

Results: Based on literature mining and bioinformatics analysis, we found several variants causative for PV in exon 12, exon 13, exon 14 and surrounding introns. One variant was found in exon 19 and another in exon 24. While only one variant was reported as causative for CE in exon 19. Sequencing of JAK2 in cohort of 27 patients suspected with CE did not reveal any of the previously reported causative variants. Nevertheless, we identified two new JAK2 variants with unknown significance in exon 13 and intron 19 in one family^1,2.

Conclusion: Variants of the JAK2 gene show considerable potential for development of erythrocytosis based on their location in previously reported regulatory regions. In future, the involvement of selected variants in erythrocytosis development will be validated by functional analysis.

References: 1. Kristan et al., 2021. J Clin Lab Anal, 35:e23715.

2. Kristan et al., 2021. Front Genet, 12:689868.

Grants: ARRS grants L3-9279 and Young Researcher funding.

Conflict of Interest: Monika Banfi: None declared, Aleša Kristan: None declared, Irena Preložnik Zupan: None declared, Andrej Vuga Kemomed, Tanja Kunej: None declared, Nataša Debeljak Leader of the project grant L3-9279..

EP08.020 Chronic idiopathic splenomegaly - a challenging autoimmune lymphoproliferative syndrome

Cristina Emilia Ursu ¹, Margit Serban¹, Adela Chirita-Emandi², Maria Puiu², Estera Boeriu³, Cristian Jinca³, Smaranda Teodora Arghirescu³

¹Romanian Academy of Medical Sciences, Onco-Hematology Research Unit, Children Emergency Hospital “Louis Turcanu”, European Haemophilia Treatment Center, Timisoara, Romania; ²Department of Genetics, Victor Babes University of Medicine and Pharmacy, Timisoara, Romania; ³Department of Pediatrics, Division of Onco-Hematology, Victor Babes University of Medicine and Pharmacy, Timisoara, Romania

Background/Objectives: Autoimmune lymphoproliferative syndrome (ALPS) is a primary immune regulatory disorder with a heterogeneous genetic background frequently confronted with diagnosis challenges. Even today with performant genetic technologies there remain some unclarified situations accepted as ALPS-like cases.

Methods: Case presentation.

Results: We bring to attention a 12 years old girl, without pathological history, who at the age of 9 presented mild splenomegaly, anaemia, and jaundice justifying a diagnosis of congenital microspherocytosis. At the age of 10, she was addressed to our clinic for skin pallor and significant splenomegaly. Laboratory investigations indicated a mild normochromic normocytic anaemia, without microspherocytosis, with important persistent reticulocytosis (>3%), mild inconstant thrombocytopenia, hyperbilirubinemia, very low haptoglobin values, direct positive Coombs test; and significantly high values (>10%) of double negative TCR αβ populations, of vitamin B12 (3757pg/ml) and IL-10 (103pg/ml) were assessed. Bone marrow biopsy revealed hypercellularity. Considering an ALPS, next-generation sequencing was performed revealing in the girl and mother a heterozygous variant of MYH13 gene, with uncertain significance. Meeting the mandatory criteria and 3 secondary accessory criteria, we initially interpreted the case as an ALPS-like syndrome. Later the necessary primary accessory biomolecular criteria were found using the whole genome sequencing which assessed a loss of the number of copies on chromosome 10q23.31, with a heterozygous deletion, including the pathogenic FAS gene, finally confirming the ALPS type I diagnosis.

Conclusion: Initially, miss-interpreted, followed by a presumption of ALPS-like, this case finally reached the right diagnosis proving the importance of a comprehensive exploratory approach.

References: NA

Grants: NA

Conflict of Interest: None declared.

EP08.022 A young adult with vasculitis and stroke at the age of 8: consider adenosine deaminase-2 deficiency

Romana Vulturar ¹, Simona Mihaela Grad², Liliana Bene², Adina Chis³, Carolina Solomon⁴, Laura Damian⁵

¹“Iuliu Hatieganu” University of Medicine and Pharmacy Cluj-Napoca, Romania, Department of Molecular Sciences, Cluj-Napoca, Romania; ²University of Medicine and Pharmacy ‘Iuliu Hatieganu” from Cluj-Napoca, Dept. of Internal Medicine, Cluj-Napoca, Romania; ³“Iuliu Hatieganu” University of Medicine and Pharmacy Cluj-Napoca, Romania, Department of Molecular Sciences, Cluj-Napoca, Romania; ⁴“Iuliu Hatieganu” University of Medicine and Pharmacy Cluj- Napoca, Radiology Department, Cluj-Napoca, Romania; ⁵Emergency Clinical County Hospital Cluj, Department of Rheumatology, Center for Rare Autoimmune and Autoinflammatory Diseases, Cluj-Napoca, Romania

Background/Objectives: The deficiency of adenosine deaminase-2 (DADA2) is a recently described autoinflammatory and metabolic disease evolving with childhood-onset polyarteritis nodosa, strokes, livedo racemosa and immune deficiency, responsive to anti-tumour necrosis factor agents.

Methods: A 26-year male patient with a vasculitis with stroke at the age of 8, presented for headache. Examination revealed livedo racemosa, a sluggish speech and splenomegaly. Apart from inflammation (CRP 22 mg/L, normal <6 mg/L) and a selective IgG deficiency (520 mg/dl, normal 700-1600 mg/dl), all immunological tests (including antinuclear, antiphospholipid and anti-neutrophil cytoplasm antibodies, complement fractions and hepatitis B, C and HIV serology) were normal.

Results: A 109 genes assessment (autoinflammatory syndromes panel, Invitae, US) revealed two heterozygous variants of unknown significance (VUS) in ADA2: a c.620T>G(p.Phe270Cys) in exon 4, suggested by predictive algorithms to be disruptive, and c.967G>A (p.Val323Ile), in exon 7, for which the predictive algorithms did not agree (SIFT: deleterious, PolyPhen-2 probably damaging, Align GVGD Class 0).

Conclusion: Nevertheless, the first enzymatic test (Synlab) did not confirm the enzyme deficiency. He was treated with glucocorticoids and azathioprine, with clinical improvement. The patient is being tested further in order to clarify a DADA2 or other type of vasculitis and treat it optimally.

References:

Grants:

Conflict of Interest: None declared.

EP08.023 Two cases of a rare association between ataxia-telangiectasia and juvenile idiopathic arthritis

Tamara Savevska ¹, Vangel Ristovski¹, Alenka Gagro², Carola Vinuesa^3;4, Marija Jelusic⁵, Todor Arsov^1;4

¹Ss. Cyril and Methodius University in Skopje, Faculty of Medicine – Skopje, Institute of Immunobiology and Human Genetics, Skopje, Macedonia; ²Department of Paediatrics, Children’s Hospital, Zagreb, Croatia; ³The Francis Crick Institute, London, United Kingdom; ⁴Centre for Personalised Immunology, Australian National University, Canberra, Australia; ⁵Department of Paediatrics, University of Zagreb School of Medicine, University Hospital Centre, Zagreb, Croatia

Background/Objectives: Ataxia-telangiectasia (AT) is an autosomal recessive condition caused by biallelic pathogenic ATM variants, presenting with cerebellar ataxia and telangiectasia, endocrine and immunological features, radiation sensitivity and cancer susceptibility. Immunodeficiency is present in 60-80% of AT patients, most frequently low immunoglobulin levels, lymphopenia, weak vaccine response and common infections. There have been rare reports of AT cases with inflammatory and autoimmune conditions such as thrombocytopenia, vitiligo and cutaneous granulomatosis.

Methods: We present two patients with AT and juvenile idiopathic arthritis (JIA), the most frequent autoimmune rheumatic disease in childhood. Both patients presenting as oligoarthritis originate from the same geographic region.

Results: Exome sequencing found compound heterozygosity in both (patient 1: c.5293C>T; p.Gln1765Ter and c.8305T>C; p.Trp2769Arg and patient 2: c.4383G>A; p. Trp1461Ter and c.6095G>A; p.Arg2032Lys). No significant known gene variants or susceptible HLA alleles associated with JIA were identified.

Conclusion: These two cases provide support of the hypothesis that loss of ATM function leads to immune deregulation and systemic inflammation. It raises questions about the clinical management of patients with dysfunctional DNA repair due to loss of ATM function in terms of systematic screening for autoimmune and inflammatory conditions in patients with AT and rational use of the imaging tools given the radiosensitivity and predisposition to cancer.

References:

Grants:

Conflict of Interest: None declared.

EP09 Intellectual Disability

EP09.001 Ververi-Brady syndrome associated with QRICH1 variants: a report of two new cases in Russia and literature review

Tatyana Kozhanova ^1;2, Svetlana Zhilina^1;2, Tatyana Mescheryakova¹, Ekaterina Luk`yanova³, Lilya Sushko³, Andrey Prityko^4;5, Nikolay Zavadenko²

¹St. Luka’s Clinical Research Center for Children, laboratory genetics, Moscow, Russian Federation; ²Pirogov Russian National Research Medical University, Neurology, Neurosurgery and Medical Genetics named after L.O. Badalyan Faculty of Pediatrics, Moscow, Russian Federation; ³St. Luka’s Clinical Research Center for Children, psychoneurology, Moscow, Russian Federation; ⁴St. Luka’s Clinical Research Center for Children, Director, Moscow, Russian Federation; ⁵Pirogov Russian National Research Medical University, Department of Maxillofacial Surgery and Dentistry Faculty of Pediatrics, Moscow, Russian Federation

Background/Objectives: Recent advances in sequencing technologies have enabled identification of multiple genes associated with intellectual disability disorders, including QRICH1. The function of QRICH1 is largely unknown but it is likely to play a key role in the unfolded response of endoplasmic reticulum stress. Ververi-Brady syndrome (VBS; MIM:#617982) is a rare developmental disorder, characterized by mild developmental delay, mildly impaired intellectual development and speech delay and mild dysmorphic facial features, and loss of-function variants in QRICH1 were implicated in its etiology.

Methods: We examined 2 patients with epilepsy, developmental and speech delay. The clinical phenotyping, video-electroencephalography, magnetic resonance imaging of the brain were carried out. All patients received informed consent for whole exome sequencing.

Results: We describe two new patients and review the clinical and genetic information on all previously reported VBS cases. Here, we present two unrelated individuals with one missense mutation (c.1711G>A; p. Asp571Asn) and one frameshift mutations (c.1963_1964insT; p.Lys655IlefsTer). These two individuals contributes to the delineation of the VBS phenotype and suggests epilepsy, developmental and speech delay, moderate motor delay, learning difficulties and mildly impaired dysmorphic features.

Conclusion: To date, thirty eight individuals have been reported with QRICH1 mutations in the world. Our new clinical cases presented in Russia also confirms that the phenotype of Ververi-Brady syndrome is relatively nonspecific and includes variable neurodevelopmental features, delayed speech, learning difficulties, epilepsy and mildly impaired dysmorphic features.

References: Kumble S, Levy AM, Punetha J, et al. The clinical and molecular spectrum of QRICH1 associated neurodevelopmental disorder. Hum Mutat. 2021 Dec 2. https://doi.org/10.1002/humu.24308.

Grants: No.

Conflict of Interest: None declared.

EP09.002 Refining the Phenotypic Spectrum of KMT5B-Associated Syndromic Developmental Delay

aviva eliyahu^1;2, Ortal Barel Barel^3;4, Lior Greenbaum^1;2, gal zaks hoffer^2;5, Yael Goldberg^2;5, annick raas-rothschild^2;6, Amihood Singer⁷, ifat bar-joseph^3;4, vered kunik⁸, elisheva javasky^3;4, orna staretz-chacham^9;10, naomi pode shakked^2;11, lily bazak^5;12;13, noa ruhrman-shahar^2;5, elon pras^1;2, moshe frydman^1;2, mordechai shohat^2;3, Ben Pode-Shakked ^2;6

¹Sheba Medical Center, The Danek Gertner Institute of Human Genetics, Ramat Gan, Israel; ²Tel-Aviv University, Sackler Faculty of Medicine, Tel-Aviv, Israel; ³Sheba Medical Center, The Genomic Unit, Sheba Cancer Research Center, Ramat Gan, Israel; ⁴Sheba Medical Center, The Wohl Institute for Translational Medicine and Cancer Research Center, Ramat Gan, Israel; ⁵Rabin Medical Center - Beilinson Hospital, The Raphael Recanati Genetics Institute, Petach Tikva, Israel; ⁶Edmond and Lily Safra Children’s Hospital, Sheba Medical Center, The Institute for Rare Diseases, Ramat Gan, Israel; ⁷Israeli Ministry of Health, Community Genetics, Public Health Services, Jerusalem, Israel; ⁸Bioinformatics consulting, Mass, Israel; ⁹Soroka Medical Center, Metabolic Clinic, Beer Sheva, Israel; ¹⁰Ben-Gurion University, Faculty of Health Sciences, Beer Sheva, Israel; ¹¹Edmond and Lily Safra Children’s Hospital, Sheba Medical Center, Department of Pediatrics, Ramat Gan, Israel; ¹²Bar Ilan University, Mina and Everard Goodman Faculty of Life Science, Ramat Gan, Israel; ¹³Rabin Medical Center, Felsenstein Medical Research Center, Petach Tikva, Israel

Background/Objectives: The role of lysine methyltransferases (KMTs) and demethylases (KDMs) in the regulation of chromatin modification is well established. Recently, heterozygous variants in KMT5B were implicated in individuals with intellectual disability (ID) and/or autism.

Methods: Trio exome sequencing (ES) was performed for three unrelated patients with global developmental delay (GDD) or ID, macrocephaly and additional features. 3D structural models of wild-type (WT) and mutated KMT5B structures were constructed, and intra-structure hydrogen bonds and the distances between atoms were calculated.

Results: Patient A presented at 12 months of age due to profound GDD, and showed macrocephaly, broad forehead, strabismus, short neck and mild hypertelorism. Patient B presented at 2.5 years with GDD and relative macrocephaly, and showed a broad forehead, conical fingers, hypotonia and joint hypermobility. Patient C presented at 35 years due to ID, seizures and overgrowth (weight, height and head circumference >97 centile). Physical examination was notable for occipital protrusion and upsweeping of frontal hair, large hands with long and lean fingers. All three probands had normal Chromosomal Microarray Analysis and normal maternal Fragile X testing. Following ES, each of the probands was found to harbor a distinct de-novo heterozygous disease-causing variant in KMT5B: c.541C>G (p.His181Asp); c.833A>T (p.Asn278Ile); or c.391_394delAAAG (p.Lys131GlufsTer6). Using 3D-computational modeling of the KMT5B protein, structural effects of the two missense variants were demonstrated.

Conclusion: Our findings support the role of de-novo heterozygous variants in KMT5B-associated GDD, and suggest that KMT5B should be considered in the differential diagnosis of neurodevelopmental disorders accompanied by macrocephaly and/or overgrowth.

References:

Grants:

Conflict of Interest: None declared.

EP09.003 Williams-Beuren region triplication syndrome in a 4-year-old girl from Ukraine

Nataliya Kitsera ^1;2, Maya Bondarenko², Ruslan Kozovyi², Roman Bahrynovskyi²

¹Institute of Hereditary Pathology of NAMS of Ukraine, Lviv, epidemiology, Lviv, Ukraine; ²Ivano-Frankivsk National Medical University, Ukraine, Ivano-Frankivsk, Ukraine

Background/Objectives: Williams-Beuren Duplication Syndrome (7q11.23) is a multisystem developmental pathology with a variety of clinical polymorphisms.

The aim of this study was to diagnose chromosomal changes at the submicroscopic level as a first-line test for patients with intelligence deficits, autism spectrum disorders.

Methods: Chromosomal micromatrix analysis was performed in the laboratory “Vista” (Tai Po Industrial Estable, N.T. Hong Kong).

Results: Parents turned to a geneticist with complaints of delayed physical, intellectual, language development in a 4-year-old daughter to clarify the diagnosis, as well as to determine the prognosis of further childbirth. The girl was born without congenital anomalies from the 1st pregnancy, premature birth within 36 weeks. At birth: parents age 27 years, body weight - 2620 g, height - 46 cm, APGAR score 7/7. Up to 6 months was diagnosed with hydrocephalus. Began sitting at 8 months, walking - 1 year 3 months. Doesn’t talk. Doesn’t enter into speech contact, eye contact is relative. Constructive tasks are sometimes performed with the help, emotionally labile. Motor activity is satisfactory.

Siblings of the proband mother’s grandfather had mental retardation in anamnesis.

Chromosomal analysis - 46, XX. Chromosomal micromatrix analysis - Duplicate fragment detected of a 1.67 Mb in the region 7q11.23q11.23 (copy number: 3).

Diagnosed Williams-Beuren triplication syndrome (OMIM: 609757) with autosomal dominant inheritance.

In literature only several descriptions of such patients were found.

Conclusion: Due to chromosomal micromatrix analysis diagnosed Williams-Beuren triplication syndrome in the proband and the mother was recommended to perform prenatal diagnosis in the next pregnancy.

References:

Grants: no.

Conflict of Interest: None declared.

EP09.004 Identification of a new frameshift deletion in the PTRHD1 gene in two affected children with juvenile-onset parkinsonism and intellectual disability

Aruna Marchetto ¹, Felicitas Maier¹, Moneef Shoukier¹, Sabine Minderer¹, Karl-Philipp Gloning¹

¹Prenatal Medicine Munich, Munich, Germany

Background/Objectives: Juvenile parkinsonism is a progressive neurodegenerative disease that starts at an early age and is characterized by tremor, muscular stiffness, unbalanced coordination and slowness of movements. While it is known that parkinsonism is associated with cognitive impairment, data on juvenile-onset parkinsonism and intellectual disability are still limited to a few case reports. We present two siblings from a consanguineous Syrian family: a 4-year-old patient with severe speech delay, behavioral problems, gait difficulties and delayed global development, and his less severely affected 14-year-old sister with delayed global development and gait difficulties.

Methods: Morphological examination, fragile X syndrome, Whole Exome Trio analysis, MRT scans and histological staining of skin biopsy were performed.

Results: A Whole Exome Trio analysis revealed a homozygous and likely disease-causing variant c.280delC, p.(Leu94TrpfsTer40) in the PTRHD1 gene of both siblings. This mutation is not listed in public databases but it is located in the functional domain PTH2 of the PTRHD1 gene, known to suppress proteolysis degradation and to be causative for neurodegenerative diseases such as the autosomal recessive juvenile-onset parkinsonism and intellectual disability (Kuipers et al., 2018; Khodadadi et al., 2017).

Conclusion: Given the role of the PTRHD1 gene in parkinsonism and intellectual disability in combination with the molecular and clinical findings of both siblings, we assume this frameshift mutation to be causative for a PTRHD1-associated juvenile-onset parkinsonism and intellectual disability supported by previous results about the function of the PTH2 domain in the PTRHD1 gene. Experimental therapy with Levodopa was successfully started in the 4-year-old patient.

References:

Grants:

Conflict of Interest: None declared.

EP09.005 Child health, parent health, and family functioning in families of children with Down syndrome, congenital heart disease and both conditions from Ecuador, Spain and USA

Marcia Van Riper ¹, George Knafl², Laura Serrano Fernández³, Blanca Egea-Zerolo⁴

¹University of North Carolina - Chapel Hill, Nursing/ Carolina Center for Genome Sciences, Chapel Hilll, United States; ²University of North Carolina - Chapel Hill, Chapel Hill, United States; ³Villanueva University, Madrid, Spain; ⁴Comillas Pontifical University, Madrid, Spain

Background/Objectives: Down syndrome (DS), the most common chromosomal cause of intellectual disability, can affect the health of the child with DS, parental health and overall family functioning. Approximately 50% of children with DS have congenital heart disease (CHD). The aim of this international collaboration was to compare parental perceptions of child health, parental health (physical and mental) and family functioning in families of children with DS, CHD and both conditions from Ecuador, Spain and USA.

Methods: Parents completed an online survey. Clinicians and group leaders of support groups and foundations shared study information with eligible families.

Results: 560 parents completed the online survey (141 Ecuador, 273 Spain, and 146 USA). One-way analysis of variance F tests were used to compare means for the four variables across the nine groups. While there were significant differences between groups for all four variables, the overall pattern was one of good health and above average family functioning. Generally, health and family functioning were less positive in families of children with both conditions and families from Ecuador.

Conclusion: Findings suggest that despite the ongoing challenges associated with living with a chronic condition, many parents reported good health and above average family functioning. More research is needed to identify families that need extra support, such as families of children with multiple conditions and families from countries known to provide less support to families. Research is also needed to understand how social determinants of health influence child health, parental health and family functioning.

References:

Grants: Dhillon Jordan Shah Innovation Fund in CHD.

Conflict of Interest: None declared.

EP09.006 Case Report: Identification of a de novo nonsense mutation in WASF1 gene in a patient with seizures and developmental delay

Hengameh Abdollahpour ¹, Pauline E. Schneeberger², Martin Merkel³

¹MVZ für Laboratoriumdmedizin und Humangenetik Hamburg, Hamburg, Germany; ²Amedes MVZ Wagnerstibbe für Laboratoriumsmedizin, Hämostaseologie, Humangenetik und Mikrobiologie Hannover, Hannover, Germany; ³Endocrinologicum, Hamburg, Germany

Background/Objectives: De novo heterozygous nonsense or frameshift mutations in the WASF1 gene have been first reported in 5 unrelated patients presenting neurodevelopmental disorder and seizures with absent language. We report on a girl with severe muscular hypotonia and global developmental delay, first observed at the age of 6 months who further developed an epilepsy. A brain MRI did not show any structural abnormalities. No Organ malformations were detected.

Methods: The karyotyping showed a normal female karyotype (46,XX), confirmed by aCGH analysis. Whole exome sequencing detected one pathogenic variant and a variant of uncertain significance in the GBA gene. Furthermore, we could detect a likely pathogenic heterozygous variant in WASF1. Using dried blood spots technique for the evaluation of beta glucocerebrosidase activity the suspicion of Gaucher disease could be ruled out. Therefore, we examined our second candidate, the variant c.1507G>T, p.(Glu503*) in WASF1.

Results: The variant p.(Glu503*) in WASF1 gene predicted to result in a premature stop codon, has not previously been reported. The variant could be excluded in both parents by Sanger sequencing, confirming de novo status of the variant in the patient.

Conclusion: This case report represents the seventh patient with developmental delay and seizure having a pathogenic variant in WASF1. Therefore, this case highlights the utility of exome sequencing for identifying rare genetic causes of developmental delay at a very early age.

References:

1-Ito, Y., et al., De novo truncating mutations in WASF1 cause intellectual disability with seizures. Am. J. Hum. Genet. 2018 Jul 5; 103(1): 144-153.

Grants:

Conflict of Interest: None declared.

EP09.007 A new patient with TLK2-associated Mental Retardation: Further delineation of the clinical and molecular phenotype of a rare Neurodevelopmental Disorder

Andreas Busche ¹, Ann-Christin Tewes¹, Axel Bohring¹

¹University of Münster, Institute of Human Genetics, Münster, Germany

Background/Objectives: The TLK2-asscociated Mental Retardation, also referred to as Autosomal Dominant Mental Retardation Type 57 (MRD57, OMIM#618050) is a rare condition characterized by mild to moderate developmental delay, behavioral disorders, and gastrointestinal feeding problems. Certain facial dysmorphic features have been observed frequently. MRD57 is caused by heterozygous mutations in TLK2 (OMIM*608439) and is inherited in an autosomal dominant pattern. Up to now, only about 40 patients have been reported.

Methods: Case report.

Results: Here we present a 9 year and 4 month old boy with MRD57 due to the heterozygous de novo variant c.2034del p.(Glu679Lysfs*9) in TLK2. This variant is classified as likely pathogenic (Class 4) according to ACMG guidelines. The patient has a moderate intellectual disability, restlessness, a short attention span, and social-emotional problems. Facial dysmorphisms include telecanthus, prominent nasal bridge, broad nasal tips, upslanting palpebral fissures, and low set ears. These features have been described before in patients with MRD57. Gastrointestinal feedings problems, seen in many other patients with MRD57, have not been observed.

Conclusion: The dymorphisms seen in the boy reported here are overlapping with the patients described before, but a distinct facial gestalt can´t be defined so far. In this report, we present detailed clinical and molecular data of a new patient with MDR57 to delineate further the clinical phenotype of TLK2-associated Mental Retardation and to point to this rare intellectual disability syndrome.

References: Reijnders et al. (2018). De Novo and Inherited Loss-of-Function Variants in TLK2: Clinical and Genotype-Phenotype Evaluation of a Distinct Neurodevelopmental Disorder. Am J Hum Genet. 102, 1195-1203..

Grants:

Conflict of Interest: None declared.

EP09.009 How DeepGestalt triggered WES re-analysis and led to the identification of a KANSL1 intragenic deletion causing Koolen-de Vries syndrome

Claudia Perne ¹, Axel Schmidt¹, Sophia Peters¹, Elisabeth Mangold¹, Kirsten Cremer¹, Tim Bender², André Heimbach¹, Sugirthan Sivalingam³, Jessica Trautmann¹, Hela Hundertmark¹, Nuria Ortega Ibanez¹, Alexej Knaus⁴, Fabian Brand⁴, Peter Krawitz⁴, Hartmut Engels¹

¹University of Bonn - University Hospital Bonn, Institute of Human Genetics, Bonn, Germany; ²University of Bonn - University Hospital Bonn, Center for Rare Diseases Bonn, Bonn, Germany; ³University of Bonn, Medical Faculty, Core Unit for Bioinformatics Data Analysis, Bonn, Germany; ⁴University of Bonn, Medical Faculty, Institute for Genomic Statistics and Bioinformatics, Bonn, Germany

Background/Objectives: Koolen-De Vries syndrome (OMIM # 610443) is caused either by recurrent heterozygous 500 - 650kb microdeletions at chromosome 17q21.31 including KANSL1 or by heterozygous intragenic pathogenic variants in KANSL1.

