Abstracts from the 57th European Society of Human Genetics (ESHG) Conference: e-Posters (2025)

EP01.001 Relevance of the gene ITIH5 in vulvar diseases

Olga Rodenko¹, Anke Esber¹, Irina Cepraga¹, Stefanie Schütze¹, Lars Jansen¹, Mieczyslaw Romuald Gajda², Nikolaus Gaßler², Ingo B. Runnebaum¹, Matthias Dürst¹, Norman Häfner¹, Claudia Backsch ¹

¹Department of Gynecology and Reproductive Medicine, University Hospital Jena, Jena, Germany; ²Section of Pathology, Institute of Legal Medicine, University Hospital Jena, Jena, Germany

Background/Objectives: The number of vulvar cancers (VC) and their precancers has increased worldwide in recent decades. To date, molecular genetic markers have not played a role in the differential diagnosis of vulvar intraepithelial neoplasias (VIN) and VC. The gene ITIH5 could be play a role as such a marker in vulvar carcinogenesis as like in other cancers. The aim of the present study is to determine the ITIH5 status on different vulvar patient material.

Methods: The copy number status of ITIH5 was analysed using fluorescence in situ hybridization in vulvar scrapes and vulvar tissues of patients with histologically confirmed VIN and VC and as well as Lichen sclerosus (LS) (scrapes n = 10, tissues n = 28). In parallel, promoter methylation was investigated by methylation specific PCR on vulvar scrapes and expression was determined using immunohistochemistry on paraffin embedded vulvar tissues.

Results: In vulvar scrapes, 60% of VIN and 60% of LS showed ITIH5 copy number loss and in 40% of VIN and in 20% of LS ITIH5 methylation were found. In paraffin sections, ITIH5 copy number losses were detected in 36.4% of VIN, in 40% of VC and in 14.3% of LS. Evaluation of ITIH5 expression in paraffin sections are still ongoing.

Conclusion: ITIH5 aberrations seems to be play a role in vulvar carcinogenesis. The use of ITIH5 as a molecular genetic marker could possibly enable a better treatment-relevant or prognostic differentiation of VIN, VC, LS and recurrences in the future.

Grants: Project is supported in part by the Brigitte and Dr. Konstanze Wegener Stiftung.

Conflict of Interest: None declared

EP01.002 Exploring the Impact of Secretome on Inflammation in Myeloproliferative Neoplasms (MPNs)

Selin Fulya Toprak Yalçın ¹, Esra Nur Demirtaş¹, Tugba Sarac-Sivrikoz², Selcuk Sözer Tokdemir¹

¹Istanbul University Aziz Sancar Experimental Medicine Research Institute, genetic, Istanbul, Türkyie; ²Istanbul University Faculty of Medicine, Department of gynecology and obstetrics, Türkyie

Background/Objectives: Myeloproliferative Neoplasms (MPNs) are characterized by a critical inflammatory component that exacerbates disease progression. This is believed to stem from alterations in the bone marrow milieu. Our research focuses on assessing the influence of the secretome, released by mutant cells, on the inflammatory processes in MPNs.

Methods: We utilized HEL cells-derived spent media, introducing it to cord blood mononuclear cells (MNCs) in co-culture systems. Following a 48-hour incubation, the supernatants from these cultures were subjected to ELISA using the LEGENDplex™ cytokine array. Additionally, RT-PCR was employed to quantify changes in IRF3 and cGAS gene expression in the affected cells.

Findings: Significant enhancements in the secretion of cytokines IFN-γ, IL-1β, IL-12p70, and IL17A (P < 0.0001) were detected from infected cells compared to control groups. Concurrently, there was a marked upregulation in IRF3 (~3-fold) and cGAS (~1.5-fold) gene expressions, relative to control groups.

Conclusions: Our findings underscore the pivotal role of the secretome in promoting the cytokine release from healthy cells which may contribute to inflammation and immune response activation in MPNs. Moreover, this research enhances our understanding of genometastasis and potentially guiding the development of more precise and effective treatment modalities for this disease.

Grant: This project is supported by Istanbul University Project Support Office with TSA-2022-39111 grant

Conflict of Interest: None declared

EP01.003 Exploring prevalence of BARD1 pathogenic variant in breast cancer patients

Albertas Valintelis ¹, Ieva Drejerienė¹, Matas Norvydas¹, Jūratė Gruodė^1;2

¹Klaipėda University Hospital, Klaipėda, Lithuania; ²University of Klaipėda, Klaipėda, Lithuania

Introduction: Founder mutations or population-specific variants are significant in disease diagnostics, especially in cancer genetics. BRCA1, BRCA2 gene mutations in Ashkenazi Jewish and Polish-Baltic populations allow for a more precise examination plan or even population-specific screening. We present a variant of the BARD1 gene identified in a cohort of Lithuanian women with breast cancer (BC), consistent with a hereditary predisposition to cancer. BARD1 is a known cancer susceptibility gene that moderately increases breast cancer risk. Among the BC patients BARD1 pathogenic variants were found to be around 0.25%. The aim of this study was to analyze pathogenic BARD1 variants among our cohort.

Methods We performed Ion Torrent on-demand panel testing for 883 patients diagnosed with BC, referred to Klaipėda University Hospital between 2018 and 2023. The library and template preparations were executed through the automated Ion Chef System or IonTorrent PGM, followed by sequencing on NGS Ion torrent platform, in accordance with the manufacturer’s guidelines. Results were analyzed using Ion Reporter Software.

Results In our patient cohort, we identified 5 unrelated patients with heterozygous pathogenic BARD1 frameshift variant (0.56%) NM_000465.4:c.2300_2301del (p.Val767AspfsTer4). No other BARD1 pathogenic variant was found. This variant was observed in the gnomAD controls 0.00002288% among European (non-Finnish) population. 3 of the 5 patients had triple negative BC, 2 patients had bilateral BC. 1 Patient also had BRCA1 pathogenic variant.

Conclusion: This study identified 1 pathogenic variant among the cohort that had high frequency rate among Lithuanian population. Further studies are required to determine this variant clinical management.

Conflict of Interest: None declared

EP01.004 Diagnostic value of NGS and Microarrays vs Conventional Karyotype and FISH in a case of hematological malignancy.

Fotini Ieremiadou¹, Kostantinos Tyrosvoutis², Aspasia Divane ³

¹Life Code, Mol. Biol., Athina, Greece; ²Life Code, Cytogenetics, Athina, Greece; ³Life Code, Director, Athina, Greece

Background/Objectives: Present case is part of a larger effort on comparing diagnostic value of molecular techniques (NGS and Microarray) versus conventional analysis (karyotyping and FISH) in hematological malignancies.

Methods: A 71 years old CLL male patient came to our Lab for genetic testing.

Cytogenetic analysis performed on G-banded chromosome preparations from unstimulated BM culture. FISH performed using ZytoLight SPEC TP53/CEN 17 Dual Color Probe.

DNA extracted from peripheral blood using NucleoSpin blood extraction kit (Macherey-Nagel). NGS performed using custom panel (Agilent Technologies) and data analyzed by seqpilot software (JSI medical systems). CytoScan® 750K Array (Afffymetrix) and ChAS software used for detection of CNVs and LOH.

Results: Cytogenetic analysis revealed a 46,XY[9] karyotype.

I-FISH revealed one hybridization signal both for 17p13.1 and centromeric region in 90%.

NGS revealed: 1. the presence of TP53 p.R273L (c.818G>T) pathogenic variant (confirmed by sanger). 2. deletion of VHL, XPC, MLH1, MYD88, RET, BMPR1A, PTEN, DICER1 and TP53 and duplication of FANCD2, SMARCA4 and CALR genes.

Microarray analysis showed that these CNVs are part of larger multiple aberrations that include 3p and 14q terminal deletions, monosomy 10, 17p deletion and 19p terminal duplication.

Conclusion: NGS and microarray can play a vital role in the analysis of hematological malignancies. NGS can provide information both for chromosomal aberrations and mutational status eliminating the need for multiple testing. Detection of fusions by NGS is also possible but its efficiency remains to be tested.

Conflict of Interest: None declared

EP01.005 Retrospective study of non-informative BRCA1/2 families using NGS. The path towards preventive and personalized medicine.

Mónica Arranz Ledo ¹, Enrique Lastra Aras², Amaya Olaverri Hernández³, Marta Orozco Belinchón³, Enrique Pérez Riesgo¹, Lara Hernández Sanz¹, Noemí Martínez Martín¹, Mar Infante Sanz¹, Mercedes Durán Domínguez¹

¹Institute of Biomedicine and Molecular Genetics, University of Valladolid-Spanish National Research Council (IBGM, UVa- CSIC), Cancer Genetics Group, Unit of Excellence, Valladolid, Spain; ²Burgos University Hospital, Unit of Genetic Counseling in Cancer, Oncology, Burgos, Spain; ³Rio Hortega University Hospital, Unit of Genetic Counseling in Cancer, Oncology, Valladolid, Spain

Background/Objectives: It is widely accepted that analysing a greater number of predisposition genes benefits more families with suspected hereditary breast and ovarian cancer (HBOC). Since 2018, our Hereditary Cancer Laboratory has routinely used multigenic panels instead of BRCA1/2 screening alone, resulting in an increased diagnostic yield from 11% to 20%. However, many families included in the HBOC prevention program prior to its implementation did not have access to a more accurate diagnosis. Therefore, we conducted a retrospective study to evaluate the usefulness of reanalysing families with non-informative results from BRCA1/2 screening.

Methods: We selected a cohort of BRCA1/2 non-informative HBOC samples for them analyse using a multigene panel of 40 predisposition genes. Genomic DNA was extracted from peripheral blood and NGS was performed using ThermoFisher Scientific technology. Library and template preparation was performed on the Ion Chef System, followed by sequencing on the Ion S5. The results were analysed using Ion Reporter Software.

Results: In our study, 19% of the non-informative BRCA1/2 samples analysed were positive for a predisposition gene. The main mutated genes were ATM, CHEK2, BRIP1, PALB2 and RAD51D.

Conclusion: Our findings highlight the need to reanalyse suspected families for HBOC by using multigenic panels. The use of hereditary cancer testing can benefit a larger number of families by providing genetic counselling, preventive medicine for healthy carriers, and personalised medicine based on the mutated gene for those who have developed cancer.

Grants: Predoctoral fellowship co-financed by European Social Fund and Junta de Castilla y León (EDU/842/2022). Research Project PID2021-125909OB-100, IP Carlos Villalobos.

Conflict of Interest: None declared

EP01.006 Fumarate hydratase gene mutation in a family with familial colorectal cancer type X

Marija Staninova Stojovska ¹, Elizabeta Krstevska Bozhinovkj¹, Nadica Matevska-Geshkovska¹, Nenad Mitrevski², Biljana Grozdanovska², Milco Panovski², Aleksandar Dimovski^1;3

¹Faculty of Pharmacy, Ss. Cyril and Methodius University, Skopje; ²Faculty of Medicine, Ss. Cyril and Methodius University, Skopje; ³Macedonian Academy for Sciences and Arts, Skopje

Background/Objectives: Familial colorectal cancer type X (FCCX) is the most heterogeneous type of hereditary cancer of the large bowl whose genetic background has still not been determined.

Methods: We performed targeted NGS sequencing of 36 FCCX families from N. Macedonia. The inclusion criteria were absence of multiple polyps, MSS tumors and Amsterdam I/II positive family history. All patients were analyzed using multigene panels covering coding and exon/intron sequences of >100 genes implicated in hereditary cancers.

Results: We identified an in-frame insertion pathogenic variant c.1431_1433dupAAA p.Lys477dup in the fumarate hydratase (FH) gene which segregated with the disease in one of the examined families with multiple affected members over three generations. The propositus was a 38y old male who was diagnosed with advanced rectal cancer. The same mutation was detected in his sister (45y) who had several distal bowel polyps and his daughter who was diagnosed with Wilms tumor (WT-1 mutation negative) at the age of 10y. The patient’s father was affected with colorectal (56y) and lung cancer (71y). His uncle (56y) and aunt had prostate cancer and breast cancer (63y) + AML (70y), respectively. These two family members were not available for study. The known clinical features of the carriers of pathogenic FH mutations (HLRCC Sy - hereditary leiomyomatosis and renal cell cancer) were not present in the affected members of this family.

Conclusion: We describe a family with pathogenic FH mutation which co-segregated with the predisposition to various cancers, including colorectal FCCX cancer.

Grants: No

Conflict of Interest: None declared

EP01.007 Molecular characterization of tumor DNA and RNA from patients with lung cancer

Ieva Drejeriene ¹, Matas Norvydas¹, Albertas Valintelis¹, Jūratė Gruodė^1;2

¹Klaipeda University Hospital, Klaipėda, Lithuania; ²Klaipeda University, Klaipėda, Lithuania

Introduction: Lung cancer stands out as the most prevalent cancer diagnosis as well as the leading cause of cancer-related deaths in the world. Targeted therapies guided by molecular diagnostics have become a standard treatment of lung cancer. On that note, molecular testing should be quick, comprehensive, and in accordance with clinically approved guidelines. The best option in lung cancer diagnostics is to use new generation sequencing (NGS) platform. It allows rapid and becomes economically viable testing of multiple targets (hotspot mutations, translocations, copy number variations) from one sample with a single assay.

Materials and Methods: DNA and RNA were extracted from 126 lung cancer formalin-fixed, paraffin-embedded (FFPE) tumour samples. Hotspot mutations, translocations and copy number variations were detected using NGS (Ion Torrent™ 5S System) Oncomine Focus Assay panel (ThermoFisher). Sequencing data were analyzed with Ion Reporter™ Software on ThermoFisher™.

Results: Hotspot mutations, translocations or copy number variations were detected in 80 (63,5%) of 126 lung cancer samples. Hotspot mutations were detected in KRAS, MAPK2K1, EGFR, BRAF, ERBB2, HRAS, MET, CTNNBI, PIK3CA genes and amounted to 50% of all variations. Translocations were identified in 16,25% and copy number variations in FGFR1, MYC, PIK3CA, BRAF, FGFR1, KRAS, AR, MET, MYCN, CCND1 genes constituted 33,75% of all genetic aberrations. Of all genetic findings, 44,5% of variants were clinically actionable driver mutations.

Conclusion: The findings underscore the practical value of our assay as a diagnostic tool capable of providing insights into cancer diagnosis, prognosis, and directing therapeutic approaches.

Conflict of Interest: None declared

EP01.008 Investigation of cancer related genes in patients with Enchondromatosis

Ian Babler-Madrid ¹, Nara Sobreira¹, Renan Martin¹, Elizabeth Wohler¹

¹Johns Hopkins University

Background/Objectives: Ollier disease (OD) and Maffucci syndrome (MS) are cancer susceptibility syndromes, and their molecular bases are not fully understood. Approximately 50% of patients with these syndromes develop a malignancy, including chondrosarcomas and gliomas. The objective is to identify the molecular bases of unsolved OD and MS cases.

Methods: A burden test was conducted with a multisample VCF file containing WGS data from 638 samples (74 cases, 564 controls) and WES data from 2141 samples (46 cases, 2095 controls). The VCF was filtered based on the gene symbols of 1202 genes described in GeneDx, COSMIC, and glioma literature. Only single nucleotide variants (SNVs) were analyzed and filtered based on RefSeq and gnomAD annotations to include functional, rare (MAF ≤ 0.01) SNVs. The variants in genes that were more mutated among patients than controls with a p-value < 0.05 were further evaluated based on their gnomAD frequency, familial segregation, variant type, in silico predictions, ClinVar, HGMD, and ACMG classifications.

Results: Out of 534 genes with at least one variant in a case, 44 were more mutated among patients than controls with a p-value < 0.05. Further analysis identified strong candidate genes that are being further evaluated, including FGFR1 and TCF3.

Conclusion: The most robust candidate genes will be further investigated functionally and in unrelated cohorts of patients with sarcoma-related diseases to determine if they are involved with these malignancies.

Grants: National Human Genome Research Institute/National Heart, Lung, and Blood Institute (NHGRI/NHLBI) grant UM1 HG006542; Gabriella Miller Kids First Pediatric Research Program grant X01HL140517; NIH/NCI grant R03CA256535.

Conflict of Interest: None declared

EP01.009 Complex diagnostics of somatic mutations in tumor cells

Gulshara Abildinova¹, Zhanna Zhabakova ¹, Anna Borovikova¹

¹Medical Center Hospital of the President’s Affairs Administration of the Republic of Kazakhstan, Laboratory genetics, Astana, Kazakhstan

Consortium: Complex diagnostics of somatic mutations in tumor cells

The molecular profile of a tumor is necessary for personalized selection of drugs for cancer patients in order to establish the risk of tumor development, clarify the prognosis of the disease and predict the response to treatment.

Aim: search for mutations in paraffin-fixed tumor tissues in order to optimize targeted treatment.

Material and research methods: The research material was DNA and RNA isolated from tumor cells.

Method: DNA and RNA library preparation followed by sequencing of amplified fragments was carried out according to the manufacturer’s protocol.

Results: The study group consisted of 240 patients with tumors of various locations (70% solid, 30% gliomas). 140 patients with solid tumors used a set of 52 and 161 genes. 80% of patients had 1 mutation and 20% had 2 or more mutations

Missense mutations were identified in 75% of cases [BRAF (Val600Glu); KRAS (Gly12Val); IDH1(R132H); JAK3 (S493C); PIK3CA (E545Q; E542K; p.(H1047L), PTEN H93Y], CDK12 c.1047-1G > A, TERT c.-124C > T for the prognosis of glioblastoma.

In 10 patients, a combination of 2 or more mutations was identified [PIK3CA (E547K), FGFR3 (G697S); PIK3CA (E542K), KRAS (Gly12Val); ATR c.7762-2A > G, ATR c.5739-14_5739-6delinsT EGFR(A289T), JAK3(S493C); BRAF (G469V), KRAS (Gly12Val); TP53, FGFR4, CREBBP, SMARCA4].

In 10% of cases, an increase in the number of copies of the EGFR and KRAS genes was detected, and in 10% of cases, a fusion of the gene (RNA) MET-MET.M13M15, TMPRSS2-ERG, RBB2 amplification was detected.

Conclusion: For personalized prescription of targeted drugs, it is more effective to use multigene diagnostics for a comprehensive study of the mutational status of the tumor.

Conflict of Interest: None declared

EP01.010 Clinical relevance of immune microenvironment and gene-expression-based biomarkers in colorectal cancer

Vincenzo Rallo ¹, Matteo Massidda², Xiaofen Wen³, Jiaxin Shen³, Manila Deiana¹, Andrea Maschio¹, Antonio Mario Scanu², Maria Rosaria De Miglio², Andrea Angius¹

¹Institute for Genetic and Biomedical Research, Italian national research council, Monserrato, Italy; ²University of Sassari, Sassari, Italy; ³Cancer Hospital of Shantou University Medical College, Shantou, China

Background/Objectives: We explored genomic and immune landscape of colorectal cancer (CRC) compared to normal adjacent tissue (NCT). Starting from differentially expressed genes (DEGs) data, protein-protein interaction (PPI) network and deconvolution analysis were employed to unravel the intricate interplay between molecular alterations and immune cell CRC composition.

Methods: The PPI network was constructed using the STRING database and Cytohubba MCC algorithm was applied to identify hub genes. Deconvolution analysis utilizing CIBERSORTx estimated the immune cells proportions between CRC and NCT. Statistical analyses, including the Wilcoxon signed rank test, effect size measurement and Cox regression, were applied to determine the magnitude of variations in cell populations and their correlation with overall survival.

Results: The PPI network exploration revealed ten hub genes upregulated in CRC, showing significant enrichment (P < 1.0e-16). KEGG pathway analysis highlighted their involvement in critical cellular processes such as cell cycle, cell senescence, and p53 signaling pathways. Deconvolution analysis disclosed a distinct immune microenvironment, with fifteen cell types exhibiting significant differences in abundance and impact between CRC and NCT. Two correlated with a favorable and three with worse prognosis.

Conclusion: We provided insight into the molecular and immune dynamics in CRC, identifying potential prognostic biomarkers and therapeutic targets. The intricate interplay between specific immune cell populations and clinical outcomes underscores the importance of genetic and immune factors in the management of CRC. These findings contribute to advancing our knowledge of cellular heterogeneity and CRC pathogenesis toward personalized patient treatment strategies.

Grants: 2022WBLA3Z and B/2020/0094 from MIUR and Intesa Sanpaolo

Conflict of Interest: None declared

EP01.011 Prostate cancer genetic screening for BRCA1 and BRCA2 mutations

Agnieszka Podgorska ¹, Ewa Kwiatkowska¹, Renata Lotocka¹, Pawel Wiechno², Katarzyna Seliga¹, Aleksandra Gos¹, Katarzyna Zielinska-Musko¹, Lukasz Fulawka¹, Aleksandra Kuzniar¹, Kinga Koszucka¹, Andrzej Tysarowski¹

¹Maria Sklodowska-Curie National Research Institute of Oncology, Cancer Molecular and Genetic Diagnostics Department, Warsaw, Poland; ²Maria Sklodowska-Curie National Research Institute of Oncology, Department of Uro-Oncology, Warsaw, Poland

Background/Objectives: Prostate cancer is the most commonly diagnosed malignancy in men. Patients with diagnosed BRCA-positive, castration-resistant, metastatic prostate cancer can qualify for PARP-inhibitor targeted therapy. Therefore, genetic testing for at least BRCA1 and BRCA2 genes should become a routine practice, however available prostate tissue material is often difficult and test results are inconclusive.

Methods: Genetic testing was performed on DNA extracted from FFPE tissue from 211 prostate cancer patients using next-generation sequencing (NGS). Sequencing was run on Ion S5 (ThermoFisher) or Miniseq (Illumina) systems.

Results: Within the analyzed patient group 10 (6%) cancer samples were harboring a pathogenic variant in BRCA1/2 gene (one of which was carrier of two mutations): BRCA1 (1%) - c.5289dup, c.181T>G, c.3629_3630del; BRCA2 (5%) - c.5471dup, c.4176del, c.7251_7252del, c.9118-2A > G, c.9154C>T, c.2095C>T, c.3847_3848del, c.5621_5624del. These mutations indicate homologous recombination repair pathway (HRR) deficiency and sensitivity to PARP-inhibitor treatment.

Eligible analysis results could be obtained for 66% of prostate cases. However, tissue material occurred to be challenging and 34% of test results were unsuitable for molecular analysis. The main cause was poor fixation of cancer tissue and insufficient tumor content.

Conclusion: Frequency of BRCA-positive prostate cancers is low and amounts to approximately 6%. Due to high percentage of inconclusive results, some prostate cancer patients might miss the opportunity to qualify for targeted therapy. In this case BRCA testing on liquid biopsy (ctDNA) might be considered. There are no characteristic BRCA1/2 hotspot mutations in prostate cancers, therefore testing of the whole coding sequence is recommended using NGS technology.

Conflict of Interest: None declared

EP01.012 Li-Fraumeni syndrome : pathways of care for cancer prevention versus cancer treatment

marion rolain¹, patricia faure¹, Nathalie Parodi¹, Maud Brauchaud¹, Margaux Clement Le Choismier¹, Stéphanie Baert-Desurmont², Edwige Kasper¹, Gaelle Bougeard², Mariette Renaux-Petel³, Thomas Vermeulin⁴, Théry Jean-Christophe⁵, Claude Houdayer ¹

¹Univ Rouen Normandie, Normandie Univ, Inserm U1245, CHU Rouen, Department of Genetics, Rouen, France; ²Univ Rouen Normandie, Normandie Univ, Inserm U1245, Rouen, France; ³CHU Rouen, Department of Child and Adolescent surgery, Rouen, France; ⁴Centre Henri Becquerel, Department of Medical Information, Rouen, France; ⁵Univ Rouen Normandie, Normandie Univ, Inserm U1245, Centre Henri Becquerel, Department of Medical Oncology, Rouen, France

Consortium: the PREVENTABLE Consortium

Background/Objectives: Li-Fraumeni syndrome (LFS) represents one of the most severe hereditary predispositions to cancer, given the broad spectrum of tumours involved and their early age of onset. The Rouen genetics department is an expert centre for LFS, part of the European Reference Network for rare tumour risk syndromes (RTRS) GENTURIS. Prevention is a major aspect of health still underestimated by stakeholders and policymakers, which is why we joined the European PREVENTABLE H2022-2025 consortium. The aim of PREVENTABLE is to assess the clinical, social and financial benefits of applying multidisciplinary and specialized care to prevent advanced disease in families suffering from RTRS.

Methods: Based on experience and expertise of our clinical teams, we have identified all possible care pathways for the LFS patients, in order to create standard matrices for children and adults. Then matrices will be filled in with clinical data, lastly prices will be allocated to clinical procedures in order to compare prevention versus treatment in medico-economic terms.

Results: Matrices contained 124 procedures divided into 6 care modules for 58 different pathways of care. All the necessary ethical approvals were obtained, as patients must be informed about the opportunity to object. Thanks to the French LFS network, clinical data could be obtained from other caregivers, and from European centres involved in the project.

Conclusion: Despite the high complexity of LFS care, we demonstrated that consensus matrices are achievable. This project aims to demonstrate the virtuous practice of oncogenetics by highlighting the benefits of prevention in a complex, costly and deadly disease.

Grants: Funded by EU

Conflict of Interest: None declared

EP01.013 Whole exome sequencing shows recurrent abnormalities in members of the WNT pathway in familial polyposis

Paula García-Vallés ^1;2;3, Jésica Pérez-García^1;2;3, Paloma Martín-Bejarano Soto^1;2;3, Rosario Vidal-Tocino^1;4, Óscar Javier Blanco Múñez⁵, Jose Perea¹, Ana-Belén Herrero^1;2;3, Rogelio González-Sarmiento^1;2;3

¹Institute of Biomedical Research of Salamanca (IBSAL), Salamanca, Spain; ²Molecular Medicine Unit, Department of Medicine, University of Salamanca, Salamanca, Spain; ³Institute of Molecular and Cellular Biology of Cancer (IBMCC), University of Salamanca-CSIC, Salamanca, Spain; ⁴Medical Oncology Service, University Hospital of Salamanca, Salamanca, Spain; ⁵Pathological Anatomy Service, University Hospital of Salamanca, Salamanca, Spain

Background/Objectives: Familial adenomatous polyposis (FAP) makes up about 1% of all colorectal cancers, which in turn are the second cause of cancer death. FAP is characterized by the early development of hundreds or thousands of adenomas throughout the colon. Germline mutations in APC or MUTYH account for the majority of FAP cases. However, there are patients with familial polyposis whose genetic cause is unknown. This project aims to analyze the whole exome of patients with polyposis without germline mutations in APC or MUTYH.