We present a fifteen-year-old girl with global developmental delay and moderate intellectual disability. She has had muscular hypotonia since early childhood. The patient had distinctive facial dysmorphism and her family described her as extremely friendly, but anxious in contact with other children. Chromosomal microarray (CMA) performed at the age of eight years were unremarkable.

Methods: Trio whole exome sequencing (WES) was performed on leukocyte-derived DNA samples. The DeepGestalt algorithm of Face2Gene was used to prioritize syndrome suggestions.

Results: The DeepGestalt algorithm scored remarkably high for Koolen-de Vries syndrome. Nevertheless, Sanger sequencing and MLPA of the KANSL1 gene gave normal results. We then performed trio WES but again no pathogenic variant was detected in KANSL1 or any other gene. Only after targeted re-analysis of KANSL1 sequencing data in the Integrative Genomics Viewer we were able to detect a 4708 bp intragenic deletion comprising parts of intron 6 and exon 7 (c.1849-4611_1895del;r.spl). The deletion was verified by qPCR and whole genome sequencing (WGS). It was not detected in the parents; hence, it is highly likely that this variant occurred de novo and is causal for the patient’s symptoms.

Conclusion: This case demonstrates the utility of Face2Gene and DeepGestalt in facilitating the diagnosis of genetic syndromes with typical facial dysmorphism and stresses the importance of accurate CNV analysis of WES and WGS data.

References:

Grants:

Conflict of Interest: None declared.

EP09.010 OrphaID: a new platform for rare intellectual disabilities in Orphanet in partnership with ERN-ITHACA

Mutaz Amin ¹, Annie Olry¹, Valerie Serriere-Lanneau¹, Christiane Zweier^2;3, Anne Hugon⁴, Bernt Popp⁵, Caterina Lucano¹, Houda Ali¹, Ana Rath¹, Alain Verloes⁴

¹INSERM, US14-Orphanet, Paris, France; ²Friedrich-Alexander-Universität Erlangen-Nürnberg, Institute of Human Genetics, Erlangen, Germany; ³University of Bern, Department of Human Genetics, Bern, Switzerland; ⁴ERN-ITHACA, APHP-Robert Debré University Hospital, Université de Paris, Genetics, Paris, France; ⁵University of Leipzig Hospitals and Clinics, Institute of Human Genetics, Leipzig, Germany

Background/Objectives: Intellectual disability (ID) is a group of heterogenous disorders affecting up to 3% of worldwide population and characterized by significant impairment in cognition and behavior which can be associated with other syndromic or dysmorphic features. The heterogeneity of intellectual disabilities and rarity of most forms render the genetic diagnosis challenging in most cases. Hence, stemmed a need for developing a comprehensive list of curated intellectual disability related genes and associated phenotypes.

Methods: Orphanet, which is the largest portal for rare diseases in the world has partnered with the European Reference Network on Rare Congenital Malformations, Autism and Rare Intellectual Disability (ERN-ITHACA) in order to develop a platform (OrphaID) for curated intellectual disability related genes and phenotypes. This list of intellectual disability-related genes is curated in partnership with ERN-ITHACA and SysID/SysNDD (www.sysid.dbmr.unibe.ch).

Results: OrphaID currently contains 728 Orpha-coded ID phenotypes linked to 1385 curated ID genes. This will allow systematic and comprehensive access to and retrieval of ID specific information within Orphanet. The list of OrphaID entries is linked to both internal (e.g. genes information, classification, HPO signs) and external resources (e.g. SysNDD, OMIM). It can be searched or filtered by gene, disease or OMIM number and the results can be downloaded in different file formats (excel, csv and txt).

Conclusion: OrphaID is a valuable tool for all users (patients, researchers and clinicians) with interest in rare intellectual disabilities. The new platform will be publicly available on Orphanet’s webpage by March 2022.

References:

Grants:

Conflict of Interest: None declared.

EP09.011 Study of IL6 and TNF genes polymorphisms as a risk factor for the development of early neurological disorders and subsequent consequences in neonates

Ana Djuranovic ¹, Nataša Cerovac², Dijana Perovic¹, Nataša Stojanovski¹, Tatjana Damnjanovic¹

¹Institute of Human Genetics, Faculty of Medicine, University of Belgrade, Belgrade, Serbia; ²Clinic of Neurology and Psychiatry for Children and Youth, Belgrade, Serbia

Background/Objectives: Intraventricular hemorrhage (IVH) and hypoxic-ischemic encephalopathy (HIE) are the most common types of early neurological disorders which are detectable by neuroimaging in newborns with perinatal asphyxia. They might be reversible, but require adequate and early treatment, otherwise their most serious consequence is cerebral palsy (CP). IVH and HIE are activating immune system and inflammation mediators like interleukin 6 (IL6) and tumor necrosis factor (TNF). Inflamation and neurodegeneration could contribute to development of CP. Aim of this study was to examine association between IL6 and TNF polymorphisms with the development of early neurological disorders and CP in children with perinatal asphyxia.

Methods: Study included 167 patients aged 1 to 16 years with perinatal asphyxia. Clinical evaluation and neuroimaging (ultrasound, magnetic resonance) were performed for all subjects. IL6 and TNF polymorphisms were genotyped using rs1800795 and rs1800629 TaqMan assays.

Results: Frequencies of IL6 polymorphisms were statistically significantly different between groups with early neurological disorders and without them (43.4% GG, 48.2% GC, 8.4% CC, versus 26.5% GG, 54.2% GC, 19.3% CC, respectively) (p = 0.027). GG+GC genotypes were significantally more frequent in patients with early neurological disorders (p = 0.043, OR = 0.386, 95%CI 0.150-0.994). There was statistical significant difference in genotype distribution of TNF polymorphism in CP onset. AA+GA genotype was more frequent in patients who developed CP (p = 0.040, OR = 0.425 95%CI 0.185-0.974).

Conclusion: IL6 gene polymorphism could be a risk factor for early neurological disorders which require tretment, while TNF polymorphism may be predictor for CP.

References:

Grants:

Conflict of Interest: None declared.

EP09.012 CCNK-intellectual developmental disorder with hypertelorism and distinctive facial dysmorphism - the impact of cyclins in neurodevelopment

Ana Raquel Gouveia Freitas da Silva ¹, Oana Moldovan¹, Ana Medeira¹, Ana Berta Sousa¹

¹Serviço de Genética Médica, Departamento de Pediatria, Hospital de Santa Maria, Centro Hospitalar Universitário Lisboa Norte, Lisboa, Portugal

Background/Objectives: Cyclin K (CCNK) integrates the same cyclin-dependent kinase complex as CDK13, which plays a role in early brain development. Variants in CCNK gene are associated with autosomal dominant intellectual developmental disorder with hypertelorism and distinctive facies (OMIM#618147). We are aware of at least 4 patients reported in the literature, 3 bearing microdeletions in 14q32.2 involving CCNK gene, and 1 with a missense variant, all de novo. The condition is characterized by developmental delay (DD) and typical dysmorphism.

Methods: We describe a 3-year-old girl, who is the only child of non-consanguineous parents with no relevant family history. At birth, she was diagnosed with a thin patent ductus arteriosus and pulmonary valve dysplasia with mild stenosis. She evolved with mild DD and mild cerebellar vermis hypoplasia. On physical examination, she presented glabellar hemangioma, high anterior hairline, hypertelorism, flattened nasal root, low-set and posteriorly rotated ears, short columella, thin upper vermilion, narrow jaw and a deep sacrococcygeal pit. ArrayCGH was normal. Sequencing of CDK13 gene found no relevant variant.

Results: Whole exome sequencing identified a heterozygous missense variant of uncertain significance in CCNK gene: c.556T>C, p.(Trp186Arg). De novo origin was confirmed by parental analysis.

Conclusion: Our patient’s facial phenotype is consistent with the few cases previously reported in the literature. Similar to the only other case caused by a missense variant, DD is milder compared to deletions, corroborating haploinsufficiency as a pathogenetic mechanism. It overlaps with CDK13-ID and shares the same episignature, highlighting their close interaction.

References:

Grants:

Conflict of Interest: None declared.

EP09.013 Pathogenic variants in nucleoporin TPR (translocated promoter region, nuclear basket protein) cause severe intellectual disability in humans

Nicole Van Bergen ^1;2, Katrina Bell^3;4, Kirsty Carey⁵, Russell Gear⁴, Sean Massey¹, Edward Murrell¹, Lyndon Gallacher^2;4, Kate Pope⁶, Paul Lockhart⁶, Andrew Kornberg^2;7;8, Lynn Pais⁹, Marzena Walkiewicz¹⁰, Cas Simons^1;10, RDNow Flagship¹, Vi Wickramasinghe^5;11, Sue White^2;4, John Christodoulou^1;2;4;12

¹Murdoch Children’s Research Institute, Brain and Mitochondrial Research Group, Parkville, Australia; ²University of Melbourne, Department of Paediatrics, Parkville, Australia; ³Murdoch Children’s Research Institute, Bioinformatics Methods, Parkville, Australia; ⁴Victorian Clinical Genetics Services, Parkville, Australia; ⁵Peter MacCallum Cancer Centre, RNA Biology and Cancer Laboratory, Melbourne, Australia; ⁶Murdoch Children’s Research Institute, Bruce Lefroy Centre for Genetic Health Research, Parkville, Australia; ⁷Royal Children’s Hospital, Neurology Department, Parkville, Australia; ⁸Murdoch Children’s Research Institute, Neurosciences Research, Parkville, Australia; ⁹Broad Institute of MIT and Harvard, Center for Mendelian Genomics, Cambridge, United States; ¹⁰Murdoch Children’s Research Institute, Translational Genomics Research Group, Parkville, Australia; ¹¹The University of Melbourne, Bio21 Molecular Science and Biotechnology Institute, Parkville, Australia; ¹²University of Sydney, Discipline of Child & Adolescent Health, Sydney Medical School, Sydney, Australia

Background/Objectives: The nuclear pore complex (NPC) is a multi-protein complex that regulates the trafficking of macromolecules between the nucleus and cytoplasm. Genetic variants in components of the NPC have been shown to cause a range of neurological disorders, including intellectual disability and microcephaly. Here we present two siblings who present with a phenotype of microcephaly, ataxia and severe intellectual disability. Whole Genome Sequencing (WGS) identified biallelic variants in TPR and we undertook functional genomics analysis to evaluate pathogenicity.

Methods: WGS and RNAseq were used to search for pathogenic variants, and identified candidate variants in TPR. Functional analysis in patient fibroblasts evaluated pathogenicity of TPR variants. We analysed protein levels by western blot and nuclear pore density and RNA intensity in the nucleus by immunostaining and confocal microscopy.

Results: The variants result in a premature truncation variant (c.6625C>T; p.Arg2209*), and a splice variant (c.2610+5G>A) leading to a 12-amino acid deletion respectively. Functional analyses in patient fibroblasts demonstrate significantly reduced TPR levels by western blot, decreased colocalization of TPR to NPCs, and decreased TPR-containing NPC density. A compensatory increase in total NPC levels was observed, whilst decreased global RNA intensity was apparent in the nucleus.

Conclusion: The discovery of variants that partly disable TPR function provide valuable insight into this protein which is essential for neurodevelopment, and our findings show that TPR variants are the cause of the siblings’ neurological disorder.

References: Van Bergen et al. (2021). Human Molecular Genetics; 31 (3): 362–375.

Grants: Royal Children’s Hospital Foundation and NHMRC.

Conflict of Interest: None declared.

EP09.014 A novel SMG9 variant enriched in the Finnish population causes intellectual disability

Elisa Rahikkala ¹, Lea Urpa², Bishwa Ghimire², Hande Topa³, Mitja I. Kurki^2;4;5, Maryna Koskela³, Mikko Airavaara^3;6, Eija Hämäläinen², Katri Pylkäs⁷, Jarmo Körkkö⁸, Helena Savolainen⁸, Anu Suoranta², Aida Bertoli-Avella⁹, Arndt Rolfs^10;11;12, Pirkko Mattila², Mark Daly^2;4;5, Aarno Palotie^2;4;5, Olli Pietiläinen^3;5, Jukka Moilanen¹, Outi Kuismin^1;2

¹Oulu University Hospital and University of Oulu, Department of Clinical Genetics, PEDEGO Research Unit and Medical Research Center Oulu, Oulu, Finland; ²Institute for Molecular Medicine Finland (FIMM), University of Helsinki, Helsinki, Finland; ³Neuroscience Center, Helsinki Institute of Life Science, University of Helsinki, Helsinki, Finland; ⁴Psychiatric & Neurodevelopmental Genetics Unit, Massachusetts General Hospital, Boston, MA, United States; ⁵The Stanley Center for Psychiatric Research, The Broad Institute of MIT and Harvard, Cambridge, MA, United States; ⁶Division of Pharmacology and Pharmacotherapy, Faculty of Pharmacy, University of Helsinki, Helsinki, Finland; ⁷University of Oulu and NordLab Oulu, Cancer and Translational Medicine Research Unit and Biocenter Oulu, Oulu, Finland; ⁸Northern Ostrobothnia Hospital District, Center for Intellectual Disability Care, Oulu, Finland; ⁹Centogene GmbH, Rostock, Germany; ¹⁰Centogene GmbH, Rostock, Germany; ¹¹University of Rostock, Medical Faculty, Rostock, Germany; ¹²Arcensus GmbH, Rostock, Germany

Background/Objectives: The SMG9 gene encodes a regulatory subunit of nonsense-mediated mRNA decay (NMD) machinery. We identified five patients from three families with a similar intellectual disability (ID) and an identical homozygous SMG9 variant.

Methods: Using exome sequencing, a novel SMG9 variant was identified. RNA sequencing of patients and age- and sex-matched healthy controls was used to assess the effect of the variant.

Results: The homozygous variant SMG9 (NM_019108.4) c.551T>C p.(Val184Ala) segregated recessively in each family. Characteristic clinical findings in patients were mild to moderate ID, intention tremor, pyramidal signs, dyspraxia, and ocular manifestations. Allele-specific expression analysis did not provide evidence that the nonsense mRNA-induced NMD was affected. Differential gene expression analysis identified a prevalent upregulation of genes in patients, including the genes SMOX, OSBP2, GPX3, and ZNF155. These findings suggest that normal SMG9 function may be involved in transcriptional regulation without affecting nonsense mRNA-induced NMD.

Conclusion: We demonstrate that the SMG9 c.551T>C variant causes a neurodevelopmental disorder and impacts gene expression. NMD components have roles beyond aberrant mRNA degradation that are crucial for neurocognitive development.

References: Eur J Hum Genet 2022 https://doi.org/10.1038/s41431-022-01046-5.

Grants: ER: Academy of Finland (338446), the Finnish Medical Foundation, AP: Academy of Finland Centre of Excellence in Complex Disease Genetics (312074, 336824) and Sigrid Juselius Foundation, EU/Horizon2020, COSYN, grant number 667301, MD: 5U01MH111660-04, 04/10/17- 01/31/22, NIH/NIMH, The Autism Sequencing Consortium: Autism Gene Discovery in >50,000 Exomes. The sequencing of the Northern Finland Intellectual Disability cohort was funded by the US National Institutes of Health Grants U54HG003067, 5U01MH105669 and 5UM1HG008895.

Conflict of Interest: Elisa Rahikkala: None declared, Lea Urpa: None declared, Bishwa Ghimire: None declared, Hande Topa: None declared, Mitja I. Kurki: None declared, Maryna Koskela: None declared, Mikko Airavaara: None declared, Eija Hämäläinen: None declared, Katri Pylkäs: None declared, Jarmo Körkkö: None declared, Helena Savolainen: None declared, Anu Suoranta: None declared, Aida Bertoli-Avella Aida Bertoli-Avella is an employee of Centogene GmbH/Germany., Arndt Rolfs Arndt Rolfs is a founder and former CEO of Centogene NV/Germany and founder and CEO Arcensus GmbH/Germany., Pirkko Mattila: None declared, Mark Daly: None declared, Aarno Palotie: None declared, Olli Pietiläinen: None declared, Jukka Moilanen: None declared, Outi Kuismin: None declared.

EP09.015 Mutation analysis of NSD1 gene in a cohort of patients with clinical suspicion of Sotos Syndrome: a 18-year single center experience

Barbara Testa¹, Giuseppina Conteduca ¹, Marina Grasso¹, Laura Andraghetti¹, Massimiliano Cecconi¹, Michela Malacarne¹, Domenico Coviello¹

¹IRCCS Istituto Giannina Gaslini, Laboratory of Human Genetics, GENOVA, Italy

Background/Objectives: Sotos Syndrome (SoS) (OMIM #117550) is a rare genetic disorder whose prevalence is estimated to be 1:14,000 live births (1). It is caused by haploinsufficiency of NSD1 (nuclear receptor-bindingSETdomain-containing protein1) gene. Most cases are sporadic with few familial cases reported. Approximately 90% of patients bear heterozygous mutation in NSD1 gene or deletions in the 5q35 region (2).

Affected children exhibit distinctive facial features, learning disability and overgrowth (height and/or head circumference ≥98th percentile) overlapping with few others conditions. No clinical diagnostic consensus criteria are still published for SoS and molecular analysis of the NSD1 gene reduced clinical diagnostic uncertainty.

Methods: Using DHPLC, Sanger sequencing, NGS and MLPA on negative samples, we screened NSD1 gene on 1530 unrelated patients enrolled from 2003 to 2021 at the Human Genetics Laboratory of Gaslini Institute of Genova.

Results: NSD1 variants were identified in 292 patients, including 9 partial gene deletions, 13 microdeletions of the entire NSD1 gene and 115 novel intragenic variants never previously described. Nine patients resulted carriers of a mutation in a different gene from NSD1 out of 239 patients analyzed through NGS.

Our approach allowed molecular confirmation for 292 patients with suspected SoS and the formulation of a differential molecular diagnosis in 9 patients of our cohort.

Conclusion: This study broadens the molecular heterogeneity of NSD1variants in Italy, improving the genotype/phenotype correlation for a more appropriate genetic counseling.

References: 1) Kurotaki N et al., 2002.

2) Tatton‐Brown K et al., 2005.

Grants: AssiGulliver and Fondazione Sardegna.

Conflict of Interest: None declared.

EP09.016 Deep intronic de novo germline EHMT1 variant in a patient with syndromic developmental delay

Sophia Peters ¹, Axel Schmidt¹, Tim Bender^1;2, Kirsten Cremer¹, Claudia Perne¹, Elisabeth Mangold¹, Heidrun Raschke¹, André Heimbach¹, Sugirthan Sivalingam³, Peter Krawitz⁴, Hartmut Engels¹

¹Institute of Human Genetics, School of Medicine, University of Bonn, University Hospital Bonn, Bonn, Germany; ²Center for Rare Diseases Bonn, University of Bonn, University Hospital Bonn, Bonn, Germany; ³Core Unit for Bioinformatics Data Analysis, Medical Faculty, University of Bonn, Bonn, Germany; ⁴Institute for Genomic Statistics and Bioinformatics, Medical Faculty, University of Bonn, Bonn, Germany

Background/Objectives: Heterozygous pathogenic variants in the EHMT1 gene cause Kleefstra syndrome 1 (OMIM # 610253), which is characterized by intellectual disability with severe expressive language delay, distinctive facial features and additional abnormalities, including organ malformations, seizures, and psychiatric disorders.

We present a six-year-old girl with developmental delay with prominent speech involvement and dysmorphic facial features that correlate well with Kleefstra syndrome.

Methods: Trio whole exome sequencing (WES) was performed on leukocyte-derived DNA samples after enrichment with Human Core Exome and RefSeq kits (Twist Bioscience) using a NovaSeq 6000 sequencer (Illumina). Alignment to the reference genome was done using the Burrow Wheeler Aligner software, and variant calling was performed according to the GATK best practice guidelines.

Results: Variant prioritization using phenotype data revealed a heterozygous deep intronic variant (NG_011776.1:c.2712+1866G>A) in EHMT1; no other (likely) pathogenic variant was identified. The variant positon was covered in 90 sequencing reads in the index; however, it was covered by only two and zero reads in the father and mother, respectively. Using Sanger sequencing, the variant was shown to be absent in both parents and confirmed in the girl. Hence, it is highly likely that it occurred de novo in the patient. In silico tools predict that this variant generates a new splice site. To analyze possible splice effects, a transcript-analysis using the patient’s cDNA is currently in progress.

Conclusion: Our findings stress the importance of deep intronic variants and highlight this pitfall of WES which can be overcome systematically by e.g. whole genome sequencing.

References:

Grants:

Conflict of Interest: None declared.

EP09.017 Widening of the mutation spectrum in KCNK9 gene for the Birk Barel syndrome

Danijela Krgović ^1;2, Iva Opalič¹, Niha Rihar³, Mario Gorenjak⁴, Špela Stangler Herodež¹, Peter Dovč⁵, Nadja Kokalj Vokač¹

¹Maribor University Medical Centre, Laboratory of medical genetics, Maribor, Slovenia; ²University of Maribor, Faculty of Medicine, Department for Molecular Biology, Maribor, Slovenia; ³University of Ljubljana, Department of Animal Science, Chair of Genetics, Animal Biotechnology and Immunology, Ljubljana, Slovenia; ⁴University of Maribor, Faculty of Medicine, Centre for Human Molecular Genetics and Pharmacogenomics, Maribor, Slovenia; ⁵University of Ljubljana, Biotechnical Faculty, Department of Animal Science, Chair of Genetics, Animal Biotechnology and Immunology, Ljubljana, Slovenia

Background/Objectives: Birk Barel syndrome (BBS) is rarely described imprinting disorder with indistinguishable clinical picture. To date, only 22 molecular diagnosed patients were described in literature, of which 20 patients share the same pathogenic missense variant p.Gly236Arg in maternally expressed KCNK9 gene. Only recently, two new patients with a novel missense mutation p.Ala237Asp and a ring chromosome 8 have expended the genetic cause of this syndrome. Here we report an additional nucleotide variant in KCNK9 gene detected in a 16-year old boy with autism spectrum disorder (ASD).

Methods: WES was performed in a cohort of 148 Slovenian children with suspected ASD. A genotype-phenotype driven analyses was performed to identify a rare variants in 1150 autism associated genes.

Results: Among analysed genes, only one variant was detected in KCNK9 gene, de novo missense c.392G>A mutation, causing the amino-acid exchange p.Arg131His in its protein product. A clinical re-examination of the patient confirmed that the boy had BBS.

Conclusion: In 16-year old boy with intellectual disability, ASD, scoliosis, long face, tented upper lip vermilion, cleft palate, strabismus, and single transverse palmar crease, a clinical diagnosis of BBS was established after determination of de novo missense mutation c.392G>A in KCNK9 gene. This variant was also previously described in one patient from a large study of 2000 patients with suspected genetic disorder but without its clinical characterization and in 5 patients in ClinVar database. Therefore, the role c.392G>A variant as possible new mutational hotspots in KCNK9 gene for BBS is further discussed.

References:

Grants:

Conflict of Interest: None declared.

EP09.018 An inherited loss of function variant in SPTAN1; support of reduced penetrance?

Katrine Aagaard ¹, Mads Thomassen¹, Maria Kibaek², Christina Fagerberg²

¹Odense University Hosptial, Department of Clinical Genetics, Odense, Denmark; ²Odense University Hosptial, H C Andersen Children’s Hospital, Odense University Hospital, Odense, Denmark

Background/Objectives: Pathogenic variants in SPTAN1 are associated with a broad phenotypic spectrum from epileptic encephalopathy and severe developmental delay to mild intellectual disability, some of whom have ataxia and/or spasticity. Most reported variants are missense variants.

Only few loss of function variants in SPTAN1 are reported – some have familial juvenile onset hereditary motor neuropathy with reduced penetrance, and some have de novo variants with variable neurodevelopmental phenotype. We present a patient with a paternally inherited nonsense mutation in SPTAN1, thus adding to the knowledge of the phenotype of SPTAN1 loss of function variants.

Methods: Whole Exome Sequencing (WES) of patient and parents followed by trio analysis for filtering causal variants. Sanger sequencing for segregation analysis.

Results: The patient was an 11 years old boy with juvenile onset of ataxia, cerebellar atrophy, hypotonia, dysarthria, mild spasticity of the lower extremities, and learning disability.

A heterozygous nonsense variant SPTAN1:c.710G>A p.(Trp237*), predicted to result in nonsense mediated decay, was identified. It was present in the father and paternal grandmother, who both were considered healthy.

Conclusion: The consequences of loss of function variants in SPTAN1 are not well described, even if hereditary motor neuropathy seems to be a recurrent feature in some families exhibiting reduced penetrance. The phenotype of our patient with an inherited loss of function variant overlaps with that of some patients with missense variants in SPTAN1. Our case might add to the knowledge of the phenotypic spectrum of SPTAN1-associated disease, and if so, it supports the existence of reduced penetrance for loss of function variants.

References:

Grants:

Conflict of Interest: None declared.

EP09.019 Interactive tools for evaluating attention capacities in patients with severe intellectual disability

Annick Toutain ¹, Anne Claire Gelineau², Solveig Heide², Cyril Mignot³, Anne Faudet³, Anne Sophie Pellen³, Sophie Gallas Lemonnier³, Gilles Rautureau⁴, Pierre Canet⁴, DELPHINE HERON², Karim Ndiaye⁴, Perrine Charles^3;5

¹reference center for developmental abnormalities and rare intellectual disabilities, Genetic Department, Tours, France; ²Salpetriere Hospital, Genetic Department and reference center for rare intellectual disabilities, Paris, France; ³Salpetriere Hospital, Genetic department and reference center for rare intellectual disabilities, Paris, France; ⁴IHU-A-ICM, Plateforme PRISME, Paris, France; ⁵Salpetrière Hospital, Genetic Department and reference center for rare intellectual disabilities, Paris, France

Background/Objectives: Intellectual disability (ID) affects approximately 2% of general population. Within reference center “intellectual disabilities from rare causes”, a consultation dedicated to ID adults has been developed since 2005 with different objectives: diagnosis, genetic counseling, treatment, follow-up, phenotypic characterization, natural evolution, research and participation in therapeutic trials. We have been involved in international, multicenter therapeutic trials in Fragile-X syndrome which did not show any benefit on all evaluation criteria used, which leads questions on their relevance… Criteria used are most often behavior scales filled in by caregiver with important subjective interpretation. We realized obvious lack of objective criteria which could be used as primary or secondary endpoints in neurodevelopmental disorders. By definition, a primary endpoint useful to demonstrate efficacy of the treatment studied must validate different conditions and allow calculation number of subjects necessary to obtain statistically significant results.

Methods: We have thus developed and validated in collaboration with PRISME platform (IHU-A-ICM, Brain and Spine Institute), interactive, quantitative tools to assess attention capacities in patients with ID of different degrees (IQ < 70 but often < 50), with no language for some of them, consisting on playful tests on touch screens for a manual response or using eye tracking for patients who have motor disorders that do not allow use of their hands.

Results: We’ll present results obtained in cohort of patient with ID of different severity.

Conclusion: Those tools could serve as quantitative attention evaluation criteria in therapeutic trials and as rehabilitation tools for patients.

References:

Grants: Grant from John Bost Foundation.

Conflict of Interest: None declared.

EP09.020 Genotype-phenotype functional correlation in KCNB1-related epilepsies

Loretta Ferrera ¹, Antonella Riva², Giulio Alberini³, Ganna Balagura², Marcello Scala², Elisabetta Amadori², Maria Stella Vari², Francesca Madia¹, Elena Gennaro⁴, Michele Iacomino¹, Irene Bagnasco⁵, Gaetano Terrone⁶, Emilia Ricci⁷, Chiara Vannicola⁷, Chiara Reale⁸, Vincenzo Salpietro¹, Elena Gardella⁸, Pasquale Striano², Federico Zara¹

¹Istituto G. Gaslini, U.O.C. Genetica Medica, Genova, Italy; ²Università di Genova, 2Unità di Neurologia Pediatrica e Malattie Muscolari, Dipartimento di Neuroscienze, Riabilitazione, Oftalmologia, Genetica e Scienze Materno-Infantili, Genova, Italy; ³Istituto Italiano di Tecnologia, Center for Synaptic Neuroscience and Technology, Genova, Italy; ⁴Istituto G. Gaslini, U.O.C Laboratorio di Genetica Umana, Genova, Italy; ⁵Ospedale Martini, Division of Child Neuropsychiatry, Torino, Italy; ⁶Università Federico II, Department of Translational Medical Sciences, Section of Pediatrics-Child Neurology Unit, Napoli, Italy; ⁷Ospedale San Paolo, Health Sciences Department, Milano, Italy; ⁸Danish Epilepsy Centre, Department of Epilepsy Genetics and Personalized Medicine, Dianalund, Denmark

Background/Objectives: Pathogenic variants in the KCNB1 gene, encoding the voltage-gated K⁺ channel α-subunit, are associated with a spectrum of phenotypes ranging from developmental and epileptic encephalopathies (DEE) to mild intellectual disability without epilepsy. KCNB1 exerts an electrical role in neurons and mutations may cause changes in its biophysical properties. We evaluated the functional effect of 6 different variants in KCNB1 and correlated the results with the electro-clinical phenotype of patients.

Methods: KCNB1 variants were identified through Next Generation Sequencing. Mutants were expressed in HEK293 cells and membrane currents were evaluated by patch-clamp technique in whole-cell configuration. Patients were deep-phenotyped through clinical charts collected from referring clinicians.

Results: We identified 6 KCNB1 variants: p.T210M; p.R293C; p.A406V; p.F416L; p.P784L; p.T804A (5 de novo, p.T804A inherited from the affected mother). The T804A mutant showed to stay open at >+50 mV potentials, whereas the R293C mutant showed a different voltage dependence and a modified reversal potential as compared to the wild-type (WT) channel (p = 9.35e-6). These variants were found in 10 patients with focal epilepsy with mild intellectual disability. The P784L mutant (1 patient with DEE) showed biophysical properties comparable to that of the WT channel. The remaining mutants showed loss of the ion current conduction and were found in 48 patients with DEE.

Conclusion: KCNB1 mutations may impact patients’ phenotypes depending on the functional effect on the channel. Functional data suggested that P784L may not be responsible for the severe phenotype of the patient, confirming that electrophysiological experiments are pivotal in validating disease-causing variants.

References:

Grants:

Conflict of Interest: None declared.

EP09.021 PSMD12 haploinsufficiency is not simply a neurodevelopmental disorder

Agnese Feresin ¹, Maria Teresa Bonati², Flavio Faletra², Elisa Rubinato², Agnese Luglio¹, Ginevra Morgante¹, Beatrice Spedicati¹, Anna Morgan², Giorgia Girotto^1;2, Paolo Gasparini^1;2

¹University of Trieste, Deparment of Medicine, Surgery and Health Sciences, Trieste, Italy; ²Institute for Child and Maternal Health, IRCCS Burlo Garofolo, Trieste, Italy

Background/Objectives: Syndromic neurodevelopmental disorders (NDDs) are mostly due to de novo variants in coding DNA, disrupting gene function. PSMD12 encodes a subunit of the proteasome and is involved in ubiquitination, thus in cellular homeostasis, brain development and interferon-mediated inflammation. PSMD12 haploinsufficiency is associated to autosomal dominant Stankiewicz-Isidor Syndrome (STIS). Up to now, 45 subjects have been described, including two families.

Methods: Detailed clinical history. X-Fragile test and SNP-array for affected members; familial WES analysis, standard filtering procedures with selection of pathogenic variants. Ongoing analysis of interferon-1 activity.

Results: We report six members (median age 29 years, 3 males) within two unrelated, Italian families with STIS due to a known (p.Arg289*) and a novel (p.Tyr111*) variant in PSMD12. They all had global developmental delay, intellectual disability (1 moderate, 5 mild), dysmorphisms and skeletal anomalies. In family 1, all individuals exhibited a good sociality, while in family 2, siblings showed apathy or irritability. 5/6 subjects developed obesity. Diffuse acne was present in 4/6. Congenital anomalies included interventricular defect (1/6) and cryptorchidism (1/6). Two subjects had strabismus, and one had a hearing loss. One of the subjects had an ovaric teratoma and presented oligodontia. In a single case there was a unilateral, ectopic nail within thumb. None presented specific autoinflammatory disorder.

Conclusion: We described two novel familiar cases of STIS, documenting intrafamilial variability. We expand the phenotype of pre-axial anomalies. We assume to prove an altered interferon signature due to PSMD12 haploinsufficiency.

References:PMIDs: 28132691; 30421579; 34906456; 35080150;.

Grants: NA

Conflict of Interest: None declared.

EP09.022 Potocki-Lupski Mimicking Dubowitz Syndrome: the Importance of Molecular Methodological Approach

Nader Khaleghi Hashemian ¹, Gioia Mastromoro¹, Daniele Guadagnolo¹, Antonio Novelli², Antonio Pizzuti¹

¹Sapienza University of Rome, Rome, Italy; ²Bambino Gesù Pediatric Hospital, Rome, Italy

Background/Objectives: Neurodevelopmental disorders are conditions characterized by cognitive impairment, that can be associated with a wide spectrum of signs and symptoms. The etiopathogenesis of these disorders represents a challenge for clinicians and molecular investigations usually play a pivotal role in obtaining the diagnosis.

Methods: We examined a patient presenting with short stature, microcephaly, bitemporal narrowing, mild micrognathia, ear lobe crease, downslanting palpebral fissures, bilateral ptosis, thinning of the lateral third of eyebrows, thin lower lip and flattening of the Cupid’s bow. At 4 months, he was referred to a geneticist due to growth retardation, hypospadias, unilateral cryptorchidism, and eczema. These characteristics lead to suspicion of Dubowitz syndrome. Standard karyotype was normal (46, XY). At 33, the man underwent genetic counselling again in order to formulate recurrent risk for his sister’s pregnancy. We requested Chromosomal Microarray Analysis (CMA) on the patient’s blood sample.

Results: CMA detected a constitutional de novo 17p11.2 triplication of 851 Kb (arr[GRCh38] 17p11.2(16,854,250-17,705,309)) encompassing three Morbid genes: TNFRSF13B, FLCN and partially RAI1. The triplication overlaps with the critical region of Smith-Magenis syndrome (17p11.2 microdeletion) and Potocki-Lupski syndrome (17p11.2 microduplication). These two conditions entail cognitive impairment, facial dysmorphisms and other structural anomalies. Intrachromosomal microtriplications are rare copy number variants (CNVs), not always associated with a recognizable phenotype. The patient we described resembled typical features of Potocki-Lupski syndrome.

Conclusion: Chromosomal microtriplications are CNVs of rare occurrence, with complex genotype-phenotype correlations. The present case is the first patient detected with 17p11.2 triplication resembling the features of Potocki-Lupski syndrome.

References:

Grants:

Conflict of Interest: None declared.

EP09.023 A small supernumerary marker chromosome resulting from a balanced maternal translocation and leading to partial trisomy of 13q12-q21.1 and 3p26.3 in a child with syndromic intellectual disability

Fatma Maazoun ¹, Mohamed Ali Bouhlel¹, Yosra Lajmi², Aziza Lebbar², Ikhlas Ben Ayed¹, Imene Boujelbene¹, Nourhene Gharbi¹, Hassen Kamoun¹, Jean-Michel Dupont², Fatma ABDELHEDI¹

¹Medical Genetics Departement, Hedi Chaker Hospital, Sfax, Tunisia; ²Cytogenetics Department, Cochin Hospital, Paris, France

Background/Objectives: Small supernumerary marker chromosomes (sSMCs) are rare structural abnormalities in the population; however, they are frequently found in patients with developmental disorders. SMCs are usually observed first by karyotype, and further analysis of their molecular origin is important in clinical practice. We report a case of a sSMC characterized by array-CGH in a 2-year-old boy with neurological disorders with an aim to establish genotype-phenotype correlations. Clinical examination revealed growth retardation, facial dysmorphism, and abnormalities of the extremities. We noted the absence of consanguinity, but rather the occurrence of several spontaneous miscarriages.

Methods: GTG-banding Karyotype carried out on peripheral blood lymphocytes, array-CGH and FISH analysis using Whole Chromosome Painting probes were performed.

Results: Karyotype showed the presence of a homogeneous sSMC. The paternal karyotype was normal, while the maternal karyotype revealed the presence of a balanced (3;13) translocation. Array-CGH analysis showed a gain of 35Mb and 944Kb in 13q12.11-q21.1 and in 3p26.3, respectively. FISH analysis confirmed the origin of sSCM from chromosomes 3 and 13. Thus, we conclude that the sSMC derived from a 3:1 segregation of a maternal balanced t(3;13)(p26;q12) translocation and resulting in partial trisomy of 13q12.11-q21.1 and 3p26.3 as showed by array-CGH.

Conclusion: Molecular characterization of sSMCs by array-CGH is essential to establish genotype-phenotype correlations leading to an appropriate genetic counseling. Our observation underlines also the importance of analyzing karyotypes of couples with a history of repeated spontaneous miscarriages, in search of a balanced parental chromosomal rearrangement which will be transmitted in an unbalanced manner to the offspring.

References: https://doi.org/10.1159/000106438.

Grants: no.

Conflict of Interest: None declared.

EP09.024 Whole DDX3X gene deletion in a female patient with DDX3X-related Neurodevelopmental Disorder

carmen palma milla ¹, Irene Gómez Manjón¹, María de los Ángeles Gómez Cano¹, Lucía Garzón Lorenzo¹, Ana Rosa Arteche López¹, María José Gómez Rodríguez¹, Jose Miguel Lezana¹, Sonia Mayo de Andrés¹, Rubén Pérez de la Fuente¹, Juan Francisco Quesada Espinosa¹, María Teresa Sánchez Calvín¹, Emma Soengas Gonda¹, Patricia Ramos Gómez¹, Alexandra Juárez Rufián¹, Olalla Sierra Tomillo¹, Marta Moreno García¹

¹Hospital 12 de Octubre, Madrid, Spain

Background/Objectives: De novo germline pathogenic variants in DDX3X gene account for 1–3% of unexplained intellectual disability (ID) in females (MIM# 300958), with an X-linked pattern of inheritance. Most affected patients are females with de novo variants, occasionally some males have been reported with hypomorphic inherited pathogenic variants. The associated phenotype is clinically variable, including ID, behaviour problems, minor dysmorphic features and various others manifestations. De novo nonsense, frameshift, splice site or missense mutations have been reported in DDX3X gene, suggesting a disease-causing mechanism via haploinsufficiency.

Here we describe for the first time a whole DDX3X deletion.

Methods: We evaluated a 17-years-old female patient, characterized by mild-to-moderate ID, long and and hypotonic facies, strabismus, recurrent otitis, primary autoimmune hypothyroidism and precocious puberty. No remarkable alteration on brain MRI or Electroencephalogram were detected.

Karyotipe, aCGH (60K) and subtelomeric MLPA were normal.

The patient underwent WES analysis. Variant interpretation was realized on candidate genes based on HPO prioritization.

Results: A whole DDX3X gene deletion was detected (chrX:41193507-41206972, hg19), confirmed by digital PCR. This variant is neither reported in HGMD professional nor in ClinVar. The absence of this gross deletion in the healthy mother is likely indicative of a de novo inheritance (analysis on patient`s father was unavailable), consistent with the hypothesis that haploinsufficiency at this locus on the X-chromosome is likely lethal in males.

Conclusion: We report a new case of DDX3X-related Neurodevelopmental Disorder expanding the knowledge about phenotypic spectrum and describe for the first time a likely de novo whole gene deletion in a female patient.

References:

Grants:

Conflict of Interest: None declared.

EP09.025 An inherited case of 3q29 microdeletion: the unlikely story of a diagnosis

Raquel Rodrigues ¹, Mariana Neves¹, Diana Gonçalves², Ana Barreta², Eva Rolo¹, Ana Sousa¹, Hildeberto Correia³, Ana Berta Sousa¹

¹Hospital de Santa Maria, Departamento de Pediatria, Serviço de Genética Médica, Lisboa, Portugal; ²Laboratório de Análises Clínicas Joaquim Chaves Saúde, Serviço de Genética Médica, Lisboa, Portugal; ³Instituto Nacional de Saúde Doutor Ricardo Jorge, Departamento de Genética Humana, Lisboa, Portugal

Background/Objectives: 3q29 microdeletion is a rare autosomal dominant disorder with heterogeneous presentation and variable penetrance of medical, neurodevelopmental and neuropsychiatric phenotypes. Typically, this microdeletion occurs de novo and, when inherited, the transmitting parent is affected to some degree. We describe a novel case, initially suspected by conventional karyotyping, inherited from an apparently unaffected mother.

Methods: A 5-year-old boy was referred to our Genetics Department for global developmental delay and behavioral problems. His father had psychiatric pathology, his mother was healthy and a 12-year-old brother had learning difficulties. The karyotype showed a chromosome 3 of atypical conformation. Further studies were performed by aCGH (180K-CGX-HD). Segregation was investigated by karyotype and aCGH.

Results: aCGH revealed a ~1.56 Mb microdeletion overlapping recurrent 3q29 syndromic region.

Parental karyotyping showed that the mother also presented an atypical 3qter. aCGH confirmed the 3q29 terminal microdeletion to be maternally inherited. The brother will be studied next.

Conclusion: G-banding karyotype resolution is in the threshold of 5-10Mb. Nevertheless, in this case karyotyping led to the first suggestion of a 3qter abnormality in the proband and in his mother. To our knowledge there are no previous studies reporting such a small deletion suspected in the karyotype.

In the rare inherited cases of 3q29 microdeletion, the overwhelming majority of parents are affected at some level. Penetrance for this deletion is not fully established. However, data from the literature combined with our report of a 3q29 microdeletion carrier unaffected mother, suggests that while penetrance is high, it is likely not 100%.

References:

Grants:

Conflict of Interest: None declared.

EP09.026 Clinical and genetic features of X-linked mental retardation 102 type caused by mutations in the DDX3X gene in adolescent-girl

Olga Gumeniuk ¹, Yuriy Chernenkov¹, Olga Groznova², Lyusya Melikyan²

¹Saratov State Medical University, Saratov, Russian Federation; ²Veltischev Research and Clinical Institute for Pediatrics of the Pirogov Russian National Research Medical University, Moscow, Russian Federation

Background/Objectives: Mutations in DDX3X are known to cause a characteristic neurodevelopmental disorder in female patients. Analysis of the clinical and genetic features in adolescent-girl with mental retardation due to mutations in DDX3X.

Methods: In this study we performed whole genome sequencing of patient (girl, 10 y.o.) by high-throughput sequencing (NGS) on DNBSEQ-G400, BGISEQ-500 and T7 platforms with the reading of 75 billion nucleotides, conclusion according to ACMG recommendations.

Results: Girl was born from 2 pregnancy, the 1pregnancy ended intrauterine death of female fetus. In the first year of life, there was a delay in psychomotor and speech development. The physical examination of the patient revealed delayed speech development, elongated faces, high forehead, lagophthalmos, upturned nose, wide filter, 2-4 finger syndactyly, Hallux valgus, scoliosis, high growth (SDS +2.1) and early sexual development (Tanner III).The patient has learning difficulties, is being monitored by a psychiatrist with an autism spectrum disorder. A variant of rs1569238002, not previously described in the literature, was found in a heterozygous state in exon 8 of 19 of the DDX3X gene, leading to a synonymous replacement of p.Gly248Gly.

Conclusion: The results obtained may testify in favor of the existence of a dependence of the severity of the phenotype on the localization and nature of mutations in the gene and determine the relevance of further research aimed at searching for clinical and genetic correlations.

References: Dadali E.L. et al. Clinical and genetic characteristics of X-linked mental retardation 102 type caused by novel mutations in the DDX3X gene. Neur Dis. 2020; 10;75–80.

Grants: None.

Conflict of Interest: None declared.

EP09.027 A recurrent de novo mutation in ZMYND11 associated with global developmental delay genocopy the 10p15.3 deletion syndrome: a case report

Alba Muñoz ¹, Maria Ballesteros¹, Teresa CARRION^1;2, Laura Torres^1;3, Victor Asencio^1;3, Rosa Martorell¹, Damian Heine^1;3, Antonia Obrador^1;3, Maria Vidal^1;3, Fernando Santos¹, Iciar Martínez^1;3

¹Hospital Universitario Son Espases, Unidad de Diagnostico Molecular y Genética Clínica, Palma de Mallorca, Spain; ²Hospital Universitario Son Espases, Pediatrics, Palma de Mallorca, Spain; ³Fundació Institut d’Investigació Sanitària Illes Balears (IdISBa), Palma de Mallorca, Spain

Background/Objectives: A 7-year-old patient initially diagnosed as Silver-Russel syndrome due to mosaicism of chromosome 7 returns to our genetic consultation for diagnostic re-evaluation. Characteristics symptoms are global developmental delay (84%), hypotonia, feeding difficulties, short stature and several facial dysmorphisms (microcephaly, depressed nasal bridge and microretrognathia).

Methods: Karyotype and CGH array were performed. MLPA assay (MS-MLPA Probemix ME032 DUP7-DUP14) was also done to test if there was maternal uniparental disomy of chromosome 7 (DUP7). Clinical exome was sequenced by Human Whole-Genome Sequencing with the Nextera^TM-DNA-Flex-Library Preparation Kit (Illumina).

Results: Karyotype and CGH array were normal. MLPA assay revealed absence of DUP7. The analysis of the clinical exome showed a heterozygous autosomal dominant missense variant: c.1798C>T p.(Arg600Trp) in ZMYND11 (NM_006624.5), classified according the ACMG guidelines as pathogenic. Genetic testing of both parents showed it was arisen de novo.

Conclusion: Zinc finger MYND-type, expressed in many human tissues, acts as a transcriptional repressor, playing an inhibitory role in the muscle and neuronal differentiation steps. Specifically, the mutated position in this case Arg600 is very conserved and essential for its binding to ligands (Kateb et al.,2013). ZMYND11 has also been proposed as a candidate gene for 10p15.3 microdeletion syndrome, which shares common clinical features with our patient (DeScipio, 2021). Moreover, other authors have described the same mutation (Cobben,2014), so that it can be considered as definitely pathogenic. Finally, the same SNP has been reported 4 times in Decipher and 8 times in ClinVar thus the variant here reported could be a hotspot mutation.

References:

Grants:

Conflict of Interest: None declared.

EP09.028 A novel intragenic deletion in oligophrenin-1 features in a mother, her two sons, and one daughter with intellectual disability, retrocerebellar arachnoid cysts, and neuropsychiatric disorders, but without cerebellar hypoplasia

Ebtesam AL-Enezi¹, Wafaa Eyaid¹

¹Department of Clinical Genetics and Precision Medicine, King Abdulaziz Medical City, Riyadh, Saudi Arabia

Background/Objectives: OPHN1 gene mutations can cause X-linked mental retardation (XLMR), cerebellar hypoplasia, and facial dysmorphism. A few cases of symptomatic females carrying a novel OPHN1 intragenic deletion have been reported. we report a Saudi family in which a mother, one daughter, and two sons were affected by this variant.

Methods: 19-year-old boy presented with attention-deficit/hyperactivity disorder, severe mental retardation, facial dysmorphism, mild cerebral volume loss, and retrocerebellar arachnoid cyst. Whole exome sequencing identified the hemizygous likely pathogenic variant in OPHN1c.1105-13_1109del, consistent with the diagnosis of XLMR with cerebellar hypoplasia and distinctive facial appearance. The family was screened and his mother, sister, and brother were found to be carriers.

Results: The mother had mild mental retardation, depression, and schizoaffective personality disorders. Her sons, 19 and 14 years of age, had attention-deficit/hyperactivity disorder, severe mental retardation, facial dysmorphism, mild cerebral volume loss, and retrocerebellar arachnoid cyst. The 7-year-old daughter shared the same phenotype as her brothers but with mild mental retardation and thinning of corpus collosum. No seizure or cerebellar hypoplasia were noted in any of them.

Conclusion: The phenotype in this family with two severely affected boys and two females with milder forms, was suggestive of XLMR. This report confirms previous findings that women carrying OPHN1 c.1105-13_1109del may be symptomatic and mildly affected by facial dysmorphic features and mild cognitive delay, with or without abnormal findings on brain imaging.

References: l-Owain M, et al. Novel intragenic deletion in OPHN1 in a family causing XLMR with cerebellar hypoplasia and distinctive facial appearance. Clin Genet. 2011;79(4):363-370. https://doi.org/10.1111/j.1399-0004.2010.01462.x.

Grants: None.

Conflict of Interest: None declared.

EP09.029 Kleefstra syndrome does not always occur de novo: A family report

ABDULLAH SEZER ¹, Abdüllatif Bakır¹

¹Dr. Sami Ulus Obstetrics and Gynecology, Children Health and Disease Training and Research Hospital, Medical Genetics, Ankara, Turkey

Background/Objectives: Kleefstra syndrome is an autosomal dominant neurodevelopmental disorder characterized by delayed psychomotor development, intellectual disability and dysmorphic features. In this syndrome, mostly de novo variants are identified in EHMT1 and KMT2C genes. Here we present an exceptional family including a mother and two daughters diagnosed with Kleefstra syndrome 2 associated with a novel missense variant in KMT2C gene.

Methods: Two sisters sharing the same mother but different fathers, with the age of 5 and 9, were evaluated due to their mild intellectual disability, speech delay, and articulation problems. The older sibling also had aggressive and self-injurious behavior. Additionally, the intellectual capacity and problem-solving skills of the 40-year-old mother were limited. The mother and daughters had similar facial dysmorphic features, which are compatible with Kleefstra syndrome. Etiological evaluations were studied by chromosomal microarray (CMA) analyses in both sisters and clinical exome sequencing (CES) analysis in the younger sister.

Results: CMA analyses were normal and CES analysis revealed a heterozygous c.584A>G (p.Gln195Arg) variant in KMT2C (NM_170606) gene. Sanger sequencing confirmed the variant as heterozygous also in the mother and the elder sister.

Conclusion: De novo variants of EHMT1 and KMT2C genes are identified in most patients with Kleefstra syndrome. It is thought that the reproductive fitness of this disease is zero, as in most autosomal dominant neurodevelopmental disorders developing by de novo variants. Along with another example of paternal transmission in the literature, this family shows that Kleefstra syndrome 2 with mild intellectual disability may be familial.

References:

Grants:

Conflict of Interest: None declared.

EP09.030 Case report of rare 3q27.1 microdeletion syndrome for patient with dysmorphic features, growth retardation and mild developmental delay

Rasa Traberg ¹, Ruta Navardauskaite², Rasa Ugenskiene¹

¹Lithuanian University of Health Sciences, Medical Academy, Department of Genetics and Molecular medicine, Kaunas, Lithuania; ²Lithuanian University of Health Sciences, Medical Academy, Department of Endocrinology, Kaunas, Lithuania

Background/Objectives: Constitutional rearrangements involving chromosome 3q are very rare and overlapping microdeletions of chromosome 3q26-3q28 have only been reported in ten individuals with severe prenatal and postnatal growth restriction and neurodevelopmental abnormalities.