Methods: Two polyps and one healthy tissue sample were analyzed from three patients. Genomic DNA was extracted from formalin-fixed paraffin-embedded (FFPE) polyps and normal adjacent tissues using the QIAamp DNA FFPE tissue kit. The SureSelect Human All Exon V8 kit was used for library preparation and sequencing was performed on NovaSeq 6000 platform. Mutations of interest identified in this study were confirmed through Sanger sequencing.

Results: All the polyps from these patients display pathogenic somatic mutations or variants of uncertain significance in some member of the WNT pathway or the mTOR pathway. Notably, healthy tissues samples already presented alterations in two of the patients, a pathogenic LRP5 mutation in the first patient and a variant of uncertain significance in LGR4 in the second patient. These genes code a WNT pathway co-receptor and receptor, respectively.

Conclusions: Mutations in members of the WNT pathway appear to play a key role in those patients with familial polyposis without germline mutations in APC or MUTYH. However, further studies are required.

Grants: Study funded by Fundación Mutua Madrileña.

Conflict of Interest: None declared

EP01.014 Assessment of EGFR mutation status in cf-DNA from patients with NSCLC in a clinical setting

Maria Michelli ^1;1, Eirini Roupou¹, Ioanna Giannopoulou¹, Niki Prekete¹, NIKOLAOS KAVANTZAS¹, Angelica a Saetta¹

¹Medical School of Athens, NKUA, 1st Department of Pathology, Athens, Greece

Background/Objectives: EGFR mutation analysis in circulating cell-free (cf-DNA) tumor DNA from plasma has shown to be a very promising approach for biomarker analysis of NSCLC patients with insufficient tumor tissue and disease monitoring due to several significant advantages (minimally invasive, potential for repeated sampling).

Methods: 595 cf-DNA samples from patients with NSCLC and 158 matched FFPE tumor tissues were analyzed using an IVD test, Cobas ® EGFR mutation v2. 8 samples were re-analyzed using Next Generation Sequencing.

Results: FFPE samples showed insufficient tumor cells in 16/158 cases (10%) and an invalid cobas test in 18/158 (11%). 3/34 total FFPE cases without a test result displayed EGFR mutation in cf-DNA. The presence of mutations in plasma from patients with primary NSCLC reached 9% (25/286). In total 153/595 cf-DNA samples (26%) displayed EGFR mutations. Exon 19 deletions were the most frequent mutations (66%), followed by point mutations in exon 21 (26%). The overall concordance of EGFR mutation status in plasma and tumor samples was 78% and the sensitivity 57%, specificity 100%, PPV 100%. The concordance of EGFR mutation status in plasma between Cobas and NGS was 100%.

Conclusion: EGFR mutation testing in cf-DNA using Cobas IVD test showed significant concordance with analysis of tissue samples and an optimal specificity and PPV. However, the sensitivity of the test has to be further improved indicating that a biopsy should be optimally analyzed for patients with an EGFR mutation-negative cf-DNA test.

Grants: no

Conflict of Interest: Maria Michelli full, Eirini Roupou full, Ioanna Giannopoulou full, Niki Prekete full, NIKOLAOS KAVANTZAS yes, Angelica a Saetta full

EP01.015 Multi-locus Inherited Neoplasia Alleles Syndromes in Cancer: An updated review and implications for clinical practice

Jeanette YUEN ¹, Malikka Venkatramani¹, Si Qin Zhou¹, Jianbang Chiang¹, Sock Hoai Chan¹, Joanne Ngeow^1;2

¹National Cancer Centre Singapore, Division of Medical Oncology, Singapore, Singapore; ²Nanyang Technological University, Lee Kong Chian School of Medicine, Singapore, Singapore

Background/Objectives: The popularity of multi-gene panel testing has identified individuals with two or more pathogenic variants (PV). These carriers are at risk of suboptimal treatment, management and inaccurate cancer risk estimations from the lack of data. We reviewed published double heterozygote cases to identify possible predictors and associations with more severe phenotypes to understand the impact on clinical management.

Methods: We conducted a systematic review of published literature tracing double heterozygotes of cancer predisposition genes. Statistical tests were conducted to assess for predictors of double heterozygotes vs. monoallelic and non-carriers. Variables examined were: first age of diagnosis, early age of diagnosis defined as <5% age-stratified risk, number and type of primary cancers, and presence of unassociated phenotype.

Results: We identified 394 cases, with 33 patients from our local service. Results suggest that double heterozygotes presented with significantly more malignancies (32.0% vs 21.5% vs 10.3%), a younger median age of diagnosis (41 vs. 45 vs. 49 years) and an early age of diagnosis (24.9% vs 7.7% vs 4.7%) vs. monoallelic and non-carriers. Carriers with two dominant PVs had more malignancies (34.4% vs. 23.9% vs. 20.0%) and un-associated phenotypes (87.5% vs. 82.1% vs. 40.0%) vs. dominant-recessive and recessive-recessive PV carriers.

Conclusion: Our findings suggest that double heterozygotes have more severe phenotypes, with more than one primary cancer and younger ages of diagnosis. The nature of genes in double heterozygotes (i.e. dominant-dominant / dominant-recessive) may also have implications on cancer risk and clinical management. We propose a framework to evaluate double heterozygotes to guide clinical management.

Grants:

Conflict of Interest: None declared

EP01.016 Genetic variability in inflammation and oxidative stress genes affects gastric cancer risk and clinical features

Pia Pužar Dominkuš ¹, Marija Rogar^1;2, Vita Dolzan¹, Petra Hudler²

¹University of Ljubljana, Faculty of Medicine, Institute of Biochemistry and Molecular Genetics, Pharmacogenetics laboratory, Ljubljana, Slovenia; ²University of Ljubljana, Faculty of Medicine, Institute of Biochemistry and Molecular Genetics, Medical Centre for Molecular Biology, Ljubljana, Slovenia

Background/Objectives: Inflammation and oxidative stress are the hallmark of Helicobacter pylori infection, which is a leading risk factor for gastric cancer. The aim of our study was to evaluate the effect of single nucleotide polymorphisms in genes involved in inflammation and oxidative stress on gastric cancer risk, clinical-histopathological features and patient’s survival.

Methods: We enrolled 248 patients with gastric adenocarcinoma and 247 healthy control subjects in the study. Genotyping for two polymorphisms in GSTP1 and KEAP1, three in NFE2L2, and one in CAT, GPX1, IL-1β, TNFα, IL6, SOD2, and IL6R was performed using KASP assays. Logistic regression and Kaplan-Meier method were used for association and survival analysis.

Results: TNFα rs1800629 was significantly associated with higher gastric cancer risk (GG/GA + AA, OR = 1.62, P = 0.011). IL-1β rs1143623 (GG/CG + CC, OR = 0.37, P = 0.017), rs16944 (GG + AG/AA, OR = 0.28, P = 0.027) and IL6 rs1800795 (GG/CG, OR = 0.38, P = 0.010 and GG/CC, OR = 0.16, P = 0.010) were associated with tumour location. NFE2L2 rs6721961 was associated with tumour grade (GG + TG/TT, OR = 0.08, P = 0.007). KEAP1 rs9676881 (GG/GA + AA, OR = 0.36, P = 0.034) and IL-6 rs1800795 (GG + CG/CC, OR = 6.40, P = 0.027) were associated with vascular and perineural invasion, respectively. The CC genotype of IL-6 rs1800795 was associated with longer overall patient survival (P = 0.021).

Conclusion: Our results may be valuable in disease modelling and for the development of personalised approach in the risk assessment and disease management of gastric cancer.

Grants: ARIS grants P1-0170 and P1-0390.

Conflict of Interest: None declared

EP01.017 Telomeres and TERT polymorphisms as biomarkers of the risk of malignant mesothelioma

Tanja Blagus ¹, Ana Mervič², Katja Goricar¹, Danijela Strbac³, Alenka Franko^2;4, Viljem Kovac^2;3, Vita Dolzan¹

¹University of Ljubljana, Faculty of Medicine, Institute of Biochemistry and Molecular Genetics, Parmacogenetics Laboratory, Ljubljana, Slovenia; ²University of Ljubljana, Faculty of Medicine, Ljubljana, Slovenia; ³Institute of Oncology Ljubljana, Ljubljana, Slovenia; ⁴University Medical Centre Ljubljana, Institute of Occupational, Traffic and Sports Medicine, Ljubljana, Slovenia

Background/Objectives: Malignant mesothelioma (MM), strongly associated with asbestos exposure, belongs to the group of telomerase-dependent cancers. The enzyme complex telomerase is activated due to increased expression of its main component, telomerase reverse transcriptase (TERT). We aimed to determine the associations between telomere length and TERT polymorphisms with the risk of developing MM.

Methods: Our retrospective case-control study, included 302 patients with MM, 386 subjects with pleural plaques (PP), and 86 subjects that were occupationally exposed to asbestos, but had no asbestos related diseases (controls). Relative telomere length was determined by MMQ-PCR, and the presence of TERT rs10069690, rs2736100, and rs2736098 was determined by KASP-PCR. Statistical analysis was performed using nonparametric tests and logistic regression.

Results: MM patients had shorter telomere length than subjects with PP (p < 0.001). Both TERT rs2736098 and rs10069690 were statistically significantly associated with a higher risk of MM in additive and dominant models (OR_add = 1.63; CI = 1.20–2.21; p = 0.002; OR_dom = 1.46; CI = 1.09–1.96; p = 0.011; OR_add = 1.52; CI = 1.08–2.12; p = 0.017, OR_dom = 2.2; CI = 1.10–4.48; p = 0.026, respectively). On the contrary, the presence of at least one polymorphic TERT rs2736100 allele reduced the risk of MM in additive and dominant models (OR_add = 0.56; CI = 0.35–0.90; p = 0.017; OR_dom = 0.66; CI = 0.46–0.95; p = 0.026).

Conclusion: Telomere length and TERT polymorphisms may serve as biological markers of MM development.

Grants: ARIS grants P1-0170 and L3-2622.

Conflict of Interest: None declared

EP01.018 Challenges of PMS2 screening: technical procedures to validate the presence of PMS2 variants.

Maria Isabel Alvarez ^1;2, Paula Trilla¹, Marta Gracia¹, Laura Muñoz¹, Maria Pellise³, Belen Pastor Piñera^3;4, Lorena Moreno^3;4, Teresa Ocaña^3;4, Francesc Balaguer^3;4, Celia Badenas^1;2

¹Hospital Clínic de Barcelona, Biochemistry and Molecular Genetics, Barcelona, Spain; ²CIBER of Rare Diseases (CIBERER), Madrid, Spain; ³Hospital Clínic de Barcelona, Department of Gastroenterology, Barcelona, Spain; ⁴Centro de Investigación Biomédica en Red en Enfermedades Hepáticas y Digestivas (CIBERehd), Madrid, Spain

Background/Objectives: In the era of next generation sequencing technology (NGS), the molecular screening for PMS2 is challenging due to the presence of a large family of pseudogenes that are highly homologous. After routine genetic study we detected 6 patients in which neither long-range PCR nor MLPA could ensure the location of the variant detected. In this work, we present additional strategies to confirm PMS2 alteration.

Methods: Patients 1 to 3 presented an unique deletion of PMS2 exon 14, patient 4 showed a duplication of exons 7 to 11, patient 5 carried a deletion encompassing exons 14 and 15, and patient 6 presented a pathogenic variant in exon 12 in which long-range PCR was not conclusive. For copy number variants specific primers were used in order to confirm their presence by PCR and/or Sanger sequencing. For patient 6, RNA was extracted and cDNA was sequenced.

Results: All patients with single deletion of exon 14 were confirmed using the primers described previously identifying a recurrent deletion of approximately 2kb (c.2276-113_2245+159del). The duplication of exons 7 to 11 was confirmed using the combination of specific primers revealing the presence of an inverted tandem duplication of these exons. In patient 6, RNA assay suggested the presence of a PMS2- PMS2CL hybrid allele. Patient 5 remains under assessment.

Conclusion: All PMS2 variants require addition confirmation with specific approaches. Interestingly, the fact that all 6 patients showed isolated PMS2 loss which indicates that it could represent a highly indicator of a germline pathogenic variant.

Grants:

Conflict of Interest: None declared

EP01.019 Extended genetic testing of KIT/PDGFRA wild-type GISTs revealed NF1-associated GIST in one patient

Alenka Bombač ¹, Mojca Unk², Vida Stegel¹, Srdjan Novaković¹

¹Institute of Oncology Ljubljana, Department of Molecular Diagnostics, Ljubljana, Slovenia; ²Institute of Oncology Ljubljana, Division of Medical Oncology, Ljubljana, Slovenia

Background/Objectives: Gastrointestinal stromal tumors (GISTs) represent a heterogeneous group of mesenchymal neoplasms with a spectrum of genetic alterations. Among these alterations, the association between GISTs and neurofibromatosis type 1 (NF1) mutation has garnered significant attention. NF1 mutation-related GISTs present distinct clinical and molecular characteristics compared to their counterparts. This abstract review a case of NF1-associated GIST from a larger study of 116 metastatic GIST patients, treated at Institute of Oncology Ljubljana.

Methods: Patient’s DNA was extracted from Formalin-fixed, paraffin-embedded (FFPE) primary tissue sample that was obtained prior to the imatinib treatment. Tumor genotyping was performed using Sanger and NGS sequencing with Illumina’s TruSight Oncology 500 DNA Kit. The expression of succinate dehydrogenase (SDH) complex was assessed by immunohistochemistry.

Results: From the initial study cohort of 116 patients twelve out of sixteen KIT/PDGFRA wild-type GISTs retained SDH expression. Additional NGS genotyping of KIT/PDGFRA wild-type GISTs revealed mutations in KIT or PDGRFA in seven patients and NF1 mutation in one patient with no prior association with Neurofibromatosis Type 1 syndrome.

Conclusion: For patients with KIT/PDGFRA wild-type GIST the reliability of direct Sanger sequencing results is insufficient therefore, it is recommended that NGS becomes a necessity for their treatment decision by its ability to detect low allele frequency mutations and potential other targetable alterations as well as identify germline mutations in genes associated with hereditary syndromes.

References: PMID: 35904169

Conflict of Interest: None declared

EP01.020 Germline genetic profiling using next-generation sequencing technology in a man with advanced prostate cancer: a case report

Anca Pavel ¹, Petruta Gurban¹, Andreea Tutulan-Cunita¹, Danai Stambouli¹

¹Cytogenomic Medical Laboratory, Molecular Genetics, Bucharest, Romania

Background/Objectives: Recent developments in next-generation sequencing (NGS) technologies have contributed to our growing understanding of the prostate cancer (PC) biology, revealing the genetic foundation of the disease’s clinical variability. The aim of the current study was to identify potential genetic risk variants for PC using whole exome sequencing (WES) and to evaluate the effect of these variations on PC aggressiveness.

Methods: We present a case of advanced PC for which WES analysis was performed for the identification of potential genetic variants involved in PC pathology. To isolate the variants significant to our case, a PC susceptibility gene panel comprised of 164 genes was used. The obtained variants were then subjected to further analysis. A brief frequency data analysis was conducted. To assess the possible contribution of genetic variants to the PC disease, genotype-phenotype analysis was performed.

Results: WES revealed 291 nonsense and missense variants in 113 different genes, previously associated with PC. The results of frequency data analysis showed that most susceptibility genes identified were located on chromosomes 2 and 11 (24%). The most frequent variants were single nucleotide variants (SNV) (89.3%). Genotype-phenotype correlation analysis highlighted four variants that had a strong contribution to the PC phenotype, namely c.8965C>G in ATM gene, c.1385G>A in RNASEL gene, c.589G>A in TMPRSS2 gene and c.1501G>A in ELAC2 gene.

Conclusion: Our results suggest that genetic variants play an important role in the progression of PC and their confirmation could have future implications in PC diagnosis and prognosis, as well as therapy.

Grants: -

Conflict of Interest: None declared

EP01.021 Downregulation of SLC22A1 expression in hepatocellular carcinoma and its prognostic significance

Hubert Chen ¹

¹Vanderbilt University, College of Arts and Science, Nashville, United States

Background/Objectives: Hepatocellular carcinoma (HCC) is the most common primary liver malignancy that occurs predominantly in patients with underlying chronic liver disease and cirrhosis. Organic cation transporter 1(OCT1), encoded by SLC22A1 gene, is mainly expressed in the liver and is located at the basolateral membrane of hepatocytes. It plays a key role in the disposition and hepatic clearance of mostly cationic drugs and endogenous compounds. We aimed to systematically analyze SLC22A1expression and its prognostic role in HCC using various open databases.

Methods: Comparison of HCC and matched normal tissue gene expression was performed with the UALCAN and GEPIA online graphical user interfaces. Correlation between SLC22A1 gene expression and patient survival was evaluated with OncoLnc. SLC22A1 genetic alterations in HCC were also explored using cBioPortal.

Results: Expression of SLC22A1 is significantly down-regulated in the clinic-pathological characteristics (cancer stages, tumor grade, nodal metastasis status, and histological subtypes) examined in HCC patients compared to normal counterparts. Clinically, low expression of SLC22A1 was correlated with shorter overall survival in HCC patients (Log rank p-value = 0.00007). Analysis of the TCGA Liver Hepatocellular Carcinoma dataset (TCGA’s Pan-Cancer Atlas) revealed a low amplification (0.57%) and deep deletion (1.71%) frequency in HCC.

Conclusion: These observations indicate that SLC22A1 may function as a potential tumor suppressor gene. In HCC patients, SLC22A1 expression levels may serve as a prognostic predictive marker.

Grants: NA

Conflict of Interest: None declared

EP01.024 Hormone-induced intracellular interactions of androgen and glucocorticoid receptors

Nuria Arroyo-Garrapucho ^1;2;3;4, Vera Sommers⁴, Hanne Willems⁴, Kaat Peperstraete⁴, Vanessa Dubois⁵, Ana-Belén Herrero^1;2;3, Rogelio González-Sarmiento^1;2;3, Frank Claessens⁴

¹Biomedical Research Institute of Salamanca (IBSAL), University Hospital of Salamanca-USAL-CSIC, Salamanca, Spain; ²Molecular Medicine Unit, Department of Medicine, University of Salamanca, Salamanca, Spain; ³Institute of Molecular and Cellular Biology of Cancer (IBMCC), University of Salamanca-CSIC, Salamanca, Spain; ⁴Laboratory of Molecular Endocrinology, Department of Cellular and Molecular Medicine, KU Leuven, Leuven, Belgium; ⁵Basic and Translational Endocrinology (BaTE), Department of Basic and Applied Medical Sciences, Ghent University, Ghent, Belgium

Background/Objectives: Androgens are crucial for male sexual development, while glucocorticoids regulate energy homeostasis and immune responses. These hormones activate the androgen receptor (AR) and glucocorticoid receptor (GR). Despite tissue-specific crosstalk between glucocorticoids and androgens, the molecular mechanisms remain unclear. We explored AR and GR cooperation in mouse Fkbp5 transcription, a well-known gene responsive to both hormones, testing the hypothesis that this collaboration occurs via the FKBP5 androgen response element (ARE), which would reveal protein-protein interactions between the receptors. Enzalutamide effect, an AR antagonist, was tested on these interactions.

Methods: Glucocorticoids and androgens were tested in chemically castrated mice, supplemented with one or both steroids. Tissue qPCR was conducted. AR:AR, GR:GR, and AR:GR dimerization were studied using the NanoBiT system in HEK293T. Transcriptional activation via FKBP5 ARE was assessed through a luciferase reporter assay. Statistical significance was determined using Friedman test with Dunn multiple comparisons.

Results: In male mice, Fkbp5 responded to androgens and glucocorticoids in the gastrocnemius and adipose tissue. Treatment with both hormones increased expression levels in these tissues. NanoBiT confirmed rapid AR dimerization with dihydrotestosterone, while dexamethasone failed to induce detectable GR dimers. Combining DHT and dexamethasone resulted in AR:GR heterodimerization. AR and GR activation also additively induced the FKBP5-ARE-luciferase reporter gene. Enzalutamide effectively suppressed both AR:AR and AR:GR dimerization, while inhibiting AR- and GR-dependent FKBP5-ARE activation.

Conclusion: Our study suggests that AR:GR interaction could be involved in the interplay between androgens and glucocorticoids, which could be highly relevant during treatment of prostate cancer with AR inhibitors and glucocorticoids.

Grants: FIS-FEDER PI20/01569, FWO-1131720N, GOA/19/100.

Conflict of Interest: None declared

EP01.025 Germline long insertions in BRCA1/PALB2 exon revealed by PCR-free short-read and long-read whole-genome sequencing

Ava Kwong^1;2;3, Chun Hang Au ⁴, Dona N Ho⁴, Elaine YL Wong⁴, Henry CM Leung⁴, Amy WS Leung⁴, Janet HY Law⁴, Fian BF Law⁴, Sze Keong Tey⁵, Cecilia YS Ho⁴, Edmond SK Ma^3;4

¹The University of Hong Kong and University of Hong Kong-Shenzhen Hospital, Department of Surgery, Hong Kong; ²Hong Kong Sanatorium & Hospital, Department of Surgery, Hong Kong; ³Hong Kong Hereditary Breast Cancer Family Registry, Hong Kong; ⁴Hong Kong Sanatorium & Hospital, Molecular Pathology Division, Department of Pathology, Hong Kong; ⁵The University of Hong Kong, Department of Surgery, Hong Kong

Background/Objectives: While to detect structural variants disrupting hereditary breast cancer genes is important, the detection of long insertion (>100 bp) is challenging.

Methods: Short-read PCR-free whole-genome sequencing (WGS) libraries were constructed using KAPA EvoPlus Kit (Roche), sequenced at 2x151 bp on NovaSeq 6000 S1 flow cells (Illumina), followed by alignment using Clara Parabricks v4.0.0 (Nvidia) and insertion detection using INSurVeyor v1.1.2. Long-read PCR-free WGS libraries were constructed using Ligation Sequencing Kit V14, sequenced on PromethION R10.4.1 flow cells (Oxford Nanopore), followed by Dorado v4.2.0 super-accurate basecalling, alignment using minimap2 v2.24 and consensus sequence generation using Flye v2.9.

Results: Under the hereditary breast cancer genetic testing program of Hong Kong Hereditary Breast Cancer Family Registry, we identified germline long insertions from two probands. Proband 1 was a Chinese female bilateral breast cancer patient with positive family history. A heterozygous 318 bp insertion with AluY and target site duplication at PALB2 exon 11 was identified by both short-read and long-read WGS. Proband 2 was a Chinese female early-onset triple-negative breast cancer patient with positive family history. An insertion at BRCA1 exon 24 was suspected by short-read WGS and fully elucidated by long-read WGS as a heterozygous complex deletion of 12 bp and insertion of 12,423 bp. It is among the longest insertions reported in hereditary breast cancer genes as deciphered by WGS.

Conclusion: Detection of the germline insertions enabled better-informed patient management and family member testing.

Grants: Dr. Ellen Li Charitable Foundation; Kerry Kuok Foundation; Hong Kong Hereditary Breast Cancer Family Registry

Conflict of Interest: None declared

EP01.026 Two cases of PTEN Hamartoma Tumor Syndrome caused by novel variants in the PTEN gene

Trayan Delchev ¹, Tsvetina Veleva¹, Maria Sredkova-Ruskova¹, Nevyana Ivanova², Kalina Mihova², Radka Kaneva², Daniela Avdjieva-Tzavella¹

¹University Children Hospital - SBALDB, Clinical Geneitcs, Sofia, Bulgaria; ²Molecular Medicine Center, Genome Diagsnostics Laboratory, Sofia, Bulgaria

Background: PTEN hamartoma tumor syndrome (PHTS) includes Cowden syndrome (CS), Bannayan-Riley-Ruvalcaba syndrome (BRRS), PTEN-related Proteus syndrome (PS), and PTEN-related Proteus-like syndrome.

PTEN (Phosphatase And Tensin Homolog) is a multi-functional tumor suppressor that is very commonly lost in human cancer. The protein encoded by this gene is a phosphatidylinositol-3,4,5-trisphosphate 3-phosphatase. It contains a tensin like domain as well as a catalytic domain. Unlike most of the protein tyrosine phosphatases, this protein preferentially dephosphorylates phosphoinositide substrates. It negatively regulates intracellular levels of phosphatidylinositol-3,4,5-trisphosphate and functions as a tumor suppressor by negatively regulating AKT/PKB signaling pathway.

We present two cases of PHTS. Case A – a 17-year-old female patient with macrocephaly, psychomotor regress, epileptic seizures, polymicrogyria and atypical autism. Case B, a 1-year-old male, who presented with psychomotor delay, muscle hypotonia, failure to thrive and macrocephaly.

Methods: Conventional cytogenetic assay, MLPA for microdeletions, subtelomeric deletions and duplications, as well as metabolic screening showed normal results in both cases. Subsequently we employed Whole Exome Sequencing in case A and B.

Results: In case A we found a novel, heterozygous, likely pathogenic variant c.158T>C (p.Val53Ala) in PTEN.

In case B we established a novel, heterozygous, likely pathogenic variant c.755A>G p.(Asp252Gly) in PTEN.

Conclusion: Two novel PTEN variants were found in two unrelated patients, who both manifested features of Cowden syndrome. These findings reaffirm the genetic and phenotypic variability of PHTS and expand our understanding of this rare condition.