Methods: The proband is 2-year-old female who was born at 38 weeks of gestation, birth weight 2060 g (-2.88 SD). Severe Intrauterine growth restriction was noticed from the begging of 3rd trimester. She failured to thrive during infancy with normal motor development. At the clinical visit her height was 79 cm (-2.44 SD), weight 8 kg (-4.26 SD). The hormonal assessment was performed and normal levels of IGF-1, TSH, FT4, basal cortisol levels were detected. Dysmorphic facial features were noticed: hyperthelorism, high anterior hairline, frontal bossing, depressed nasal bridge, short nose with anteverted nares, low set ears. Developmental delay was assed by scale of Diagnostic inventory for screening children, 1984 (DISC). Test value was 50-77%, especially language delay with feeding difficulties.

Results: Whole Genome NGS-based Large Copy Number Variation Analysis (Centogene, Germany) was performed and 867 kb one copy loss within chromosome region 3q27.1 was detected. The deletion covered 21 genes and 9 of them were OMIM-associated with disease, particularly DVL3, AP2M1 and PARL genes. Frameshift variants in DVL3 gene are associated with autosomal dominant type III Robinow syndrome (MIM#: 616894). AP2M1 (MIM#: 601024) has role in poor speech, developmental delay, hypotonia and seizures. PARL (MIM#: 607858) possibly associated with growth restriction.

Conclusion: We suggest that haploinsufficiency of DVL3, AP2M1 and PARL genes can cause 3q27.1 microdeletion phenotype.

References:

Grants:

Conflict of Interest: None declared.

EP09.031 DNA methylation signature associated with Bohring-Opitz syndrome: A new tool for functional classification of variants in ASXL genes

Zain Awamleh ¹, Eric Chater-Diehl¹, Sanaa Choufani¹, Valerie Arboleda², Bianca Russell³, Rosanna Weksberg¹

¹The Hospital for Sick Children, Genetics and Genome Biology Program, Toronto, Canada; ²University of California, Los Angeles, UCLA, Department of Human Genetics, Los Angeles, Canada; ³University of California, Los Angeles, UCLA, Department of Pediatrics, Los Angeles, United States

Background/Objectives: The additional sex combs-like (ASXL) gene family—encoded by ASXL1, ASXL2, and ASXL3—is crucial for mammalian development. Pathogenic variants in ASXL genes are associated with three phenotypically distinct neurodevelopmental syndromes. Our previous work has shown that syndromic conditions caused by pathogenic variants in epigenetic regulatory genes show consistent patterns of genome-wide DNA methylation (DNAm) alterations (DNAm signature) in peripheral blood. Given the role of ASXL1 in chromatin modification, we hypothesized that pathogenic ASXL1 variants underlying Bohring-Opitz syndrome (BOS) have a unique DNAm signature.

Methods: We profiled whole-blood DNAm for 17 ASXL1 variants, and 35 sex- and age-matched typically developing individuals, using Illumina’s Infinium EPIC array.

Results: We identified 763 differentially methylated CpG sites in individuals with BOS. Differentially methylated sites overlapped 323 unique genes, including HOXA5 and HOXB4, supporting the functional relevance of DNAm signatures. We used a machine-learning classification model based on the BOS DNAm signature to classify variants of uncertain significance in ASXL1, as well as pathogenic ASXL2 and ASXL3 variants. The DNAm profile of one individual with the ASXL2 variant was BOS-like, whereas the DNAm profiles of three individuals with ASXL3 variants were control-like. We also used Horvath’s epigenetic clock, which showed acceleration in DNAm age in individuals with pathogenic ASXL1 variants, and the individual with the pathogenic ASXL2 variant, but not in individuals with ASXL3 variants.

Conclusion: These studies enhance our understanding of the epigenetic dysregulation underpinning ASXL gene family associated syndromes.

References:

Grants: (ARRE, MC2015-16), CIHR (IGH-155182 and MOP-126054) to R.W. ARRE (20201705) and DP5OD024579 (NIH) to V.A.

Conflict of Interest: None declared.

EP09.032 MECP2 duplication syndrome: report of an affected female

Patrícia Costa ¹, Gabriela Fernandes¹, Pedro Vale¹, Cintia Ventura¹, Rita Cerqueira¹, Joaquim Sá¹, Miguel Gonçalves-Rocha²

¹CGC Genetics, Unilabs, Porto, Portugal; ²Hospital de Braga, Braga, Portugal

Background/Objectives: MECP2 duplication syndrome also known as Intellectual developmental disorder, X-linked syndromic, Lubs type, is a neurodevelopmental disorder characterized by early-onset hypotonia, delayed psychomotor development leading to severe intellectual disability, poor speech development, mild dysmorphic features, seizures, progressive spasticity, recurrent infections, autistic features, feeding difficulty and gastrointestinal manifestations^1,2.

This syndrome has complete penetrance in males. Female carriers may have a mild phenotype, as neuropsychiatric features. Due to the skewing of X inactivation against the duplicated X chromosome, females are rarely affected. However, in rare cases, females have the same severe clinical findings as males³. Reviewing the literature, at least 23 affected females were reported⁴.

We report a case of a 40 years old female with severe intellectual disability, epilepsy, facial dysmorphism, morbid obesity and mild strabismus. She has two sisters with overlapping phenotype and a female cousin with intellectual disability.

Methods: Array CGH was performed with the Affymetrix Cytoscan 750K. The results were analyzed with ChAS software.

Results: We found a duplication from the Xq28 region of 334 Kb, in the genomic coordinates 153254852-153588530 (GRCh37), involving the IRAK1, MECP2, OPN1LW, OPN1MW2, OPN1MW, OPN1MW3, TEX28, TKTL1 and FLNA protein coding genes.

Array CGH analysis of the other affected female relatives was recommended and it is ongoing.

Conclusion: This is a rare case of an Xq28 duplication detected in a severely affected female.

This case demonstrates the importance of the correlation between genetic findings with the patients’ phenotype and the family history.

References: ¹https://omim.org/entry/300260.

²https://search.clinicalgenome.org/kb/gene-dosage/region/ISCA-46304.

³https://www.ncbi.nlm.nih.gov/books/NBK1284/.

⁴ PMID: 27761913.

Grants:

Conflict of Interest: None declared.

EP09.033 A novel KCNK9 missense variant adjacent to the typical amino acid substitution causes Birk-Barel syndrome

Sophie Uyttebroeck¹, Tessa Wassenberg², Alex Michotte³, Rani Cooreman¹, Kathelijn Keymolen¹, Frederik Hes¹, Laura Pölsler ¹

¹Universitair Ziekenhuis Brussel, Center for Medical Genetics, Brussels, Belgium; ²Universitair Ziekenhuis Brussel, KidZ Health Castle, Brussels, Belgium; ³Universitair Ziekenhuis Brussel, Department of Neurology, Brussels, Belgium

Background/Objectives: A 17-year-old girl presented with intellectual disability, and facial dysmorphisms. At birth, she was severely hypotonic requiring tube feeding, and had retromicrognathia, cleft palate, and bilateral club feet. She developed postnatal microcephaly, delay of developmental milestones, and scoliosis, and has hypotonia with areflexia, spasticity, and contractures of the legs. A muscular biopsy at age 4 years showed unspecified myopathy with an excess of type 1 fibres and atrophy of type 2 fibres.

Methods:.

Results: We report a heterozygous novel, maternally inherited, likely pathogenic variant in the KCNK9 gene: c.710C>T;p.(Ala237Val).

Pathogenic variants in the KCNK9 maternal allele cause Birk-Barel syndrome, characterized by congenital central hypotonia, developmental delay, and dysmorphic features^1,2. Almost all patients carry KCNK9 p.Gly236Arg^1,2.

One additional patient carries the substitution p.(Ala237Asp)³. The novel variant in our patient involves a different substitution at the same position (p.(Ala237Val)), adding strength to the interpretation of both variants as likely pathogenic. In addition to the clear phenotypic match, both patients show selective atrophy of type 2 fibres and predominance of type 1 fibres on muscle biopsy³, which is similar to neurogenic changes seen in spinal muscular atrophy (SMA). Two patients of the originally described group showed signs of SMA on muscle biopsy¹.

Conclusion: Our patient demonstrates the typical phenotype of Birk-Barel syndrome, likely caused by the novel KCNK9 variant p.(Ala237Val). This case provides further support that substitutions at both positions p.Gly236 and p.Ala237 are to be considered pathogenic.

References: ¹Barel et al, https://doi.org/10.1016/j.ajhg.2008.07.010.

²Graham et al, 10,1002/j.ajmg.a,37740.

³Šedivá et al, https://doi.org/10.1016/j.ejmg.2019.01.009.

Grants:

Conflict of Interest: None declared.

EP09.034 Biallelic MAPKAPK5 pathogenic variants are associated with a distinct syndromic neurodevelopmental disorder with craniofacial, digital and neuroradiological abnormalities

Stephanie Efthymiou ¹, Reza Maroofian¹, mohnish suri², fatima rahman³, Maha Zaki⁴, shazia maqbool³, najwa anwar³, victor l. ruiz-pérez⁵, sniya sudhakar⁶, Kshitij Mankad⁶, Tamar Harel⁷, Henry Houlden¹

¹UCL Queen Square Institute of Neurology, London, United Kingdom; ²Nottingham University Hospitals NHS Trust - City Hospital Campus -Nottingham Mobility Centre, Nottingham, United Kingdom; ³University of Child Health Sciences, Lahore, Department of Developmental-Behavioral Pediatrics, Lahore, Pakistan; ⁴National Research Centre, Human Genetics and Genome Research Institute, Cairo, Egypt; ⁵Instituto de Investigaciones Biomédicas “Alberto Sols”. IIBm (CSIC-UAM), Madrid, Spain; ⁶Great Ormond Street Hospital for Children, Department of Radiology, London, United Kingdom; ⁷Hadassah Ein Kerem, Jerusalem, Israel

Background/Objectives: MAPK activated protein kinase 5 (MAPKAPK5) is an essential enzyme for diverse cellular processes. Dysregulation of the pathways regulated by MAPKAPK enzymes can lead to the development of variable diseases. Recently, three cases from two families presented with a distinct syndromic neurodevelopmental disorder with two different homozygous frameshift variants in MAPKAPK5 were reported.

Methods: We evaluated three individuals from three consanguineous south-Asian, middle eastern and north-African families. The study was approved by the institutional ethics committees of University College London (UCL) and additional local ethics committees of the participating centres. Exome sequencing (ES) and Sanger segregation analysis were performed separately at two different laboratories.

Results: In the present study, we identified three types of homozygous MAPKAPK5 variants, frameshift, nonsense, and missense by using exome sequencing, in three unrelated individuals from three consanguineous families. All affected individuals exhibited a syndromic neurodevelopmental disorder characterized by severe global developmental delay, intellectual disability, distinct dysmorphic features, brachycephaly, digits abnormalities and abnormal brain MRIs (cerebellar hypoplasia, hypomyelination) as well as variable vision and hearing impairment. The additional features are failure to thrive, hypotonia and microcephaly.

Conclusion: In this study, we consolidate the causality of loss of MAPKAPK5 function and further delineate the clinical features associated with biallelic MAPKAPK5 variants and expand the molecular and phenotypic spectrum of this new ultra-rare neurodevelopmental syndrome.

References:

Grants: MRC (MR/S01165X/1, MR/S005021/1, G0601943).

Conflict of Interest: None declared.

EP09.035 Chromosomal Microarray Analysis in children with unexplained neurodevelopmental disorder

Enise AVCI DURMUŞALİOGLU ¹, Erhan Pariltay², Durdugul Ayyildiz Emecen¹, Berk Ozyilmaz³, Tuba Sozen Turk³, Haluk Akin², Ozgur Cogulu¹, Ferda Ozkinay¹, Tahir Atik¹

¹Ege University, Pediatric Genetics, Izmir, Turkey; ²Ege University, Medical Genetics, Izmir, Turkey; ³Tepecik Education and Research Hospital, Medical Genetics, Izmir, Turkey

Background/Objectives: Neurodevelopmental disorders are a heterogeneous group that includes developmental delay (DD), intellectual disability (ID) and autism spectrum disorder (ASD). Recently chromosomal microarray analysis (CMA) has been recommended as the first-tier genetic test in children presenting with an unexplained neurodevelopmental disorder.

Herein, it was aimed to expand the phenotypic spectrum of copy number variations (CNVs) detected in patients with DD/ID/ASD and evaluate the clinical benefits of CMA.

Methods: The size, number of genes, inheritance, and classification (according to ACMG guidelines) of CNVs detected in 31 patients with DD/ID/ASD were evaluated retrospectively along with phenotypic characteristics of the patients.

Results: A total of 41 CNVs were detected in 31 patients, including 24 deletions and 17 duplications.12/31 of patients had at least one congenital anomaly accompanying DD/ID/ASD. The mean age at which CMA performed was 4.1 years. The size of the largest CNV was 51 megabases, the smallest one was 58 kilobases. 17/41 of CNVs were de novo, 5/41 were inherited in the same manner from one of the parents, and 4/41 CNVs were inherited to a larger extent. 22/41 of CNVs were classified as ‘pathogenic’, 2/41 as ‘likely pathogenic’, 16/41 as ‘variant of uncertain significance (VUS)’ and 1/41 as ‘benign’. The most affected chromosomal regions were 22q11.2 (4/41) and 15q11.2 (4/41). 10/41 of CNVs were consistent with a previously described microdeletion/duplication syndrome (2q37,7q31, 15q11.2, 16p11.2, 17p11.2, 17q21.31, 22q11.2).

Conclusion: CMA may reveal underlying molecular defects in children presenting with DD/ID/ASD. Knowing the phenotypic features of CNVs enables better patient management and genetic counseling.

References:

Grants:

Conflict of Interest: None declared.

EP09.036 A novel loss-of-function SEMA3E mutation causes severe intellectual disability and cognitive regression

Alyssa Paganoni ¹, Federica Amoruso², Javier Porta Pelayo³, Valeria Vezzoli⁴, Alessia Caramello⁵, Roberto Oleari², Alberto Fernández-Jaén⁶, Anna Cariboni²

¹Università degli Studi di Milano, Milan, Italy; ¹Università degli Studi di Milano, Milan, Italy; ³Genologica Medica, Malaga, Spain; ⁴IRCCS Istituto Auxologico Italiano, Milan, Italy; ⁵Imperial College London, London, United Kingdom; ⁶Hospital Universitario Quirónsalud, Madrid, Spain

Background/Objectives: Intellectual Disability (ID) is a neurological disorder arising from early neurodevelopmental defects characterized by subaverage intellectual and adaptive functioning due to abnormalities of brain structure and function¹. The underlying genetic and molecular mechanisms are complex, but thought to involve, among others, alterations in genes involved in axon guidance and/or neural circuit formation as demonstrated in the developing mouse brain². Here, by combining exome sequencing with in silico analyses, we identified a patient affected by severe ID and cognitive regression, carrying a novel loss-of-function mutation of the semaphorin 3E (SEMA3E) gene, which encodes for a key secreted cue that controls brain development in mouse.

Methods: ad hoc in vitro and in vivo experiments were performed. Protein secretion and binding were assessed by western blot, immunocytochemistry and binding assays, while human SEMA3E expression was analysed by immunohistochemistry.

Results: we found that the identified variant impairs protein secretion and hampers the binding to embryonic mouse neuronal cells and tissues. Further, we revealed SEMA3E expression during human brain development.

Conclusion: overall, our findings demonstrate the pathogenic impact of identified SEMA3E variant and provide evidence that clinical neurological features of the patient might be due to a defective SEMA3E signalling in the brain.

References:1 Purugganan O. Intellectual Disabilities. Pediatrics In Review 2018; 39: 299–309. 2 Steele JL, Morrow MM, Sarnat HB et al. Semaphorin-Plexin Signaling: From Axonal Guidance to a New X-Linked Intellectual Disability Syndrome. Pediatric Neurology 2022; 126. https://doi.org/10.1016/j.pediatrneurol.2021.10.008.

Grants: Ministry of Health and CHARGE Syndrome Foundation to A.C.

Conflict of Interest: None declared.

EP09.037 Investigation of serotonin, histamine and diamine oxidase in the improvement of child development in ASD: benefits from improved nutrition and diet

Tanya Kadiyska^1;2;3, Ivan Tourtourikov^1;2;4, Zornitsa Pavlova^1;2;4, Dilyana Madzharova ^1;2, Maria Savcheva⁵, Stanislava Ciurinskiene⁶

¹Genetic Medico-Diagnostic Laboratory “Genica”, Sofia, Bulgaria; ²IMDL Genome Center “Bulgaria”, Sofia, Bulgaria; ³Medical University of Sofia, Department of Physiology and Patophysiology, Sofia, Bulgaria; ⁴Medical University of Sofia, Department of Medical Chemistry and Biochemistry, Sofia, Bulgaria; ⁵Vita Hospital, Bio Medical Ltd., Sofia, Bulgaria; ⁶Early Intervention Service “Vsyaka Duma”, Sofia, Bulgaria

Background/Objectives: Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by social impairment, repetitive behavior and impaired communication. Alternations in vital neurotransmitters are frequently found in children with ASD, which can be further complicated in cases with the additive phenotype of histamine intolerance.

As diet and nutrition play an important role in maintaining the proper functions of the brain, our aim was to determine if nutritional improvements can alter the symptoms of ASD and improve general development. We analyzed the changes in the levels of serotonin, histamine and diamine oxidase (DAO) activity together with DAO polymorphisms (rs2052129, rs2268999, rs10156191 and rs1049742).

Methods: A cohort of 117 patients with ASD was tested for histamine, serotonin and DAO activity via ELISA prior to and following an appropriate diet. Genotyping was performed for DAO polymorphisms using Sanger sequencing. Statistical analysis was performed using SPSS v26.0.0.0. and Pearson Chi Square Test.

Results: We observed statistically significant changes in all measured parameters including an improvement in the development of the patients following the assigned diet. Furthermore, we observed a correlation between DAO genotypes and enzymatic activity.

Conclusion: Our study discovered a positive effect of an individualized, clinically-based diet approach on the general condition and development of children with ASD. Further clinical studies are required to validate the correlation between DAO genotype and enzymatic activity.

References:

Grants:

Conflict of Interest: None declared.

EP09.038 Neuroanatomical studies identify VPS13B as an important regulator of brain architecture and hippocampal formation

Charlotte Montillot ¹, Sylvie Nguyen¹, Stephan Collins¹, Binnaz YALCIN¹

¹Inserm UMR1231, Genetics of Developmental Disorders Laboratory, Dijon, France

Background/Objectives: Vacuolar Protein Sorting 13 homolog B (VPS13B) is a highly conserved protein through evolution, but its function is not well understood yet. Human mutations in VPS13B cause Cohen Syndrome, a rare recessive developmental disease characterized by intellectual disability, acquired microcephaly and hypotonia.

Methods: Using a knockout mouse model approach, we set out to identify the role of VPS13B in the brain and animal behavior.

Results: We first showed that homozygous mutant mice of Vps13b are sub-viable, half dying during the first week of life. The survivor homozygous mutant mice exhibit growth delay and microcephaly (-27% for weight, and -20% for microcephaly in adult male mice). Using systematic neuroanatomical studies at multiple ages (embryonic E18.5 and postnatal 5, 7, 18 and 33 weeks), we then found severe neuroanatomical changes appearing within the first weeks after birth in homozygous mutant mice when compared to controls. The hippocampal formation is the most affected region with a reduction of 34% of the size of the dentate gyrus. In addition, we performed a battery of behavioral tests and showed hyperactivity, hypotonia and altered memory but enhanced sociability and resilience to anxiety and depression in Vps13b homozygous mutant mice. It is noteworthy that heterozygous mutant mice did not show any apparent phenotype.

Conclusion: Together, these findings indicate a highly specific role of VPS13B in the regulation of brain structure and an association with previously unreported features of Cohen Syndrome.

References: Grants: Jérôme Lejeune Foundation and ANR JCJC Young Team, to BY.

Conflict of Interest: None declared.

EP09.039 Two novel variants in POLA1 and TBC1D8B identified in a Japanese patient with failure to thrive, mild intellectual delay, skin pigmentation and renal failure

Kumiko Yanagi ¹, Yasutsugu Chinen², Kazuhito Satou¹, Arisa Igarashi¹, Kenji Naritomi³, Koichi Nakanishi², Yoichi Matsubara⁴, Tadashi Kaname¹

¹National Center for Child Health and Development, Genome Medicine, Tokyo, Japan; ²University of the Ryukyus, Pediatrics, Graduate School of Medicine, Okinawa, Japan; ³Okinawa Nanbu Habilitation and Medical Center, Okinawa, Japan; ⁴National Research Institute for Child Health and Development, Tokyo, Japan

Background/Objectives: Whole-exome sequencing (WES) analysis has revealed two pathogenic variants in some patients of rare diseases. Here we report a 23-year-old Japanese patient who was made dual diagnosis by the WES analysis.

Methods: The proband was born to non-consanguineous parents at 41 weeks with birth weight of 2,687g (-0.76 SD), height of 48 cm (-0.48 SD) and head circumstance of 34.0 cm. (+0.5 SD). Failure to thrive was clearly observed at 1 year of age (weight; -2.4 SD, height; -1.4 SD, head circumstance; -2.2 SD). He shows mild intellectual disability and skin pigmentation. Immune dysregulation was not pointed out in his medical history. Proteinuria was observed from 8- years-old and pathologically diagnosed with membranoproliferative glomerulonephritis. He reached renal failure by 23-year-old. After obtaining written informed consent, trio-based WES analysis.

Results: WES analysis revealed two novel pathogenic variants in TBC1D8B (NM_017752:c.1711T>C:p.[Tyr571His]) and POLA1 (c.3082G>A:p.[Gly1028Arg]), on X chromosome in the patient and his mother.

Conclusion: His glomerulonephritis may be caused by the variant in TBC1D8B that is known to causative gene for nephrotic syndrome, type 20. POLA1 is known to causative gene for two allelic disorders with different molecular pathogenesis. One is X-linked reticulate pigmentary disorder, which is characterized by reticular pigmentation abnormalities of the skin and immune dysregulation. Another is van Esch-O’Driscoll syndrome (VEODS), characterized by severe growth retardation, microcephaly and hypogonadism without pigmentation abnormalities. The patient shows both VEODS and skin pigmentation phenotypes. We concluded that he is an intermediate type of the two disorders.

References: AJHG 104, 957-967, 2019.

AJHG 104, 348–355, 2019.

Grants:

Conflict of Interest: None declared.

EP09.040 Genetic diagnosis in Bulgarian autistic spectrum disorder patients

Tihomir Todorov ¹, Slavena Atemin^1;2, Petia Dimova³, Daniela Avdjieva-Tzavella⁴, Iliyana Pacheva^5;6

¹Genetic Medico-Diagnostic Laboratory “Genica”, Sofia, Bulgaria; ²Medical University of Sofia, Department of Medical Chemistry and Biochemistry, Sofia, Bulgaria; ³University Hospital “Saint Ivan Rilski”, Department of Neurosurgery, Sofia, Bulgaria; ⁴University Pediatric Hospital Sofia, Department of Clinical Genetics, Sofia, Bulgaria; ⁵Medical University - Plovdiv, Department of Pediatrics and Medical Genetics, Plovdiv, Bulgaria; ⁶University Hospital “St. George”, Department of Pediatrics, Plovdiv, Bulgaria

Background/Objectives: Methods for discovering point variants by using next generation sequencing (NGS), include analysis of the genome, exome, diagnostic panels and single genes. Methods for discovering deletions and duplications include array-CGH and NGS with low coverage which allows for fast scanning of the genome for copy number variations (CNVs). The percentage of genetically confirmed cases with autism spectrum disorder (ASD) is not very high, no matter of the available technologies for diagnostic purposes.

The aim of the study was to screen 12 ASD patients with different diagnostic approaches.

Methods: NGS with low coverage and WES were performed for all 12 patients. Segregation analysis by Sanger sequencing was performed in the families.

Results: Two de novo pathogenic CNVs were detected: duplication on the short arm of chromosome 8 and deletion on the long arm of chromosome 20. In addition, 2 de novo pathogenic missense variants were found: DDX3X gene (de novo X-linked) - c.857C>A, p.Ala286Asp and HIVEP2 gene (de novo autosomal dominant) - c.7310A>C; p.Asp2437Ala. Two more patients turned out to be double heterozygous: SPATA5 gene c.554G>A, p.Gly185Glu (maternal origin) and c.1831C>T, p.Pro611Ser (paternal origin); UNC80 gene - c.29A>G, p.Gln10Arg (maternal origin) and c.2338A>G, p.Ser780Gly (paternal origin). Altogether 6 point mutations were detected in 4 ASD patients.

Conclusion: Altogether, 50% of our patients with ASD were genetically diagnosed. When the genetic target is clear, Sanger sequencing or MLPA analysis is applicable. If the genetic target is unclear, WES and whole genome analysis for CNVs are of first choice.

References:

Grants:

Conflict of Interest: None declared.

EP09.041 A novel variant in BRWD3 gene in four related male patients with intellectual disability

Marta Soares ¹, Marcia Rodrigues², Ana Berta Sousa²

¹Serviço de Genética Médica, Departamento de Pediatria, Hospital de Santa Maria, Centro Hospitalar Universitário Lisboa Norte, Lisboa, Portugal, Lisboa, Portugal; ²Serviço de Genética Médica, Departamento de Pediatria, Hospital de Santa Maria, Centro Hospitalar Universitário Lisboa Norte, Lisboa, Portugal, Lisbo, Portugal

Background/Objectives: BRWD3 gene encodes a chromatin modifying protein, acting in the epigenetic regulation of the nervous system development. Intellectual developmental disorder X-linked type 93 (OMIM#300659) is caused by pathogenic variants in BRWD3 and was reported in 36 patients (35 males and 1 female). The clinical picture includes developmental delay (DD)/intellectual disability (ID), macrocephaly, and facial dysmorphisms (long face, broad forehead, prominent ears, pointed chin).