Grants: N/A

Conflict of Interest: None declared

EP01.027 Gene expression profiling in porocarcinoma indicates heterogeneous tumour development and substantiates poromas as precursor lesions

Svenja Holst¹, Friedegund Meier², Jörg Otte³, Patrick Petzsch⁴, Julia Reifenberger⁵, Thorsten Wachtmeister⁴, Dana Westphal², Mirjana Ziemer⁶, Wasko Wruck³, James Adjaye³, Regina C. Betz⁷, Harald Surowy¹, Arno Ruetten⁸, Silke Redler ¹

¹University Hospital Düsseldorf, Institute of Human Genetics, Düsseldorf, Germany; ²University Hospital Dresden GmbH, Department of Dermatology, Dresden, Germany; ³University Hospital Düsseldorf, Institute for Stem Cell Research and Regenerative Medicine, Düsseldorf, Germany; ⁴Heinrich Heine University Düsseldorf, Biological and Medical Research Centre (BMFZ), Düsseldorf, Germany; ⁵University Hospital Düsseldorf, Department of Dermatology, Düsseldorf; ⁶University Medical Center Leipzig, Department of Dermatology, Leipzig; ⁷University of Bonn, Institute of Human, Bonn, Germany; ⁸Dermatopathology, Bodensee, Dermatopathology, Bodensee, Friedrichshafen

Background: Malignant sweat gland tumours are rare, with the most common form being eccrine porocarcinoma (EP). Its benign counterpart is the eccrine poroma (EPO). Approximately 18% of EPO cases transit to EP, suggesting that EPOs represent a transitional state in EP development.

Objectives: Generate in depth insights into EP biology via comprehensive analysis of tumour-specific gene expression properties. Generate insights into potential drivers of malignant transformation from EPO to EP.

Methods: Transcriptome profiling of 23 FFPE samples of primary EP and available SHT. Genome-wide transcriptome analysis in an exploratory cohort of 10 EPs. Validation via qPCR in an independent cohort of the remaining 13 primary EPs. All findings from the EP samples were then tested in a cohort of 17 EPOs.

Results: Transcriptome profiling revealed an overall diversity in gene expression, and indicated the existence of biologically heterogeneous sub-entities. Hierarchical clustering identified two key matrisomal clusters, which probably represent two major EP tumour subtypes. In both clusters, widespread gene downregulation was observed in EP tumours compared to SHT. Expression levels of CD74, NDGR1, SRRM2, ANXA2, and NOP53 showed a stepwise-reduction in expression from SHT via EPO to EP, thus supporting the hypothesis that EPO represents a transitional state in EP development. Besides providing further evidence implicating the p53 axis and the EGFR pathway, our results suggest new mechanisms of tumorigenesis, including the VEGF/VEGFR axis and the Endothelin pathway.

Conclusions: Larger samples are warranted to generate deeper insights into intra- and inter tumour heterogeneity.

Conflict of Interest: Svenja Holst: None declared, Friedegund Meier: None declared, Jörg Otte: None declared, Patrick Petzsch: None declared, Julia Reifenberger: None declared, Thorsten Wachtmeister: None declared, Dana Westphal: None declared, Mirjana Ziemer: None declared, Wasko Wruck: None declared, James Adjaye: None declared, Regina C. Betz: None declared, Harald Surowy: None declared, Arno Ruetten: None declared, Silke Redler Wilhelm Sander-Stiftung (Nr. 2015.042.1)

EP01.028 Blocking LSD1 and GCN5 enhances retinoid therapy in high RARA AML

Faezeh Ghazvini Zadegan ¹, Franziska Fiedler¹, Tino Schenk¹

¹Jena University Hospital, Jena, Germany

Objectives: Acute Myeloid Leukemia (AML) is characterized by a blockage in myeloid cell differentiation, primarily regulated by retinoic acid receptor transcription factors, including RARA, RARB, and RARG. While RARA is necessary for the induction of granulocytic differentiation, RARG plays a central role in maintaining the self-renewal of hematopoietic stem cells. To prevent the activation of RARG by ATRA, specific RARA-agonists, such as Am80, have been developed. Our results indicate that substituting ATRA with Am80, which specifically targets RARA, produces remarkably similar outcomes.

Methods: A promising approach involves using Am80, a specific RARA agonist, in combination with LSD1 and GCN5 inhibitors. The study conducted experiments on both AML cell lines and primary AML samples to investigate the effects of ATRA, Am80, and the inhibitors on myeloid differentiation and treatment response.

Results: The findings revealed that AML cell lines with higher RARA expression showed improved myeloid differentiation, including increased CD11b expression and reduced CD117 levels, with ATRA or Am80, and combining these retinoids with LSD1 and GCN5 inhibitors enhanced the response. In primary AML samples, a significant proportion exhibited positive responses, correlating with higher RARA levels, indicating better outcomes. The research underscored the importance of low LSD1 and GCN5 levels for RARA activity and myeloid differentiation.

Conclusion: The combination of Am80, LSD1 inhibitors, and GCN5 inhibitors holds promise in overcoming ATRA resistance in non-APL AML, particularly in cases with high RARA expression and low levels of LSD1 and GCN5. This approach presents potential for improved treatment responses, necessitating further clinical investigation. Grant: SCHE1909/2–3.

Conflict of Interest: Faezeh Ghazvini Zadegan SCHE1909/2–3, Franziska Fiedler: None declared, Tino Schenk SCHE1909/2–3, Universitätsklinikum Jena

EP01.030 BRCA1 mutations in family members with breast cancer history

Merita Xhetani ¹, Albina Hasa², Blerta Laze³

¹University of Tirana, Faculty of Natural Sciences, Department of Biology and Center for Molecular Diagnostics and Genetic Research, Tirana, Albania; ²University of Tirana, Faculty of Natural Sciences, Department of Biology, Tirana, Albania; ³University of Vlora “Ismail Qemali”, Department of Biology, Vlora, Albania

Consortium: NanoAlb

Background/Objectives: Mutations in BRCA1 and BRCA2 genes are considered with a potential increased risk for developing early-onset breast cancer and familial ovarian cancer. The incidence of early-onset breast cancer depends largely on the type of mutation that causes it. This type of breast cancer follows an autosomal dominant inheritance pattern and tends to present as an early, high-intensity, bilateral form of the disease. We aim to investigate the BRCA 1 gene in a family with a breast cancer history.

Methods: Here we present a family study of 6 members with breast cancer history for detection of deletions or duplications in the human BRCA1 gene in genomic DNA isolated from human peripheral whole blood specimens. Copy Number Variations (CNVs) were detected by MLPA using P002 BRCA1 probemix and confirmation test with P087. Coffalyser.Net software was used for fragment analysis.

Results: We detected in all family members the heterozygous 40 nt deletion in BRCA1 exon 11. Two women age 32 and 35 yo (siblings) both without breast cancer clinical outcome, one women is 68 yo (mother) with early-onset breast cancer, her sister 58 years old diagnosed with breast cancer, mastectomy performed. Two other women (age 28 and 33 yo) first degree relatives, were diagnosed with breast cancer, has also heterozygous 40 nt deletion in BRCA1 exon 11.

Conclusions: Analysis of BRCA1 cancer families revealed a correlation between the mutation site and the relative risk of breast cancer development. We highlight the importance of screening for BRCA genes mutations in hereditary breast cancer.

Conflict of Interest: None declared

EP01.033 Advanced molecular diagnosis for type X adenomatous polyposis.

Beatriz Hidalgo Calero ¹, José Manuel Sánchez Zapardiel¹, Sonia Rodriguez Novoa², Montserrat de Miguel¹, Ricardo Blázquez Martín², Marina Ibáñez Vizcaíno¹, Francisca Luengo Sainz de Baranda¹, Victoria Carrero Blázquez¹, Ainhoa Almeida Santiago², Anna Esteve-Codina³, Luis Robles Díaz⁴

¹Hospital Universitario 12 de Octubre, Hereditary cancer laboratory, Madrid, Spain; ²Instituto de investigación sanitaria hospital 12 de Octubre (imas12), Hereditary cancer laboratory, Madrid, Spain; ³Centre Nacional d’Analisi Genòmica (CNAG), Barcelona, Spain; ⁴Hospital Universitario 12 de Octubre, Medical Oncology, Madrid, Spain

Background/Objectives: Adenomatous polyposis (AP) are diseases that predispose to develop tens to hundreds colorectal adenomatous polyps and thereby increase the risk of colorectal cancer. Many cases within hereditary cancer syndromes, are explained by the presence of deleterous variants in genes such as APC and MUTYH, among others. AP without identified mutation are called X adenomatous polyposis (XAP).

We performed multigenic NGS panel, targeting several genes involved in AP in 40 XAP cases. The unresolved XAP cases will be studied through a RNAseq approach.

The present Project will evaluate the relevance of implementing advanced molecular diagnosis through multigenic panels and RNAseq transcriptome sequencing in XAP cases.

Methods: NGS was performed using the Hereditary Cancer Solution kit (Sophia Genetics) and sequenced on the NextSeq platform (Illumina). Results were analyzed using Sophia-DDM software.

Detection of aberrant gene expression, aberrant splicing and monoallelic expression events in RNA sequencing data was carried out following DROP protocol. Also, fusion detection was carried out with starfusion

Results:

Conclusion: The use of multigenic panels in the study of XAP can improve diagnostic performance. RNAseq analysis as a second-line studies can help in the interpretation and classification of variants.

Grants: Project “PI19/00340” to L.R., funded by Instituto de Salud Carlos III (ISCIII) and co-funded by the European Union.

Conflict of Interest: None declared

EP01.034 Identification of Pathogenic BRCA1/BRCA2 Variants in Hereditary Breast Cancer Patients: Initial In-House NGS Results from the Northern Region of Morocco

Jaouhara MAAMAR ^1;2, Rajaa Chahboune¹, Ouafae Kaissi², Hasna Hamdaoui², Badreddine Nouadi², Fatima Zahra Laarabi^1;2, Houda Jelti^1;2, Sabrine Bouressas^1;2, Abdelhaq Lamaibdel^1;2, Oussama Kettani², Rabie Rahhali³, Nabila Sellal^4;5, Mounia Amzerin^3;4, Mohamed El Hfid^4;5, Fatima Zahra El m’rabet^3;4, Afaf Lamzouri^1;2

¹Life and Health Sciences Laboratory, Faculty of Medicine and Pharmacy of Tangier, Abdelmalek Essaâdi University, Morocco; ²Department of Medical Genetics and Oncogenetics, Mohammed VI University Hospital, Tangier, Morocco; ³Department of Medical Oncology, Mohammed VI University Hospital, Tangier, Morocco; ⁴Faculty of Medicine and Pharmacy of Tangier, Abdelmalek Essaâdi University, Morocco; ⁵Department of Radiotherapy, Mohammed VI University Hospital, Tangier, Morocco

Background/Objectives: Hereditary breast cancer (HBC) is characterized by the notable likelihood of being inherited across generations. Next-generation sequencing (NGS) has become an essential tool in the identification of pathogenic mutations within the responsible genes. The majority of HBC cases are associated with genetic mutations in the BRCA1/BRCA2 genes. With the establishment of the NGS platform in the northern region of Morocco, our study focuses on evaluating the mutational status of these two genes in women from this area, who present with suspected hereditary predisposition to breast cancer.

Methods: To achieve this objective, a targeted NGS panel focusing on BRCA1/2 genes was applied. This study was conducted with a cohort of eight patients from the northern region of Morocco, all of whom had undergone oncogenetic consultation. These women had been diagnosed with breast cancer and were deemed to be at significant hereditary risk.

Results: Among the 8 patients with HBC, 3 had pathogenic variants detected in BRCA1/2 genes (more than one-third). The identified mutations included c.798_799del (p.Ser267fs) and c.5309G>T (p.Gly1770Val) in the BRCA1 gene, as well as the c.1310_1313del (p.Lys437fs) mutation in the BRCA2 gene.

Conclusion: Our study emphasizes the high prevalence of BRCA mutations among patients with HBC, underscoring the necessity to establish at least one NGS platform in the Northern region of Morocco. This approach facilitates personalized management for these patients and enables presymptomatic diagnosis for at-risk family members.

Conflict of Interest: None declared

EP01.035 Pediatric cancer predisposition syndromes (pCPS) in an acute lymphoblastic leukemia cohort with no familial cancer history

Clara Venegas ^1;2;3, Elisa Izquierdo^1;2;3, Maria Pilar Casares¹, Beatriz Ruz^1;2;3, Alicia de Pablo¹, Elixabet Lopez-Lopez⁴, Angela Gutierrez-Camino⁵, Antonio Pérez^2;3;6, Alvaro Martinez¹, ADELA ESCUDERO^1;2;3

¹Institute of Medical and Molecular Genetics (INGEMM), La Paz University Hospital, Madrid, Spain; ²Hospital La Paz Institute for Health Research - IdiPAZ, Madrid, Spain; ³Spanish National Cancer Research Centre (CNIO), Madrid, Spain; ⁴IIS Biobizkaia, Biochemistry & Molecular Biology, Barakaldo, Spain; ⁵IIS Biobizkaia, Pediatric Oncology, Barakaldo, Spain; ⁶Paediatric Haemato-Oncology Department, La Paz University Hospital, Madrid, Spain

Background/Objectives: Acute leukemia is the most frequent neoplasm in childhood. Although its etiology is largely unknown, genetic factors predisposing to childhood acute leukemia development have been described in 4-5% of patients. The identification of these pediatric cancer predisposition syndromes (pCPS) is crucial for the correct patient management and follow-up; however, most of them do not have recognizable clinical signs leading to underdiagnosis. This study aims to improve the diagnosis of pCPS associated with acute lymphoblastic leukemia (ALL) using a custom NGS panel.

Methods: Three hundred and fifteen patients diagnosed with ALL between 2014 and 2023, lacking familial cancer history, were sequenced using a hybridization customized NGS panel (Mut4Child) targeting 352 genes associated with pCPS. Bioinformatics analysis, classification, and interpretation of SNVs, indels and CNVs was performed in 264 patients using a tailor-made pipeline, VarSeq tool (Golden Helix), and the ACMG guidelines.

Results: The findings revealed 13 germline SNVs/CNVs classified as pathogenic or likely pathogenic (4.9%) in PAX5, TP53, MLH1, BRCA1, BRCA2, CHEK2, TSC2, BARD1 genes. These results allowed the diagnosis of 9 pCPS (3.4%), one of them predisposing exclusively to leukemia and the others to both leukemia and solid tumors.

Conclusion: These findings facilitated genetic counseling and individualized follow-up for patients based on their genetic diagnosis. Importantly, this analysis demonstrated the feasibility of implementing an NGS custom panel and in-house bioinformatics pipelines for the diagnosis of pCPS associated with ALL in routine clinical practice and highlights the relevance of the clinical management and follow-up of patients and their families.

Grants: Cris-Cancer and PMP21/00073.

Conflict of Interest: None declared

EP01.036 Genomic exploration of small renal masses in Lithuanian cohort

Ieva Vaicekauskaitė^1;2, Raimonda Kubiliūtė^1;2, Kristina Žukauskaitė^1;2, Algirdas Žalimas^1;2, Albertas Ulys¹, Rasa Sabaliauskaite^1;2, Sonata Jarmalaite ^1;2

¹National Cancer Institute, Vilnius, Lithuania; ²Vilnius University Life Sciences Centre, Institute of Biosciences, Vilnius, Lithuania

Background/Objectives: Small Renal Masses (SRMs) are a heterogeneous group of kidney lesions made up of about 20% of benign lesions, and 80% renal cell carcinoma (RCC) cases. 25% of RCCs are aggressive tumors. Currently, there are no reliable biomarkers for SRMs. The aim of the study was to explore the genomic landscape of SRMs to identify possible diagnostic and prognostic genetic biomarkers.

Methods: 52 SRMs biopsies (38 RCC and 14 benign) collected at the National Cancer Institute of Lithuania were first analyzed for common CHEK2 mutations by qPCR and then by targeted-next generation sequencing encompassing an expanded hotspot mutation gene panel made up of 50 commonly altered genes in cancer. The NGS was conducted on the Ion Torrent platform.

Results: Non-synonymous alterations were detected in 78% of SRMs in 16/51 genes. The presence of mutations listed as pathogenic in ClinVar database correlated with smaller tumor volume (p = 0.02). Mutations in KRAS, VHL, HNF1A, TP53, and ATM genes were predominantly detected in RCC cases rather than benign tumors. Together with BMI and artery hypertension status these mutations were predictive for RCC and rapidly growing tumors (AUC = 0.78 and AUC = 0.63 respectively).

Conclusion: This pilot study of Lithuanian patients with SRMs gave unique insights into the mutational landscape of these tumors. Mutational analysis of SRMs may provide an avenue for detection and prognosis of RCC cases.

Conflict of Interest: None declared

EP01.037 Exploring apoptotic responses and oxidative stress regulation by curcumin and a novel curcumin analogue (B3) in glioblastoma: Insights for therapeutic strategies

varol güler ¹, Funda Özkök², Başak Günçer³, Sacide Pehlivan⁴

¹Institute of Graduate Studies in Health Sciences, Istanbul University, Medical Biology, istanbul, Türkyie; ²Istanbul University-Cerrahpasa, Department of Chemistry, İstanbul, Türkyie; ³Istanbul Faculty of Medicine, Istanbul University, Department of Biophysics, İstanbul, Türkyie; ⁴Istanbul Faculty of Medicine, Istanbul University, Medical Biology, İstanbul, Türkyie

Background / Objectives: Glioblastoma multiforme (GBM) presents a significant oncological challenge due to its aggressiveness and limited treatment options. This study investigates B3, a curcumin derivative, as a potential antitumor agent against U87 glioblastoma cells. Objectives encompass understanding B3’s molecular mechanisms in influencing cell proliferation, migration, apoptosis, and its synergistic potential with Temozolomide (TMZ).

Materials and Methods: We evaluated the impact of B3 on U87 cells through various assays. Proliferation was assessed using the MTT assay, and the DCFH-DA assay measured reactive oxygen species (ROS) elevation. Apoptosis induction was studied through Acridine Orange/Propidium Iodide dual staining, while migration was analyzed using the wound healing assay. Quantitative real-time PCR was employed to examine changes in RNA expression.

Results: B3 treatment demonstrated a significant inhibition of U87 cell proliferation, accompanied by elevated ROS levels. The compound exhibited anti-migratory effects, triggered apoptosis, and showed synergy with TMZ, rendering U87 cells less resistant. Molecular analysis revealed downregulation of cancer markers (Ki67, c-MYC, CYDD1) and upregulation of the tumor suppressor p53 at the RNA level. Additionally, migration-related genes (NFκB, MMP-2, MMP-9) showed decreased expression in B3-treated cells. Apoptosis pathway activation was confirmed with increased caspase-3 expression and an elevated Bax/Bcl-2 ratio at the RNA level.

Conclusion: B3 shows strong anti-tumor effects, impacting proliferation, migration, and apoptosis. Combined with TMZ, it enhances therapeutic potential. These results highlight B3’s promise for cancer treatment, urging further investigation into its mechanisms for translation to clinical use.

Grants: This study was supported by the Scientific Research Project Coordination Unit of Istanbul University under Grant [number 39220].

Conflict of Interest: None declared

EP01.038 Whole exome sequencing in a family with familial colorectal cancer

Hafdis T Helgadottir ^1;2, Pär Lundin³, Annika Lindblom^1;2

¹Karolinska University Hospital, Clinical genetics and genomics, Stockholm, Sweden; ²Karolinska Institutet, Department of Molecular Medicine and Surgery, Stockholm, Sweden; ³Stockholm University, Science for Life Laboratory, Department of Biochemistry and Biophysics, Stockholm, Sweden

Background/Objectives: Colorectal cancer (CRC) is a common cancer type with environmental and genetic risk factors. Family history is a strong risk factor where close relatives of CRC patients have an increased risk of CRC. Here, a family with several CRC patients was exome sequenced to identify genetic risk factors contributing to the disease.

Methods: Exome sequencing was performed on a large Swedish family with several individuals with CRC and polyps. In total, 16 family members from three generations were sequenced where seven had CRC. The sequencing data was analysed and downstream filtering was performed to identify candidate variants and genes, where only rare coding variants with severe in silico prediction were included.

Results: Several variants with allele frequency in gnomAD<0.1% and CADD > 20 were found to be shared by at least six of the CRC patients. All variants could be identified in at least one of the other family members, most of whom had been diagnosed with polyps. When less stringent filtering with gnomAD<1% and CADD > 20 was used, additional variants were found to be shared by at least six of the CRC patients. The most notable candidate variant was a frameshift variant in the AKR1B10 gene, but several additional candidate genes were identified.

Conclusion: In the search for a genetic cause for CRC in the family, several high-risk variants were found that could contribute to the disease.

Grants: The Nilsson-Ehle Endowments, Cancerfonden

Conflict of Interest: None declared

EP01.039 The effect of transcriptomic annotations in breast cancer DGE study

Magda Mielczarek ¹, Bartosz Czech¹, Rafał Stępień¹, Joanna Szyda¹

¹Wrocław University of Environmental and Life Sciences, Wrocław, Poland

Background/Objectives: Gene expression profiling is crucial for understanding breast cancer biology and treatment individualization. The aim of this study was to elucidate transcriptome annotation effect on differential gene expression (DGE) and breast cancer survival prognosis.

Methods: DGE analyses were performed for MCF7 breast cancer (case) and normal tissues (control). The pipeline consisted of quality control, data editing, expression quantification, and DGE analysis. Two quantified transcripts expression outputs were used to apply four approaches defining DGE between (A1) case and control samples quantified based on GRCh37 assembly, (A2) case and control on GRCh38, (A3) case on GRCh37 and case on GRCh38 and (A4) control on GRCh37 and control on GRCh38.

Results: Identical Hallmark pathways resulted in Gene Set Enrichment Analysis for both A1 and A2, except Pancreas beta cells presented in A1 only. The Kyoto Encyclopedia of Genes and Genomes pathways presented only in one approach involved: Melanoma and Prostate cancer (A1) and ABC transporters, Acute myeloid leukaemia, Glycerophospholipid and retinol metabolism, Hedgehog and p53 signalling (A2). PCA determined that the greatest variability (97%) was found between cancer and normal samples (A1, A2) and GRCh37 and GRCh38 annotations (A3). For A4 the variability determined by the annotations was lower (40%). The difference between the average expression of prognostic genes associated with survival in breast cancer (NADERI) between GRCh37 and GRCh38 was not statistically significant (P-value = 0.91).

Conclusion: The overall DGE outcomes were not identical between GRCh37 and GRCh38 annotations, however, the transcriptome annotation had no effect on genes realted to survival prognosis in breast cancer.

Grants: No grant.

Conflict of Interest: None declared

EP01.041 Comparison of morphological patterns and genetic markers in non-small-cell lung cancer (NSCLC)

Anzhelika Chegodar ¹, Natalia Bodunova¹, Natalia Oparina^1;2, Chinchi Babadzhanova^1;3, Larisa Tsapkova¹, Nikolay Karnaukhov¹, Ainash Altayeva⁴

¹Тhe Loginov Moscow Clinical Scientific Center, Moscow, Russian Federation; ²Petrovsky National Research Centre of Surgery Rams, Medical Genetic, Moscow, Russian Federation; ³City Clinical Oncological Hospital No. 1, Moscow, Russian Federation; ⁴Peoples’ Friendship University of Russia named after Patrice Lumumba, Moscow, Russian Federation

Background/Objectives: Histological types of NSCLC differ in genesis, genetic characteristics and clinical course, which affects the choice of treatment. We investigated histological types and several genetic markers of NSCLC.

Methods: Were examined FFPE tumor tissue samples of NSCLC from 182 patients. The patients’ ages were between 35 and 88 (median 66). There were 113 males and 69 females in our sample. Antibodies used for IHC studies: ALK(D5F3), PD-L1(SP263, SP142, 22C3), TTF-1(8G7G3/1), CK7 (SP52), p40 (BC28). RT-PCR was used to detect mutations in the EGFR (exons 18,19,20,21) and BRAF (V600E) genes. FISH identified ROS1 rearrangements.

Results: Histological types of NSCLC: adenocarcinoma - 161 (88.5%) cases, squamous cell carcinomas - 18 (10%) cases, NSCLC, NOS - 3 (1.5%) cases.

TTF-1 and CK7 positive adenocarcinomas were detected in 96/125 (77%) cases. All cases of squamous cell carcinomas were p40 positive. 44/121 (36%) adenocarcinomas and 10/121 (8%) squamous cell carcinomas were PD-L1 positive. 4/139 (2.8%) adenocarcinomas were ALK positive.

The BRAF mutation was detected in 5/125 (4%) adenocarcinomas.

42/161 (26%) adenocarcinomas had the EGFR mutation: 26 of Ex19del, 10 of L858R, 2 of Ex20ins, 1 of L861Q. 3 adenocarcinomas had double EGFR mutations: Ex19del and Ex20ins - 1; L861Q and G719X - 1; L858R and T790M - 1. ROS1 rearrangement - 2/132 (1.5%).

Conclusion: The EGFR L858R mutation was detected in lepidic and acinar types of adenocarcinomas only. A rare case of BRAF V600E and EGFR L858R co-mutation was discovered in G2 TTF-1 positive adenocarcinoma. The histological types and molecular genetic markers of NSCLC requires further in-depth study.

Conflict of Interest: None declared

EP01.042 PIK3CA expression as a prognostic factor for bladder cancer

Paulina Slawnikowska¹, Tomasz Konecki², Piotr Kutwin^2;3, Zbigniew Jablonowski², Agnieszka Zmysłowska¹, Edyta Borkowska ¹

¹Medical University of Lodz, Department of Clinical Genetics, Łódź, Poland; ²Medical University of Lodz, Ist Clinic of Urology, Łódź, Poland; ³

Background & Objectives: Bladder cancer is the sixth most common cancer in Poland among men.The PI3K/AKT/mTOR signaling pathway, responsible for cell survival, growth and proliferation is one of the main pathways of intracellular transmission. Its regulation involves the proteins encoded by PIK3CA gene, the altered expression of which may contribute to the development of cancer, including bladder cancer. The aim of the study was to evaluate the expression of the PIK3CA in bladder cancer and to determine its correlations with clinicopathological parameters.

Materials & Methods: Tumor sections from 189 patients (81% of tissues came from patients with non-invasive cases) were examined. The control group consisted of 21 cases of normal urothelium from patients without cancer diagnosis (histopathologically confirmed). Real-time PCR reactions were performed using two TaqMan probes for PIK3CA and GAPDH. Expressions were assessed using the double delta method.

Results: The analysis showed that reduced expression PIK3CA dominates in the non-invasive stage, while in the invasive stage more often overexpression is observed (p = 0,003). There was also a correlation between the malignancy potential and the level of PIK3CA expression in the non-invasive stage (p = 0.01). Moreover, patients with reduced expression had a chance to live longer (p = 0,0003) and also had a greater chance of not having progression (p = 0,026).

Conclusions: Reduced expression of PIK3CA contributes to a milder course of diseases, while increased expression promotes tumor infiltration and corelates with an unfavorable prognosis.

Conflict of Interest: None declared

EP01.043 SEHOP-PENCIL project: Current status of the diagnosis and management of pediatric cancer predisposition syndromes in Spain

Elisa Izquierdo^1;2, Clara Venegas^1;2, Beatriz Ruz^1;2, Antonio Pérez^1;3, Ana Patiño^4;5, Teresa Imizcoz⁶, Gorka Alkorta⁶, Elena Panizo⁷, Sergio Manresa⁸, Lucas Moreno Martin Retortillo^9;10, Adela Cañete¹¹, Maria Teresa Tormo¹², Josep Escriva¹², Piedad Alba Pavón¹³, Angela Gutierrez-Camino¹³, Olatz Villate¹³, Manuel Ramirez Orellana¹⁴, Ana Chamorro¹⁴, Cristina Saiz¹⁵, Andrea Vilaplana⁸, Asbleidy Carolina Torres⁸, Aroa Soriano⁸, Hector Salvador¹⁶, Laura Martí¹⁷, Diana Salinas-Chaparro¹⁷, Estela Carrasco López^18;19, ADELA ESCUDERO ^1;2

¹Hospital La Paz Institute for Health Research - IdiPAZ, Translational Research in Pediatric Oncology Hematopoietic Transplantation & Cell Therapy, Madrid, Spain; ²La Paz University Hospital, Institute of Medical and Molecular Genetics (INGEMM), Madrid, Spain; ³La Paz University Hospital, Department of Pediatric Hematology and Oncology, Madrid, Spain; ⁴University Clinic of Navarra, Department of Pediatrics and Medical Genomics Unit, Pamplona, Spain; ⁵Center for Applied Medical Research and IdiSNA (CIMA), Solid Tumor Program, Pamplona, Spain; ⁶University of Navarra, CIMA-LAB Diagnostics, Pamplona, Spain; ⁷University Clinic of Navarra, Department of Pediatrics, Madrid, Spain; ⁸Vall d’Hebron Research Institute, Universitat Autònoma de Barcelona., Personalised Medicine Programme, Childhood Cancer and Blood Disorders Research Group, Barcelona, Spain; ⁹Vall d’Hebron Research Institute, Universitat Autònoma de Barcelona., Childhood Cancer and Blood Disorders Research Group, Barcelona, Spain; ¹⁰Vall d’Hebron University Hospital, Pediatric Oncology and Hematology Department, Barcelona, Spain; ¹¹Hospital Universitari i Politècnic La Fe, Valencia, Spain; ¹²Instituto de Investigación Sanitaria La Fe, Valencia, Spain; ¹³Biobizkaia Health Research Institute, Pediatric Oncology Group, Barakaldo, Spain; ¹⁴Hospital Infantil Universitario Niño Jesús, Madrid, Spain; ¹⁵Fundación para la Investigación Biomédica del Hospital Infantil Universitario Niño Jesús., Madrid, Spain; ¹⁶Hospital Sant Joan de Déu, Pediatric Oncology Unit, Barcelona, Spain; ¹⁷Hospital Sant Joan de Déu, Genetic Unit, Barcelona, Spain; ¹⁸Vall d’Hebron Institute of Oncology, Herediatry Cancer Genetics, Barcelona, Spain; ¹⁹Vall d’Hebron University Hospital, Herediatry Cancer Genetics, Barcelona, Spain

Consortium: On behalf of the Group of Pediatric Cancer Predisposition Syndromes of SEHOP-PENCIL study- Personalised mEdicine for Cancer in children in Spain.

Background/Objectives: SEHOP-PENCIL is a nationwide project aiming to improve the genetic characterization of pediatric cancer in Spain. This includes optimizing the diagnosis of pediatric cancer predisposition syndromes (pCPS) caused by germline mutations detected in 10-12% of patients. pCPS identification is crucial to ensure effective patient management and personal and familial genetic counseling. However, the diagnosis of pCPS can be challenging as these patients are very heterogeneous and might not have a recognizable phenotype. This study aims to analyze the current status of pCPS diagnosis and management in Spain in order to improve the current diagnostic rate and to facilitate access to genetic testing and counselling by creating seven reference genomic centers focused on pCPS in Spain.

Methods: A national survey was conducted using the REDCap platform, targeting the oncohematology services of all SEHOP hospitals in Spain between 2022 and 2023.

Results: A total of 78% (35/45) Spanish centers completed the survey. Of these, 66% (23/35) perform germline genetic analyses related to pCPS. Jongmans/Ripperger/MIPOGG genetic testing criteria were followed by only 30% (7/23) of the centers in their clinical practice. The use of NGS panels was the most chosen technique by most centers, 52% (12/23). Surprisingly, less than 50% (11/23) referred patients and families to a genetic counseling unit.

Conclusion: The diagnosis and management of pCPS is already being implemented in some Spanish hospitals; however, it is necessary to standardise patient selection criteria, methodology and genetic counseling in order to offer a robust and uniform pCPS diagnosis across the country.

Grants: PMP21/00073.

Conflict of Interest: None declared

EP01.044 Improving the diagnosis of hereditary retinoblastoma using Mut4Child, a high depth NGS hybridization custom panel

Maribel Soler¹, Clara Venegas^1;2;3, Beatriz Ruz^1;2;3, Borja González¹, Virginia Contreras¹, Alicia de Pablo¹, Núria Rodríguez-Salas¹, Dolores Corral¹, Sonsoles San Roman¹, Jesus Peralta^1;2, Susana Noval^1;2, Antonio Pérez^1;2;3, ADELA ESCUDERO^1;2;3, Elisa Izquierdo ^1;2;3

¹La Paz University Hospital, Madrid, Spain; ²Hospital La Paz Institute for Health Research - IdiPAZ, Madrid, Spain; ³Spanish National Cancer Research Centre (CNIO), Madrid, Spain

Background/Objectives: Retinoblastoma is the most frequent childhood eye malignancy. This tumor type usually appears in the first three years of life, with an overall incidence of 1 in 14000 live births in Spain. Retinoblastoma can occur either as a hereditary or nonhereditary form, depending on whether patients are carriers of a RB1 germline variant. This study includes a cohort of 317 pediatric patients diagnosed of retinoblastoma in a national reference hospital in Spain between 1998 and 2023. Of those, 176/317 (59%) and 130/317 (41%) patients were diagnosed with unilateral and bilateral retinoblastoma, respectively. The average age of unilateral and bilateral patients was 15 and 9 months, respectively (IQR = 17).

Methods: Conventional methods, such as Sanger and MLPA were performed in 238 patients, whereas 79 patients were anlyzed using a NGS hybridization custom panel at high depth (Median: 834X).

Results: Hereditary retinoblastoma was confirmed in 115/238 patients (48.6%), in which a RB1 germline variant was detected (87% SNVs, 13% deletions). Of note, 11/79 (14%) genetic alterations were detected by the custom NGS approach panel in patients who could not had been diagnosed by conventional methods, including two translocations and nine germline mosaic variants.

Conclusion: These results demonstrate the use of a high depth NGS custom panel improved the identification of heritable retinoblastoma. In addition, this retrospective study highlights the need for an accurate RB1 germline variant detection in the routine care of a public hospital to improve diagnosis, management, and outcome of children with retinoblastoma and their families.

Grants: Spanish Association of Retinoblastoma (AER), Cris-Cancer and PMP21/00073

Conflict of Interest: None declared

EP01.045 Lynch syndrome: one disease with different phenotypes or several different diseases?

Laura Roht ^1;2, Piret Laidre², Mikk Tooming^1;3, Hanno Roomere³, Kadri Rekker³, Kadri Toome³, Olga Fjodorova³, Ülle Murumets³, Jaan Soplepmann^4;5, Katrin Ounap^1;2, Tiina Kahre^1;3

¹University of Tartu, Institute of Clinical Medicine, department of Clinical Genetics, Tartu, Estonia; ²Tartu University Hospital, Genetics and Personalized Medicine Clinic, Clinical Genetics, Tartu, Estonia; ³Tartu University Hospital, Genetics and Personalized Medicine Clinic, Laboratory Genetics, Tartu, Estonia; ⁴University of Tartu, Institute of Clinical Medicine, department of Hematology and Oncology, Tartu, Estonia; ⁵Tartu University Hospital, Surgery Clinic, Institute of Clinical Medicine, department of Hematology and Oncology, Tartu, Estonia

Background/Objectives: Lynch syndrome (LS) is the most frequent inherited colorectal cancer syndrome. The main cause of LS is the loss of function of DNA mismatch repair (MMR) genes. There are four key MMR genes: MLH1, MSH2, MSH6, PMS2, and one non-MMR gene (EPCAM). Recently, it has been hypothesized, that LS is not one disease, but encompasses different diseases.

Methods: We collected clinical data of 134 LS variant carriers during twelve years (2012-2023). There were 66 cancer patients and 68 healthy carriers of MMR genes disease-causing variants alone or in seven cases together with EPCAM gene deletion. Individuals were identified by Next-Generation Sequencing, using either Illumina’s TruSight Cancer (94 genes) or Trusight Hereditary Cancer (113 genes) panel. In case of family members, Sanger sequencing was used to detect the variant already known in the family.

Results: We identified 41 MLH1, 27 MSH2, 7 MSH2 + EPCAM deletion, 23 MSH6 and 36 PMS2 disease-causing variant carriers. In case of MLH1, MSH2 alone or together with EPCAM deletion and MSH6 pathogenic variants, colorectal cancer (CRC) or concurrent CRC only or together with other cancer types was the most common phenotype. The mean age of the first cancer diagnosis was 40.2, 49.2, 35.8 and 40.6 years, respectively. PMS2 mutated individuals had either CRC, breast or gynaecological cancers, the mean age of the first cancer was 54.5 years.

Conclusion: The phenotypic diversity of Lynch syndrome stems from the specific gene affected, causing variations in cancer risks, types, and mean ages of diagnosis.

Grants: PRG471

Conflict of Interest: None declared

EP01.046 Combining germline, tissue and liquid biopsy analysis by comprehensive genomic profiling to improve the yield of actionable variants in a real-world cancer cohort.

Irene Vanni¹, Lorenza Pastorino^1;2, Virginia Andreotti¹, Danila Comandini³, Giuseppe Fornarini³, Massimiliano Grassi³, Alberto Puccini³, Enrica Teresa Tanda^2;4, Alessandro Pastorino³, Valentino Martelli³, Luca Mastracci^5;6, Federica Grillo^5;6, Francesco Cabiddu⁶, Antonio Guadagno⁶, simona coco⁷, Eleonora Allavena², Francesca Barbero¹, William Bruno^1;2, Bruna Dalmasso¹, Sara Erika Bellomo⁸, Caterina Marchiò^8;9, Francesco Spagnolo^10;11, Stefania Sciallero³, Enrico Berrino^8;9, Paola Ghiorzo ^1;2

¹IRCCS Ospedale Policlinico San Martino, Genetica dei Tumori Rari, Genoa, Italy; ²University of Genoa, Department of Internal Medicine and Medical Specialties, Genoa, Italy; ³IRCCS Ospedale Policlinico San Martino, Oncologia Medica 1, Genoa, Italy; ⁴IRCCS Ospedale Policlinico San Martino, Oncologia Medica 2, Genoa, Italy; ⁵University of Genoa, Dipartimento di Scienze Chirurgiche e Diagnostica Integrata, Genoa, Italy; ⁶IRCCS Ospedale Policlinico San Martino, Anatomia Patologica, Genoa, Italy; ⁷IRCCS Ospedale Policlinico San Martino, Tumori Polmonari, Genoa, Italy; ⁸Oncological Institute Candiolo, FPO-IRCCS, Pathology Unit, Candiolo, Italy; ⁹University of Turin, Department of Medical Sciences, Torino, Italy; ¹⁰IRCCS Ospedale Policlinico San Martino, Oncologia Medica 2; ¹¹University of Genoa, Dipartimento di Scienze Chirurgiche e Diagnostica Integrata

Consortium: none

Background/Objectives: Comprehensive next-generation sequencing is widely used for precision oncology and precision prevention approaches. We aimed to determine the yield of actionable variants, the capacity to uncover hereditary predisposition and liquid biopsy appropriateness instead, or in addition to, tumor tissue analysis, in a real-world cohort of cancer patients, who may benefit the most from comprehensive genomic profiling.

Methods: Twenty-three patients were consecutively enrolled at our center, according to at least one of the following criteria: no available therapeutic options; long responding patients potentially fit for other therapies; rare tumor; suspected hereditary cancer; primary cancer with high metastatic potential; tumor of unknown primary origin. Matched germline/tumour/circulating-free DNA (cfDNA) profiling using the Hereditary Cancer Panel (germline) and the TruSight Oncology 500 panel (tissue/cfDNA) (Illumina) was performed. Variants were mapped by OncoKB and AMP/ASCO/CAP classification.

Results: The overall yield of actionable somatic and germline variants was 57% (13/23 patients), or 43.5%, excluding variants previously identified by routine testing. The accuracy of tumor/cfDNA germline-focused analysis was demonstrated by overlapping results of germline testing. Five germline variants in BRCA1, VHL, CHEK1, ATM genes would have been missed without extended genomic profiling. A previously undetected BRAF V600E mutation was emblematic of the clinical utility of this approach in a patient with a liver undifferentiated embryonal sarcoma responsive to BRAF/MEK inhibition.

Conclusion: Our study confirms the clinical relevance to perform extended parallel tumor DNA and cfDNA testing to broaden therapeutic options and to uncover hereditary predisposition following tumor sequencing in patient care.

Grants: Italian Ministry of Health-IRCCS Ospedale Policlinico San Martino, MUR-PRIN 2022.

Conflict of Interest: None declared

EP01.048 Familial leiomyosarcoma due to a pathogenic TP53 variant

Maria Hellberg ¹

¹, Department of Clinical Genetics, Pathology, and Molecular Diagnostics, Lund

Background/Objectives: Pathogenic variants in TP53 can cause Li-Fraumeni syndrome which is a condition that confers a high risk of many types of cancer. Sarcoma, breast cancer, adrenal cancer and brain cancer are considered the core cancers. Certain pathogenic variants in TP53 are known to confer a high risk of breast cancer but significantly less risk for other TP53-related cancers. Here we present a family with a cluster of four first degree relatives with leiomyosarcoma and one case of a malignant peripheral nerve sheath tumour (MPNST) but no other known malignancies.

Methods: Information regarding cancer diagnoses in the family was collected from family members. Germline whole genome sequencing was performed with DNA extracted from peripheral blood from one of the individuals with leiomyosarcoma. The data was filtered using a gene list consisting of four genes: FH, NF1, PTEN and TP53.

Results: Four family members with leiomyosarcoma were confirmed from the Swedish National Cancer Register. One of the individuals with leiomyosarcoma had previously been diagnosed with a MPNST. A pathogenic frameshift variant was detected in TP53 c.330del;p.(Leu111Trpfs*12) NM_000546.6 in the bloodsample from the individual with leiomyosarcoma and MPNST.

Conclusion: In this unusual family with four closely related individuals who had leiomyosarcoma but no other TP53-associated cancers a pathogenic TP53-variant was detected.

Conflict of Interest: Maria Hellberg Employed full time at the Department of Clinical Genetics, Pathology, and Molecular Diagnostics in Lund

EP01.049 Development of a targeted RNA sequencing panel to reclassify VUS in cancer predisposing genes

Larissa Dias de Souza¹, Alexandre Defelicibus², Diogo Soares³, José Rocha³, Daniele Paixão Pereira³, Dirce Carraro¹, Giovana Torrezan ¹

¹A.C.Camargo Cancer Center, Clinical and Functional Genomics Group; ²A.C.Camargo Cancer Center, Bioinformatics facility; ³A.C.Camargo Cancer Center, Oncogenetics Department

Genetic tests often yield uncertain results, particularly with the identification of variants of unknown clinical significance (VUS), which poses a dilemma in clinical practice. This study aims to validate a set of computational and experimental tools to prioritize and reclassify predicted spliceogenic or non-coding VUS. Rare exonic and non-coding variants from Brazilian cancer patients who underwent DNA multigene panel testing were selected for this purpose. In silico tools (MaxEntScan, dbscSNV, SpliceAI and UTRannotator) were used for variant prioritization and targeted RNA-sequencing (RNA-cap) of 27 cancer predisposition genes was performed on predicted spliceogenic variants. Raw genetic test data from 2,082 patients revealed 1,954 rare non-benign or unclassified variants; among these 148 are predicted spliceogenic and 32 of impacting the 5’ UTR. We performed RNA-cap sequencing on 37 samples: 14 with Pathogenic/Likely Pathogenic (P/LP) variants, 11 with VUS and 12 negative controls. Splicing profiles were analyzed and compared using the rMATS software. Our findings validated the splicing effect in 12 out of 14 patients with P/LP variants. For the VUS group, only one significant splicing event was detected out of the total cases (10/11), suggesting that most may be downgraded to Benign/Likely Benign variants. This study has established criteria and an automated pipeline for selecting rare variants predicted to alter splicing, which contributes to standardized tools for differential splicing analysis. The anticipated data outcomes are aimed at improving clinical interpretation of hereditary cancer-related variants, thereby enhancing diagnostic accuracy and reducing risks for cancer patients.

Conflict of Interest: None declared

EP01.050 The most frequent germline pathogenic variants in Russian patients with breast cancer: whole genome sequencing results

Maria Makarova ¹, Marina Nemtsova¹, Maria Byakhova², Anastasia Danishevich³, Denis Chernevskiy¹, Olesya Sagaidak¹, Maria Vorontsova⁴, Maxim Belenikin¹, Anastasia Krinitsina¹, Aleksey Antonenko¹, Anna Semenova², Natalia Bodunova³, Igor Khatkov³, Vsevolod Galkin²

¹EVOGEN LLC, Moscow, Russian Federation; ²City Clinical Oncological Hospital №1, Moscow, Russian Federation; ³Тhe Loginov Moscow Clinical Scientific Center, Moscow, Russian Federation; ⁴Lomonosov Moscow State University, Moscow, Russian Federation

Objective: To analyze pathogenic variants (PV) in breast cancer patients (BC) and propose a novel target screening panel for individuals with suspected hereditary cancer syndromes (HCS).

Methods: 1514 patients with BC and suspected HCS (patient group, PG) and 5163 individuals without cancer (control group, CG) whole genome sequencing (WGS) were performed.

WGS: 30х, DNBseq-T7, EVOGEN LLC; bioinformatics analysis accelerators: EVA Pro, EVOGEN, Russia; MegaBOLT, MGI, China.

Results: 324 pathogenic variants (PV) in cancer-associated genes in 1514 BC patients were identified (21.4%).

Using PG ang CG WGS results 21 genes were determined for the most effective NGS-panel for suspected hereditary BC in Russia including ATM, BARD1, BRCA1, BRCA2, BRIP1, CDH1, CHEK2, FANCC, FANCM, FANCA, FANCI, FANCL, MLH1, MSH2, MSH6, PMS2, PALB2, PTEN, RAD51C, RAD51D, TP53. A significant association with BC was established for PV in ATM (p < 0.001), BARD1 (p = 0.003), BRCA1 (p < 0.001), BRCA2 (p < 0.001), BRIP1 (p = 0.013), MLH1 (p = 0.010), PALB2 (p < 0.001), TP53 (p < 0.001).

The most frequent PV in the PG were identified (hg38): BRCA1 (NM_007300.4) – c.181T>G, c.1961del, c.5329dup, c.4327C>T, c.5215+1G > T; BRCA2 (NM_000059.4) – c.7007G>A, c.658_659del; ATM (NM_000051.4)c.8147T>C; PALB2 (NM_024675.4) – c.172_175del, c.509_510del. Variants CHEK2 (NM_007194.4) c.444+1G > A, c.1100del, NBN (NM_002485.5) c.657_661del, did not demonstrate significant frequency differences between the PG and CG.

Conclusion: WGS results make it possible to assess the structure and frequency of the PV in BC patients and propose novel screening PCR- and NGS-based target panels.

Grants: Moscow City Health Department financial support.

Conflict of Interest: None declared

EP01.051 Expanding the possibilities for diagnosis, staging, prognosis and follow-up of patients with neuroblastoma through molecular genetic research

Gergana Stancheva ¹, Ivan Boronsuzov², Veronika Petkova¹, Kalina Mihova¹, Margarita Kamenova³, Prof. Dobrin Konstantinov^2;4, Maya Yordanova^2;4, Radka Kaneva¹

¹Medical University of Sofia, Medical Chemistry and Biochemistry, Molecular Medicine Center, Sofia, Bulgaria; ²University Hospital “Queen Johanna-ISUL”, Pediatric Oncohematology Department, Sofia, Bulgaria; ³University Multiprofile Hospital for Active Treatment and Emergency Medicine “N.I.Pirogov”, Department of Pathology, Sofia, Bulgaria; ⁴Medical University of Sofia, Department of Pediatrics

Background/Objectives: The relevance of the research topic is determined by the high incidence of paediatric solid tumours, their great malignancy potential, the high mortality rates and the possibility to offer the patients reliable therapy when timely and accurate diagnosis is made. Neuroblastoma(NB) is the most common solid extracranial tumor in children with incidence in Europe of 10/1 000 000. Approximately 40% of NB patients have high-risk disease with dissemination in bone marrow(BM), bone, distant lymph nodes, liver, and other organs. PCR-based detection of minimal residual disease(MRD) in NB patients can be used for initial staging and monitoring therapy response in BM and peripheral blood(PB).

Methods: The expression of TH and PHOX2B mRNA was analysed in materials from 11 patients(19BM, 11PB) and 7 healthy people by real-time quantification PCR(RQ-PCR).B2M was used as endogenous control gene.

Results: The expression of TH was confirmed in 14 of 19 BM and in 7 of 11 PB samples. The PHOX2B expression was detected in 15 of 19 BM and in 6 of 11 PB samples. Both BM and PB materials have been collected from nine patients. Combination of TH and PHOX2B expression in BM confirmed dissemination in 89% of the patients. It is interesting to note that in patients with metastatic disease, the studied markers are also found in the PB, which shows the potential of the method as non-invasive and gentle.

Conclusion:The combination of TH and PHOX2B expression has been identified as more sensitive and specific marker for monitoring MRD in NB than the routine investigations.

Grants: MES/Contracts D01-302/17.12.2021,D01-165/28.07.2022;MES,NSF/Contract-KP-06-N63/4-13.12.2022

Conflict of Interest: None declared

EP01.052 Eventual founder effect of a mutation in MLH1 gene within a large Tunisian family with CMMRD

Rasene Gereisha ¹, Gabteni SANA^1;2, Ahlem Achour^1;2, Firas Akrout³, Rym Meddeb^1;2, Carli Tops⁴, Richard Gallon⁵, Mariem Ben Rekaya^6;7, Soumaya Rammeh^6;7, Ridha Chkili³, Nada Mansouri⁸, Neila Belguith^1;9, Ridha Mrad^1;2

¹Charles Nicolle Hospital, Department of Congenital and Hereditary Diseases, Tunis, Tunisia; ²Faculty of Medicine of Tunis, Laboratory of Human Genetics, Tunis, Tunisia; ³Military Hospital of Tunis, Department of Neurosurgery, Tunis, Tunisia; ⁴Leiden University Medical Center, Department of Clinical Genetics, Leiden, Netherlands; ⁵Faculty of Medical Sciences, Newcastle University, Translational and Clinical Research Institute, Newcastle upon Tyne, United Kingdom; ⁶Charles Nicolle Hospital, Department of Pathology, Tunis, Tunisia; ⁷Faculty of Medicine of Tunis, Research Unit of Onco-theranostic Biomarkers UR17ES15, Tunis, Tunisia; ⁸Military Hospital of Tunis, Department of Pathology, Tunis, Tunisia; ⁹Faculty of Medicine of Sfax, Laboratory of Human Molecular Genetics, Tunis, Tunisia

Background/Objectives: Constitutional mismatch repair deficiency (CMMRD) syndrome is a rare autosomal recessive genetic disorder caused by biallelic germline mutations in one of the mismatch repair genes.

Our study explores distinctive clinical, pathological, and genetic aspects of CMMRD, emphasizing the importance of comprehensive management for families meeting CMMRD criteria.

Methods: A 38 years old tunisian patient was referred to our genetic consultation for a suspicion of cancer predisposition syndrome. He presented at the age of 18 a colonic oligopolyposis and adenosquamous carcinoma of the caecum. He later developed an undifferentiated carcinoma of the parotid, an astrocytoma, and an ampulla of Vater adenocarcinoma.

Family history was significant for early-onset colorectal cancer and Lynch syndrome.

A CRC predisposition gene panel including MMR genes was performed.

Results: Molecular analysis identified a homozygous germline missense variant in the MLH1 gene: c.1918C>A; p.(Pro640Thr).

Functional impact assessment, immunohistochemistry and the detection of increased cMSI confirmed its pathogenicity.

The variant was also identified in a heterozygous state in the eldest brother and his non-consanguineous wife who also presented a family history of colorectal cancer. This finding suggests a potential hereditary risk for the couple’s offspring to develop the disease.

The incidence of this variant in Tunisian families with LS and CMMRD could be consistent with a founder effect.

Conclusion: Even though CMMRD syndrome is a rare cause of early-onset malignancy, the diagnosis of CMMRD is crucial for early cancer detection, effective cancer therapy, accurate genetic counseling, and the implementation of targeted preventive surveillance programs.

Grants: None

Conflict of Interest: None declared

EP01.053 Somatic mosaicism for NF1 pathogenic variant detected by peripheral-blood exome sequencing in a patient with colon and lung cancers and no apparent clinical signs of Neurofibromatosis type 1.