Methods: We report a 18-year-old male, first child of non-consanguineous parents, with family history of ID (three maternal uncles) and learning difficulties (mother and two maternal half-sisters). Pregnancy, delivery and growth were normal. He has moderate ID, behavioural issues, and unilateral cryptorchidism. Physical examination revealed relative macrocephaly, dysmorphic features (long face, bulbous nose, prognathism), and bilateral clinodactyly of the 5th finger. Previous investigation included metabolic studies, head MRI, karyotype, array-CGH and molecular study for fragile X syndrome (PCR), all normal.

Results: Whole-exome sequencing with copy number variations analysis identified a novel hemizygous variant in BRWD3 gene: c.331+5G>C. Cascade genetic testing revealed maternal inheritance and segregation with the phenotype. Genetic testing of unaffected family members is still ongoing.

Conclusion: The reported variant is predicted to disrupt the highly conserved donor splice site, is absent in control population databases and co-segregates with the phenotype, which is suggestive of causality. This case illustrates that segregation analysis is a powerful tool in variant classification. Further analysis of X-chromosome inactivation pattern in a carrier female may help establish variant pathogenicity. Molecular diagnosis allows proper clinical follow-up and genetic counselling.

References:

Grants:

Conflict of Interest: None declared.

EP09.042 Genetic study of intellectual disability in children with Duchenne muscular dystrophy

MAHA BEN JEMAA ^1;2, najla soyah³, ahlem msakni², dorra hmida², ali saad², ilhem ben youssef⁴, moez gribaa²

¹Sfax Faculty of Medicine, Department of Cytogenetics and Biology of Reproduction,Histology-embryology laboratory, Sfax, Tunisia; ²Hospital Farhat Hached, Department of Cytogenetics and Biology of Reproduction, sousse, Tunisia; ³Hospital Farhat Hached, Pediatrics department, sousse, Tunisia; ⁴National institute Mongi ben hmida of neurology, neuropediatrics, Tunis, Tunisia

Background/Objectives: Duchenne muscular dystrophy (DMD) is a severe dystrophinopathy linked to mutations of the dystrophin gene. The disease mainly affects the muscles; however, intellectual disability (ID) is present in approximately one third of DMD cases due to dystrophin isoforms’ expression in the central nervous system.

We aim to analyze the mutational profile and the dystrophin isoforms in patients with DMD associated with ID.

Methods: Nineteen DMD cases associated with ID are included. DMD was genetically confirmed by MLPA, and we explored the relationship between ID and affected isoforms.

Results: Family history of ID is found in 15.79% of our patients. The age of onset of the disorders is 3.2. Gait disturbances and increased CPK are constant in all our patients. 79% of patients carry deletions between exons 45 and 55. The Dp427 isoform is absent in all patients. Dp140pc is absent in 58% of cases, against 32% for the Dp140utr isoform, while Dp71 is absent in 5% of cases.

Conclusion: Molecular diagnosis helps predict the association of DMD with ID and even have an idea of the severity of ID. This is crucial for adequate care, aiming for a better quality of life of patients.

References: 1. Sequence and Structure Characteristics of 22 Deletion Breakpoints in Intron 44 of the DMD Gene Based on Long-Read Sequencing.Geng C et al. Front Genet. 2021.

2. Timing and localization of human dystrophin isoform expression provide insights into the cognitive phenotype of Duchenne muscular dystrophy. Natatlie et al.Sci Rep. 2017.

Grants: The authors received no financial support for the research.

Conflict of Interest: None declared.

EP09.043 Inherited Maternal Duplication at 15q11.2-q13.1: A new case, detected by Whole Exome Sequencing (WES)

Danai Veltra ^1;1, Faidon-Nikolaos Tilemis^1;2, NIKOLAOS MARINAKIS¹, marina katsalouli³, Jan Traeger-Synodinos¹, Christalena Sofocleous^1;2

¹Laboratory of Medical Genetics,Medical School, National and Kapodistrian University of Athens, St. Sophia’s Children’s Hospital, ATHENS, Greece; ²Research University Institute for the Study and Prevention of Genetic and Malignant Disease of Childhood, National and Kapodistrian University of Athens, St. Sophia’s Children’s Hospital, ATHENS, Greece; ³Pediatric Neurology Department, St. Sophia’s Children’s Hospital, ATHENS, Greece

Background/Objectives: 15q Duplication Syndrome (Dup15q) is caused by one extra copy of the 15q11.2-q13.1 Prader–Willi/Angelman critical region, subjected to imprinting. Genes like UBE3A, GABRA5, GABRB3, GABRG3 and SNRNP are crucial for development and synaptogenesis and normally present differential monoallelic expression. In maternal Dup15q, inherited in 15% of all cases, allelic balance is disrupted and patients present hypotonia and psychomotor delays, intellectual disability, autism spectrum disorder (ASD), and epilepsy. An interstitial Dup15q was revealed by ExomeDepth during Whole Exome (WES) analysis and is described in the context of intrafamilial clinical heterogeneity, ambiguous presentations, and challenging differential diagnosis.

Methods: A 4-year-old female with low-level 45,X(7)/46,XX(66) mosaicism was referred for developmental delay. From the family history her 10-year-old brother had speech delay and autistic behavior and their mother was reported as socially distant. Following clinical evaluation, pre-test counselling and signed consent, WES was performed with xGen Exome Research v2 kit (Integrated DNA Technologies) on Illumina NextSeq-500 system. Bioinformatic analysis on VarSome Clinical platform, included CNV detection by ExomeDepth. Variant classification followed ACMG and ClinGen recommendations. Mehylation Specific -MLPA (MRC ME028-C1) was applied for further investigation.

Results: A 5.2 Mb heterozygous pathogenic duplication of 15q11.2-q13.1 was detected by ExomeDepth. Subsequent MS-MLPA allowed confirmation and characterization of parental origin, detecting the same maternally-derived duplication (15:23.060.849-28.277.167/breakpoints BP1-BP3) in both the affected proband and her mother.

Conclusion: ExomeDepth algorithm enables detection of Copy Number Variants and may be used for the elucidation of complex neurodevelopmental disorders like Dup15q syndrome.

References: PMID: 23633446, 22942019.

Grants: None.

Conflict of Interest: None declared.

EP09.044 The divergent pattern of inheritance in siblings with novel homozygous DMN1 gene variant

Ahmad Alaqeel ¹, Lova Matsa², Issa Almashmoom¹, Hari-Chandan Appikonda², Neetha John²

¹KOC Hospital, Paediatric Department, Ahmadi City, Kuwait; ²Igenomix - UAE, Genomic Precision Diagnostics, Dubai, United Arab Emirates

Background/Objectives: We present two affected siblings (a brother and a sister) with developmental delay, poor scholastic performance, difficulty to remember, recurrent seizures attacks, abnormal muscle movements, increased deep tendon reflexes, and mild spasticity. EEG showed epileptiform activity. 3-generation pedigree revealed consanguineous parents.

Methods: Whole-exome sequencing (WES) analysis quadro-test was done for both affected siblings plus their phenotypically healthy parents using the Illumina platform at Igenomix laboratory, Dubai.

Results: A novel homozygous missense variant in exon 1 of the DNM1 gene [NM_004408.4:c.107T>G, p.(Val36Gly)] was identified. This variant has interestingly been detected in a homozygous state in both affected siblings, while it was found to be heterozygous in both phenotypically normal parents.

Conclusion: Although to date the DNM1 gene has only been associated with an autosomal dominant disorder, i.e., developmental and epileptic encephalopathy-31 (DEE31); the above results clearly show autosomal recessive (AR) inheritance. These results also consistent, though different variants; with Goekhan et al. (2021) that showed biallelic variants in individuals with developmental delay and epilepsy [1]. Our case also clearly shows the importance of testing multiple family members at the same time as in this family where WES-quadro was done; in order to reach a definitive diagnosis in a precise manner. Genetic testing of other siblings is on-going.

References: Gökhan Yigit, Ruth Sheffer, Muhannad Daana, Yun Li, Emrah Kaygusuz, et al. Loss-of-function variants in DNM1 cause a specific form of developmental and epileptic encephalopathy only in biallelic state. J Med Genet. 2021 Jun 25;jmedgenet-2021-107769.

Grants: No grants.

Conflict of Interest: None declared.

EP09.046 Diagnostic yield of exome trio analysis to identify the genetic etiology in 804 previously undiagnosed cases

Irene Diez ¹, Monica Martinez-Garcia¹, Celia Rodriguez¹, Marta Hervás¹, Andrea de la Fuente¹, Guillermo Martin^1;1, Ángel Carro¹, Pablo Solar¹, David Rodriguez¹, Maria Calvente¹, Paolo MAIETTA¹, Sara ALVAREZ¹, María García¹

¹NIMGenetics S.L, Madrid, Spain

Background/Objectives: Exome trio analysis is an effective strategy to identify potentially causal variants, along with their inheritance pattern, on rare genetic disorders. This approach has entered the medical practice as an effective diagnostic test transforming the molecular diagnosis and clinical management of undiagnosed genetic diseases.

Methods: We performed exome sequencing using SureSelectXT Human All Exon V6 technology (Agilent Technologies) and Comprehensive Exome Panel technology (Twist Bioscience). Sequencing reads were analyzed using DRAGEN software. Trio annotation and prioritization was performed with an in-house pipeline that allowed the detection of candidate SNVs and CNVs, based on genetic and phenotypic priorization.

Results: We present the analysis of 804 trios referred to our lab since 2018. Patients were mainly children with neurodevelopmental disorder (92%). The genetic etiology was potentially elucidated in 344 probands, achieving a 43% genetic diagnostic rate. Among these patients, 176 harbored de novo variants, 66 hemizygous maternally inherited variants, 45 in compound heterozygous variants, 27 newly homozygous variants and 30 variants inherited from parents. In two cases, clinically relevant variants detected were pathogenic CNVs.

Conclusion: In our cohort exome trio analysis provides a diagnostic yield of 43% in patients whom traditional molecular diagnostics strategies were uninformative. This analysis also allows CNV screening, but we only detected two CNVs due to CGH array was previously performed in the vast majority of cases. The implementation of exome trio analysis as a first-tier diagnostic approach will provide a higher diagnostic yield and a cost-efficient option particularly specially in patients with neurodevelopmental disorder.

References:

Grants:

Conflict of Interest: None declared.

EP09.049 PHF8-related X-linked phenotype and genotype - review of literature

Alice Porto Vasconcelos ¹, Manuela Rodrigues², Mafalda Sampaio³, Carmen Silva⁴, João Parente Freixo⁵, Renata Oliveira¹

¹Centro Hospitalar Universitário de São João (CHUSJ) EPE, Medical Genetics Service, Porto, Portugal; ²Centro Hospitalar Universitário de São João (CHUSJ) EPE, Neonatology Unit, Department of Paediatrics, Porto, Portugal; ³Centro Hospitalar Universitário de São João (CHUSJ) EPE, Neuropediatrics Unit, Department of Pediatrics, Porto, Portugal; ⁴Centro Hospitalar Universitário de São João (CHUSJ) EPE, Neurodevelopment Unit, Department of Paediatrics, Porto, Portugal; ⁵Institute for Research and Innovation in Health (i3s), Centro de Genética Preditiva e Preventiva (CGPP), Porto, Portugal

Background/Objectives: X-linked intellectual disability, Siderius type (MIM #300263) is a genetic disorder caused by pathogenic loss-of-function variants in the PHF8 gene, with X-linked recessive inheritance pattern. The main clinical features reported include the association of cognitive impairment, cleft lip and/or palate and facial dysmorphisms. Up to date, 16 variants submitted in ClinVar database were classified as pathogenic or likely pathogenic and 44 as uncertain significance. Regarding phenotypic descriptions other than intellectual disability, nine patients from five families have been published.

Methods: We summarize the clinical features and variants of the patients previously reported in the literature and compare them with an additional patient from our clinic.

Results: We report a 4-year-old male patient with mild global developmental delay, dysmorphic facial features, brain encephaloclastic lesions and symptomatic focal epilepsy. There is a maternal family history of epilepsy. Clinical exome analysis revealed a maternally inherited truncating variant in the PHF8 gene. Unlike those previously reported, there is no personal or family history of cleft lip/palate.

Conclusion: Features like autism spectrum disorder, brain encephaloclastic lesions and epilepsy could be part of the phenotypic spectrum of Siderius type X-linked intellectual disability. There is a high number of uncertain significance variants in the PHF8 gene published in public databases, therefore further phenotype characterization of these patients could be helpful to interpret such variants and provide an accurate diagnosis, that can be beneficial for surveillance, prognosis assessment and familial genetic counselling.

References:

Grants:

Conflict of Interest: None declared.

EP09.050 Case study: deep phenotyping of a case with paternally inherited 16p11.2 duplication and 22q11.2 duplication

Jente Verbesselt¹, Inge Zink^2;3, Jeroen Breckpot ^1;4, Ann Swillen^1;4

¹KU Leuven, Department of Human Genetics, Leuven, Belgium; ²KU Leuven, Department of Neurosciences, Research Group Experimental Oto-Rhino-Laryngology, Leuven, Belgium; ³UZ Leuven, Department of Oto-Rhino-Laryngology, Head & Neck Surgery, MUCLA, Leuven, Belgium; ⁴UZ Leuven, Centre for Human Genetics, Leuven, Belgium

Background/Objectives: Some recurrent copy number variants (CNVs), such as 16p11.2dup and 22q11.2dup, are associated with significant risk for neurodevelopmental disorders (NDD), although associated phenotypes are heterogeneous and familial transmission rates are high. We report for the first time co-occurrence of these NDD susceptibility CNVs in one patient.

Methods: Using standardized instruments, clinical and neurodevelopmental features were assessed in a 9-year-old boy, carrying a paternally inherited 16p11.2dup ([hg19](29,652,360-30,195,608)x3) and 22q11.21dup ([hg19](18,765,102-21,808,998)x3).

Results: This boy is the first and only child of unrelated parents. He was born at 36 weeks PMA following a pregnancy complicated by pre-eclampsia and IUGR (birth weight:1.9kg;length:42.5cm). At age 2, he was referred to the genetics clinic due to nutritional problems, failure to thrive, strabismus, congenital defects such as cryptorchidism and ankyloglossia, and developmental delay. At age 9, he attends special education, received the diagnosis of DCD. Longitudinal follow-up shows a relative stable cognitive trajectory within the borderline range (IQ76), whereas language skills show a growing into deficit trajectory. His father experienced nutritional problems in infancy and attended vocational education. His mother had learning problems, and only completed primary school. Compared to an in-house reference set of unrelated index patients with either 22q11.2dup or 16p11.2dup, scores for IQ, language and behaviour in this boy were within the same range.

Conclusion: No clear additive effect of the co-occurrence of 22q11.2dup and 16p11.2dup was observed on neurodevelopmental outcome of this boy. Systematic familial phenotyping and genotyping is required to explain intra- and interfamilial phenotypic variability of these CNVs.

References:

Grants: NIMH (U01MH119759).

Conflict of Interest: None declared.

EP09.051 Identification through aCGH of CNVs involved in the genetic etiology of intellectual disability - data from a small Romanian cohort

Alexandru Caramizaru ^1;2, Ioana Streata^1;3, Andrei Pirvu^1;3, Simona Serban-Sosoi^1;2, Alexandra Dumitra⁴, Ovidiu Voican¹, Anca Riza^1;3, Razvan Plesea^1;3, Mihai Cucu^1;2, Monica-Laura Cara^1;5, Amelia Dobrescu^1;2, Florin Burada^1;2, Mihai Ioana^1;3

¹Regional Center for Medical Genetics Dolj, Craiova, Romania; ²University of Medicine and Pharmacy of Craiova, Medical Genetics Department, Craiova, Romania; ³University of Medicine and Pharmacy of Craiova, Cellular and Molecular Biology Department, Craiova, Romania; ⁴University of Medicine and Pharmacy of Craiova, Craiova, Romania; ⁵University of Medicine and Pharmacy of Craiova, Public Health Department, Craiova, Romania

Background/Objectives: According to WHO Europe, intellectual disability (ID) is a significantly reduced ability to understand new or complex information, to learn and apply new skills (impaired intelligence), and to cope independently (impaired social functioning). There are various causes which could lead to intellectual disability, including genetic abnormalities such as CNVs (copy number variations). The association between CNVs and patients suffering from ID has been acknowledged through multiple studies. The aim of our study is to present the results of aCGH (array comparative genomic hybridization) testing from a small Romanian cohort of patients with intellectual disability, most of which were syndromic.

Methods: Purified genomic DNA from peripheral blood was examined for copy number variations (CNVs) using Agilent Cytogenomics 4x180K/8x60K or OGT Cytosure 8x60K ISCA design oligonucleotide microarrays. Copy number data was analyzed with Agilent Cytogenomics and OGT Cytosure Interpret software, respectively.

Results: From a total of 366 patients with syndromic ID tested through aCGH, 76 presented one CNV and 6 presented two CNVs each, resulting in a diagnostic yield of 22.4%. The group of patients was clinically heterogeneous, as were the identified microdeletions and microduplications.

Conclusion: This study demonstrated the importance of CNVs testing through aCGH in the management of patients with unexplained ID. These results could contribute to the existing data and knowledge regarding the utility of aCGH in the diagnosis of ID.

References:

Grants:

Conflict of Interest: None declared.

EP09.052 Experience to integrate exome sequencing and reanalysis into clinical practice in a tertiary hospital of the public healthcare system

Marta Codina-Sola ¹, Berta Campos¹, Paula Fernández-Álvarez¹, Mar Costa-Roger¹, Alejandro Moles-Fernández¹, Irene Valenzuela¹, Anna Maria Cueto-González¹, Laura Carmen Trujillano Lidon¹, Anna Abulí¹, Eulàlia Rovira¹, Patricia Muñoz-Cabello¹, Jordi Leno-Colorado¹, Ida Paramonov¹, Ivon Cuscó¹, Elena Garcia Arumi¹, Eduardo Tizzano¹

¹Vall d’Hebron University Hospital, Department of Clinical and Molecular Genetics, Barrcelona, Spain

Background/Objectives: The introduction of exome sequencing (ES) into clinical practice has increased the rate of molecular diagnosis in patients affected with rare disorders. The integration of genomics into the public healthcare system requires a multidisciplinary approach, as well as the implementation of strategies for systematic reanalysis.

Methods: We report our strategy and results of implementing singleton ES in our current diagnostic practice in a cohort of 1111 patients with a suspicion of a genetic condition, evaluated from June 2017 to November 2021 in a specialized genetics consultation with a team including clinical geneticists, genetic counselors, laboratory geneticists, bioinformaticians and other specialists. We also report the results of introducing systematic reanalysis of unsolved cases, combined with a translational research approach and the use of collaborative platforms such as GeneMatcher.

Results: Our overall diagnostic yield was 29% (333/1111) in the first analysis. In a second step, we reanalysed 298 cases. We obtained 20% (60/298) additional diagnoses: 53 through the usual diagnostic process (23 candidate genes/HPOs, 21 improved pipelines, 6 new publications, 2 initially misclassified, and 1 copy-number variant), and 7 through translational research by international data sharing. Our final diagnostic yield was 35% (34% due to a traditional diagnostic approach, and 1% through an additional research strategy).

Conclusion: Our diagnostic yield was similar to that previously described in the literature. We show that the periodic reanalysis of ES allows additional diagnosis in approximately 20% of patients and that it can be integrated into the diagnostic routine.

References:

Grants:

Conflict of Interest: None declared.

EP09.053 KBG syndrome in the Portuguese population: clinical and molecular characterization of 41 patients

Mariana Neves ¹, Patricia Dias¹, pedro louro², Catarina Silva Rosas², sofia fernandes², maria abreu³, Susana Ferreira⁴, mafalda melo⁴, oana moldovan¹, Juliette Dupont¹, André Travessa¹, João Alves¹, Ana Medeira¹, heloisa santos¹, pedro almeida², joaquim sá², Fabiana Ramos², Ana Carvalho², Sérgio Sousa², Lina Ramos², ana soares³, celia soares³, gabriela soares³, Natalia Tkachenko³, Marta Amorim⁴, Diana Antunes⁴, João Parente Freixo⁵, Ana Maria Fortuna³, Cláudia Falcão Reis³, Jorge M Saraiva², Ana Berta Sousa¹

¹Hospital de Santa Maria, Serviço de Genética Médica, Departamento de Pediatria, Hospital de Santa Maria, Centro Hospitalar Universitário Lisboa Norte, Lisboa, Portugal, Lisboa, Portugal; ²Centro Hospitalar e Universitário de Coimbra - Hospital Pediátrico, Serviço de Genética Médica, Hospital Pediátrico, Centro Hospitalar Universitário de Coimbra, Coimbra, Portugal, Coimbra, Portugal; ³Hospital Geral de Santo António, Unidade de Genética Médica, Centro de Genética Médica Doutor Jacinto Magalhães, Centro Hospitalar Universitário do Porto, Porto, Portugal, Porto, Portugal; ⁴Dona Estefânia, Serviço de Genética Médica, Hospital Dona Estefânia, Centro Hospitalar Universitário Lisboa Central, Lisboa, Portugal, Lisboa, Portugal; ⁵Centro de Genética Preditiva e Preventiva, Centro de Genética Preditiva e Preventiva, Instituto de Biologia Molecular e Celular, i3S, Porto, Portugal, Porto, Portugal

Background/Objectives: KBG syndrome (MIM#148050) is an autosomal dominant syndromic developmental delay/intellectual disability (DD/ID) disorder caused by haploinsufficiency variants in ANKRD11 gene, characterized by a typical gestalt, macrodontia and short stature. It is presumed to be a frequent underdiagnosed aetiology of syndromic DD/ID. We present the clinical and molecular characterization of 41 Portuguese patients with KBG.

Methods: Retrospective review of clinical and molecular data from KBG patients diagnosed in Portuguese medical genetics centres. The study was approved by the Ethics Committee of the Lisbon Academic Medical Centre.

Results: We collected data from 41 patients (21 females, 20 males). All patients had DD/ID and learning difficulties. The typical gestalt was recognized in 73%, and in 11% Cornelia de Lange syndrome was first considered. Common findings were 5th finger clino/brachydactyly (86%), macrodontia (79%), attention deficit hyperactivity disorder (ADHD) (64%) and hearing loss (53%). Short stature (<3rd centile) was observed in 38%. 25 different ANKRD11 sequence variants were identified: 19 frameshift [pathogenic (P)/likely pathogenic (LP)]; 4 missense [2 (P/LP), 2 uncertain (VUS)]; and 1 affecting splicing (P). Four unrelated patients had large deletions involving ANKRD11 gene, including one case without the typical KBG phenotype. Variants were found in all but two patients, who both had a high KBG Face2Gene score.

Conclusion: This data is consistent with previous literature. KBG syndrome has a distinctive gestalt which is easier to recognize later in childhood when macrodontia becomes evident. DD/ID is usually mild; behaviour disorders, particularly ADHD, are common. Target gene sequencing should confirm most clinical diagnoses.

References:

Grants:

Conflict of Interest: None declared.

EP09.054 Beyond craniosynostosis: can TCF12 gene be responsible for a wider clinical spectrum?

Susana Ferreira ¹, João Parente Freixo², Marta Amorim¹

¹Centro Hospitalar Universitário de Lisboa Central, Serviço de Genética Médica, Lisboa, Portugal; ²CGPP – Centro de Genética Preditiva e Preventiva, IBMC – Instituto de Biologia Molecular e Celular, i3S – Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal

Background/Objectives: TCF12 gene encodes transcription factor 12 (tcf12), a member of the basic helix-loop-helix (bHLH), and plays a key role in neurogenesis, mesoderm formation, and cranial suture development. TCF12 haploinsufficiency results in coronal craniosynostosis type 3 (CRS3, #615314) and hypogonadotropic hypogonadism 26 (#619718). Several cases of CRS3 presented with other comorbidities such neurodevelopmental impairment, dysmorphims and other congenital anomalies.

Methods: We report a new patient with a likely pathogenic variant in TCF12 gene aiming to further expand its clinical spectrum.

Results: A 30-year-old female was first referred to our outpatient genetic department at 16-years-old with a central nervous system malformation (focal cortical dysplasia with left­side frontal polymicrogyria), nystagmus, right hemiparesis, epilepsy, hearing loss and intellectual disability. At our observation, she showed low anterior hairline, thin upper lip, and a prominent chin. Extensive investigation was performed with normal karyotype, FRAXA, metabolic investigation, microarray analysis. Exome sequencing identify a de novo heterozygous variant in TCF12 (NM_207036.1):c.1453C>T (p.(Arg485*)), classified as likely pathogenic, previously described as a cause of CRS3.

Conclusion: Although, our patient does not have a craniosynostosis, this result can potentially explain the phenotype and be clinically relevant. We propose that TCF12 could be responsible for other craniofacial abnormalities and neurodevelopmental disorders. It is important to note that genetic research suggests that mutations in regulatory elements of genes can cause developmental defects. Therefore, more investigation is necessary to understand the role of tcf12 as transcriptional regulation factor in other tissues besides cranial suture development, and its impact in additional developmental defects.

References:

Grants:

Conflict of Interest: None declared.

EP09.056 Exploring the phenotypic spectrum of CHD4 gene variants: Case report and literature review

Mafalda Melo ¹, Ana Isabel Dias², João Parente Freixo³, Diana Antunes¹

¹Centro Hospitalar Universitário de Lisboa Central, Serviço de Genética Médica, Lisboa, Portugal; ²Centro Hospitalar Universitário de Lisboa Central, Serviço de Neurologia Pediátrica, Lisboa, Portugal; ³CGPP – Centro de Genética Preditiva e Preventiva, IBMC – Instituto de Biologia Molecular e Celular, i3S – Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal

Background/Objectives: CHD4 is a chromatin remodeler involved in epigenetic regulation of gene transcription. Pathogenic variants in CHD4 gene are associated with Sifrim-Hitz-Weiss syndrome (OMIM#617159), a well described chromatinopathy characterized by global developmental delay, mild to moderate intellectual disability, brain anomalies, congenital heart defects, and dysmorphic features. Additional abnormalities include hypogonadism, skeletal and limb anomalies, hearing impairment, and ophthalmic abnormalities. Recently, missense variants in CHD4 were identified in patients with a milder phenotype consisting of epilepsy with sinus arrhythmia.

Methods: We report a case of a novel missense variant in CHD4 that presented with an intermediate phenotype, aiming to contribute to the clinical and genotypic spectrum characterization.

Results: A 9-year-old boy was referred for developmental delay/mild intellectual disability and epilepsy evaluation. He was the oldest son of a non-consanguineous healthy couple. Prenatal and neonatal history were unremarkable. At our observation, he had no dysmorphisms nor congenital anomalies. FRAXA and microarray were normal. A WES-based gene panel identified a missense variant in the CHD4 gene, NM_001273:c.2660G>A (p.Arg887Gln). The variant was not previously described in literature, nor in gnomAD population. It occurs at a position that is conserved across species, in the same codon as a known pathogenic variant, and in silico analysis predicts a probably deleterious effect in protein. Finally, familial studies showed a de novo origin.

Conclusion: This report contributes to the expansion of the phenotypic spectrum of CHD4-related disorders. It highlights it can be a less obvious diagnosis since there is a wide phenotypic heterogeneity, without known genotypic-phenotypic correlations.

References:

Grants:

Conflict of Interest: None declared.

EP09.057 Heterozygous HMGB1 loss-of-function variants are associated with developmental delay and microcephaly

Kevin Uguen ^1;2, Kilannin Krysiak³, Severine Audebert Bellanger², Sylvia Quemener^1;2, Caroline Benech¹, Eléonore Viora-Dupont⁴, Frédéric Tran Mau-Them^5;6, Sophie Rondeau⁷, Ibrahim Elsharkawi⁸, Jorge Luis Granadillo De Luque⁸, Julie Neidich³, Celia Azevedo Soares^9;10, Natalia Tkachenko⁹, Shivarajan, M Amudhavalli¹¹, Kendra Engleman¹¹, anne boland¹², Jean-François Deleuze¹², stéphane bezieau¹³, Sylvie Odent¹⁴, Annick Toutain¹⁵, Dominique Bonneau¹⁶, Brigitte Gilbert-Dussardier¹⁷, Laurence Faivre^4;6, Marlene Rio⁷, Cédric Le Maréchal^1;2, Claude Férec¹, Elena Repnikova¹⁸, Yang Cao³

¹University of Brest, Inserm, EFS, UMR 1078, GGB, Brest, France; ²CHU de Brest, Service de génétique médicale et biologie de la reproduction, Brest, France; ³Washington University in St. Louis School of Medicine, Department of Pathology and Immunology, Saint Louis, United States; ⁴FHU TRANSLAD, Hôpital d’Enfants, CHU Dijon, Centre de Référence Anomalies du Développement et Syndromes Malformatifs, Dijon, France; ⁵Pôle de Biologie, CHU Dijon Bourgogne, Unité Fonctionnelle 6254 d’Innovation en Diagnostic Génomique des Maladies Rares, Dijon, France; ⁶Université de Bourgogne, UMR1231 GAD, FHU-TRANSLAD, Dijon, France; ⁷AP-HP, Hôpital Necker-Enfants Malades, Fédération de Génétique Médicale, Paris, France; ⁸Washington University in St. Louis School of Medicine, Department of Pediatrics, Saint Louis, United States; ⁹Centro de Genética Médica Jacinto Magalhães, Centro Hospitalar Universitário do Porto, Serviço de Genética Médica, Porto, Portugal; ¹⁰Instituto de Ciências Biomédicas Abel Salazar/Universidade do Porto, Unit for Multidisciplinary Research in Biomedicine, Porto, Portugal; ¹¹Children’s Mercy Hospital, Department of Clinical Genetics, Kansas City, United States; ¹²Université Paris-Saclay, CEA, Centre National de Recherche en Génomique Humaine, Evry, France; ¹³Centre Hospitalier Universitaire de Nantes, Service de Génétique Médicale, Nantes, France; ¹⁴Centre Hospitalier Universitaire de Rennes, Service de Génétique Clinique, Centre Référence Déficiences Intellectuelles de Causes Rares, Centre de Référence Anomalies du Développement, Centre Labellisé pour les Anomalies du Développement (CLAD) Ouest, Rennes, France; ¹⁵Centre Hospitalier Universitaire de Tours, Service de Génétique, Tours, France; ¹⁶Centre Hospitalier Universitaire de Angers, Département de Biochimie et Génétique, Angers, France; ¹⁷Centre Hospitalier Universitaire de Poitiers, Service de Génétique, Poitiers, France; ¹⁸Children’s Mercy Hospital/University of Missouri Kansas City Medical School, Department of Pathology, Kansas City, United States

Background/Objectives: The 13q12.3 microdeletion syndrome is a rare cause of syndromic intellectual disability for which two candidate genes were previously proposed (KATNAL1 and HMGB1). However, to date, no single point mutation or deletion of either of these genes has been described in patients.

Methods: Thanks to exome sequencing and international data sharing, we report here 6 patients with de novo loss of function (n = 5), or missense (n = 1) variations in the HMGB1 gene.

Results: The clinical signs found in these patients are consistent with 13q12.3 microdeletion, namely intellectual disability, language delay, microcephaly, dysmorphic features, obesity and atopic dermatitis. In silico data in line with intolerance to loss of function of the gene and previous in vitro studies showing its importance in neurodevelopment support the involvement of these variations in the neurodevelopmental phenotype of the patients. The role of the HMGB1 gene in the inflammatory process could explain cutaneous signs such as atopic dermatitis. Finally, the predicted interaction between HMGB1 and other genes involved in neurodevelopmental disorders (SMARCA4, SMARCA2, SMARCD1) raises the hypothesis of a common epigenetic signature.

Conclusion: Thus, as for many microdeletion syndromes, the search for the genetic basis and the genes responsible for the pathology has been greatly facilitated by high-throughput exome and genome sequencing. Indeed, this cohort allows us to propose HMGB1 as a major gene of the 13q12.3 microdeletion syndrome and as a new gene for intellectual disability. Finally, this study shows once again the importance of international data sharing in the context of rare diseases.

References:

Grants:

Conflict of Interest: None declared.

EP10 Neurogenetic and Psychiatric Disorders

EP10.001 Pipelines to investigate both small and large variants from a small amyotrophic lateral sclerosis family

Sandrine Chan Moi Fat ¹, Emily McCann¹, Lyndal Henden¹, Kelly Williams¹, Dominic Rowe², Jennifer A Fifita¹, Ian P Blair¹

¹Macquarie University Centre for Motor Neuron Disease Research, Department of Biomedical Sciences, Faculty of Medicine, Health and Human Sciences, Sydney, Australia; ²Faculty of Medicine, Health and Human Sciences, Macquarie University, Department of Clinical Medicine, Sydney, Australia

Background/Objectives: Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease. To date, 90% of ALS cases still have an unknown genetic cause. Most research has focused on small nucleotide level variants only. This project extends ALS gene discovery to consider large structural variation (SVs) by using bioinformatic pipelines to identify both small and large genetic alterations in a small ALS family with limited power for traditional gene discovery methods.

Methods: Whole genome sequencing (WGS) was performed on two affected individuals from a family with ALS history (MQ52). To identify candidate small nucleotide variants, shared variant analysis was carried out to filter for nonsynonymous and indel variants absent/extremely rare from public controls. To identify SVs, we have developed a comprehensive pipeline that calls SVs from WGS data using multiple tools such as Manta, Lumpy and MetaSV. The Duphold tool is then used to filter SVs based on quality scores, followed by annotation using Reciprocal Overlap Annotator and AnnotSV to generate a list of shared or overlapping SVs between the two affected individuals.

Results: Initial analysis of family MQ52 identified 14 small variants and 80 novel/rare SVs, of which 44 SVs lie in coding regions. This SV pipeline is undergoing refinement and resultant SVs will be validated using established PCR techniques.

Conclusion: Our gene discovery pipelines can successfully identify both small and large variants that potentially cause disease within small ALS families. Given that SVs have been largely understudied in ALS, discovery of ALS-linked SVs could help to elucidate the missing genetic architecture of ALS.

References:

Grants:

Conflict of Interest: None declared.

EP10.002 Clinical spectrum and genetic characteristics of four patients with autism spectrum disorders and 15q11-q13 duplication syndrome

Daniela Avdjieva-Tzavella ¹, Stoyan Bichev², Tsvetina Veleva³, Trayan Delchev³, Maria Sredkova-Ruskova³, Savina Hadjidekova⁴

¹University Pediatrics Hospital, Medical University, Department of Clinical Genetics, Sofia, Bulgaria; ²University Hospital “Majcin Dom”, National Genetic Laboratory, Sofia, Bulgaria; ³University Pediatrics Hospital, Medical University, Department of Clinical Genetics, Sofia, Bulgaria; ⁴Medical University, Department of Medical Genetics, Sofia, Bulgaria

Background/Objectives: Chromosomal copy number variants (CNV) are the most common genetic lesion found in 5–10% of all patients with autism spectrum disorder (ASD). The 15q11.1-13.1 duplication syndrome characterized by ASD, hypotonia, intellectual disability, motor delays and epilepsy. Disease frequency in the population is 1: 30,000 to 1: 60,000. As many as 1–3% of all ASD cases may be the result of duplications of the 15q11.2-q13 region. The duplication described as supernumerary isocentric chromosome in 80% of cases and as an interstitial duplication in 20% of cases.

Methods: We described the clinical picture in 4 children with 15q11-q13 duplication syndrome. Conventional chromosomal analyses at 550 G-band resolution were performed on all 4 of our patients. We used MLPA P245 Microdeletion Syndromes for screening of the most common microdeletion syndromes and MLPA P036 Subtelomeres Mix 1 for screening of subtelomeric deletions/duplications in 3 patients. Array CGH was performed in one case - OGT 4x44k format oligonucleotide microarray with targeted CN resolution of 1 probe every 52kb and backbone CN resolution of 1 probe every 81kb. The slides were scanned on a GenePix 4100A two-colour fluorescent scanner (Axon Instruments, Union City, CA, U.S.A.). The arrays were analyzed by CytoSure Interpret Software.

Results: The chromosomal analysis yielded a supernumerary marker chromosome in 3 children. MLPA analysis demonstrated the presence of chromosome 15 (15q11-q13) duplication in 3 patients. By array CGH we determined arr15q11.2q13.1(22,765,658-29,030,488)x3.

Conclusion: Our results strongly support the implication of 15q11-13 rearrangements as a predisposing factor for autism.

References:

Grants:

Conflict of Interest: None declared.

EP10.003 Perceptions of causal attribution and attitudes to genetic testing among people with schizophrenia and their first-degree relatives: A qualitative study

Bettina Meiser ¹, Melissa Cullen², Kristine Barlow-Stewart³, Melissa Green², Paul Appelbaum⁴, Vaughan Carr², Murray Cairns⁵, Matthew Lebowitz⁴, Rajneesh Kaur²

¹University of New South Wales, School of Clinical Medicine, Sydney, Australia; ²University of New South Wales, Sydney, Australia; ³University of Sydney, Sydney, Australia; ⁴Columbia University, New York, United States; ⁵University of Newcastle, Newcastle, Australia

Background/Objectives: Rapid advances in the genetics of psychiatric disorders mean that diagnostic and predictive genetic testing for schizophrenia risk may one day be a reality. This study examined how causal attributions for schizophrenia contribute to interest in a hypothetical genetic test.

Methods: People with schizophrenia and first-degree relatives of people with schizophrenia were recruited through a schizophrenia research bank and mental health organisation. Semi-structured telephone interviews were conducted with 13 individuals with schizophrenia and 8 first-degree relatives. Transcripts were subjected to a qualitative analysis using the thematic analysis framework.

Results: Five themes were developed: (i) “It is like a cocktail”, with most participants aware that both genetic and environmental factors contributed to causation, and many mentioning the positive impact of genetic causal explanations; (ii) “Knowledge is power” (i.e., in favour of genetic testing); (iii) Genetic testing provides opportunities for early intervention and avoiding triggers, with participants citing a wide range of perceived benefits of genetic testing but few risks; (iv) Views on reproductive genetic testing for schizophrenia risk with a few participants viewing it as “playing God” but not necessarily being against it; and (v) “It snowballs”, whereby participants’ understanding of genetics was sophisticated with most believing that multiple rather than single genes contributed to schizophrenia.

Conclusion: Many individuals had a sound understanding of the role of genetic testing if it were to become available, with evidence of insight into the role of multiple genes and the contribution of other risk factors that may interact with any inherited genetic risk.

References:

Grants:

Conflict of Interest: None declared.

EP10.004 Re-analysis of exome sequencing data of undiagnosed epilepsy cases

Natalie Trump ¹, Andrea Haworth¹, John Filby¹, Anthony Rogers¹, Charles Steward¹, Sandro Morganella¹, Laura Ponting¹, Meriel McEntagart², Frances Elmslie²

¹Congenica, Hinxton, United Kingdom; ²South West Thames Regional Genetics, St George’s University Hospitals NHS Trust, London, United Kingdom

Background/Objectives: The rapidly changing landscape of epilepsy genetics makes it essential to establish new strategies for genetic testing that aim to increase the diagnostic yield. Salinas et al. (2021) have demonstrated the importance of periodic re-interpretation and re-analysis of genetic data to achieve this. Re-analysis of exome data of previously unsolved developmental and epileptic encephalopathy cases was shown to further increase the diagnostic yield by ~15%. The objective of our study is to evaluate the diagnostic potential of exome sequence re-analysis in our cohort of undiagnosed epilepsy cases.

Methods: DNA extracted from blood was enriched using Agilent SureSelect Clinical Research Exome V2 (CRE V2) or Nonacus ExomeCG and sequenced on Illumina NextSeq 500 or NovaSeq. Secondary and tertiary analysis of DNA sequences and review of SNVs and CNVs was undertaken using the Congenica clinical decision platform. Re-analysis was performed 6 - 48 months after initial interpretation, using 1) an updated curated epilepsy gene panel, and 2) gene agnostic prioritisation using Congenica AI.

Results: Through original exome sequencing analysis, a diagnosis was achieved in 34/129 patients (26%). Of 59 cases assessed for CNVs, 2 had pathogenic variants (3.4%). Re-analysis was performed on 95 unsolved cases. Updated figures will presented.

Conclusion: This study illustrates the diagnostic utility of re-analysing exome sequencing data in previously unsolved epilepsy cases.

References: Salinas et al. (2021) Clinical next generation sequencing in developmental and epileptic encephalopathies: Diagnostic relevance of data re-analysis and variants re-interpretation’. Eur. J. Med. Genet. 64(12):104363.

Grants:

Conflict of Interest: None declared.

EP10.005 A novel homozygous nonsense CCDC186 variant that causes neurodevelopmental delay, microcephaly, and seizures. New syndrome?

ALPER GEZDIRICI ¹, Pınar Arıcan²

¹Basaksehir Cam and Sakura City Hospital, Medical Genetics, Istanbul, Turkey; ²Basaksehir Cam and Sakura City Hospital, Pediatric Neurology, Istanbul, Turkey

Background/Objectives: CCDC186 is hypothesized to play an important role in the biogenesis of dense-core vesicles in neurons and endocrine cells.

Methods: Here, we report a 2 years female Syrian patient who has microcephaly, hypotonia, neurodevelopmental delay, and seizures. She is the 3rd child of a consanguineous couple. There was no significant dysmorphic finding in our patient. No abnormality was detected as a result of routine chromosome analysis and microarray analysis. Exome sequencing was planned in order to identify the genetic etiology that may be responsible for the clinical findings.

Results: Exome sequencing identified a homozygous c.535C>T; p.(Arg179Ter) loss-of-function variant in CCDC186(NM_018017.4).

Conclusion: Any disease phenotype related to the CCDC186 has not been identified in OMIM. There are only two patients with CCDC186 biallelic variants have been reported in the literature so far. Neurodevelopmental delay and some additional anomalies were described in both patients. In addition to all these data, the fact that our patient had clinical findings similar to the other 2 cases in the literature supports that the CCDC186 gene is responsible for a new autosomal recessive neurodevelopmental disease.

References:

Grants:

Conflict of Interest: None declared.

EP10.006 Target next-generation sequencing as a comprehensive test for genetics epilepsy

Mariagrazia Talarico ¹, Radha Procopio¹, Monica Gagliardi², Donatella Malanga^3;4, Antonio Gambardella¹, Grazia Annesi²

¹Institute of Neurology, Department of Medical and Surgical Sciences, Catanzaro, Italy; ²Institute for Biomedical Research and Innovation, National Research Council, Cosenza, Italy; ³Laboratory of Molecular Oncology, Department of Experimental and Clinical Medicine, Catanzaro, Italy; ⁴Laboratory of Molecular Oncology, Interdepartmental Center of Services (CIS), Catanzaro, Italy

Background/Objectives: Next-generation sequencing (NGS) has contributed to the identification of many monogenic epilepsy syndromes and is favouring earlier diagnosis in a subset of paediatric patients with epilepsy. The cumulative information emerging from NGS studies is rapidly changing our comprehension of the epileptic disorders. The aim of this study was to identify new variants, potentially pathogenic, in candidate genes. We performed NGS on 72 epileptic patients.

Methods: We selected 38 epilepsy causative genes for the targeted sequencing panel and performed the analysis using Ion Torrent Proton Sequencer. Raw data were analysed to select the potential pathogenic variants using bioinformatics tools (Mutation Taster, PolyPhen-2). All candidate variants were validated in Sanger sequencing.

Results: The pathogenic significance variants observed were 26: 11 variants were identified in genes encoding ion channels (KCNA2, KCNQ2, KCNQ3, KCNT1, SCN1A, SCN1B, and SCN2A). Other variants were found in genes that encodes for proteins involved in different cell functions: receptor subunits (CHRNA2, CHRNA4, GRIN1, GABRD), enzymatic activity (POMGNT1, PNKP), transmembrane proteins (PRRT2), nucleotide exchange factor (ARHGEF9) and others (RELN, CDKL5, FLNA, DCX).

Conclusion: Our findings provide several potential insights to understand the complex molecular mechanism underlying the epilepsy disorder.

References: J. Symonds, A. McTague, “Epilepsy and developmental disorders: Next generation sequencing in the clinic” Eur J Paediatr Neurol. 2021.

Grants: PRIN Project “Genetic Epileptic channelopathies as disease models for drug discovery toward personalized treatment: an integrated bench-to-bedside and backward approach” - CUP B64I19001030001 – funded by MIUR identification code 2017ALCR7C - Sector LS5 line C – in the following topic: “Epilepsy Genetics”.

Conflict of Interest: None declared.

EP10.007 A novel splicing mutation in ANO10 is responsible for Spinocerebellar ataxia, autosomal recessive 10 (SCAR10) in a large kindred in Northern Israel

Morad Khayat ¹, Olfat Aboleil Zoubi¹, Sleman Artul², Elana Chervinsky¹, Stavit A. Shalev^1;3

¹Ha`Emek Medical Center, The Institute for Genetics, Afula, Israel; ²Kupat Holim Clalit North District, Family Medicine Department, Magar, Israel; ³Technion - Israel Institute of Technology, Rappaport Faculty of Medicine, Haifa, Israel

Background/Objectives: The hereditary cerebellar ataxias are a heterogeneous group of conditions that is commonly classified according to their mode of inheritance. At least 20 different forms of autosomal recessive cerebellar ataxias (ARCAs) are known to date. Mutations in ANO10 causing slowly progressive spastic ataxia have so far been identified in several families from different ethnicities.

Methods: Five affected individuals from a large Arab-Christian family living in north Israel presented with progressive ataxia, variable degree of pyramidal signs and learning disabilities were studied. Using whole genome homozygosity mapping and targeted gene Sanger sequencing we identify the genetic cause of the disease.

Results: a novel homozygous variant c.139+1G>T was detected at the first nucleotide of the consensus donor splice site of exon 2 in the ANO10 gene, which is fully segregated in the family. This change leads to a frame shift and to a formation of stop codon after 17 amino acids p.G47Efs*9.

Conclusion: To date, at least 26 different pathogenic variants of ANO10 have been reported. These variants are spread throughout the gene and associated with variable phenotypes, with no clear genotype-phenotype correlation. Moreover, we report phenotypic heterogeneity within the same family that strongly suggests the existence of additional environmental and/or genetic factors which modify the phenotype induced by ANO10 variants. Our finding supports the assumption that there is a relationship between ANO10 coded protein TMEM16K, which is important for brain tissue development, and learning disabilities.

References: None.

Grants: None.

Conflict of Interest: None declared.

EP10.008 The diagnostic yield of genetic testing for middle-aged and elderly patients with neurological conditions

noga lempel^1;2, elon pras^1;2, Lior Greenbaum ^1;2

¹Sheba Medical Center, Tel Hashomer, Ramat Gan, Israel; ²Tel Aviv University, Tel Aviv, Israel

Background/Objectives: The use of genetic testing for neurological disorders is rapidly expanding in clinical practice. However, its yield in middle-aged and elderly population is still unclear.

Methods: We evaluated the diagnostic yield of genetic workup for patients aged 50 years and above, who referred for a specialized neurogenetics clinic within a tertiary medical center in Israel, during the period of 2017-2020.

Results: The study included 156 consecutive Jewish Israeli patients (61% males). All had neurological disorder, without a previous molecular diagnosis. The mean age of first genetic counselling at the clinic was 61.1±7.4 years (range: 50-85), and the mean age of disease onset was 47.1±15.9 years (range: infancy to 75). 43.8% were of Ashkenazi origin, and 41.7% had a positive relevant family history. The main indications for referral were neuromuscular disorders, cerebrovascular disorder/white matter hyperintensities, movement disorders and cognitive impairment/dementia. In total, 60.9% of the patients performed genetic testing, including one or more of the following: tests for specific mutations, gene panel, whole-exome sequencing (WES) or chromosomal microarray (CMA). Costs were covered by public health services, or by patients themselves. Overall, 21.7% of patients received a molecular diagnosis (30.0% of the patients who performed any genetic testing). The highest yield was for neuromuscular disorders (37.9%), and substantially smaller for other indications.

Conclusion: Our experience, in the setting of a specialized neurogenetics clinic, demonstrates that genetic workup for neurological disease among individuals aged ≥ 50 years is beneficial mainly for neuromuscular disorders, with relatively high diagnostic yield.

References:

Grants:

Conflict of Interest: None declared.

EP10.009 Functional genomics and transcriptomics further characterise and potentially improve diagnostic yield of hereditary ataxias

Zhongbo Chen ¹, Valentina Cipriani², Arianna Tucci³, Emil Gustavsson⁴, David Zhang⁴, Regina H. Reynolds⁴, Letizia Vestito⁴, Smedley Damien³, Henry Houlden⁵, Juan Botia⁶, Mina Ryten⁴

¹Queen Square Institute of Neurology, UCL, Department of Neurodegenerative Disease, London, United Kingdom; ²Queen Mary, University of London, Centre for Translational Bioinformatics and Genomics England, London, United Kingdom; ³Queen Mary, University of London, William Harvey Research Institute and Genomics England, London, United Kingdom; ⁴University College London, Great Ormond Street Institute of Child Health, London, United Kingdom; ⁵Queen Square Institute of Neurology, UCL, Department of Neuromuscular Disease, London, United Kingdom; ⁶University of Murcia, Computer Science, Murcia, Spain

Background/Objectives: Up to 80% of hereditary ataxia (HA) patients remain molecularly undiagnosed even following high-depth whole genome sequencing. We aimed to leverage detailed multi-omics data to further characterise the genetic architecture of HA and to increase the diagnostic yield of patients.

Methods: We generated 284 genic features without a priori assumption, capturing information about a gene’s structure; genetic variation; tissue-specific, cell-type-specific and temporally-relevant expression and protein products. Using modified PanelApp age-of-onset information, we categorised 318 HA-associated genes as childhood-onset, adult-onset and those overlapping both. We then compared these individual genomic features across gene categories and collectively through unsupervised machine learning.

Results: By comparing these genic properties, we demonstrated: (i) an unexpectedly high short tandem repeat density within childhood-onset genes suggesting that we may be missing pathogenic repeat expansions in this cohort; (ii) cell-type-specific expression differentiates childhood- and adult-onset ataxias with CNS glial-specific expression in childhood-onset and Purkinje cell-specific expression in overlap-onset genes; (iii) significant similarities in annotation across the groups using unsupervised analysis suggesting adult- and childhood-onset patients should be screened using a common gene set. We tested the latter hypothesis within the 100,000 Genomes Project by assessing the burden of potentially pathogenic variants among childhood-onset genes in adult-onset HA patients and vice versa. This demonstrated a significantly higher burden of rare, potentially pathogenic variants in certain childhood-onset HA genes among adult-onset HA patients.

Conclusion: Our analysis highlights genic features of importance to investigate in an unsolved cohort and suggests that a broader testing strategy for HA could increase diagnostic yield.

References:

Grants:

Conflict of Interest: None declared.

EP10.011 Burden of functional variants in epilepsy patients using a deep learning approach

Alexandre Girard ¹, Claudia Moreau¹, Jacques Michaud^2;3, Berge Minassian^4;5, Patrick Cossette^6;7, Simon L. Girard^1;8

¹Université du Québec à Chicoutimi, Centre Intersectoriel en Santé Durable, Saguenay, Canada; ²CHU Sainte-Justine, Montréal, Canada; ³University of Montreal, Department of Neurosciences and Department of Pediatrics, Montréal, Canada; ⁴The Hospital for Sick Children, Department of Pediatrics, Toronto, Canada; ⁵University of Texas Southwestern Medical School, Department of Pediatrics, Dallas, United States; ⁶Research Center Du Chum ., Montréal, Canada; ⁷University of Montreal, Department of Neurosciences, Montréal, Canada; ⁸Laval University, CERVO Research Center, Québec, Canada

Background/Objectives: Epilepsy is a neurological disease with a strong genetic component. Nevertheless, classical methods, like GWAS, are not as effective at detecting new causal loci as in other diseases. We used a deep learning approach to predict the tissue specific functional effect of variants in epilepsy patients.