Miriam Carriero ¹, Ludovico Graziani¹, Emanuele Agolini², Mario Bengala³, Antonio Novelli², Michela Biancolella⁴, Giuseppe Novelli^1;3

¹Tor Vergata University of Rome, Department of Biomedicine and Prevention, Rome, Italy; ²Bambino Gesù Children’s Hospital, Translational Cytogenomics Research Unit, Rome, Italy; ³Tor Vergata University Hospital, Medical Genetics Unit, Rome, Italy; ⁴Tor Vergata University of Rome, Department of Biology, Rome, Italy

Background/Objectives: Mosaic neurofibromatosis type 1 (MNF1) is an uncommon form of neurocutaneous disorder caused by postzygotic mutation in the neurofibromin gene (NF1). MNF1 is characterized by clinical manifestations typical of neurofibromatosis type 1 (NF1), although localized to one or more body segments. Patients with NF1 pathogenic variants have an increased risk for various types of tumors; however, adenocarcinomas involving the colon or the lungs have rarely been reported.

Methods: A 64-year-old man was referred to genetic counselling for previous synchronous diagnosis of lung and rectal adenocarcinoma. The patient also had multiple colorectal hyperplastic/adenomatous polyps. Physical examination revealed asymptomatic brownish soft papules across the chest. Several other lesions, surgically excised, were reported to first appear after pulmonary lobectomy grouped in the surgical wound region. There was no evidence of typical features of NF1 and no family history of pigment disorders.

Results: Custom phenotype-focused exome sequencing detected an NF1 pathogenic variant (NM_001042492.3: c.61-2A > G) at a frequency of 33% in the blood. Functional mRNA studies in an individual suspected to have NF1 showed that the c.61-2A > G intronic variant causes aberrant splicing and partial deletion of exon 2.

Conclusion: MNF1 phenotypic expression is sometimes subtle and internal malignant tumors may be diagnostic clues to previously undiagnosed MNF1. Molecular studies in paraffin-embedded tumor and skin samples are needed to understand the association between MNF1 and lung/colorectal cancer or polyposis. Regardless, this finding highlights that NF1 screening should be considered in patients suspected of having cancer genetic susceptibility syndrome even in the absence of clinical symptoms of neurocutaneous disorder.

Conflict of Interest: None declared

EP01.055 Quality of life in children with cancer and their carers

Joshua Kraindler ¹, Rupendra Shrestha¹, Owen Tan¹, Jayamala Parmar¹, Reanu Gopal¹, Natalie Hart¹, Vanessa Tyrrell^2;3, Tracey O’Brien^3;4, Deborah Schofield¹

¹GenIMPACT: Centre for Economic Impacts of Genomic Medicine, Macquarie University, Australia; ²Children’s Cancer Institute, Kensington, Australia; ³School of Clinical Medicine, UNSW Medicine & Health, Sydney, Australia; ⁴Kids Cancer Centre, Sydney Children’s Hospital, Randwick, Australia

Background/Objectives: Cancer is the leading cause of disease related death in children. While there have been significant improvements in survival rates for many childhood cancers, similar progress has not occurred in the most aggressive and high-risk cancers, with survival rates of only 30%. Precision medicine brings hope for patients and families. However, to ensure funding is accessible, evidence of cost-effectiveness is needed, which in addition to survival rates, will require data on quality of life measured in health utilities.

Methods: Patients are those with aggressive and high-risk child cancer enrolled in the Zero Childhood Cancer National Clinical Trial in Australia. 105 carers completed the tailored questionnaire, which collected information on QoL and health utilities through the Assessment of Quality of Life (AQoL)-8D and the EQ-5D-Y for children, both validated measures of QoL.

Results: Both EQ-5D-Y values for children and AQoL-8D values for carers were lower than population norms. Bivariate and multivariate regression analysis will also report some factors associated with QoL in children with aggressive and high-risk cancer and for their carers.

Conclusion: Childhood cancer has a substantial impact on both patients and families. Caring for a child with cancer has substantial impacts on the carer’s quality of life. This paper will present QoL data measured in health utilities for both children and carers. These are key inputs for cost-effectiveness of precision medicine for childhood cancer.

Grants: National Health and Medical Research Council (NHMRC) (Partnership Grant ID: 1159004).

Conflict of Interest: None declared

EP01.056 miRNA expression testing could improve presurgical diagnosis of follicular thyroid neoplasm

Deimante Usaite ¹, Julija Simiene¹, Kestutis Suziedelis^1;2

¹National Cancer Institute, Laboratory of Molecular Oncology, Vilnius, Lithuania; ²Vilnius University, Department of Biochemistry and Molecular biology, Vilnius, Lithuania

Background/Objectives: Discrimination between follicular thyroid cancer and benign adenoma is the most difficult aspect of thyroid pathology. Only 30% of patients who are reported as Follicular neoplasm by cytopathology and have thyroidectomy, ends up as Follicular carcinoma by postsurgical histopathology. The aim of this study was to identify miRNAs differentially expressed in human thyroid carcinoma line cells established from metastases of a follicular thyroid carcinoma (RO82-w-1), lymph node metastasis (FTC-133) or normal thyroid follicular epithelial cells (Nthy-ori-3-1), cultivated as 3D in vitro cell cultures.

Methods: Triplicates of human thyroid FTC-133, RO82-w-1 and Nthy-ori-3-1 (Sigma-Aldrich, USA) cell line 3D in vitro cultures were used in this analysis. RNA has been extracted and sequenced by NextSeq 500/550. Analysis of differentially expressed miRNA between different cell lines have been performed using R package DESeq2 v1.40.2. Only highly abundant (base mean ≥ 100) with threshold of Bonferroni adjusted p value < 0.01 and absolute log2 fold change (FC) |log2FC | > 3 were selected as differentially expressed miRNAs.

Results: We have identified 44 differiantially expressed miRNAs. 12 of them were downregulated and 2 of them were upregulated in both FTC-133 and RO82-w-1 cell lines if compared to Nthy-ori-3-1. By raising the threshold to |log2FC | > 5, 4 miRNAs were chosen: hsa-miR-10b-3p, hsa-miR-10b-5p, hsa-miR-196b-5p, hsa-miR-126-5p.

Conclusion: We have found that 4 miRNAs were significantly differentially expressed in follicular thyroid cells compared to benign cells. These data suggest that follicular thyroid adenoma can be distinguished from follicular thyroid carcinoma by miRNA expression analysis.

Grants: EU grant no: J05-LVPA-K-01-0122

Conflict of Interest: Deimante Usaite J05-LVPA-K-01-0122, Julija Simiene: None declared, Kestutis Suziedelis: None declared

EP01.058 (Epi)genetic Biomarkers for High-Grade Serous Ovarian Carcinoma

Ieva Vaicekauskaitė^1;2, Paulina Kazlauskaitė^1;2, Rugilė Gineikaitė^1;2, Ruta Ciurliene³, Juozas Rimantas Lazutka², Rasa Sabaliauskaite ^1;2

¹National Cancer Institute, Genetic diagnostic laboratory, Vilnius, Lithuania; ²Vilnius University, Life Sciences Center, Vilnius, Lithuania; ³National Cancer Institute, Oncogynecology, Vilnius, Lithuania

Background/Objectives: Ovarian Cancer (OC) is the second deadliest oncogynecologic disease in the world. High-grade serous ovarian cancer (HGSOC) is the most common type of OC with the highest rates of mortality. Currently, there is no specific means of OC detection recommended for use in the clinical setting, thus accurate OC diagnostic biomarkers are in high demand.

Methods: Tissues from 51 patients with gynecologic tumors (32 HGSOC, 10 other gynecologic tumors, 9 benign) were analyzed for the NOTCH pathway (NOTCH1-4, DLL1, JAG2, and HES1), WNT pathway (CTNNB1, FBXW7) and ARID1A gene expression by RT-qPCR, and HOX family related gene (HOPX, ALX4, CDX2), and ARID1A gene promoter methylation status by MSP.

Results: A significant decrease in ARID1A, WNT pathway, NOTCH1-4, DLL1, and HES1 gene expression in HGSOC patients in comparison with the benign cases was detected (p < 0.02). The HOPX gene promoter methylation was significantly increased in HGSOC cases when compared to benign (p = 0.02), ALX4 and CDX2 showed tendency for promoter hypermethylation (p = 0.06), while ARID1A had no significant differences in promoter methylation.

Conclusion: Gene expression and promoter methylation status could be useful for HGSOC diagnosis. More extensive research on early stage patients and non-invasive biosample specimens are needed for biomarker validation for clinical use.

Conflict of Interest: None declared

EP01.060 Clinical significance of germline variants in MLH1, MSH2, MSH6 and PMS2 genes and their association with prostate cancer risk in Polish men - preliminary results.

Marta Heise ¹, Piotr Jarzemski², Aneta Bąk¹, Anna Junkiert-Czarnecka¹, Olga Haus¹

¹Collegium Medicum in Bydgoszcz, Nicolaus Copernicus University in Toruń, Department of Clinical Genetics, Faculty of Medicine, Bydgoszcz, Poland; ²Jan Biziel University Hospital, Department of Urology, Faculty of Health Science, Bydgoszcz, Poland

Background/Objectives: Prostate cancer (PC) is a complex disease that affects millions of men globally. The relationship of PC with Lynch syndrome is unresolved though molecular investigations and epidemiologic studies have suggested a potential link to the syndrome. Thus, we searched for germinal variants in MLH1, MSH2, MSH6 and PMS2 mismatch-repair genes in Polish prostate cancer patients and controls and analyzed their impact on clinical course of the disease, including overall survival time.

Material: DNA from 97 men with PC from all over Poland and DNA from 100 men - volunteers, healthy at the time of the study.

Methods: NGS, Sanger sequencing.

Results: In 11/97 (11,3%) PC patients 11 variants were detected. The MSH2 c.744G>A was predicted as likely pathogenic and MSH6 c.1969C>T as pathogenic. The MSH2 c.337A>G, MSH6 c.2017C>G, c.3257C>A, c.3557-7_3557-4del, c.1714C>G and PMS2 c.1268C>G, c.1162T>A were predicted as VUS. The MSH6 c.3257-65_3257-61del and c.2267-97T > C were predicted as benign. Two PC patients were carriers of PMS2 c.1268C>G. One PC patient was the carrier of two variants in MSH6. No variant was detected in MLH1 gene. All detected variants were described previously. Bioinformatic analysis of all changes was performed using Franklin database.

Conclusions: The results of the preliminary investigation point at the need to study germinal variants of tested genes to help fully understand the pathogenesis of prostate cancer, identify men at PC high risk and predict the disease recurrence risk after radical prostatectomy.

Grant: This study was supported by the fund of the CM UMK, Bydgoszcz, Poland.

Conflict of Interest: None declared

EP01.061 PDCDL1 Single Nucleotide Variants (SNVs) in relation with the evolution of Cutaneous Malignant Melanoma (CMM)

Elizabeth Córdoba-Lanús^1;2;3, Omar García-Pérez ^1;2, Leticia Melgar-Vilaplana^2;4, Noelia Hernández Hernández⁵, Ricardo Fernández-de-Misa^2;5;6

¹Instituto Universitario de Enfermedades Tropicales y Salud Pública de Canarias (IUETSPC), San Cristóbal de La Laguna, Spain; ²Universidad de La Laguna, San Cristóbal de La Laguna, Spain; ³Consorcio Centro de Investigación Biomédica en Red (CIBER) de Enfermedades Infecciosas (CIBERINFEC). Instituto de Salud Carlos III, Madrid, Spain; ⁴Departamento de Anatomía Patológica. Hospital Univ. Ntra. Sra. de Candelaria, Santa Cruz de Tenerife, Spain; ⁵Departamento de Dermatología, Hospital Univ. Ntra. Sra. de Candelaria, Santa Cruz de Tenerife, Spain; ⁶Unidad de Investigación. Hospital Univ. Ntra. Sra. de Candelaria, Santa Cruz de Tenerife, Spain

Background/Objetives: The search for more precise biomarkers to improve knowledge about the prognosis of cutaneous malignant melanoma (CMM) patients in routine clinical practice is still an important challenge. PD-L1 protein exerts a regulation of the antitumor response acting as a good prognostic marker for the response to immunotherapy treatment. Different single nucleotide variants (SNV) in its coding gene, PDCDL1, have been associated with a greater susceptibility to developing other tumors and can operate as prognostic biomarkers in the early neoplasm phases.

Methods: Allelic and genotype discrimination of five SNVs rs4143815, rs822336, rs822337, rs2297136, rs822338 in the PDCDL gene was performed in 338 patients with CMM by qPCR. Hardy-Weinberg equilibrium was tested. Clinical variables were recorded.

Results: rs2297136 harboring the minor allele homozygous genotype AA in the 3’-UTR of the gene is associated with a higher Breslow index (OR = 1.15;95%CI = 1.042-1.267;p = 0.005), presence of ulceration (OR = 1.77;95%CI = 1.00-3.13;p = 0.05) and stage II-IV (OR = 2.10;95%CI = 1.03-4.29;p = 0.04). However, Cox regression analysis revealed that none of the SNVs were associated with melanoma-specific survival or disease-free survival (p > 0.05). As expected, the clinical variables, age at diagnosis (>50 years) [HR = 1.04(1-02-1.06)], gender (male) [2.59(1.48-4.51)], higher tumor thickness (mm) [HR = 1.17 (1.12-1.22)], presence of ulceration [HR = 6.31(3.55-11.23) and stage (III-IV) [HR = 3.16(1.62-6.15)] significantly (p < 0.001) associated with an increased risk of death.

Conclusions: In CMM patients, SNV rs2297136 in PDCL1 gene is associated with a higher Breslow and ulceration, both risk factors of poor prognosis. Further studies are needed to confirm this novel finding.

Grants: PIFIISC21/15, FIISC; CB21/13/00100, CIBERINFEC, Instituto de Salud Carlos III, Spain; Cabildo de Tenerife 2023-2028 (PI-CC202302222, Cabildo.23), Spain.

Conflict of Interest: Elizabeth Córdoba-Lanús Centro de Investigación Biomédica en Red de Enfermedades Infecciosas, Omar García-Pérez Financiado por Cabildo Insular de Tenerife 2023-2028, PROYECTO CC20230222, Leticia Melgar-Vilaplana: None declared, Noelia Hernández Hernández: None declared, Ricardo Fernández-de-Misa: None declared

EP01.063 Evaluation of the anticancer effect of Spirulina platensis on the MCF7 human breast cancer cell line

olfa tahri¹, Sabriye Kocatürk Sel ², oya ışık³, leyla uslu³, choubaila reddad¹, Bertan YILMAZ²

¹Çukurova University, Institute of Natural and Applied Sciences, Biotechnology, Adana, Türkyie; ²Çukurova University, Faculty of Medicine, Medical Biology, Adana, Türkyie; ³Çukurova University, Faculty of Fisheries, Marine Biology, Adana, Türkyie

Background: Spirulina platensis (Arthrospira platensis), a blue-green alga, is extensively studied for its anti-inflammatory and antioxidant properties. It is rich in vitamins, minerals, protein, and pigments (phycocyanin). C-phycocyanin (C-PC) is a natural blue colorant extracted from Spirulina, which is used in food and cosmetics. It has been found to have a strong anti-cancer effect on various cancer types such as breast cancer, liver cancer, lung cancer, and colon cancer, both in vitro and in vivo. In this study, the effect of C-PC derived from Spirulina produced at Çukurova University, Adana, Turkey on human breast cancer cell line MCF7 was tested.

Methods: In this study, we evaluated the effects of both Spirulina aqueous extract and its active ingredient C-PC on the viability of NIH3T3 and MCF7 cells using the MTT assay. Different doses (5, 25 ve 45 μg/ml) of C-PC were exposed to MCF7 for 24 hours. Expression levels of apoptotic genes (caspase-3, -8, -9) were evaluated by Real-Time PCR.

Results: According to the results of the MTT test, it was observed that C-PC significantly reduced the viability of both NIH3T3 and MCF7 cells. Additionally, it was found that C-PC caused significant increases in the expression levels of apoptotic genes.

Conclusion: The number of natural and synthetic substances that can exhibit chemotherapeutic effects for the treatment of breast cancer is increasing every day. According to our results, it is considered that C-PC may be a natural and alternative therapeutic agent in cancer treatment.

Conflict of Interest: None declared

EP01.064 Detection of PIK3CA mutations on breast cancer: Comparison of NGS and Cobas mutation test to Sanger sequencing in a clinical setting

Niki Prekete ¹, Eirini Roupou¹, Maria Michelli¹, Vasiliki Chaidou¹, Aphrodite Nonni¹, NIKOLAOS KAVANTZAS¹, Angelica a Saetta¹

¹1st Department of Pathology, Medical School, National and Kapodistrian University of Athens, Athens, Greece

Background/Objectives: The PI3K pathway is involved in breast tumourigenesis and PIK3CA mutations mainly found in exons 9 and 20, are currently used as therapeutic biomarkers. We compared Next Generation Sequencing (NGS) and Cobas mutation test to Sanger sequencing for detecting PIK3CA mutations (exons 9 and 20) to determine the best approach for the analysis of HR + /HER2- advanced breast cancer patients.

Methods: 160 samples from women previously diagnosed with breast cancer were analysed through NGS and Sanger sequencing whereas a second cohort of 69 FFPE samples were analysed through Cobas and Sanger. The same extracted DNA was used for the methods in comparison to avoid biased results.

Results: 53 cases (33%) were excluded from NGS analysis after not meeting the pre-analytical quality criteria or having a non-informative result and thus were only analysed through Sanger sequencing. 28% and 26% of the cases were found mutated with NGS and Sanger respectively. The PIK3CA mutated cases in exons 9 and 20 detected with Cobas (42%) were also detectable with Sanger analysis. The most frequently mutated codons were 545 in exon 9 (NGS/Sanger 33.3%, Cobas/Sanger 29.4%) and 1047 in exon 20 (NGS/Sanger 50%, Cobas/Sanger 41%) for both cohorts.

Conclusion: NGS and Sanger sequencing showed an overall 94% concordance, whereas Cobas and Sanger sequencing showed 100% concordance for exons 9 and 20. In conclusion, all methods are useful in detecting SNVs in PIK3CA and can be complementary in everyday practice depending on clinical requirements.

Grant References: No

Conflict of Interest: None declared

EP01.065 Chromosomal instability precede microsatellite instability in Lynch syndrome-associated colorectal tumorigenesis

Marjaana Pussila ¹, Aleksi Laiho², Petri Törönen², Pauliina Björkbacka³, Sonja Nykänen¹, Kirsi Pylvänäinen⁴, Liisa Holm², Jukka-Pekka Mecklin⁵, Laura Renkonen-Sinisalo⁶, Taru Lehtonen⁶, Anna Lepistö⁶, Jere Linden³, Satu Mäki-Nevala⁷, Päivi Peltomäki⁷, Minna Nyström⁸

¹University of Helsinki, Molecular and Integrative Biosciences Research Programme, Faculty of Biological and Environmental Sciences, Helsinki, Finland; ²University of Helsinki, Organismal and Evolutionary Biology Research Program, Faculty of Biosciences, and Institute of Biotechnology, Helsinki Institute of Life Science (HiLIFE), Helsinki, Finland; ³University of Helsinki, Department of Veterinary Biosciences, and Finnish Centre for Laboratory Animal Pathology (FCLAP), Helsinki Institute of Life Science (HiLIFE), Helsinki, Finland; ⁴University of Jyväskylä, Faculty of Sports and Health Sciences, Jyväskylä; ⁵University of Jyväskylä, Faculty of Sports and Health Sciences, Jyväskylä, Finland; ⁶University of Helsinki, Applied Tumor Genomics, Research Programs Unit, Helsinki, Finland; ⁷University of Helsinki, Department of Medical and Clinical Genetics, Helsinki, Finland; ⁸University of Helsinki, Molecular and Integrative Biosciences Research Programme, Faculty of Biological and Environmental Sciences, Helsinki, Finland

Background/Objectives: In Lynch syndrome (LS) inherited mutations in DNA mismatch repair (MMR) genes cause MMR deficiency and microsatellite instability (MSI) in tumors. Regular surveillance is crucial for cancer prevention due to high risk; however, frequent interval cancers suggest limitations of colonoscopy-based methods alone. This study aimed to identify precancerous functional changes in colonic mucosa that could facilitate the monitoring and prevention of cancer development in LS.

Methods: We examined 71 colon biopsy specimens from individuals with LS, either tumor-free, diagnosed with adenoma/carcinoma, along with a control group of sporadic cases without LS or neoplasia. The majority (80%) of LS carriers had an MLH1 mutation. RNA-sequencing was used to analyse the transcriptomes, followed by GOrilla ontology analysis, and Reactome Knowledgebase and Ingenuity Pathway Analysis to identify changes potentially linked to neoplastic initiation in LS.

Results: Using pathway and gene ontology analyses along with measuring mitotic perimeters from colonic mucosa and tumors, we discovered a tendency to chromosomal instability (CIN), evident already in colonic mucosa. Our findings imply that CIN precedes MSI and could be the primary driver of cancer initiation, with MSI speeding up tumor formation. Moreover, our results indicate that MLH1 deficiency contributes to CIN development.

Conclusion: The findings confirm our earlier discoveries in mice and underscore the significance of early CIN as a crucial factor and precancerous indicator in the development of colorectal tumors in Lynch syndrome.

Grants: This work was supported by grants from the Jane and Aatos Erkko Foundation, the Academy of Finland; Cancer Foundation Finland sr, and the Sigrid Juselius Foundation.

Conflict of Interest: Marjaana Pussila: None declared, Aleksi Laiho: None declared, Petri Törönen: None declared, Pauliina Björkbacka: None declared, Sonja Nykänen: None declared, Kirsi Pylvänäinen: None declared, Liisa Holm: None declared, Jukka-Pekka Mecklin: None declared, Laura Renkonen-Sinisalo: None declared, Taru Lehtonen: None declared, Anna Lepistö: None declared, Jere Linden: None declared, Satu Mäki-Nevala: None declared, Päivi Peltomäki: None declared, Minna Nyström Minna Nyström is a member of the board of directors and a shareholder in LS CancerDiag Ltd

EP01.066 Non-invasive detection of bladder cancer by digital PCR analysis of urine samples for the presence of the most common mutations: pilot study for the detection of S249C FGFR3 mutation

Sanja Kiprijanovska ¹, Hristina Dichevska¹, Aleksandar Trifunovski², Ognen Ivanovski², Skender Saidi², Zivko Popov^1;2;3, Aleksandar Dimovski^1;4

¹Research Center for Genetic Engineering and Biotechnology, Macedonian Academy of Sciences and Arts, Skopje, N.Macedonia; ²University Clinic for Urology, Faculty of Medicine, St. Cyril and Methodius University, Skopje, N.Macedonia; ³Clinical Hospital “Acibadem Sistina”, Skopje, N.Macedonia; ⁴Faculty of Pharmacy, University “St. Cyril and Methodius”, Skopje, N.Macedonia

Background/Objectives: Bladder cancer (BlCa) is one of the most common types of carcinomas which is characterized by a high level (>75%) of mutations in the CDKN2A/2B and FGFR3 genes. The presence of these mutations in early stages of the disease makes them an ideal target for the development of a non-invasive assay for their detection in urine.

Methods: Tissue samples from 254 early stage (BlCa) were included in the study. Urine samples were available for most patients. The CDKN2A/2B and FGFR3 mutation status of each tissue sample was determined previously. A specific digital PCR assay was developed for the detection of the most common FGFR3 S249C mutation, while the development of methods for the detection of other common FGFR3 mutations and CDKN2A/2B deletions is in progress.

Results: A total of 31 urine samples were analysed by digital PCR, of which 23 samples were from patients with S249C mutant tumours. The average age of patients was 64.5 ± 11.1 years (20 male and 3 female) and 2/3 had early-stage disease (Ta-T1). The S249C mutation was detected in urine of all patients with mutation positive and was absent in the urine of all mutation negative tumours (specificity/sensitivity of 100%). Serial dilution analysis revealed that the mutation could be detected at the frequency of 0.1%.

Conclusion: Digital PCR analysis for the presence of the common mutations in urine is a promising strategy for non-invasive early detection and monitoring of BlCa.

Grants:

Conflict of Interest: None declared

EP01.067 Could VUS be important in AML?

Nuket Y Kutlay ¹, Halil Gürhan Karabulut¹, TIMUR TUNCALI¹, Hatice Ilgın Ruhi¹, Sadiye Ekinci¹, Şule Altıner¹, Arzu Vicdan¹, Ezgi Gökpınar İli¹

¹Ankara University Medical School, Medical Genetics, Ankara, Türkyie

Background/Objectives: Acute myeloid leukemia (AML) includes a heterogeneous group of immature myeloid cells. In addition to recurrent cytogenetic abnormalities, European LeukemiaNet and National Comprehensive Cancer Network guidelines also recommend the use of sequencing variants in key genes in classification of AML. At this point, considering different variants in the same gene or a combination of different mutated genes, VUS (Variant of Uncertain Significance) variants may also be important in classification.

Methods: NGS results covering IDH1, IDH2, TP53, RUNX1, ASXL1, WT1, KIT, FLT3, NPM1 and CEBPA genes were evaluated in 108 AML patients with at least one variant. Variant frequencies, different variants in the same gene and variants occurring together in different genes were determined. Only pathogenic (P), likely pathogenic (LP) and VUS variants with VAF scores greater than 1% were analyzed.

Results: Approximately half of the total 253 variants were VUS (128), whereas 80 P and 45 LP variants were detected. The two most frequent variants were in FLT3 > WT1 genes for P, CEBPA > RUNX1 genes for LP and RUNX1 > WT1 genes for VUS variants. In 31 cases, more than one variant was detected in one gene, and in three of them, the same condition was detected for two different genes. Repeated variants were found in all genes.

Conclusion: VAF values in some of the repeated VUS variants suggest the possibility of germline susceptibility. Considering that leukemia is a somatic, clonal and heterogeneous disease, it would be useful to evaluate variant combinations on patient basis to interprete data in diagnosis and prognosis.

References:

Grants: No

Conflict of Interest: None declared

EP01.068 Application of Next-Generation Sequencing-based panels to improve the diagnosis, prognosis, and treatment of pediatric sarcomas.