Methods: Whole genome sequence data are available for 493 epilepsy patients and 201 controls. We computed the predicted expression change of variants using the deep learning algorithm ExPecto¹. We used python’s statsmodels to conduct logistic regressions by comparing patients and controls functional variants’ burden among different groups of genes such as known epilepsy genes and genes intolerant to loss-of-function variants.

Results: Variants were filtered based on an expression change threshold. It corresponds to the value at which directionality prediction becomes perfect when compared to known eQTLs from GTEx. Using only variants that passed the threshold, we performed a logistic regression to compute the functional variants’ burden in known epilepsy genes. Preliminary results show no difference between groups. Nevertheless, future regressions will focus on single genes and the magnitude of the expression change.

Conclusion: Deep learning algorithms are incredibly powerful tools to predict variant functional effects. Those methods are especially useful in neurology since brain tissues can only be acquired post-mortem. ExPecto is an important asset in our study of epilepsy, and it has the potential to be so for many other illnesses.

References: 1. Zhou, J. et al. 2018. PMCID: PMC6094955.

Grants: IVADO.

Conflict of Interest: None declared.

EP10.012 18-year-old patient with progressive respiratory insufficiency due to two variants in GLDN

Britta Hanker ¹, Inga Nagel², Guido Stichtenoth³, Malte Spielmann^1;2, Irina Hüning¹

¹Institute of Human Genetics, University of Lübeck, Lübeck, Germany; ²Institute of Human Genetics, University of Kiel, Kiel, Germany; ³Department of Pediatrics, University of Lübeck, Lübeck, Germany

Background/Objectives: GLDN codes for gliomedin which is required for the formation of the nodes of Ranvier and the development of the peripheral nervous system. Biallelic variants in GLDN cause lethal congenital contracture syndrome 11. Wambach et al. (2017) reported that biallelic variants in GLDN are not necessarily lethal in the neonatal period.

Methods: We report on an eighteen-year-old male patient, born at 33 weeks of gestation with normal body measurements. He presented with hypotonia and arthrogryposis. Cerebral MRI and testing for metabolic diseases gave normal results. Karyotyping and SNM1 gene analysis returned normal results. He showed mild motor development delay, learned to walk after 2 years of age and developed progressive thoracolumbar kyphoscoliosis with consequent spondylodesis at 10 years of age. Due to diaphragmatic hypomotility, he suffered respiratory insufficiency requiring non-invasive home ventilation since the age of 16.

Results: At the age of 17, exome sequencing revealed two heterozygous variants in GLDN (NM_181789.4: c.82G>C, p.(Ala28Pro) and c.1178G>A, p.(Arg393Lys)). The variant p.(Arg393Lys) was maternally inherited. DNA of the father was not available for molecular analysis.

Conclusion: Systematic review of the literature showed 16 patients with pathogenic variants in GLDN reported so far. Only six of them survived the first months of life (Wambach et al., 2017), the oldest being 17 years old. The phenotype of our patient is consistent with the literature data. We assume that the variants found are compound heterozygous. Long-term outcomes for patients with GLDN variants are still unknown. Here we present the oldest known patient with “non-lethal” congenital contracture syndrome.

References:

Grants:

Conflict of Interest: Britta Hanker: None declared, Inga Nagel: None declared, Guido Stichtenoth: None declared, Malte Spielmann Illumina.

Novartis, Irina Hüning: None declared.

EP10.014 Validity of candidate gene association studies in restless legs syndrome

Barbara Schormair¹, Chen Zhao ¹, Aaro Salminen¹, Konrad Oexle¹, Juliane Winkelmann¹

¹Helmholtz Zentrum München GmbH - German Research Center for Environmental Health, Institute of Neurogenomics, Neuherberg, Germany

Background/Objectives: To date, 14 candidate gene case-control association studies have been published for restless legs syndrome (RLS) in populations of European ancestry. The reported associations have not been validated by replication efforts so far. Therefore, we decided to check them in a large genome-wide association study (GWAS) dataset for RLS.

Methods: Candidate gene studies were extracted from PubMed. GWAS data was available from the International-EU-RLS-GENE-Consortium¹ which included 17,220 individuals of European ancestry.² Single variant association tests were performed with SNPTEST software. Study power was calculated with the genpwr package in R.

Results: Across all identified studies, five different variants located in the genes ADH1B, GABRR3, HMOX1, MAOA, and VDR were reported as significantly associated with RLS. We did not replicate these associations in our dataset and no other variants located in these genes reached genome-wide significance either. Power calculations showed that our study had sufficient power (close to or 100%) to detect the reported associations of these variants, even when accounting for biased effect size estimates due to winner’s curse.

Conclusion: Our results indicate that the reported associations were rather false-positive than true-positive associations to RLS. Moreover, our observations are clearly in favour of performing future association studies as large-scale GWAS rather than small-scale studies.

References: 1.International-EU-RLS-GENE-Consortium: https://www.helmholtz-munich.de/en/ing/rls-gene-consortium/about/index.html.

2.Schormair B, Zhao C, Bell S, et al. 2017, Lancet Neurol. https://doi.org/10.1016/S1474-4422(17)30327-7.

I.

Grants:

Conflict of Interest: Barbara Schormair European patent office - WO2021185936A1, Chen Zhao European patent office - WO2021185936A1, Aaro Salminen: None declared, Konrad Oexle European patent office - WO2021185936A1, Juliane Winkelmann European patent office - WO2021185936A1.

EP10.015 LOSS OF SERYL tRNA SYNTHETASE (SARS1) CAUSES COMPLEX SPASTIC PARAPLEGIA AND CELLULAR SENESCENCE

Edgard Verdura ^1;2, Bruno Senger³, Miquel Raspall^4;5, Agatha Schlüter^1;2, Nathalie Launay^1;2, Montserrat Ruiz^1;2, Carlos Casasnovas^1;6, Agustí Rodriguez-Palmero^1;7, ALFONSO MACAYA RUIZ^4;5;8, Hubert Becker³, Aurora Pujol^1;2;9

¹IDIBELL, Barcelona, Spain; ²CIBERER, Madrid, Spain; ³University of Strasbourg, Strasbourg, France; ⁴Pediatric Neurology Research Group, Vall d’Hebron Research Institute, Barcelona, Spain; ⁵Department of Paediatric Neurology, Vall d’Hebron University Hospital, Barcelona, Spain; ⁶Bellvitge University Hospital, L’Hospitalet de Llobregat, Barcelona, Spain; ⁷Pediatric Neurology Unit, Department of Pediatrics, Hospital Universitari Germans Trias i Pujol, Badalona, Barcelona, Spain; ⁸Institut de Neurociències, Universitat Autònoma de Barcelona, Barcelona, Spain; ⁹Catalan Institution of Research and Advanced Studies (ICREA), Barcelona, Spain

Background/Objectives: Germline mutations in aminoacyl-tRNA synthetase genes are associated with diverse neurological diseases. Recently, patients affected with microcephaly, intellectual disability and ataxia harbouring biallelic variants in the seryl-tRNA synthetase 1 gene encoded by SARS1 were reported.

Methods: We used exome sequencing to identify the causal variant in a patient affected by complex spastic paraplegia with ataxia, intellectual disability, and seizures, but without microcephaly. Functional testing using patient’s fibroblasts and S. cerevisiae strains was performed to examine this variant’s pathogenicity.

Results: A de novo splice site deletion in SARS1 was identified in our patient, resulting in a 5-amino acid in-frame insertion near its active site. Complementation assays in S. cerevisiae and serylation assays in both yeast strains and patient fibroblasts proved a loss-of-function, dominant negative effect. Fibroblasts showed an abnormal cell shape, arrested division, and increased beta-galactosidase staining along with a senescence-associated secretory phenotype (raised IL-6, p21, p16 and p53 levels).

Conclusion: We refine the phenotypic spectrum and modes of inheritance of a newly described, ultrarare neurodevelopmental disorder, while unveiling the role of SARS1 as a regulator of cell growth, division and senescence.

References:

Grants: URD-Cat [SLT002/16/00174] Generalitat de Catalunya, CIBERER [ACCI14-759], ASL-HSP France, Hesperia Foundation, Instituto de Salud Carlos III (FIS PI20/00758), [Sara Borrell, CD19/00221], ‘La Marató de TV3’ Foundation (202006-30). French National Programme Investissement d’Avenir administered by the “Agence National de la Recherche” (ANR), “MitoCross” Laboratory of Excellence (Labex) (ANR-10-IDEX-0002-02), the University of Strasbourg and CNRS.

Conflict of Interest: None declared.

EP10.017 Genetic and phenotypic associations of serum dicarbonyl metabolites in restless legs syndrome

Philip Harrer ^1;2, Julica Folberth³, Chen Zhao¹, Barbara Schormair^1;2, Erik Tilch¹, Christian Gieger⁴, Annette Peters⁴, Konrad Oexle^1;2, Markus Schwaninger³, Juliane Winkelmann^1;2

¹Helmholtz Zentrum München, German Research Centre for Environmental Health, Institute of Neurogenomics, Munich, Germany; ²Technical University of Munich, Institute of Human Genetics, School of Medicine, Munich, Germany; ³University of Lübeck, Institute of Experimental and Clinical Pharmacology and Toxicology, Lübeck, Germany; ⁴Helmholtz Zentrum München, German Research Centre for Environmental Health, Institute of Epidemiology, Munich, Germany

Background/Objectives: Altered levels of dicarbonyls such as methylglyoxal (MG) have been linked to several diseases. Little is known about the impact of genetic variation on dicarbonyl metabolism. Genome-wide association studies of the sensorimotor disorder restless legs syndrome (RLS) have identified significant association signals in and close to GLO1, which encodes the key MG-detoxifying enzyme, glyoxalase-1 (GLO1). Therefore, we decided to study dicarbonyl levels and their genetic basis in RLS patients and controls.

Methods: Three dicarbonyls (MG, GO, 3-DG) were measured in serum of 246 RLS patients and 482 controls from a population-based cohort (KORA) using liquid-chromatography-mass-spectrometry. Phenotypic information included demographic data, laboratory parameters, and comorbidities. Samples were genotyped on the Axiom® array. Association analyses were done using regression models, MDS and bidirectional stepwise RDA analysis.

Results: Validating our dataset, we replicated known associations of the dicarbonyls to clinical phenotypes, including cardiovascular disease, diabetes, age, obesity, impaired liver, and renal function. These were in accordance with significant associations to objective disease parameters such as waist-to-hip ratio and glomerular filtration rate amongst others. As a novel finding, MG levels were significantly lower in RLS cases compared to controls across all age and sex groups (p = 4×10^-23). GWAS on dicarbonyl levels revealed only signals below the genomewide significance-threshold, likely due to the limited sample size.

Conclusion: Our study provides first evidence for a role of dicarbonyls in RLS, indicating potential new treatment options. However, our study was underpowered to detect genetic effects. We are currently conducting a replication study to confirm the results and to increase study power.

References:

Grants:

Conflict of Interest: Philip Harrer: None declared, Julica Folberth: None declared, Chen Zhao European patent office - WO2021185936A1, Barbara Schormair European patent office - WO2021185936A1. Erik Tilch: None declared, Christian Gieger: None declared, Annette Peters: None declared, Konrad Oexle European patent office - WO2021185936A1, Markus Schwaninger: None declared, Juliane Winkelmann European patent office - WO2021185936A1.

EP10.018 Mutational screening of Greek patients with axonal Charcot-Marie-Tooth disease using targeted Next-Generation Sequencing

Zoi Kontogeorgiou ¹, CHRISOULA KARTANOU¹, 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, Athens, Greece

Background/Objectives: 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 20 genes. The aim of this study was to decipher the genetic landscape of axonal CMT in the Greek population.

Methods: Forty three index patients with CMT2, DI-CMT, HSAN or dHMN, negative for mutations causing CMT1A and CMTX, were screened by an NGS custom gene panel (IoN Torrent) that covers 24 of the most commonly mutated genes in cases of axonal CMT. The study was carried out in the Neurogenetics Unit of the 1st Department of Neurology, NKUA, Eginitio Hospital.

Results: Overall, we identified 9 causative heterozygous mutations corresponding to 9 index cases, representing 20.9% of the cohort. Those were 3 mutations in MPZ (c.103G>T p.Asp35Tyr, c. 449-1G>A, c.186C>G p.Ile62Met),in dHMN, CMT2 and HSAN patients respectively, 3 in MFN2 (c.1070A>C p.Lys357Thr, c.281G>A p.Arg94Gln, c.310C>T p.Arg104Trp), all in CMT2 patients, 1 in BSCL2 (c.263A>Gp.Asn152Ser), in a dHMN patient, 1 in GDAP1 (c.715C>T p.Leu239Phe), in a CMT2 patient, and 1 in DNM2 (c.1072G>A p.Gly358Arg),in a DI-CMT patient. All mutations were found in heterozygosity in accordance with dominant inheritance.

Conclusion: A 24-gene NGS panel designed to screen a Greek CMT2 cohort had a diagnostic yield of 20.9%, in accordance with results of similar studies in other European populations.

References:

Grants: 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.

EP10.019 Screening of the FMR1 premutation in Greek patients with movement disorders

CHRISOULA KARTANOU ¹, Maria Seferiadi¹, Stella Pomoni¹, Constantin Potagas², Christalena Sofocleous³, Jan Traeger-Synodinos³, Georgios Koutsis¹, Georgia Karadima¹

¹Neurogenetics Unit, 1st Department of Neurology, School of Medicine, Eginition Hospital, National and Kapodistrian University of Athens, Athens, Greece; ²1st Department of Neurology, School of Medicine, Eginition Hospital, National and Kapodistrian University of Athens, Athens, Greece; ³Department of Medical Genetics, School of Medicine, University of Athens, Aghia Sophia Children’s Hospital, Athens, Greece, Athens, Greece

Background/Objectives: Fragile X-associated tremor/ataxia syndrome (FXTAS) is a late-onset, X-linked, neurodegenerative disorder that affects premutation carriers of the FMR1 gene. Clinical features include gait ataxia and action tremor, while some patients demonstrate parkinsonism, cognitive deficits and peripheral neuropathy. Consequently, FXTAS is often misdiagnosed as spinocerebellar ataxia (SCA) or Parkinson’s disease (PD). Herein, we sought to investigate the frequency, genotypic and phenotypic profile of FXTAS in two cohorts of Greek patients with movement disorders, one with late-onset cerebellar ataxia and the other with PD.

Methods: In total, 92 index patients with late-onset cerebellar ataxia (negative for SCA1, 2, 3, 6, 7 repeat expansions) and 171 with PD (negative for p.A53T in SNCA) were selected. All cases had no male-to male transmission. Genetic screening for the FMR1 premutation was performed using fluorescent polymerase chain reaction (PCR) followed by capillary gel electrophoresis.

Results: The FMR1 premutation was detected in 2 ataxia patients (2.2%), slightly above the range reported by multiple studies (0-1.9%) and 1 PD patient (0.6%), in line with previous studies (<1%). Both FXTAS patients from the ataxia cohort had neuropathy along with cerebellar ataxia, while one patient had mild parkinsonism and cognitive impairment and the other pyramidal signs. The FXTAS patient from the PD cohort had typical PD.

Conclusion: We conclude that, in the Greek population, the FMR1 premutation is rare in PD, but should be considered in SCA panel-negative hereditary ataxia cases with supportive clinical features. Our study highlights the importance of genetic testing in the differential diagnosis and early management of FXTAS.

References:

Grants: None.

Conflict of Interest: None declared.

EP10.023 Variants inferring high risk for frontotemporal dementia (FTD) in Bulgarian patients

Dragomira Nikolova ¹, Marta Mihaylova¹, Dimitar Serbezov¹, Lubomir Balabanski¹, Vera Damyanova¹, Rada Staneva¹, Olga Antonova¹, Victoria Spasova¹, Mihail Ganev¹, Desislava Nesheva¹, Blaga Rukova¹, Diana Belejanska², Shima Mehrabian², Mariya Petrova², Sena Karachanak-Yankova¹, Latchezar Traykov², Savina Hadjidekova¹, Draga Toncheva¹

¹Medical University - Sofia, Department of Medical genetics, Sofia, Bulgaria; ²Medical University - Sofia, UH “Alexandrovska”, Department of Neurology, Sofia, Bulgaria

Background/Objectives: Frontotemporal dementia (FTD) is a heterogeneous disorder with changes in behavior, language, executive control, and often motor symptoms. FTD is difficult to be diagnosed due to the lack of recognition and the overlap with other psychiatric disorders.

Our study aims to reveal the causative genes, and genetic risk variants in a well selected group of Bulgarian patients with FTD using whole exome sequencing approach.

Methods: Genomic DNA was isolated from peripheral blood samples of 140 patients, diagnosed with FTD. WES was performed on two pooled samples. Sequence reads were aligned to the reference genome (GRCh37/hg19), vcf files were generated and annotated with wANNOVAR. The interpretation of the variants was made according to their clinical significance (Varsome and ClinVar database) and frequency (gnomeAD Exomes).

Results: We identified nine variants related to FTD: PSEN1 rs63751316 (MAF = 0.015) and rs63751287 (MAF = 0.0044); SQSTM1 rs181263868 (MAF = 0.0044), rs761423892 (MAP = 0.0086), rs535932454 (MAF = 0.0026), GRN rs63750116 (MAF = 0.0052), rs774128685 (MAF = 0.0065), TREM2 rs145080901 (MAF = 0.0025), rs147564421 (MAF = 0.0024). Variant rs63751287 is pathogenic, while the rest are variants of uncertain significance (VUS).

Conclusion: Due to geographical and ethnic variability, the prevalence of the causative genes or high risk variants for FTD may be different. We identified several genetic variations conferring risk for FTD in patients of Bulgarian origin. Their combination with a variety of environmental exposures may result in increased susceptibility to FTD.

References:

Grants: KP-06-N33/5 from 13.12.2019 - National Science Fund of Bulgaria.

Conflict of Interest: None declared.

EP10.024 El-Hattab-Alkuraya syndrome caused by biallelic WDR45B pathogenic variants: further delineation of the phenotype and genotype

Mohammed Almannai ¹, Dana Marafi^2;3, Ghada Abdelsalam⁴, Maha Zaki^4;5, Ruizhi Duan², Daniel Calame^2;6;7, Isabella Herman^2;6;7, Felix Levesque⁸, Hasna Elbendary⁴, Ibhrahim Hegazy⁴, Wendy Chung⁹, Haluk Kavus⁹, Kolsoum Saeidi¹⁰, Reza Maroofian¹¹, Aqeela Alhashim¹², Ali Al-Otaibi¹², Asma Al Madhi¹², Hager Aboalseood¹³, Ali Alasmari¹³, Henry Houlden¹¹, Joseph Gleeson¹⁴, Jill Hunter^7;15, Jennifer Posey², James Lupski^2;7;16, Ayman El-Hattab¹⁷

¹1. Genetics and Precision Medicine department (GPM), King Abdullah Specialized Children’s Hospital (KASCH), King Abdulaziz Medical City, Ministry of National Guard Health Affairs (MNG-HA), Riyadh, Saudi Arabia; ²Baylor College of Medicine, Department of Molecular and Human Genetics, Houston, United States; ³Kuwait University, Department of Pediatrics, Safat, Kuwait; ⁴Human Genetics and Genome Research Institute National Research Centre, Clinical Genetics Department, Cairo, Egypt; ⁵Armed Forces College of Medicine (AFCM), Genetics Department, Cairo, Egypt; ⁶Baylor College of Medicine, Section of Pediatric Neurology and Developmental Neuroscience, Department of Pediatrics, Houston, United States; ⁷Texas Children’s Hospital, Houston, United States; ⁸University of Saskatchewan, Division of medical genetics and metabolic, Department of Paediatrics, Saskatoon, Canada; ⁹Columbia University Irving Medical Center, Departments of Pediatrics and Medicine, New York, United States; ¹⁰Kerman University of Medical Sciences, Neuroscience Research Center, Institute of Neuropharmacology, Kerman, Iran; ¹¹University College London, UCL Queen Square Institute of Neurology, London, United Kingdom; ¹²King Fahad Medical City, Department of Pediatric Neurology, National Neuroscience Institute, Riyadh, Saudi Arabia; ¹³King Fahad Medical City, Section of Medical Genetics, Children’s Hospital, Riyadh, Saudi Arabia; ¹⁴University of California, San Diego, Howard Hughes Medical Institute, San Diego, United States; ¹⁵Baylor College of Medicine, Department of Radiology, Houston, United States; ¹⁶Baylor College of Medicine, Human Genome Sequencing Center, Houston, United States; ¹⁷University of Sharjah, Department of Clinical Sciences, College of Medicine, Sharjah, United Arab Emirates

Background/Objectives: Homozygous pathogenic variants in WDR45B were first identified in six subjects from with global development delay, refractory seizures, spastic quadriplegia, and brain malformations. Since the initial report in 2018, no further cases have been described. In this report, we present 12 additional individuals from seven unrelated families.

Methods: Retrospective chart review of 12 individuals from seven families was conducted. The molecular diagnosis of WDR45B-related disorder, El-Hattab-Alkuraya syndrome, was made by clinical or research exome sequencing.

Results: Six different variants in WDR45B were identified, including five novels. Microcephaly and global developmental delay were observed in all subjects, and seizures and spastic quadriplegia in most. Common findings on brain imaging include cerebral atrophy, ex-vacuo ventricular dilatation, brainstem atrophy, and symmetric under-opercularization.

Conclusion: El-Hattab-Alkuraya syndrome is characterized by early onset cerebral atrophy resulting in microcephaly, developmental delay, spastic quadriplegia, and seizures. The phenotype appears to be more severe among individuals with loss-of-function variants suggesting a potential genotype-phenotype correlation in this disorder. A brain imaging pattern emerges which is consistent among individuals with loss-of-function variants and could potentially alert the neuroradiologists or clinician to consider WDR45B-related El-Hattab-Alkuraya syndrome.

References: Suleiman J, Allingham-Hawkins D, Hashem M, Shamseldin HE, Alkuraya FS, El-Hattab AW. WDR45B-related intellectual disability, spastic quadriplegia, epilepsy, and cerebral hypoplasia: A consistent neurodevelopmental syndrome. Clin Genet. 2018;93(2):360-364.

Grants: U.S. National Human Genome Research Institute (NHGRI) and National Heart Lung and Blood Institute (NHBLI) to the Baylor-Hopkins Center for Mendelian Genomics (BHCMG, UM1 HG006542), NHGRI Baylor College of Medicine Genomics Research Elucidates Genetics of Rare (BCM-GREGoR; U01 HG011758).

Conflict of Interest: Mohammed Almannai: None declared, Dana Marafi: None declared, Ghada Abdelsalam: None declared, Maha Zaki: None declared, Ruizhi Duan: None declared, Daniel Calame: None declared, Isabella Herman: None declared, Felix Levesque: None declared, Hasna Elbendary: None declared, Ibhrahim Hegazy: None declared, Wendy Chung paid consultant for Regeneron Genetics Center., Haluk Kavus: None declared, Kolsoum Saeidi: None declared, Reza Maroofian: None declared, Aqeela Alhashim: None declared, Ali Al-Otaibi: None declared, Asma Al Madhi: None declared, Hager Aboalseood: None declared, Ali Alasmari: None declared, Henry Houlden: None declared, Joseph Gleeson: None declared, Jill Hunter: None declared, Jennifer Posey: None declared, James Lupski U.S. National Human Genome Research Institute (NHGRI) and National Heart Lung and Blood Institute (NHBLI) to the Baylor-Hopkins Center for Mendelian Genomics (BHCMG, UM1 HG006542), NHGRI Baylor College of Medicine Genomics Research Elucidates Genetics of Rare (BCM-GREGoR; U01 HG011758)., has stock ownership in 23andMe, paid consultant for Regeneron Genetics Center.

Scientific Advisory Board (SAB) of Baylor Genetics, Ayman El-Hattab: None declared.

EP10.025 Genetic susceptibility to telomere shortening through the rs2293607 polymorphism is associated with a greater risk of alcohol use disorder

Hernán Llorente^1;2, Daniel Salete Granado ^2;3, Pérez Rivera José Ángel^4;5, María de los Ángeles Pérez Nieto², Clara Cieza Borrella⁶, Isabel Pastor Encinas^1;2;3, ignacio novo⁷, Javier Fernández Mateos^2;3, Antonio Javier Chamorro^1;2, Patricia Crecente Otero¹, Francisco Javier Laso Guzmán^1;2, Rogelio González-Sarmiento^2;3, Miguel Marcos^1;2;3

¹University Hospital of Salamanca, Department of Internal Medicine, Salamanca, Spain; ²Institute of Biomedical Research of Salamanca-IBSAL, Salamanca, Spain; ³University of Salamanca, Department of Medicine, Salamanca, Spain; ⁴University Hospital of Burgos, Department of Cardiology, Burgos, Spain; ⁵Universidad Isabel I, Burgos, Spain; ⁶Centre for Biomedical Education/Cell Biology and Genetics Research Centre, London, United Kingdom; ⁷University Hospital of Santiago de Compostela, Department of Internal Medicine, A Coruña, Spain

Background/Objectives: Alcohol use disorder (AUD) is associated with shortened telomere length (TL), and single nucleotide polymorphisms (SNPs) in the telomerase complex may modulate telomere length (TL). This study was conducted to examine the relationship of TL to AUD and the role of SNPs in TERC and TERT for this association.