Piedad Alba Pavón ¹, Lide Alaña¹, Teresa Imizcoz², Miriam Gutierrez-Jimeno³, Aizpea Echebarria-Barona^1;4, Ricardo López Almaraz^1;4, Itziar Astigarraga^1;4;5, Ana Patiño⁶, Olatz Villate¹

¹Biobizkaia Health Research Institute, Pediatric Oncology Group, Barakaldo, Spain; ²University of Navarra, CIMA LAB Diagnostics, Pamplona, Spain; ³Hospital Clínico Universitario de Valladolid, Servicio de Pediatría, Valladolid, Spain; ⁴Hospital Universitario Cruces, Pediatrics Department, Barakaldo, Spain; ⁵University of the Basque Country, UPV/EHU, Pediatrics Department, Faculty of Medicine and Nursing, Leioa, Spain; ⁶University Clinic of Navarra, CUN and Cima/Solid Tumor Area, Dpt. Of Pediatrics and Medical Genomics Unit, Pamplona, Spain

Background/Objectives: Sarcomas are conventionally diagnosed according to morphological features and immunohistochemical techniques. However, some of them exhibit features that may overlap with other entities. Therefore, identifying tumor-specific genetic alterations is necessary for the classification of this group of tumors. The objective was to assess the implications of two NGS-based genomic panels in the diagnosis, prognosis, and treatment of pediatric patients diagnosed with sarcomas.

Methods: Forty-two tumor samples of pediatric and young adult patients diagnosed with sarcomas were sequenced using Oncomine Childhood Cancer Research Assay panel (OCCRA panel) (Life Technologies) and/or Fusion Plex Sarcomas Panel (FPS panel) (Invitae).

Results: Thirty tumor samples were sequenced using OCCRA Panel and 74 clinically relevant genetic alterations were identified. The most frequent genomic alterations were EWSR1::FLI1 fusion gene (20%), alterations in FGFR4 and TP53 genes, and PAX3::FOXO1 fusion gene (13% each).

Fourteen samples were sequenced with the FPS panel. Eleven patients were fusion-positive, of which two patients had been previously sequenced with the OCCRA panel. In these patients, TGF::ADGRG7 and TPR::NTRK1 fusion genes were identified.

The NGS sequencing tools used in this study identified a genetic alteration with potential clinical implications in 74% of patients. A variant with implication in the diagnosis or potential therapeutic value was identified in 27 and 12 of sequenced tumors, respectively.

Conclusion: NGS-based panels are tools that complement morphology, immunohistochemistry and FISH techniques to obtain accurate data for diagnosis, prognosis and the identification of potential therapeutic targets.

Grants: EITB Media AND BIOEF, SAU (BIO20/CI/011/BCB), Basque Government (2021111030).

Conflict of Interest: None declared

EP01.069 Rare germline chromosome 1 duplication identified in young male with colon cancer: a case report investigating causality

Anna Byrjalsen ¹, Sara Garcia², Line Borgwardt¹, Karin Wadt¹, Anne-Marie Gerdes¹, Thomas van Overeem Hansen¹

¹Copenhagen University Hospital Rigshospitalet, of Clinical Genetics, Copenhagen east, Denmark; ²Danish Technical University

Background/Objectives: Colorectal cancer (CRC) occurrence among young adults is increasing. In the following we present geno- and phenotype of a patient with colon cancer (CC).

Methods and Results: A 24-year-old male presented with abdominal pain, and a CT-scan revealed a pre-stenotic iliac dilatation. A subsequent laparoscopy identified a tumor in the cecal wall. The tumor was an adenocarcinoma, and immunohistochemistry and somatic gene testing came back ia.

The patient underwent genetic testing with an in-house panel: APC, AXIN2, BMPR1A, EPCAM, GREM1, MLH1, MSH2, MSH3, MSH6, MUTYH, NTHL1, PMS2, POLD1, POLE, PTEN, SMAD4 and STK11. No pathogenic variants were detected. Subsequent, whole genome sequencing revealing a duplication on chromosome 1 spanning 200 kb and covering CD101, TTF2, MIR942, TRIM45, and parts of PTGFRN and VTCN1. The duplication had not previously been described in gnomAD/the literature. However, a similar duplication covering an overlapping area (TTF2, MIR942, and TRIM45) had been reported in another family with CRC.

Segregation analysis revealed the duplication was maternally inherited. On the maternal side of the family, there had been one case of a tubular adenoma with high-grade neoplasia and one case of CRC. This led to genetic testing of the patient’s maternal grandparents. The duplication proved to be inherited from an unaffected relative.

We calculated a polygenic risk score using 95 SNPs correlated to the risk of CRC. The PRS was 7.87, indicating an average risk of CRC compared to others with CRC.

Conclusion: Our findings do not support that this variant is the sole reason for early onset CC in our patient.

Conflict of Interest: None declared

EP01.070 The CDKN2A Gene Variants related with Cancer Predisposition Syndrome

Roya Gasimli ¹, Asli Subasioglu²

¹Ege University, Medical Biology, Izmir, Türkyie; ²Izmir Katip Çelebi University, Medical Genetics, Izmir, Türkyie

Background/Objectives: The CDKN2A gene, situated on chromosome 9p21, encodes vital proteins, namely p16^INK4a and p14^ARF, pivotal in regulating cell proliferation. Mutations in CDKN2A result in uncontrolled cell growth and cancer progression. Understanding its implications not only aids in diagnostics and therapies but also enhances our understanding of cancer. Our objective was to identify gene variants, particularly within CDKN2A, among 3036 genomic DNA samples obtained from individuals with a predisposition to hereditary cancer or with affected first-degree relatives.

Material and Methods: The study collected peripheral blood samples from 3036 individuals with hereditary cancer backgrounds or predisposition. DNA extraction utilized Qiaseq Targeted Panels kits, followed by sequencing on the Illumina Miseq platform. FastQ data were analyzed, resulting in the generation of a variant call format (VCF) file using QIAGEN CLC Genomics Workbench. The Qiagen Clinical Insight (QCI Interpret) system identified variants associated with familial cancer predisposition.

Results: The analysis, conducted from the VCF file using the QCI Interpret system, identified germline likely pathogenic (LP) mutations in 5 patients and variants of uncertain significance (VUS) in 20 patients, associated with melanoma, colorectal, and pancreatic cancers. Mutations primarily localize to exon 1, 2 and intron 1.

Conclusion: In light of the potential consequences at the post-transcriptional level, variants classified as LP, VUS variants, and even those reported to have no effect at the protein level hold significant importance with various genetic and epigenetic mechanisms. Additionally, with supporting evidence from in vitro and in vivo functional studies, these variants could potentially emerge as therapeutic targets for various cancers.

Grants: None.

Conflict of Interest: None declared

EP01.072 Autophagy and P62: A Collaborative Effort in CRC Chemoresistance to 5-FU Uncovered by Combined Bioinformatics and Laboratory Tests

Sara Ebrahimi ¹, Haniye Rahimi Kolour¹, Ehsan Nazemalhosseini Mojarad², maryam Ghanbari-Safari³

¹Research Institute for Gastroenterology and Liver Diseases, 1. Basic and Molecular Epidemiology of Gastrointestinal Disorders Research Center, tehran, Iran; ²Research Institute for Gastroenterology and Liver Diseases, Gastroenterology and Liver Diseases Research Center, tehran, Iran; ³Tehran North Branch, Islamic Azad University, 3. Department of Microbial Biotechnology, Faculty of Biological Science, tehran, Iran

Background: In contemporary research, autophagy is recognized as a significant pathway implicated in chemoresistance. The P62/SQSTM1 gene, which encodes the P62 protein, is a key player in this pathway. Inhibition of this gene presents a potential strategy to enhance the anticancer efficacy of chemotherapy drugs. This investigation aimed to explore the association between autophagy and resistance to 5-FU chemotherapy in colorectal cancer by identifying a lncRNA and a miRNA linked to the P62 gene and autophagy.

Material and Methods: For the in-silico analysis, a specific lncRNA and miRNA associated with the P62 gene and autophagy pathway were chosen from databases. Subsequently, two cell lines, SW480 and Caco2, were chosen to assess resistance to the drug 5-fluorouracil (5-FU). Multiple laboratory assays were utilized to validate resistance to chemotherapy and autophagy. Finally, the expression levels of the target gene, lncRNA, and miRNA were quantified by realtime-PCR.

Results: The in-silico analysis identified LINC01962 and miR-17-5p. Western blot and immunocytochemistry demonstrated an increase in autophagy in chemoresistance cells compared to the untreated cell line. The study revealed an upregulation of P62 expression in both cell lines, a decrease in LINC01962 expression in Caco2/5-FU, and an increase in its expression in SW480/5-FU, as well as an increase in miR-17-5p expression in both cell lines.

Conclusion: In conclusion, the findings indicate that LINC01962 may modulate 5-FU drug resistance in metastatic CRC cells by influencing the P62 gene. Additionally, upregulation of miR-17-5p expression may enhance autophagy in these cells, suggesting a potential therapeutic target to enhance the effectiveness of 5-FU-based chemotherapy in CRC.

Conflict of Interest: None declared

EP01.073 Comprehensive Exploration of Nasopharyngeal Carcinoma through Whole Transcriptomic Sequencing and Single-Cell RNA Sequencing

Che Kang Lim ^1;2, Yi Tian Png³, Lee Yew Mun³, Zhi Yong Lim³, Wei Ying Sim¹, Chwee Ming Lim^1;2;3

¹Singapore General Hospital, Clinical Translational Research, Singapore; ²Duke-NUS Medical School, Singapore; ³Singapore General Hospital, Otorhinolaryngology-Head&Neck Surgery, Singapore

Background: Nasopharyngeal carcinoma (NPC), an epithelial malignancy prevalent in Southeast Asia and Southern China, is closely associated with Epstein-Barr virus (EBV). Despite therapeutic advancements, late diagnosis and drug resistance often lead to unsatisfactory clinical outcomes. Recent advances highlight single-cell RNA sequencing (scRNA-seq) as pivotal in oncology research, offering insights into cellular heterogeneity. Integrating scRNA-seq with bulk RNA-seq data holds promise to transform NPC research by providing a comprehensive understanding of gene expression patterns.

Methods: Whole transcriptomic sequencing was performed in eight NPC patients and three controls. Additionally, single cell RNA sequencing was conducted in the selected samples to assess the tumor microenvironment.

Results: From the whole transcriptomic sequencing analysis, PLLP, S100A9, and PLCG2 genes emerged as the top three significantly expressed genes, exhibiting notably elevated expression levels in the NPC patient cohort. Additionally, in conjunction with the epithelial cluster (EPCAM + , KRT5 + , KRT8 + , KRT13 + , KRT19 + ), single-cell RNA sequencing analysis revealed diverse immune cell clusters, comprising four subtypes of B cells (including plasma B cells), four subtypes of T cells (including NKT cells), NK cells, and mast cells. These findings indicate a complex and heterogeneous tumor microenvironment.

Conclusion: Our study offers a comprehensive perspective on Nasopharyngeal Carcinoma (NPC), shedding light on its cellular heterogeneity and molecular pathways. Further research is warranted to validate our findings and explore the interplay between NPC tumors and immune cells.

Grant: SRG-OPN-007

Conflict of Interest: None declared

EP01.074 Performance of the polygenic risk score PRS313 for breast cancer prediction in patients with atypical breast lesion

Manon Reda¹, Anes Hadjadj¹, Juliette Sauge¹, Laurent Arnould¹, Juliette Albuisson ¹

¹Centre Georges François Leclerc, Département de Biologie et Pathologie des Tumeurs, DIJON, France

Breast cancer (BC) genetic includes polygenic factors, combined in recently characterized polygenic risk scores (PRS). SPORA is a study based on PRS₃₁₃, the most used PRS in breast cancer (Mavaddat et al, 2019).

SPORA aimed at determining PRS₃₁₃ performance to predict the occurrence of breast cancer in patients with atypical breast lesions (ABL), presenting with intermediate BC risk (13-20%).

We conducted a monocentric retrospective study of PRS₃₁₃ performances in cases and controls (patients with ABL and BC within 5 years, n = 73, and without BC within 5 years, n = 154, respectively). Genotyping was performed on FFPE normal tissues, using a microfluidic platform.

PRS₃₁₃ could be calculated, with a median of 271 SNPs per patient. Mean PRS scores for cases and controls are respectively 0.046 and -0.1. The PRS threshold with the best sensitivity and specificity was -0.05 (AUC 0.73 [0,66 – 0,8], sensitivity 0.7, specificity 0.63, odds-ratio 3.94 [2.171 - 7.170] (p < .0001)). At 20% threshold (high risk of BC), PRS is -0.125 (AUC 0.6, sensitivity 0.83, specificity 0.38, NPV 0.82, odds-ratio 3 [1,526 - 6,182]).

PRS₃₁₃ could represent valuable tool to detect high risk patients in this group with intermediate risk.

These results will be consolidated through the analysis of NOMAT study patients, including the evaluation of PRS performance in combination with NOMAT model (predictive model of BC risk based on radiological criteria and age, Uzan et al, 2021). This new combination of biomarkers, in the scope of personalized medicine, could help selecting patients who require breast surgery.

Grants: AOR Bourgogne-Franche-Comté 2020 ; AOI Biocollection 2020.

Conflict of Interest: Manon Reda AOR Bourgogne-Franche-Comté 2020 ; AOI Biocollection 2020.

, Anes Hadjadj: None declared, Juliette Sauge: None declared, Laurent Arnould: None declared, Juliette Albuisson: None declared

EP01.075 MicroRNAs as dual action players: Tumor suppressors and differential diagnosis markers in head and neck tumors

Nadja Nikolic ¹, Jelena Carkic¹, Violeta Sango², Nicole Riberti³, Boban Anicic⁴, Jelena Milasin⁵

¹University of Belgrade, School of Dental Medicine, Implant Research Center, Belgrade, Serbia; ²University of Belgrade, Faculty of Biology, Belgrade, Serbia; ³University G. d’Annunzio of Chieti-Pescara, Department of Neuroscience, Imaging and Clinical Sciences, Chieti, Italy; ⁴University of Belgrade, School of Dental Medicine, Clinic for Maxillofacial Surgery, Belgrade, Serbia; ⁵University of Belgrade, School of Dental Medicine, Human Genetics, Belgrade, Serbia

Background/Objectives. Belonging to the broader category of head and neck tumors along with oral squamous cell carcinomas, salivary gland tumors (SGTs) are a heterogenous group of pathologies which still represents a challenge regarding differential diagnosis and therapy. Detection of molecular alterations is emerging as an effective diagnostic tool. We aimed to analyze the relative expression levels of micro RNAs miR-26a, miR-26b and miR-191, and pro-oncogenic molecular markers PLAG1, MTDH and HIF2 in SGTs and normal salivary gland (NSG) tissues, and evaluate them as potential differential diagnosis markers.

Methods. This cross-sectional study included 58 patients with SGTs (23 pleomorphic adenomas, 27 Warthin tumors and 8 malignant salivary gland tumors) and 10 controls (normal salivary gland tissues). Relative gene expression levels of all investigated molecules were determined by reverse transcriptase-real-time polymerase chain reaction.

Results. All three micro RNAs exhibited highest expression levels in benign SGTs, while miR-26a and miR-191 were significantly more expressed in PAs compared to WTs (P = 0.045 and P = 0.029, respectively). PLAG1 and HIF2 were both overexpressed in WTs compared to PAs (P = 0.048 and P = 0.053, respectively). Bioinformatic analysis suggested that all investigated micro RNAs function as negative regulators of MTDH.

Conclusion. All three micro RNAs have a negative impact on MTDH oncogene expression in malignant tumors, while the differences between levels of mir-26a, miR-191, PLAG1 and HIF2, in PA and WT represent possible differential diagnosis markers.

Grants: Science Fund of the Republic of Serbia, GRANT 7750038.

Conflict of Interest: None declared

EP01.076 Correlation between relative telomere length and specific mutations of the KRAS gene in patients with metastatic colorectal carcinoma

Dragana Jugović ¹, Marija Vukelic-Nikolic², Višnja Madić³, Ljiljana Branković¹, Radovan Milićević¹, Perica Vasiljević⁴

¹University Clinical Center Niš, Center for Medical and Clinical biochemistry, Niš, Serbia; ²University of Niš, Faculty of Medicine, Department of Biology and Human Genetics, Niš, Serbia; ³University of Niš, Faculty of Sciences and Mathematics, Department of Biology and Ecology, Niš, Serbia; ⁴University of Niš, Faculty of Sciences and Mathematics, Department of Biology and Ecology, Niš, Serbia

Background/Objectives: Telomeres, specialized repeat DNA sequences, functioning as a “protective cap” at the ends of linear chromosomes maintain chromosomal stability and genomic integrity. Telomere length anomaly is one of the earliest genetic changes in malignant transformation that may be accelerated by specific alterations in the genes involved in colorectal cancer (CRC). This study aimed to evaluate the possible relationship between the relative telomere length and specific KRAS mutations in CRC patients with developed metastases as well as with their clinical-pathological characteristics.

Methods: The genomic DNA of 89 CRC patients with mutated KRAS were extracted from the formalin-fixed paraffin-embedded tumor tissue sections while the relative telomere length was measured by real-time PCR and calculated using the formula T/S = 2^-ΔΔCt.

Results: The most frequent types of mutations were G12D (31,4%), G12V (22,4%), and G12А (11,2%) in codon 12. There was no significant correlation between relative telomere length and clinical-pathological characteristics of CRC patients. However, patients with mutation in G12V had significantly longer telomeres than patients with other mutations (p < 0.05).

Conclusion: The longer telomeres length was associated with the presence of a G12V mutation in KRAS, suggesting that this mutation coupled with the length of telomeres might play an important role in the biological behavior of CRC.

Grants: This work was supported by the Ministry of Education, Science and Technological Development of the Republic of Serbia (Grant No. 451-03-47/2023-01/ 200124).

Conflict of Interest: None declared

EP01.078 Integrated transcriptomic analysis reveals blood-based gene signature with diagnostic and prognostic potential for patients with breast cancer

Mustafa Kaya¹, Ibrahim Kaya ², Dilek Colak³

¹Hacettepe University Medicine Faculty, Türkyie; ²Al Faisal University, Riyadh, Saudi Arabia; ³King Faisal Specialist Hospital and Research Centre (KFSHRC), Department of Molecular Oncology, Riyadh, Saudi Arabia

Introduction: Breast cancer (BC) continues to be one of the most malignant tumors despite the advances in cancer therapies and raising awareness. It has utmost importance to detect the disease reliably and as early as possible in order to reduce the risk of mortality.

Materials and Methods: Integrated genomic analysis of BC was performed using genome-wide gene expression profiling datasets from both tissue and blood samples from patients with BC that were probed using Affymetrix HGU133 Plus 2.0 array. The diagnostic and prognostic potential of the identified gene signature are validated using transcriptomic datasets from patients with BC with detailed clinical information.

Results: The integrated analysis revealed a 60-gene signature that are significantly expressed in both tissue and blood of BC patients compared to healthy controls. Hierarchical clustering using 60 genes clearly separated patients from normal controls validating the gene signature’s diagnostic potential. The investigation of the 60-geneset score with the BC clinical and pathological features, including age, grade and HER2 indicated that high geneset score was significantly associated with aggressive disease characteristics. Moreover, high gene-set score is associated with a poor disease outcome.

Conclusions: The results suggest that integrated genomic analysis may provide a robust methodology to diagnose patients with BC non-invasively and lead to improved diagnosis and early detection for BC.

Acknowledgment: We would like to thank King Faisal Specialist Hospital and Research Center for supporting this study (RAC#2110006 to DC).

Conflict of Interest: None declared

EP01.079 Metastasizing colorectal cancer: intra-tumor heterogeneity of cancer stem cells related genes identified by bioinformatics analysis of publicly available RNAseq data

Emanuela Bostjancic ¹, Kristian Urh¹, Nina Zidar¹

¹University of Ljubljana, Faculty of Medicine, Institute of Pathology, Ljubljana, Slovenia

Background: Molecular pathways in colorectal cancer (CRC) are well understood; however, development of metastases and intra-tumor heterogeneity (ITH) are still largely unexplained. Our previous study using bioinformatics analysis of publicly available RNAseq data (GEO) suggest involvement of cancer stem cells (CSCs) related genes in metastasizing CRC. We therefore analyzed identified genes and their potentially regulatory miRNAs in metastasizing CRC in context of ITH, which is related to cancer progression, therapy resistance and recurrences.

Methods: Nineteen patients and 63 formalin-fixed paraffin-embedded tissue samples were included (central part and invasive front of primary tumor, lymph node and liver metastases). After RNA isolation, mRNAs and miRNAs expression was analyzed using qPCR. Expression gradient of six CSC-related genes (ANLN, CDK1, ECT2, PDGFD, TNC, TNXB) and their potential regulatory miRNAs (miR-199a-3p, miR-200b-3p, miR-217-5p, miR-223-3p, miR-335-5p, miR-425-5p) was investigated.

Results: We observed differential expression of PDGFD and TNXB (and miR-199a) at the invasive front in comparison to central part of tumor. Significant differences in expression of ANLN, CDK1 and ECT2 (and miR-199a, miR-200b-3p, miR-223-3p, miR-425-5p) was identified in lymph node metastasis when compared to central part of tumor, whereas ANLN, CDK1 and ECT2 as well as TNXB were differentially expressed in liver metastases (and miR-199a-3p, miR-425-5p). Meta-analysis of publicly available sequencing data (TCGA) confirmed correlation between miR-425-5p and TNC.

Conclusion: Our results suggest that expression of CSC‑related genes and their potential regulatory miRNAs contribute to ITH in CRC, lymph node and liver metastases.

Grant references: Slovenian Research Agency (ARRS) J3-1754, J3-3070, P3-0054.

Conflict of Interest: Emanuela Bostjancic Slovenian Research Agency (ARIS)

Project number: J3-1754, Kristian Urh Slovenian Research Agency (ARIS)

Project number: J3-1754, Young Researcher, Nina Zidar Slovenian Research Agency (ARIS)

Project number: J3-1754

Program number: P3-0054

EP01.082 Detection of a novel RUNX1::FOXJ2 fusion in acute myeloid leukemia

yiannos kyprianou ¹

¹Karaiskakio Foundation, Molecular Pathology-Oncology Department, Nicosia, Cyprus

Background/Objectives: Adult acute myeloid leukemia (AML) is a type of blood and bone marrow cancer characterized by the uncontrolled proliferation of myeloid precursor cells in the bone marrow, leading to the inhibition of normal hematopoiesis.

Methods: We herein describe a case of a 60-year-old female patient with AML relapse post-transplant. Upon diagnosis, molecular investigation was carried out on peripheral blood and an FLT3 ITD and NPM1, TET2 and DNMT3A mutations were detected.

The patient relapsed 8 months following diagnosis. Molecular investigation was repeated on bone marrow confirming the presence of the mutations also detected in the diagnostic sample. In addition, RNA NGS was also repeated.

Molecular RNA investigation employed the Archer® FusionPlex® Pan-Heme Kit for Illumina platform according to manufacturer’s recommendations.

Results: RNA NGS investigation revealed the RUNX1::FOXJ2 fusion. The identified fusion partners were RUNX1 exon 6 chr21 and FOXJ2 exon 2 chr12.

Conclusion: RNA sequencing revealed the RUNX1::FOXJ2 fusion gene transcript in the relapse sample. This fusion was not detected in the diagnostic sample.

FOXJ2 (Forkhead Box J2) is a transcriptional activator localized constitutively at the nucleus of the cell.

RUNX1, is a transcription factor that plays a crucial role in hematopoietic differentiation. Chromosomal rearrangements with RUNX1 have been identified with uncommon partners in various hematologic malignancies.

Well-powered studies indicate that patients with a RUNX1 alteration, experience a less favourable prognosis in alignment with the patient’s disease progression.

Accurate detection of RUNX1 alterations is essential for unravelling the etiology of blood disorders and determining disease prognosis.

Grants:

Conflict of Interest: None declared

EP01.083 Novel mutation in CHEK1 gene in Bulgarian patient with Poorly cohesive gastric carcinoma

Maria Glushkova ¹, Tihomir Todorov¹, Svetlana Gacheva², Albena Todorova^1;3

¹GMDL Genica, Sofia, Bulgaria; ²Аджибадем Сити Клиник УМБАЛ Младост, Sofia, Bulgaria; ³Medical University of Sofia, Sofia, Bulgaria

Background/Objectives: Here we present a 48-years old Bulgarian woman referred for testing of oncogene panel by NGS in relation to future therapy.

The diagnosis is Poorly cohesive gastric carcinoma with metastases in retroperitoneum and peritoneum. Immunohistochemistry shows high-grade adenocarcinoma, HER2- and PD-L1(CPS > 10). She underwent two courses of treatment with Oxaliplatina, Fluorouracil and Nivolumab. Cytopenia and inflammatory infectious changes are observed.

Methods: Genetic testing includes generally 688 genes: 175 genes related to target therapy, 69 genes for hereditary cancer and 7 genes are drug-metabolizing enzymes.

Results: The genetic test show pathogenic germline mutation in CHEK1 gene (p.Y334Ter;c.1002C>G). The mutation has not been reported in the literature.

Four somatic clinically significant variations were found in the FFPE: TP53 (p.K164E;c.490A>G), KRAS (CopyNumberGain) and two variants in RHOA. Based on the somatic genetic variants there are some FDA approved therapies but for the other types of tumors (Level C and D).

Conclusion: CHEK1 gene is protein kinase and plays central role in DNA damage, replication, repair, checkpoint activation and it is essential for cell proliferation and survival. It belongs to DNA damage response and repair (DDR) related genes and the detected variant is from inactivation type with possible benefit of immunotherapy. Tumors with mutations in DDR demonstrated a higher survival rate with ICB (ImmuneCheckpointBlockade) therapy. In this case immunotherapy would be possible opportunity.

We recommended segregation analysis for patient’s relatives in order to understand the risk of the corresponding tumors. The genetic testing of oncogene panel is important for the genetic counseling and correct therapeutic approach.