Methods: A total of 308 male patients with AUD and 255 sex-matched healthy controls were included. TL was measured in 99 patients and 99 controls paired by age and smoking status and all individuals were genotyped for allelic discrimination of TERC SNPs rs2293607, rs12696304, and rs16847897 and TERT SNPs rs2735940, rs2736100, and rs2736098. Univariate and multivariable logistic regression analyses were performed. A receiver operating characteristic (ROC) curve was used to analyse differences in TL.

Results: The mean TL was shorter in patients with AUD than in controls. The area under the ROC curve was 0.70 (P < 0.001). The GG genotype of TERC rs2293607 was more common among AUD patients than among controls (9.8% vs. 5.1%; P = 0.038). No difference was found for the other SNPs. Carriers of the GG genotype of rs2293607 had shorter telomeres than did allele A carriers.

Conclusion: Patients with AUD had shorter telomeres. Genetic susceptibility to telomere shortening through the rs2293607 polymorphism is associated with a greater risk of AUD, although mechanisms are not yet established.

References:

Grants: Instituto de Salud Carlos III and the European Union FEDER funds, “Una manera de hacer Europa” (PI20/00743) and Junta de Castilla y León, Spain (GRS 2388/A/21).

Conflict of Interest: None declared.

EP10.026 Molecular diagnosis of Kufor-Rakeb Syndrome in a 36-year-old patient

Rubén Pérez de la Fuente ¹, Jose Miguel Lezana¹, Eva María Sánchez Morla², Víctor Blanco Palmero³, Sonia Mayo de Andrés¹, Carmen Palma Milla¹, Ana Rosa Arteche López¹, Emma Soengas Gonda^4;5, Juan Francisco Quesada Espinosa¹, María Teresa Sánchez Calvín¹, Irene Gómez Manjón¹, María José Gómez Rodríguez¹, Jordi Corral Seijas⁶, Albert Ferran Martin⁶, Patricia Ramos Gómez¹, Olalla Sierra Tomillo¹, Alexandra Juárez Rufián¹, Marta Moreno García¹

¹Hospital 12 de Octubre, Genetics, Madrid, Spain; ²Hospital 12 de Octubre, Psychiatry, Madrid, Spain; ³Hospital 12 de Octubre, Neurology, Madrid, Spain; ⁴Centro de Investigación Biomedica en Red en Enfermedades Raras (CIBERER), Madrid, Spain; ⁵Instituto de investigación del Hospital 12 de Octubre (imas12), Madrid, Spain; ⁶Reference Laboratory, Genetics, Barcelona, Spain

Background/Objectives: Patient involuntarily hospitalized due to psychotic episodes with hallucinations and behavioral alteration. Personal history: normal psychomotor development, cannabis and alcohol consumption. Since 32 years old she showed a gait disturbance and a decline in cognitive functions. The neurological evaluation showed mild parkinsonian signs, moderate pyramidalism and supranuclear gaze palsy. MRI revealed severe cerebral atrophy for her age, suspecting an underlying genetic cause.

Methods: Whole exome sequencing was performed (xGen Exome Panel v2.0 kit). 15 genes were subsequently analyzed related to early-onset hereditary Parkinson’s disease.

Results: Two heterozygous variants in the ATP13A2 gene (NM_022089.3) were detected: c.917G>A, p.(Trp306Ter) and the deletion of exon 1 (ExomeDepth tool). Both variants have not been described to date and were classified as pathogenic variants. Bi-allelic loss-of-function variants are known as a pathogenic mechanism associated with Kufor-Rakeb syndrome and complex hereditary spastic paraparesis 78. In order to confirm the deletion, we designed a long range PCR (GoTaq, Promega) from the last coding exon of the upstream gen (SDHB) to exon 2 of ATP13A2 gene. The amplification was sequenced by NGS (SureSelect, Agilent) and allowed to confirm a 8.8kb deletion fully involving the entire coding region of exon 1 (chr1:17336716-173344506; hg19).

Conclusion: The patient is heterozygous for two pathogenic variants in the ATP13A2 gene that confirm the diagnosis of Kufor-Rakeb Syndrome with a autosomal recessive inheritance. Family studies (unaffected father and sister) are currently in progress.

References:

Grants:

Conflict of Interest: None declared.

EP10.027 A novel SACS variant identified in a patient with autosomal recessive spastic ataxia of Charlevoix-Saguenay

Birutė Burnytė ¹, Raminta Masaitiene², Karolis Baronas¹, Algirdas Utkus¹

¹Institute of Biomedical Sciences, Faculty of Medicine, Vilnius University, Vilnius, Lithuania; ²Institute of Clinical Medicine, Faculty of Medicine, Vilnius University, Vilnius, Lithuania

Background/Objectives: Spastic ataxia of Charlevoix–Saguenay is a rare progressive neurodegenerative disease caused by mutations in the SACS gene. Clinical phenotype is characterized by onset in the first decade with a triad of cerebellar ataxia, peripheral neuropathy, and pyramidal tract signs, with a significant disability on disease progression in adulthood. Additionally, other symptoms have been reported, such as deafness and generalized seizures.

Methods: The proband was identified by next-generation sequencing performed in 14 patients with early onset ataxia between 2019 and 2021.

Results: A now 33-year-old female presented with unstable gait and frequent falls when first walking at 12 months. In the first decade, she was diagnosed with early-onset ataxia, polyneuropathy and mixed hearing loss. Since age of 26 she was requiring two canes for walking, which has been disturbed by spasticity in lower limbs. Neurological examination revealed distal weakness of legs (particularly in feet dorsiflexion), decreased vibration sense, absence of tendon reflexes and loss of balance and coordination accompanied by slurred speech. Intragenic deletion NM_014363.6:c.12851_12854del (NP_055178.3:p.(Glu4284Alafs*23), rs786204628) and novel nonsense variant NM_014363.6:c.2764C>T (NP_055178.3:p.(Gln922*)) in the SACS gene, inherited from her father and mother, respectively was identified using NGS analysis and targeted multigene panel. Based on in silico analysis, identified variant is classified as pathogenic, and truncates SACS protein in highly conservative DNA sequence.

Conclusion: The identification of novel loss-of-function variant in described patient results the complex disease phenotype.

References:

Grants:

Conflict of Interest: None declared.

EP10.028 Trajectory of Neuroligin/Neurexin dysregulation associates with the establishment of an ASD-like phenotype in Tuberous Sclerosis

Jennifer Krummeich ¹, Cosima Caliendo¹, Annabelle Arlt², Kamil Rolski³, Kevin Vincze³, Rainer Schneider³, Susanne Gerber¹, Michael Schmeisser⁴, Susann Schweiger¹

¹Institute of Human Genetics, University Medical Centre Mainz, Mainz, Germany; ²Institute of Human Genetics, University Hospital Münster, Münster, Germany; ³Institute of Biochemistry, University of Innsbruck, Innsbruck, Austria; ⁴Institute of Microscopic Anatomy and Neurobiology, University Medical Centre Mainz, Mainz, Germany

Background/Objectives: Tuberous sclerosis (TS) is an autosomal dominant disorder caused by heterozygous mutations in either Tsc1 or Tsc2, that both negatively regulate mTOR (mammalian target of rapamycin). At the synapse, mTOR is a key enzyme controlling the local synthesis of proteins. Its dysfunction leads to mis-regulation of cell growth and proliferation. The sequential appearance of TAND-symptoms in children with TS suggests a dynamic rather than a static process in disease development.

Methods: In a heterozygous Tsc2 knock-out model we have longitudinally analyzed behavior at different time points after birth and have matched this with molecular signatures throughout brain development, to carefully characterize the cascade of cellular processes leading into a disease phenotype. To elucidate the molecular causes underlying the behavioral deficits, comparative proteome analysis of cortical homogenate and synaptosomes at different time points from early to late postnatal stages was carried out using serial Western blot and mass spectrometry analysis.

Results: We found that, similar to patients, TAND symptoms develop stepwise in a time-dependent manner. We show that Tsc2 is reduced only at very early stages and is fully compensated at later time points when behavior aberrations occur, suggesting that the behavior phenotype develops independent of the primary defect. Furthermore, we found that the formation of behavior aberrations correlates with a window of Neuroligin and Neurexin mis-expression in cortical but not hippocampal tissue.

Conclusion: Together our data suggests substantial homeostatic dynamics of gene expression underlying the TS phenotype and a correlation of ASD-like disease symptoms with cortical dysregulation of Neuroligins and Neurexin.

References:

Grants:

Conflict of Interest: None declared.

EP10.029 Genetic characterization of patients with bipolar disorder and controls for the generation of induced pluripotent stem cells

Karola Hünten ¹, Friederike David¹, Sugirthan Sivalingam², Bettina Burger³, Eva Beins¹, Lea Sirignano⁴, Fabian Streit⁴, Frederike Stein⁵, Tamara Krutenko⁶, Petra Ina Pfefferle⁷, Stefan Herms^1;3, Per Hoffmann^1;3, Marcella Rietschel⁴, Michael Peitz⁶, Sven Cichon³, Oliver Brüstle⁶, Markus M Nöthen¹, Andreas Forstner^1;8

¹Institute of Human Genetics, University of Bonn, Bonn, Germany; ²Core Unit for Bioinformatics Data Analysis, University of Bonn, Bonn, Germany; ³Department of Biomedicine, University of Basel, Basel, Switzerland; ⁴Department of Genetic Epidemiology in Psychiatry, Central Institute of Mental Health, Mannheim, Germany; ⁵University Hospital for Psychiatry and Psychotherapy, Philipps University of Marburg, Marburg, Germany; ⁶Institute of Reconstructive Neurobiology, University of Bonn, Bonn, Germany; ⁷Comprehensive Biobank Marburg (CBBMR), Philipps University of Marburg, Marburg, Germany; ⁸Institute of Neuroscience and Medicine (INM-1), Research Center Jülich, Jülich, Germany

Background/Objectives: Bipolar disorder (BD) is a psychiatric disorder characterized by recurrent episodes of (hypo-)mania and depression. Recent genome-wide association studies support a strong polygenic influence, but little is known about its functional effects in neurons. In this study, we performed genetic characterization of patients and controls for subsequent analyses of the contribution of polygenic factors to neurodevelopmental processes in induced pluripotent stem cell (iPSC)-derived neurons.

Methods: Patients with BD type 1 (BD1) and controls from the FOR2107 cohort (https://for2107.de/) were selected for iPSC generation according to the following criteria: male sex, availability of PBMCs and genome-wide genotype data, high polygenic risk score (PRS) for BD1 in patients and low PRS in controls (Stahl et al., 2019; PRS-CS), and no copy number variants significantly associated with BD or schizophrenia (cnvPartition). 10 patients (highest PRS) and 10 controls (lowest PRS) were whole-genome sequenced, and individuals with rare variants identified in previous BD sequencing studies (MAF < 1%, gnomAD, non-neuro dataset; PHRED-scaled CADD score>20; not present in patients and controls) were excluded.

Results: From 16 BD1 patients and 55 controls with PBMCs and genotype data, one patient was excluded due to a microdeletion on chromosome 2, and three BD1 patients (highest PRS) and three controls (lowest PRS) with no rare variants implicated in BD were selected for iPSC generation.

Conclusion: We describe a systematic procedure to characterize patients and controls at the genetic level prior to iPSC generation, leading to the exclusion of rare variants that might have influenced subsequent analyses.

References: Stahl et al., 2019, PMID:31043756.

Grants: NO246/15-1.

SC310030L_182731.

BR1337/4-1.

Conflict of Interest: None declared.

EP10.030 Associative analysis of 27 genetic variants (SNPs) with the variability in total cognitive scores defined by the MoCA (Montreal Cognitive Assessment) test in older people

Andrey Marusin ¹, Makeeva Oksana², Anna Bocharova³, Stepanov Vadim³

¹Tomsk National Research Medical Center of the Russian Academy of Sciences, Evolutionary genetics, Tomsk, Russian Federation; ²OOO “Nebbiolo”, Tomsk, Russian Federation, Tomsk, Russian Federation; ³Tomsk National Research Medical Center of the Russian Academy of Sciences, Tomsk, Russian Federation

Background/Objectives: Cognitive decline with age is an important social and medical problem that leads to a significant reduction the quality of life of the elderly. Major cognitive function genes, as well as their relationship with neuropsychiatric diseases, have not been identified.The aim of this work was the association analysis of 27 SNPs with the variability in cognitive function, defined by the MoCA test, in the elderly.

Methods: The study was carried out on a population sample of the 241 elderly Russian individuals (64 men and 177 women). The mean age was 72.5±0.4 years (from 57 to 88 years). On the basis of the selected 27 markers was formed multiplexed SNPs panel Genotyping by MALDI-TOF mass spectrometry on Sequenom MassArray platform was performed. The relationship between the studied polymorphic variants and the MoCA scores were analyzed by using the nonparametric Kruskal-Wallis test.

Results: Four SNPs associated with the total MoCA test score were identified. These were polymorphic variants: 1) rs11191580 in the NT5C2 gene (p = 0.037); 2) rs1635 (NKAPL, p = 0.042). 3) rs17693963 (MHC, p = 0.042) and 4) rs2075650 (TOMM40, p = 06034). Previously, the first three genes were found to be associated with schizophrenia according to GWAS*. For the TOMM40 gene, GWAS also revealed an association with Alzheimer’s disease.

Conclusion: The data obtained during the implementation of the project expand understanding of the neuropsychiatric diseases inheritance, as well as the biological processes underlying the decline in cognitive abilities in the elderly.

References: *https://www.ebi.ac.uk/gwas/.

Grants: The reported study was funded by RFBR, project number 20-015-00397.

Conflict of Interest: None declared.

EP10.032 Candidate regulatory variants in SNARE complex genes and their involvement in migraine susceptibility

Daniela Felício ^1;2, Sandra Martins^1;2, Mariana Santos^2;3, Alexandra M. Lopes^1;2;4, Carolina Lemos^2;3;5, Nádia Pinto^1;2;6, Miguel Alves-Ferreira^2;3;5

¹IPATIMUP - Institute of Molecular Pathology and Immunology of the University of Porto, Porto, Portugal; ²i3S - Instituto de Investigação e Inovação em Saúde, Porto, Portugal; ³UnIGENe - Unit for Genetic and Epidemiological Research in Neurological Diseases, IBMC - Instituto de Biologia Molecular e Celular, Universidade do Porto, Porto, Portugal; ⁴CGPP - Centre for Predictive and Preventive Genetics, IBMC - Instituto de Biologia Molecular e Celular, Universidade do Porto, Porto, Portugal; ⁵ICBAS - Instituto Ciências Biomédicas Abel Salazar, Universidade do Porto, Porto, Portugal; ⁶CMUP - Centro de Matemática da Universidade do Porto, Porto, Portugal

Background/Objectives: Migraine is a disabling and multifactorial neurological disease, remaining unexplained most of its heritability and susceptibility. Some migraine risk loci have been shown to reside in non-coding regions, which may alter gene expression and epigenetic regulation (1,2). Our aim was to select the best SNP candidates to study cis-regulation of genes previously associated with migraine susceptibility in the Portuguese population: SYN1, SNAP25, VAMP2, STXBP1, STXBP5, SYN2, UNC13B, GABRA3, GABRQ, and STX1A (3,4,5).

Methods: From a set of 22 tagSNPs within the candidate genes, we prioritized variants based on an integrative evaluation of their potential functional impact and regulatory features, by using Variant Effect Prediction, SNPinfo, SNPnexus, and HaploReg tools. We selected variants with at least 3 scores predicting deleteriousness (CADD, FunSeq2, DANN, ReMM, FATHMM, GWAVA, and RegulomeBD) and analysed regulatory features, DNA accessibility and histone modifications.

Results: Seven SNPs met our criteria, priority being given to the three SNPs located in major regulatory regions: rs6951030 (STX1A; promotor), rs2327264 (SNAP25; enhancer) and rs1150 (VAMP2; 3’UTR). Interestingly, both variants in SNAP25 and VAMP2 genes are in CTCF binding sites. We proceeded with functional validation of the regulatory effect of selected SNPs through luciferase reporter gene expression assays with migraineurs’ DNAs.

Conclusion: In silico analyses suggested possible alterations in gene regulation of SNARE complex proteins implicated in exocytotic neurotransmitter release in migraine pathophysiology.

References: 1-Neurogenetics.2020;21:149–157. 2-BMC Medical Genetics.2010;11(1):103. 3-Headache.2020;60(10):2152-2165. 4-PLoS ONE.2013;8(9):e74087. 5-Arch Neurol.2010;67(4):422-427.

Grants: FEDER-COMPETE 2020 (POCI), Portugal 2020; Interreg V-A Spain–Portugal POCTEP 2014–2020: 0702_MIGRAINEE_2_E; FCT grants: POCI-01-0145-FEDER-029486(PTDC/MEC-NEU/29486/2017), CEECIND/00684/2017.

Conflict of Interest: None declared.

EP10.034 The advantage of high throughput sequencing in the diagnosis of tuberous sclerosis. Clinical case

Larysa Sheiko ¹, Iryna Lastivka², Vita Antsupova³, ljudmila Brisevac¹, Ludmila Khlunovska², Anastasiya Babintseva², Iryna Malieieva³, Iana Ushko³, Oleksiy Godovanets², Olexiy Shapovalov⁴, Olha Polodienko⁴, Volodymyr Davidiuk⁵

¹Shupyk National Healthcare University of Ukraine, Kyiv, Ukraine; ²Bukovinian State Medical University, Chernivtsi, Ukraine; ³Bohomolets National Medical University, Kyiv, Ukraine; ⁴Children’s Consultative and Diagnostic Center named after academician Reznik, Odessa, Ukraine; ⁵Pirogov National Memorial Medical University, Vinnytsya, Ukraine

Background/Objectives: Tuberous sclerosis refers to phakomatoses, determined by mutations in the TSC1 and TSC2 genes. Mutations in the TSC1 gene are clinically manifested by a milder course, with TSC2 mutations, the symptoms are more severe and not amenable to therapy. Identification of the mutation in a patient is important.

Methods: Targeted high-throughput sequencing, Sanger sequencing.

Results: The 4-year-old boy with symptomatic epilepsy, frequent polymorphic seizures that first appeared at the 16th week of life. The child from the second pregnancy proceeded against the background of termination of pregnancy in the 1st trimester. Objectively: the skin of the proband’s buttocks and torso has multiple dense white matte and depigmented spots that were present from birth. MRI of the brain: cortical and subcortical focal changes in the brain is characteristic of Tuberous sclerosis. The family history of mother and father is aggravated by oncopathology. High-throughput targeted sequencing of clinically important genes has been recommended. The proband was found to have a de novo mutation of the TSC2 gene c.1869del(p.Asp624Thrfs*74). The diagnosis «Tuberous sclerosis» was confirmed. In addition, the patient and his mother were found to be carriers of the pathological mutation of the SGSH c.220C>T(p.Arg74Cys) gene responsible for the development of type IIIa mucopolysaccharidosis.

Conclusion: Using modern sequencing methods, a mutant gene was identified in the patient. In the proband and his mother, a pathological mutation of the SGSH gene was found in the heterozygous state. The information obtained is important for the prognosis of the child’s life and for medical genetic counseling of the family.

References:

Grants:

Conflict of Interest: None declared.

EP10.036 The other side of ACOX1 variation and a case of Mitchell syndrome

Daniela Iancu ¹, Gabriella Gazdagh¹, Giles Atton¹, Jaspal Singh², Hannah Robinson³

¹Southampton General Hospital, Wessex Clinical Genetics Servicce, Southampton, United Kingdom; ²Southampton General Hospital, Paediatric Neurology, Southampton, United Kingdom; ³Exeter Molecular Genetics Laboratory, Exeter, United Kingdom

Background/Objectives: Homozygous loss-of-function variants in ACOX1 cause peroxisomal acyl-CoA oxidase deficiency. Here we describe the case of a child with a recurrent heterozygous gain-of-function variant, recently proven to cause Mitchell syndrome.

Methods: A 5-years old boy was admitted following an acutely presenting and rapidly progressive ataxia. He was born to unrelated parents, after an uneventful pregnancy and had a normal early development. Around the age of 2 he was diagnosed with mild hearing loss which subsequently progressed to severe/profound hearing loss around the age of 5 years of age. Clinical examination on admission showed ataxia, lower limb spasticity, slurred speech, and developmental regression. Ophthalmological examination revealed corneal erosions, hypermetropia and astigmatism. The MRI showed atypical changes of the dentate nucleus and excluded a brain tumour. The metabolic investigations showed slightly increased lactate level in blood and cerebrospinal fluid, and normal very long chain fatty acids.

Results: The array CGH and mitochondrial sequencing were normal. With parents’ consent, we carried out rapid trio exome sequencing which identified a de novo, heterozygous missense variant in ACOX1. This variant had been reported in three other patients with Mitchell syndrome, a newly described neurodegenerative condition with a different molecular mechanism and response to antioxidant therapy than ACOX1 deficiency.

Conclusion: The association of hearing loss, rapidly progressive ataxia, eye abnormalities including corneal lesions with normal metabolic tests in a chid with previously normal development should suggest testing for Mitchell syndrome. Early diagnosis can orient management and genetic counselling.

References: Chung HL, et al., Neuron. 2020 May 20;106(4):589-606.e6.

Grants:

Conflict of Interest: None declared.

EP10.037 Two novel variants in a transmembrane domain of GRID2 confirm the phenotype of GRID2-related dominant non-progressive congenital ataxia

Alexandra Afenjar ¹, Emma Perrier¹, LEILA QEBIBO¹, Malek Louha², Bruno Delobel³, Catherine Garel², lydie BURGLEN¹

¹APHP.Sorbonne Université, Hôpital Trousseau, Reference Center for Cerebellar Malformations and Congenital diseases, Department of Medical Genetics, PARIS, France; ²APHP.Sorbonne Université, Hôpital Trousseau, Service de Radiologie, PARIS, France; ³GHICL, Hôpital Saint Vincent de Paul, Centre de Génétique Chromosomique, Lille, France

Background/Objectives: The GRID2 gene codes for the delta-2 subtype of the glutamate ionotropic receptors (GluD2), the ion channel responsible for rapid excitatory synaptic transmission in the central nervous system. Autosomal recessive ataxia due to loss of function have been described, while a gain of function is suspected in dominant, progressive adult ataxia (inherited missense) or congenital ataxia (CA) (de novo missense, two patients reported).

Objective: to describe the phenotype and genotype of two new patients with congenital ataxia related to dominant pathogenic variants in a transmembrane domain of GRID2.

Methods: Using a NGS panel of CA genes including GRID2, we identified two novel heterozygous variants in two CA patients.

Results: The variants (p.Ala653Asp and p.Gly841Arg) affect the transmembrane domain of the ion channel, like the two dominant mutations previously published. One was de novo, and the second was a somatic mosaic in the asymptomatic mother.

The phenotype combines hypotonia and cerebellar ataxia in children who make progress, and on MRI, global cerebellar atrophy or a more severe pontocerebellar hypoplasia. The cognitive phenotype of patient 1 appears more severe than that of patient 2, whose marked motor involvement contrasts with better preserved cognitive functions, as described in our 2 previously published patients.

Conclusion: These 2 new patients confirm the nonprogressive CA phenotype with cerebellar atrophy or pontocerebellar hypoplasia, associated with de novo dominant mutations, in the transmembrane domain of GRID2.

Reassuring genetic counselling for a de novo mutation must be tempered by the risk of parental, germ-line or somatic mosaic, as observed in family 2.

References:

Grants:

Conflict of Interest: None declared.

EP10.039 Childhood onset chorea: an overview of genetic etiologies in a series of 85 patients

lydie BURGLEN ¹, Claudia Ravelli², Malek Louha¹, LEILA QEBIBO¹, Alexandra Afenjar³, Cyril Mignot³, Diana Rodriguez², Diane Doummar²

¹APHP.Sorbonne Université, Hôpital Trousseau, Reference Center for Cerebellar Malformations and Congenital diseases, Department of Medical Genetics, PARIS, France; ²APHP.Sorbonne Université, Hôpital Trousseau, Service de Neurologie Pédiatrique, Centre de Référence Neurogénétique, PARIS, France; ³APHP.Sorbonne Université, Hôpital Trousseau, Department of Medical Genetics, PARIS, France

Background/Objectives: Chorea is a hyperkinetic movement disorder (MD), the third most frequent MD in children, after tics and dystonia. It is characterized by random, continuous and brief involuntary movements, and may be acquired or genetic. Autosomal dominant NKX2-1 related chorea is the most frequent cause in children, in which the MD may be associated with hypothyroidism or pulmonary disorders. More recently, other genes have been identified (ADCY5, GNAO1, etc.).

Objectives: To assess the genetic epidemiology of childhood onset chorea.

Methods: We reviewed the clinical history of 85 patients initially referred for NKX2-1 analysis (2011-2019). When early onset chorea was confirmed, negative NKX2-1 patients were analysed using a NGS panel (104 MD genes), and then exome or genome sequencing.

Results: 75 patients were included. 34 were related to NKX2-1 anomalies. Panel analysis allowed the identification of causal variants in 45% non-NKX2-1 patients (16/38), and exome/genome sequencing in 4. ATM, ADCY5 and GNAO1 were the most frequent genes after NKX2-1. A diagnosis of glutaric aciduria was made in a 70-year-old patient emphasing the need to carefully look at the MRI. Chorea was a milder phenotype or onset presentation of known pathologies related to KMT2B, GNB1 (mosaicism), PDE10A for example.

Conclusion: We identified a genetic cause in 72% patients of the series, NKX2-1 being the major gene (45%). NGS panel was a performant tool, allowing also diagnosis of mosaicism and deletions, simpler than exome analysis. Etiological diagnosis of chorea is important for genetic counseling, etiological tr