Grants: No

Conflict of Interest: None declared

EP01.084 Investigation of the effects of dietary supplement Methylsulfonylmethane on apoptosis signals in acute myeloid leukemia cell lines

Yalda Hekmatshoar ¹, Arzu Zeynep Karabay², Asli Koc², Tulin Ozkan³, Asuman Sunguroglu³

¹Altinbas University, Department of Medical Biology, Faculty of Medicine, Istanbul, Türkyie; ²Ankara University, Department of Biochemistry, Faculty of Pharmacy, Ankara, Türkyie; ³Ankara University, Department of Medical Biology, Faculty of Medicine, Ankara, Türkyie

Background/Objectives: Acute myeloid leukemia (AML) is a heterogeneous disease in both biological and clinical concepts. Methylsulfonylmethane (MSM), an organic sulfur-containing dietary supplement, utilized for improving various clinical conditions particularly osteoarthritis. MSM can exert antitumor activity in various types of cancers. However, the molecular mechanisms of action underlying antileukemic activity of MSM remain unclear.

Methods: In this study, we explored the anticancer effect of MSM on human AML cell lines (U937 and HL60) with focus on underlying death mechanism. Anticancer activity of the MSM was examined employing MTT assay, Annexin V-PE/7AAD staining and real-time q-PCR. Both cell lines were treated with different concentrations (50-400 mM) of MSM for 24h.

Results: Results of MTT assay revealed that in both cell lines, the MSM markedly reduced cell viability in comparison to the control cells. Additionally, findings of Annexin V-7AAD staining represented that MSM triggered apoptosis in both cell lines significantly. Apoptotic genes expression levels were assessed by real-time PCR. Based on the real time q-PCR data, MSM increased the mRNA expression levels of BAX and BIM genes in treated cells.

Conclusion: Over all, our results indicated that MSM could induce apoptosis in AML cell lines in a dose-dependent manner, therefore could be utilized as antileukemic agent.

Grants: -

Conflict of Interest: None declared

EP01.085 Gorlin-Goltz syndrome in six Polish patients

Renata Posmyk ¹, Joanna Karwowska¹, Klaudia Berk¹

¹Medical University in Bialystok, Department of Clinical Genetics, Bialystok, Poland

Background/Objectives: Gorlin-Goltz syndrome (GGS) (OMIM#109400), (Basal Cell Nevus Syndrome or Nevoid Basal Cell Carcinomas Syndrome) is a rare genetic disorder characterized by basal cell carcinomas and nevi, pits of palms and soles, calcification of falx cerebri, odontogenic keratocysts of jaws, bone abnormalities and dysmorphic features. Evans diagnostic criteria are very useful for clinical diagnosis. GGS is inherited in autosomal dominant manner and caused by mutations in PTCH1, PTCH2 or SUFU genes.

Methods: We present the phenotypes of Polish (N = 6) participants of the project „Genetic Background of Overgrowth Syndromes in Polish and Lithuanian Populations: Basis for Rapid Genetic Testing to Prevent Neoplasms” with cardinal features of GGS.

Results: In 4 patients the diagnosis was molecularly confirmed by the pathogenic mutations in PTCH1 gene, and in 1 the small deletion in the same gene was found. Despite the wide molecular testing in one patients, the genetic confirmation has not been made.

Conclusions: All patients fulfill the diagnostic criteria of GGS, but their symptoms are of wide spectrum and molecular results are variable. Lack of detection of pathogenic variants in genes correlated with clinical symptoms of GGS may indicate the unknown molecular cause of the disorder which may be a field for the future research.

Grants: NCN/1/DA/21/001/1106

Conflict of Interest: Renata Posmyk NCN/1/DA/21/001/1106, Full-time Medical University in Bialystok, Joanna Karwowska: None declared, Klaudia Berk: None declared

EP01.086 First association of Heyn-Sproul-Jackson syndrome and paraganglioma in one patient

Anna Maria Cueto-González ^1;2, Alejandro Moles-Fernandez^1;2, Maria Camprodón-Gómez³, Anna Casteras-Román⁴, Teresa Vendrell-BAyona¹, Elena García-Arumí^1;2, Eduardo Tizzano^1;2

¹Vall d’Hebron Barcelona Hospital Campus (Barcelona), Clinical and Molecular Genetics Area., Barcelona, Spain; ²Vall d’Hebron Institut de Recerca (VHIR), Vall d’Hebron Barcelona Hospital Campus, Medicine Genetics Group, Barcelona, Spain; ³Vall d’Hebron Barcelona Hospital Campus (Barcelona), Rare diseases Unit., Barcelona, Spain; ⁴Vall d’Hebron Barcelona Hospital Campus (Barcelona), Endocrinology Unit., Barcelona, Spain

Introduction: autosomal dominant gain-of-function mutations (GoFm) in DNMT3A gene (Heyn-Sproul-Jackson syndrome (HESJAS); OMIM618724) have recently been described with reciprocal phenotype to Tatton-Brown-Rahman syndrome (OMIM615879) and it is characterized by microcephalic dwarfism.

At present only 3 patients have been reported with HESJAS (Heyn et al.,2019), all without oncological pathology. On the other hand, 3 cases with somatic GoFm in the DNMT3A gene associated with paragangliomas have also been reported, none with HESJAS diagnosis, and only one with intellectual disability (Remacha et al., 2018; Mellid et al., 2020).

Methods: we report a 34-year-old patient with global developmental delay, mild intellectual disability, postnatal short stature, microcephaly, Chiari malformation type I, and particular phenotypic characteristics corresponding to HESJAS. Multiple asymptomatic carotid gangliogliomas were detected in a control MRI scan.

Results: Having a previously had an inconclusive array-CGH and exome, but after exome reanalysis revealed a GoFm in the DNMT3A gene not described in population databases, which was also found in the tumour tissue of one of the paragangliomas.

Conclusions: So far, GoFm in the DNMT3A gene have been described at the germline level in 3 patients with HESJAS and in 3 patients with paraganglioma without diagnosis of HESJAS, only one with isolated intellectual disability. To our knowledge, this is the first patient with HESJAS and GoFm in the DNMT3A gene confirmed at both, germline and tumour. Documentation of paragangliomas in patients with HESJAS should be incorporated into the follow-up protocol. On the other hand, a complete anamnesis and physical examination of patients with paraganglioma to discard HESJAS is also necessary.

Conflict of Interest: None declared

EP01.088 Is the obvious enough when considering Lynch Syndrome diagnosis? Rare cancers in index cases and diagnostic challenges

Iulia Simina ^1;2, Iulia Teodora Perva², Catalin-Vasile Munteanu^1;3, Oana Cristina Voinea^4;5, Adrian Trifa^1;6;7

¹Victor Babeş University of Medicine and Pharmacy, Department of Genetics, Center of Genomic Medicine, Timișoara, Romania; ²Oncohelp Medical Center, Genetics, Timișoara, Romania; ³Emergency Hospital for Children Louis Ţurcanu, Medical Genetics, Timișoara, Romania; ⁴Cantacuzino National Institute of Research, București, Romania; ⁵Carol Davila University of Medicine and Pharmacy, Pathology, București, Romania; ⁶Victor Babeș Hospital, Medical Genetics, Timișoara, Romania; ⁷The Oncology Institute “Prof. Dr. Ion Chiricuţă” Cluj-Napoca, Breast Cancer Center, Cluj-Napoca, Romania

Background: When an index case presents with a rare cancer absent from the tumour spectrum of the most prevalent cancer predisposition, Lynch syndrome (LS), several additional information can improve diagnosis timing and prevention strategy. We raise an issue that emphasizes these diagnostic challenges by presenting a case series.

Methods: The clinical workup and family history was performed for the affected individuals. MMR studies have completed the analysis. Sequence analysis and deletion/duplication testing of an extended panel of genes for cancer predisposition syndromes was performed, using Illumina’s sequencing-by-synthesis method.

Results: We identified 3 case-index patients with rare cancers consecutive to high susceptibility of LS, and only because we chose to perform extended panel sequencing due to their personal or family history: P1 presented with an extrahepatic biliary duct adenocarcinoma at age 42yo, heterogeneous family history, and a CNV in MLH1 gene; P2 had a breast luminal cancer at age 47yo, a paternal cousin with endometrial cancer and a PMS2 pathogenic variant; P3 had breast bilateral adenoma at age 34yo with positive familial history for both breast and endometrial cancers and a likely pathogenic variant in PMS2. Other 2 cases who both presented early with thyroid cancer were not diagnosed until they have developed a suggestive colon cancer, empowering the need for an improved diagnosis strategy in uncommon LS cancers.

Conclusion: Oncogenetics has an essential role in identifying individuals at-risk for Lynch syndrome and planning a preventive strategy using family history. Sometimes, thinking outside the patterns can offer a diagnosis.

Conflict of Interest: None declared

EP01.089 Oncogenic miR-21 and miR-31 clinical relevance in oral cancer: tissue specimens vs. exosomes

Milos Lazarevic¹, Dijana Trisic¹, Milica Jaksic-Karisik¹, Drago Jelovac¹, Nadja Nikolic¹, Jelena Carkic¹, Jelena Milasin ¹

¹University of Belgrade, School of Dental Medicine, Belgrade, Serbia

Background/Objectives: Oral cancers may be very aggressive, and new predictors of their behavior are needed. MicroRNAs (miRNAs) are small, non-coding RNAs involved in gene expression regulation; their altered levels have been linked to cancerogenesis. Exosomes, l extracellular vesicles that carry different cargos including nucleic acids, proteins, etc. are important mediators of intercellular communication. We aimed to investigate two oncogenic miRNAs (miR-21 and miR-31) levels in oral squamous cell carcinomas (OSCCs) specimens and exosomes, and to establish whether they may predict OSCC behavior.

Methods: RNA was extracted from 20 OSCC tissue specimens and from exosomes, using trizol. Exosomes were previously isolated from cell cultures obtained from OSCC patients, by magnetic separation, and characterized by flow-cytometry, TEM and nanoparticle tracking analysis. Reverse transcription and qPCR were applied to determine micro RNAs levels, followed by statistical analysis.

Results: MiR-21 levels were significantly higher in tissue specimens of OSCC patients compared to control tissues (p < 0.0001). The levels were also higher in T3 compared to T1 (p < 0.05) and T2 (p < 0.01) stages. Surprisingly, miR-21 levels were significantly lower in cancer exosomes compared to control exosomes. MiR-31 levels were also higher in OSCC than in controls (0.05) and exosomes followed the same pattern. MiR-31 also showed increase with increasing stages. In cancer tissue there was no significant difference of miRNAs expression, while exosomes contained much higher levels of miR-21 than miR-31 (<0.01).

Conclusion: Tumor tissues’, rather than exosomes’ miR-21 and miR-31 levels might indicate OSCC course.

Grants: Science Fund of the Republic of Serbia, GRANT 7750038

Conflict of Interest: Milos Lazarevic School of Dental Medicine, Dijana Trisic School of Dental Medicine, Milica Jaksic-Karisik: None declared, Drago Jelovac School of Dental Medicine, Nadja Nikolic School of Dental Medicine, Jelena Carkic School of Dental Medicine, Jelena Milasin Science Fund of the Republic of Serbia, GRANT 7750038, School of Dental Medicine

EP01.090 Unveiling novel genetic variants in Peutz-Jeghers syndrome: A case report of a de novo mutation in the STK11 gene

Jorge Docampo-Cordeiro ¹, Fransico Jose Garcia-Iñigo¹, Elena Jaime-Lara¹, Marta Casas-Rodriguez¹, Rosa DeFrutos-Curiel¹, Ana Santos-Corral¹, Maria Luisa Casas-Losada¹

¹Hospital Universitario Fundación Alcorcón, Analisis Clínicos, Alcorcon, Spain

Background/Objectives: Peutz-Jeghers syndrome (PJS) is a rare autosomal dominant disorder characterized by the development of hamartomatous polyps in the gastrointestinal tract and distinctive mucocutaneous pigmentation. Genetic mutations in the serine/threonine kinase 11 (STK11) gene have been implicated in the pathogenesis of PJS. Here, we present a case report of a novel de novo mutation in the STK11 gene associated with PJS, highlighting the importance of genetic testing in diagnosis and management.

Methods: Sophia Genetics Custom Hereditary Cancer Solution targeted genomic sequencing (TS) was performed on Illumina MiSeq and the resulting data was processed and analyze using Sophia DDM software.

Results: A novel loss-of-function mutation was identified, NM_000455.4:c.701del p.(Phe234Serfs*53), in heterozygosity, in exon 5 of STK11 gene. Further analysis confirmed de novo nature of the mutation.

Conclusion: We report the case of an 16-year-old female patient presenting with hamartomatous polyps and mucocutaneous pigmentation consistent with a clinical diagnosis of PJS.

The identification of a novel de novo mutation in the STK11 gene expands the spectrum of genetic variants associated with PJS. This case underscores the importance of considering de novo mutations in the genetic evaluation of patients with suspected PJS, especially in the absence of familial history. Additionally, our findings highlight the utility of TS in uncovering rare genetic variants underlying rare hereditary syndromes like PJS, facilitating accurate diagnosis and personalized management strategies.

Grants:

Conflict of Interest: None declared

EP01.091 The Association between MUTYH Gene Variants and Colorectal Cancer

Asli Subasioglu ¹, Roya Gasimli²

¹Izmir Katip Çelebi University, Faculty of Medicine, Department of Medical Genetics, İzmir, Türkyie; ²Ege University, Faculty of Medicine, Department of Medical Biology, Izmir, Türkyie

Background/Objectives: Familial cancers arise from intricate genetic and epigenetic factors, with notable focus on MUTYH gene variants. This gene encodes the DNA glycosylase enzyme involved in DNA base excision repair and is specifically associated with colorectal cancer and adenomatous polyposis. Our primary objective is to systematically document varied pathogenicity profiles of MUTYH gene variants, aiming to identify specific variants for advancing diagnostic, therapeutic, and prognostic strategies in familial cancers, with a specific focus on colorectal cancers. Disseminating these findings in scholarly literature aims to enrich the scientific discourse on molecular markers indicative of familial cancer predisposition.

Methods: The study utilized peripheral blood samples from 3036 individuals with a hereditary cancer and colorectal cancer history. Library preparations involved DNA samples from peripheral blood, utilizing Qiaseq Targeted Panels kits under optimal conditions. After quality assessments meeting concentration criteria, sequencing occurred on the Illumina Miseq platform. Raw data underwent analysis, producing a variant call format (vcf) file via the QIAGEN CLC Genomics Workbench. The Qiagen Clinical Insight (QCI Interpret) system discerned variants potentially implicated in familial cancers.

Results: In accordance to our findings, a comprehensive analysis revealed a total of 155 mutations in the MUTYH gene, comprising 79 pathogenic (P), 5 likely pathogenic (LP), and 71 variants of uncertain significance (VUS).

Conclusion: These results offer valuable insights into the genetic landscape, deepening our understanding of the potential implications of MUTYH gene variations in the context of familial cancers and colorectal cancer susceptibility.

Grants: None.

Conflict of Interest: None declared

EP01.092 Actionable secondary findings in genes related to cancer phenotypes in 2020 whole exome sequenced Turkish participants

AYBERK TURKYILMAZ ¹, KUBRA ADANUR SAGLAM¹, Oguzhan Demir¹, Mustafa Yilmaz¹, Tuna Apuhan¹, Alperhan Cebi¹

¹Karadeniz Technical University, Medical Genetics, Trabzon, Türkyie

Abstract

Background: Big data generated from whole exome sequencing (WES) and whole genome sequencing (WGS) analyses can be used to detect actionable and high-penetrance variants that are not directly associated with the primary diagnosis of patients but can guide their clinical follow-up and treatment. Variants that are classified as pathogenic/likely pathogenic and are clinically significant but not directly associated with the primary diagnosis of patients are defined as secondary findings (SF). The aim of this study was to examine the frequency and variant spectrum of cancer related SF in the Turkish population and to discuss the importance of the presence of cancer related SF in at-risk family members in terms of genetic counseling and follow-up.

Methods: A total of 2020 patients from 2020 different families were evaluated by WES.

Results: SF were detected in 20 unrelated cases (0.99%), and variants in BRCA2 (11 patients) and BRCA2 (2 patients) genes were observed most frequently. A total of 17 different variants were identified, with 4 of them (c.9919_9932del and c.3653delG in the BRCA2 gene, c.2002A>G in the MSH2 gene, c.26_29delTGCC in the TMEM127 gene) being novel variations. In 3 different families, c.1189C>T (p.Q397*) variation in BRCA2 gene was detected, suggesting that this may be a common variant in the Turkish population.

Conclusion: This study represents the largest cohort conducted in the Turkish population, examining the frequency and variant spectrum of cancer-related SF. Genetic testing conducted in family members is presented as real-life data, showcasing the implications in terms of counseling, monitoring, and treatment through case examples.

Grants: None.

Conflict of Interest: None declared

EP01.093 Exploring the Influence of HER2 Negative Breast Cancer Cell Secretome on Healthy Epithelial Cells

Busra Yasa Cevik ^1;2, gülşah tuna^1;2, fadime didem can trabulus³, Selcuk Sözer Tokdemir^1;2

¹Institute of Health Sciences, Istanbul University, Istanbul, Türkyie; ²Aziz Sancar Institute of Experimental Medicine, Istanbul University, Department of Genetics, Istanbul, Türkyie; ³Bahçeşehir University Faculty of Medicine, Department of General Surgery, Istanbul, Türkyie

Background/ Objectives: Breast cancer ranks as a leading cancer diagnosis among women. A key aspect of cancer progression is the ability of cancer cells to manipulate their surrounding micro and macroenvironments, facilitating metastasis. This study explores the functional impacts exerted by the secretome of the HER2 negative breast cancer cell line, MCF7, on healthy breast epithelial cells (MCF10A).

Methods: In our experimental setup, one-fifth of the spent media from MCF7 cells, known for their aggressive proliferation and secretion of various compounds, was introduced to MCF10A cultures. Post 48 hours of co-culturing, MCF10A cells were assessed for alterations in functional characteristics. We employed xCELLigence for real-time proliferation tracking (every 15 minutes over 144 hours), scratch assays for migration analysis (every 12 hours for 48 hours), LEGENDplex™ ELISA for cytokine profiling, and 7AAD-AnnexinV staining to evaluate apoptosis. All observations were benchmarked against control MCF10A cells.

Results: Notably, the MCF10A cells exhibited a marked increase in proliferation and IL-6 secretion (P < 0.0001). Initial 24-hour migration rates were significantly heightened. Interestingly, an increase in apoptosis rates (from 1.79% to 3.89%) was also observed.

Conclusions: The enhanced proliferation, migration, and IL-6 release in MCF10A cells suggest a potential transition towards a malignant state under the influence of the MCF7 cell secretome. Future research focusing on the composition of this spent media could illuminate the underlying mechanisms of cancerogenesis.

Grant: The study financially supported by Health Institutes of Turkey (TUSEB). (Project No: 27803)

Conflict of Interest: Busra Yasa Cevik 40, TUSEB, gülşah tuna: None declared, fadime didem can trabulus 20, TUSEB, Selcuk Sözer Tokdemir 40, TUSEB

EP01.095 uORF creation by 5’UTR variants: a new mechanism of TP53 loss-of-function

marie bequin ^1;2, Luisa Vergori^1;2, Vincent Milon^1;2, Omar Soukarieh^3;4, Caroline Meguerditchian⁴, Fida Khater^1;2, Jonathan Dauvé^1;2, Yves Delneste¹, Louise-Marie Chevalier^1;2, David-Alexandre Tregouët⁴, Gaelle Bougeard^5;6, Alain Morel^1;2, Isabelle Tournier^1;2

¹University of Angers, UMR Inserm 1307 - CNRS 6075, CRCI2NA, Innate Immunity and Cancer, Angers, France; ²Integrative Center for Oncology, Functional Genomics Unit, Angers, France; ³University of Bordeaux, INSERM, U1034, Biology of Cardiovascular Diseases, Pessac, France; ⁴University of Bordeaux, INSERM, UMR 1219, Bordeaux Population Health Research Center, Bordeaux, France; ⁵University of Rouen, UMR Inserm 1245, Cancer and Brain Genomics, Rouen, France; ⁶Rouen University Hospital, Genetics Department, Rouen, France

Background/Objectives: The TP53 gene is a major player in oncology, both in sporadic cancers and in hereditary cancer syndromes such as Li-Fraumeni (LFS). It is essential for cancer patients and their relatives to be able to detect and interpret all the variants detected in this gene, including alterations in non-coding regulatory regions such as the 5’ or 3’ untranslated regions (5’UTR, 3’UTR). Such variants can be pathogenic but can also correspond to genetic modifying factors. In particular, variants in the 5’UTR can affect mRNA stability, subcellular localization or its translation efficiency. They can also create upstream open reading frames (uORFs) that can compete with the main ORF and lead to haploinsufficiency by reducing the protein levels. Here, we set up a functional assay for TP53 UTR variants, and characterized the functional impact of 32 variants.

Methods: Using our luciferase reporter assay dedicated to p53, we analyzed 32 5’UTR variants detected in cancer patients or in LFS-evocating patients for their impact on protein levels.

Results: Six variants impacted the protein levels, two of them by creating uORFs. This impact could be predicted by bioinformatics tools. To evaluate these in silico predictions, we also tested a selection of artificial variants predicted to create different types of uORFs.

Conclusion: Our study highlights the need to explore non-coding regulatory regions of cancer genes both for genetic counselling and personalized medicine. It also underscores the necessity to integrate uORF prediction tools in diagnostic annotation pipelines.

Grants : La ligue contre le cancer

MB-LV-VM Equal contribution

Conflict of Interest: None declared

EP01.096 Lynch syndrome caused by di-genic events – inherited MSH6 pathogenic variant and mosaic MLH1 methylation

Natali Schachter- Safrai^1;2, Inbal Kedar¹, lihi atzmoni^3;4, adel Shalata⁵, Dvora Kidron^4;6, lina basel-salmon^1;4;7, Zohar Levi^4;8, Yael Goldberg ^1;4

¹Raphael Recanati Genetic Institute, Rabin Medical Center, Beilinson Hospital, Petach Tikva, Israel; ²Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel; ³Department of Dermatology, Rabin Medical Center, Beilinson Hospital, Petach Tikva, Israel; ⁴Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel; ⁵The Simon Winter Institute for Human Genetics, The Bnai Zion Medical Center Bruce Rappaport Faculty of Medicine, Technion, Haifa, Israel; ⁶Department of Pathology, Meir Hospital, Kfar Saba, Israel; ⁷Felsenstein Medical Research Center, Petah Tikva, Israel; ⁸The Gastroenterology Department, Rabin Medical Center, Beilinson Hospital, Petach Tikva, Israel

Background/Objectives: Lynch syndrome (LS) is a cancer predisposition syndrome, associated with increased risk mainly for mismatch repair deficient (MMRd)-colorectal and endometrial cancer. LS results from a monoallelic germline mutations in one of four MMR-genes: MLH1, MSH2, MSH6 and PMS2. Inactivation of the MMR genes can also be mediated by epigenetic mechanisms, mainly EPCAM deletions or MLH1 methylation. We here report on a patient with co-occurrence of inherited MSH6 pathogenic variant and MLH1 germline methylation

Methods: A 28 y/o female presented with MMRd right CRC. She reported on a neurofibroma previously removed from her skull and had five café au lait spots. Family history was significant for cancer on both sides. Molecular analysis of the tumor included MMR immunohistochemistry, MSI analysis and NGS. Germline analysis included multigene panel testing and MLH1 promoter methylation analysis by MS-MLPA, done on two independent DNA extractions.

Results: Tumor molecular analysis showed loss of MLH1 and PMS2 expression with intact MSH2 and MSH6. Tumor molecular profile showed MSI-H, mutation burden of 22 (m/mb), wild-type BRAF, and MSH6 c.755C>G; p.Ser252* variant in 46% of the reads. There were no pathogenic variants in MLH1, PMS2 and NF1. Germline testing revealed the MSH6 c.755C>G; p.Ser252* variant, inherited from the proband’s father, along with increased MLH1 methylation, indicating mosaic germline methylation.

Conclusion: The discrepancy between tumor IHC for MMR and germline status was explained by an epigenetic event of MLH1 methylation. The combined comprehensive tumor-germline analysis contributed to understanding of the underlying genetic diagnosis.

Conflict of Interest: None declared

EP01.097 Ten years investigation of EGFR mutation screening in Non-Small Cell Lung Cancer Comparison of Real-time PCR and Reverse Strip Assay methods

Maryam Safa Isini ¹, Hossein Najmabadi², ali Salehzadeh¹, Farzam Ajamian³

¹Islamic Azad University, Rasht Branch, Department of Biology, Rasht, Iran; ²Faculty of Social Welfare and Rehabilitation Sciences - National University, Tajrish, Iran; ³University of Guilan - Faculty of Sciences, Department of Biology, Rasht, Iran

Background: Non-Small Cell Lung Cancer (NSCLC) is one of the cancers whose prevalence is increasing day by day. Based on studies, it has been shown that EGFR mutation is one of the main factors in the pathogenesis of NSCLC. Strip Assay method is usually used to detect EGFR mutation. Based on recent studies, it has been determined that the use of cell free DNA (cfDNA) in peripheral blood samples can detect mutations using Reverse Strip Assay (RSA), just like biopsy samples. Considering that cfDNA samples are easier to access than biopsy samples, therefore, in this study, the sensitivity of EGFR mutation detection was compared using cfDNA and biopsy samples though RSA.

Methods: Biopsy samples of 306 patients with NSCLC and 120 cfDNA sample of patients who had mutations in the EGFR gene were collected and using cfDNA by RSA and RT-PCR methods.

Results: The EGFR RSA analysis from biopsy samples, revealed 82 (26.8%) FFPE samples carry an EGFR mutation, of which the most prevalent was p.L858R (c.2573T>G) in exon 21 (5.9%), p.E746-A750del(c.2235-2249del)(5.2%) and p.E746-A750del(c.2235-2250del)(3.9%) in exon 19. One hundred and twenty cfDNA samples of affected patients were used to evaluate EGFR mutations. Also, mutations were re-checked by RT-PCR in parallel. The results showed that cfDNA StripAssay and RT-PCR methods had the same sensitivity by using cfDNA instead of biopsy.

Conclusion: In general, it can be said that cfDNA instead of biopsy samples are more suitable for EGFR mutation screening using StripAssay, which cfDNA is more convenient, non-invasive and economical for patients.

Conflict of Interest: None declared

EP01.098 Evaluation of brain and whole-body MRI surveillance service for adult individuals with pathogenic TP53 variants: Data from a regional clinical genetics service in London

Arwa Babai ¹, Vicky Goh^2;3, Sabrina Talukdar^1;4, vishakha tripathi^1;4, Anjana Kulkarni^1;4, Louise Izatt^1;4

¹South East Thames Regional Genetics Service, Guy’s and St Thomas’ NHS Foundation Trust, London, United Kingdom; ²Guy’s and St Thomas’ NHS Foundation Trust, Department of Radiology, London, United Kingdom; ³King’s College London, School of Biomedical Engineering & Imaging Sciences, London, United Kingdom; ⁴Guy’s and St Thomas’ NHS Foundation Trust, Hereditary Breast and Ovarian Cancer Service, London, United Kingdom

Background/Objectives: Germline pathogenic variants in TP53 are associated with Li-Fraumeni syndrome and a higher lifetime risk of developing various types of cancers. The current UK Cancer Genetics Group (CGG) guidelines for carriers of clinically-actionable TP53 germline variants (‘TP53 carriers’) recommend annual screening, consisting of brain MRI (B-MRI) and whole-body MRI (WB-MRI). This retrospective service evaluation, aimed at understanding the factors that influence attendance for annual MRI screening and to assess the detection rate of cancer and incidental findings (IF) at our Centre.

Methods: The timeline to diagnosis and previous MRI reports for 26 adult TP53 carriers (7 males, 19 females) at Guy’s Hospital from 2020 to 2023 were reviewed. Data about the intervals between successive MRI scans, report findings and any extra investigations required were collected.

Results: Overall, 10 out of 26 TP53 carriers (38%) attended for screening at the recommended 12-month interval. Delayed screening was mostly due to patient-related factors, including appointment rescheduling requests, non-response to invitation letters or not tolerating the scan. IF were reported in 15 out of 26 TP53 carriers (57%). 87% of the detected IF were benign and 13% were malignant. The malignant IF were due to recurrence or metastatic disease in TP53 carriers with prior cancer diagnosis. 30% of IF required further investigations, to evaluate the nature of the screen-detected lesions.

Conclusion: A larger-scale study is recommended to further assess the detection of malignant versus benign lesions through annual MRI screening in TP53 carriers and the cost-effectiveness of annual brain and WB-MRI.

Conflict of Interest: None declared

EP01.099 Genetic Landscape of Hereditary Colorectal Cancer: Insights from Next-Generation Sequencing Analysis in a Retrospective Study

Hakan Tomac ¹, Aysel Kalayci², Deniz Agirbasli²

¹Istanbul University-Cerrahpasa, Cerrahpasa Faculty of Medicine, Istanbul, Türkyie; ²Istanbul University-Cerrahpasa, Cerrahpasa Faculty of Medicine, Department of Medical Genetics, Istanbul, Türkyie

Background/Objectives: Colorectal cancer is the third most common cancer worldwide. It is closely linked to hereditary cancer syndromes, with distinct molecular mechanisms. Our study retrospectively examined genetic variations in colorectal cancer patients, utilizing Next-generation sequencing (NGS) based-multigene panel data. We aim to uncover key genetic insights and demographic patterns, contributing to a better understanding of hereditary colorectal cancer in our population and facilitating personalized approaches for diagnosis and familial screening.

Methods: The NGS genetic panel results of 42 patients diagnosed with colorectal cancer or patients having polypoid lesions with a strong family history of hereditary colorectal cancer were retrospectively analyzed. Clinical and demographic data, along with family history, were obtained from patient reports. Variants are analyzed using the Cytoscape application to identify affected molecular networks.

Results: This study included 42 patients (n = 45.2% female and n = 54.8 % male with a mean age at diagnosis 46.3 ± 16.3 years). No identifiable variants were detected in 25 patients (59.5 %). Our findings reveal that 7 patients (16.7%) carried pathogenic variants, while 10 (23.8%) carried Variants of Unknown Significance (VUS). 5 patients (11.9%) carried multiple variants. Pathogenic/likely pathogenic variants were detected in GALNT12, MUTYH, APC, MSH6, CTNNB1 and VUS were detected in EGFR, BRCA1, APC, MLH1, NTHL1, STK11, PALB2, MUTYH genes.

Conclusion: As panel testing statistically significantly increases VUS rates, variant interpretation and appropriate genetic counseling are important for clinical decision-making. The identified variants were linked to pathways related to chromosomal and microsatellite instability, as well as the Wnt and base excision repair pathways.

Conflict of Interest: None declared

EP01.100 BRCAness Phenotype and Immune Microenvironment in Triple-Negative Breast Cancer

LUISA AYELEN RAMOS NAVAS ^1;2, Beatriz Esquivel Vázquez³, Eduardo Salido Ruiz^1;2;3

¹Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), San Cristóbal de La Laguna, Spain; ²Universidad de La Laguna, Escuela de Doctorado y Estudios de Posgrado. Ciencias médicas y farmacéuticas de la vida, San Cristóbal de La Laguna, Spain; ³Complejo Hospitalario Universitario de Canarias, Anatomía Patológica, San Cristóbal de La Laguna, Spain

Background: Breast cancer, with over 2.26 million new cases in 2021, stands as the most prevalent cancer globally. Triple-negative breast cancers (TNBCs), constituting 10–20% of cases, present aggressive features, and a challenging prognosis, prompting intensive research into their characterization and identification of therapeutic targets. Recent studies have explored “BRCAness,” indicating defects in homologous recombination repair (HRR) in tumors. Notably, inactivation of BRCA1 may result not only from genetic mutations but also epigenetic alterations like promoter methylation. Simultaneously, other studies have delved into the tumor immune microenvironment’s crucial role, with immunotherapy emerging as a breakthrough in modern oncology. Understanding TNBC immune infiltrate aids in identifying immunotherapy beneficiaries and resistance factors as potential therapeutic targets.

Methods: We analyze BRCA1 gene promoter methylation by pyrosequencing, as a potential marker of BRCAness to assess its clinical relevance in therapeutic stratification. Additionally, we explore immune biomarkers (TILs, and CD8+ spatial distribution) and their potential correlation with BRCA1 methylation and HRD-related mutations.

Results: Our pyrosequencing analysis of 100 triple-negative breast cancer patients revealed that 65% of the samples exhibited methylation CpG island methylation above 15% (range 15-80%), which could indicate BRCAness phenotype. Also, preliminary findings suggest a lack of significant correlation between Tumor Infiltrating Lymphocytes (TILs) abundance and BRCA1 methylation in TNBC.

Conclusion: In our series, more than half TNBC samples show methylation of BRCA1 promoter and a similar percentage have medium-high TIL infiltration, but statistical analysis revealed no association. Despite the recognized importance of both factors, initial results hint at a complex relationship requiring further analysis.

Conflict of Interest: LUISA AYELEN RAMOS NAVAS Proyecto PMP22/00054, Beatriz Esquivel Vázquez: None declared, Eduardo Salido Ruiz Proyecto PMP22/00054

EP01.101 Detection of PDL1 by FISH and IHC in triple negative breast cancer

Beatriz Esquivel Vázquez ¹, LUISA AYELEN RAMOS NAVAS², Eduardo Salido Ruiz^1;2

¹Complejo Hospitalario Universitario de Canarias (CHUC), Anatomía Patológica, La Laguna; ²Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), U740, San Cristóbal de La LAguna, Spain

Background/Objectives: the aim of this study was to compare results obtained analyzing gene and protein status of PD-L1, by fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) techniques in 50 breast cancer samples previously categorized as triple negative. This is an aggressive and heterogeneous subtype, characterized by the absence of expression of HER2 and hormone receptors, estrogen and progesterone. PD-L1, as programmed death ligand 1, has been identified as a potential marker of response to immune checkpoint inhibitors in several cancer types, including triple-negative breast cancer. Then, it would be a prognostic and predictive biomarker.

Methods: for the FISH technique, the ZytoLight SPEC CD274, PDCD1LG2/CEN9 Dual Color Probe (Zytovision) was used, while IHC was performed using specific antibodies for PD-L1 (Roche, both SP142 and SP263). PD-L1 expression levels were evaluated and the concordance between both techniques will be considered.

Results: preliminary results showed high overall agreement between FISH and IHC in the detection of PD-L1. Only cases were observed where it was possible to differentiate by FISH whether it was a true amplification or polysomy of chromosome 9. We would like to check if the response to immunotherapy varies in PD-L1 positive due to amplification or positive due to polysomy.

Conclusion: it is concluded that IHC is an economical and sensible enough technique to be used in routine diagnostics. The study of this molecule using FISH could become clinically useful if prognostic or treatment variations are demonstrated depending on the biological event responsible for PD-L1 overexpression.

Conflict of Interest: None declared

EP01.102 HOXB13 new founder mutation in galician (North West Spain) prostate cancer patients?

Javier Manuel Galego Carro ^1;2, Marta Santamariña^1;2;3, Miguel E. Aguado-Barrera^1;2, Jorge Amigo-Lechuga^1;2, Olivia Fuentes^1;2, Ana Crujeiras^1;2, Carlos López^1;2, Carla Coedo-Costa^1;2, Antonio Gomez-Caamaño^1;4, Ana Vega^1;2;3

¹Instituto de Investigación Sanitaria de Santiago de Compostela (IDIS); ²Fundación Pública Galega de Medicina Xenómica (FPGMX); ³Centro de Investigación en Red de Enfermedades Raras (CIBERER); ⁴Servicio de Oncología Radioterápica, Complejo Hospitalario Universitario de Santiago (CHUS)

Background/Objectives: Recent studies have associated the HOXB13 gene with a significantly increased risk of hereditary prostate cancer and different variants have been identified in various populations. However, there is only evidence regarding the pathogenicity of the c.251G>A variant, and the clinical significance of the rest remains uncertain. In this study, we propose to investigate the HOXB13 gene in a cohort of 835 high-risk prostate cancer patients.

Methods: We studied the HOXB13 gene (NM_006361.6) in a cohort of 835 galician (North West Spain) patients diagnosed with high-risk prostate cancer. We employed next-generation technologies for DNA sequencing (Illumina Novaseq6000). Variant detection, annotation and classification was performed through the use of GATK, ANNOVAR and ACMG/AMP guidelines respectively.

Results: The pathogenic variant HOXB13 (NM_006361.6): c.251G>A, p.(Gly84Glu) was found in three patients.

We have also detected the c.383C>A, p.(Ala128Asp) variant in one patient, the c.649C>T, p.(Arg217Cys) variant in another, and the c.853T>C, p.Ter285Glnext* variant in nine patients.

Patients carrying c.853T>C variant had a significantly earlier age and advanced disease at diagnosis compared to patients without pathogenic germline cancer-predisposing variants. Moreover, we found this variant to be significantly associated with the presence of family history of prostate cancer.

Haplotype and functional analysis are ongoing and will be presented at the meeting.

Conclusion: We have identified a possible first-to-date galician founder mutation in prostate cancer. The identification of these variants opens the door to personalized risk assessment and expands the possibilities of targeted therapies.

Grants: PI19/01424,INT20/00071,IN607B,PRYES211091VEGA,FPU2022-04048.

Conflict of Interest: None declared

EP01.103 Unveiling novel antitumor properties of an anthraquinone derivative: In vitro and in silico investigations

Başak Günçer ¹, varol güler², Sacide Pehlivan³, Ahmet Mesut Şentürk⁴, Bilge Özerman Edis¹

¹Istanbul Faculty of Medicine, Istanbul University, Department of Biophysics, İstanbul, Türkyie; ²Institute of Graduate Studies in Health Sciences, Medical Biology, İstanbul, Türkyie; ³Istanbul Faculty of Medicine, Istanbul University, Medical Biology, İstanbul, Türkyie; ⁴Faculty of Pharmacy, Istanbul Biruni University, Pharmaceutical Chemistry, İstanbul, Türkyie

Background/Objectives: Anthraquinone derivatives have gained traction in cancer research due to their potent anticancer properties. Our study aims to explore the antitumor potential of compound 3, a newly synthesized anthraquinone derivative, specifically targeting MDA-MB-231 breast cancer cells.

Methods: We initiated our investigation by assessing compound 3’s drug-likeness through in silico ADME analysis. Furthermore, we investigated its interaction with therapeutic target proteins via molecular docking. To evaluate its antitumor effects, we conducted proliferation assays using MTT, ROS elevation assays using DCFH-DA, migration assays employing wound healing techniques, and cytoskeleton analysis via immunofluorescence microscopy.

Results: Compound 3 demonstrated lower energy scores compared to reference drugs for targets PCNA, CK2, LC3, and MMP2, indicating its potential efficacy. In vitro analysis revealed significant inhibition of cellular proliferation, accompanied by a notable increase in reactive oxygen species (ROS) generation. Furthermore, the derivative displayed a marked reduction in migration capability and induced morphological alterations in the actin filament of the cell cytoskeleton. These findings collectively highlight the robust antitumor effects of compound 3, suggesting its promising therapeutic potential in combating cancer progression.

Conclusion: The comprehensive results underscore compound 3’s multifaceted mode of action, revealing promising therapeutic mechanisms. These findings position it as a compelling candidate for further development as a targeted anticancer agent, with broad potential applications across various cancer types. Compound 3 shows promise in hindering proliferation, inducing ROS, and impeding migration, warranting deeper investigation in preclinical and clinical settings.

Grants: This study was supported by the Scientific Research Project Coordination Unit of Istanbul University under Grant [number 39220].

Conflict of Interest: None declared

EP01.104 PALB2 variants among suspected hereditary breast cancer patients in Pakistan

Shamila Ladak ^1;1, Fizza Akbar², alizeh fatimi¹, bassim zahid¹, Salman Kirmani²

¹Medical College, Aga Khan University Hospital, Karachi, Pakistan; ²Division of Women and Child Health, Aga Khan University Hospital, Karachi, Pakistan

Background/Objectives: Partner and localizer of BRCA2 (PALB2) is a moderate-risk gene in hereditary breast and ovarian cancer (HBOC) syndromes, with an estimated penetrance at 41-60%. We retrospectively reviewed PALB2 variants and their clinicopathological features among suspected hereditary breast cancer patients at the Aga Khan University Hospital, Pakistan.

Methods: Patients were identified from a cohort of 751 individuals attending Genetics clinics between 2017 and 2023, who underwent multi-gene panel testing for breast and gynecologic cancers. Testing was outsourced to certified commercial genetics laboratories – Prevention Genetics and Invitae Genetics.

Results: 18 female patients (2.4%) had a PALB2 variant, of which eight (44%) harbored pathogenic/likely pathogenic (P/LP) variants while 10 (66%) carried Variants of Uncertain Significance (VUSs). Mean age at diagnosis was 51 ± 17 years. Among P/LP variants, 56% of tumors were HR+ Her2-, 22% were triple negative, and the rest exhibited varying immunohistochemistry patterns. Histologically, most prominent was invasive ductal carcinoma (62.5%), followed by invasive lobular carcinoma (25%). Most tumors were Grade 2 (56%),followed by Grades 3 (33%) and 1 (11%). Patients harboring P/LP variants were more likely to have Grade 2 or above cancers than those with VUSs (p < 0.05). Family history was positive among 75% for breast and/or gynecological cancers.

Conclusion: PALB2 variants constituted 5% of breast cancer patients with positive genetic results, the highest statistic ever reported from Pakistan. Including PALB2 in gene testing panels for breast cancer patients, for tailored surveillance and targeted interventions, will lead to a more comprehensive understanding of breast cancer susceptibility across various populations.

Grants: Non-funded.

Conflict of Interest: None declared

EP02.001 Screening for pharmacogenetic defects in men with azoospermia

Svetlana Yovinska ¹, Mariela Hristova-Savova², Yuri Buchvarov², Petya Andreeva², Tanya Timeva², Atanas Shterev², Rumen Nikolov¹, Ivanka Dimova³

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

Background/Objectives: Non-obstructive azoospermia is determined in 5-10% of the infertile men, being the most severe form of male factor infertility. Evidence is currently accumulating for the impact of pharmacogenetic defects on spermatogenesis, contributing for idiopathic azoospermia. The aim of our study was to provide a screening for pharmacogenetic defects in infertile men and to determine the relationship between pharmacogenetic polymorphisms and non-obstructive azoospermia among Bulgarian population.

Methods: We performed a real-time PCR for the detection of the following pharmacogenetic polymorphisms: CYP2C19 c.681G>A (n = 20), ABCB1 c.3435 C > T (n = 16), and MTHFR c.677C>T and c.1298A>C (n = 71) in men with non-obstructive azoospermia. Allele and genotype frequencies were compared between our patients and European population database.

Results: The results revealed no significant difference in the frequency of CYP2C19 and MTHFR C677T polymorphisms (alleles and genotypes) between Bulgarian azoospermia patients and European population. We detected statistically higher frequency for the ABCB1 c.3435 T/T homozygotes – 56% in our patients vs 27% in control European population (p < 0.01). Regarding MTHFR c.1298A>C polymorphism, there was a trend for significantly higher frequency of C/C genotype in patients compared to controls (17% vs 10%, p < 0.08).

Conclusion: The ABCB1 gene encodes the intestinal efflux transporter P-glycoprotein, which prevents the accumulation of toxic substances in different organs, including gonads. Its connection to male infertility is worthy of investigation. Methylenetetrahydrofolate reductase has a crucial role in the folate and homocysteine metabolism. Future studies are recommended to understand better the influence of MTHFR activity and its relation to azoospermia.

Grants:

Conflict of Interest: None declared

EP02.002 Association of autosomal recessive Von Willebrand Disease (VWD) and Recurrent Miscarriage (RM)

Emanuele Micaglio¹, Arianna Riva², Alice Tamma ², Marco Villa¹, Sara Benedetti¹, Carlo Pappone¹, Ugo Sorrentino², Daniela Zuccarello²

¹IRCCS Policlinico San Donato, Arrhythmia and Electrophysiology Department, Milan, Italy; ²Padua University Hospital, Women’s and Children’s Health Department, Padua, Italy

Background/Objectives: During pregnancy, VWF plays crucial roles in hemostasis and placentation. Its reduced availability can disrupt these processes in women affected by VWD. However, little is known regarding the association between VWD and RM.

Methods: We followed up 30 women (aged 18 - 34 years old) clinically affected by VWD, genetically confirmed in 28. We found 25 cases of two or more consecutive miscarriages, 20 in the first and 5 in the second trimester of pregnancy. Patients were matched with a control population of 30 either non-carrier or heterozygous women in a comparable age interval. Couple consanguinity, twin pregnancies, gluten sensitivity, Leiden F V, G20210A variant and common causes of autoimmunity have been ruled out. Only the five women with second-trimester miscarriages performed karyotyping, showing chromosomal abnormalities in two unrelated cases.

Results: Six recurrent pathogenic variants in the VWF gene (c.50dup, c.257T>A, c.2561G>A, c.3507T>G, c.6890C>T, c.7940delC) in 28 patients, either in homozygous or in compound heterozygous state, appeared to be correlated with miscarriages at an earlier age and time of pregnancy than in controls. Moreover, 11 out of 30 women showed thrombocytopenia associated with specific truncating mutations of the VWF gene (c.50dup, c.2435delC, c.7940delC).

Conclusion: Our results indicate that VWD seems to be a risk factor for RM, suggesting that early diagnosis and treatment could improve reproductive outcomes in affected women. However, genetic and chromosomal abnormalities could not be ruled out in all women because karyotyping was not available, and thus we could not exclude other causes of abnormal placentation.

Conflict of Interest: None declared

EP02.003 Investigating the association between DMPK CTG repeats and female hypogonadism in patients affected by Type 1 Myotonic Dystrophy

Emanuele Micaglio ¹, Arianna Riva², Alice Tamma², Marco Villa¹, Ludovico Sabatelli¹, Rosanna Cardani¹, Sara Benedetti¹, Carlo Pappone¹, Ugo Sorrentino², Daniela Zuccarello²

¹IRCCS Policlinico San Donato, Arrhythmia and Electrophysiology Department, Milan, Italy; ²Padua University Hospital, Women’s and Children’s Health Department, Padua, Italy

Background/Objectives: Type 1 Myotonic Dystrophy (DMT1) is a degenerative genetic disorder caused by CTG repeats in the DMPK gene. While DMT1 is a well-known cause of male hypogonadism, the relationship between DMPK CTG repeats and female hypogonadism is almost unexplored.

Methods: We performed a cross–sectional risk study in 60 women, with a mean age of 31.5 years (18 to 45 years), with either suspected DMT1 or hypogonadism. We carried out a DMPK–CTG analysis to investigate the correlation between CTG and hypogonadism. Based on the analysis, 11 women were diagnosed with DMT1 with associated hypogonadism, 11 with DMT1 without hypogonadism, 22 did not show DMPK expansion but were affected by hypogonadism, while the remaining 16 showed neither DMPK expansion nor hypogonadism. The patients affected by DMT1 performed standard karyotyping, FRAXA and FRAXE genetic testing to exclude alternative genetic causes of premature ovarian failure.

Results: Our results indicate that hypogonadism severity in DMT1 patients may correlate with CTG repeat size. DMPK CTG repeats ranging from 150–1000 (range E2) were associated with milder forms of hypogonadism, while 1000 CTG repeats or more (range E3) were correlated with hypergonadotropic hypogonadism with both infertility and assisted reproduction failure. However, no statistical significance was reached.

Conclusion: To the best of our knowledge, this is the first study to show a possible correlation between female hypogonadism and DMPK CTG repeat size in female patients with DMT1, representing a first step in understanding a hitherto misrecognized clinical consequence of the disease.

Conflict of Interest: None declared

EP02.004 Extended DNA screening for HPV infection in Bulgarian females and males

Slavena Davidova ^1;2, Mariela Hristova-Savova², Kalina Belemezova^2;3, Petya Andreeva^2;4, Atanas Shterev², Jasmina Jeleva⁵, Ivanka Dimova^2;3

¹New Bulgarian University, Sofia, Bulgaria; ²Medical complex “Dr. Shterev”, Sofia, Bulgaria; ³Medical University of Sofia, Sofia, Bulgaria; ⁴South-West University Neofit Rilski, Blagoevgrad, Bulgaria; ⁵SMDL Kandilarov, Sofia, Bulgaria

Background/Objectives: Human papillomavirus (HPV) infection is the cause for more than 95% of cervical cancers. Vaccination can significantly reduce the number of HPV-related cancers in the future, but the vaccine does not protect against all types of HPV. With the extended HPV genotyping we can cover many more infected individuals and provide better prevention and early treatment.

Methods: We genotyped 21 HPV types by real-time polymerase chain reaction, using HPV Quant-21 kit (DNA Technology LCC), in 1000 patients, attended reproductive clinic – 931 women and 69 men.

Results: Totally, we detected a positive result for HPV in 40.9% of women and 36.2% of men. There were patients positive for multiple types of HPV: 19.4% of women and 28% of men were positive for two types; for more than three types - 17.9% and 20%, respectively. The most prevalent types in women were HPV 31, 16 and 45, and in men – HPV 16, 52 and 51. The types, included in the vaccine Cadasil-9, comprise 88% of male cases and 85% of female cases.

Conclusion: HPV affects men and women at almost the same rate. About half of the positive men and 37% of positive women are affected by more than one HPV type. Around 12-15% of positive cases are affected by types of HPV, not included in the current vaccines. HPV genotyping is an invaluable tool for prophylaxis of cervical cancer.

Conflict of Interest: None declared

EP02.005 Identification and characterisation of a novel pathogenic KISS1R in-frame deletion in two siblings with Idiopathic Hypogonadotropic Hypogonadism

Clayton Axiak ¹, Francesca Borg-Carbott¹, Ritienne Attard¹, Clara Lazzaretti², Carmela Perri², Lara Baschieri², Karen Cassar³, Mark Gruppetta^3;4, Josanne Vassallo^3;4;5, Livio Casarini², Stephanie Bezzina Wettinger^1;5, Rosienne Farrugia^1;5

¹L-Università ta’ Malta, Department of Applied Biomedical Science, Msida, Malta; ²University of Modena and Reggio Emilia, Department of Biomedical, Metabolic and Neural Sciences, Modena, Italy; ³L-Università ta’ Malta, Department of Medicine, Msida, Malta; ⁴Mater Dei, Division of Endocrinology and Diabetes, L-Imsida, Malta; ⁵L-Università ta’ Malta, Centre for Molecular Medicine and Biobanking, Msida, Malta

Background/Objectives: Idiopathic Hypogonadotropic Hypogonadism (IHH) is a rare genetic disorder characterized by delayed or absent puberty due to impaired gonadotropin-releasing hormone signalling. Pathogenic variants in KISS1R, a key regulator of reproductive function that plays a crucial role in the activation of the hypothalamic-pituitary-gonadal axis, cause autosomal recessive IHH. Our objective was to identify and functionally characterise IHH variants in two Maltese male siblings.

Methods: Illumina HTS data for known IHH genes was generated for the probands. Bioinformatic analysis and in silico predictions were employed to prioritize variants. Bioluminescence Resonance Energy Transfer (BRET) and Homogenous Time-Resolved Fluorescence (HTRF) assays for IP1, Ca²⁺ mobilisation and cAMP were used for functional characterisation in transfected HEK293 cells after metastin stimulation.

Results: We identified a novel 10 amino acid in-frame deletion: KISS1R p.Y190_A199del (rs770541847). In silico modelling suggested a deleterious effect on protein function. One proband is homozygous for the variant and with hCG treatment successfully achieved transient fertility, whilst the other brother is heterozygous and has not had offspring despite treatment. Functional studies confirmed impaired receptor signalling. These findings establish a direct link between the novel KISS1R variant and the pathogenesis of IHH in these siblings.

Conclusion: The KISS1R p.Y190_A199del is a novel in-frame pathogenic variant which deletes half of the third extracellular domain, altering the ligand binding cavity and ablating a critical disulphide bridge. The heterozygous sibling potentially has still unidentified variant(s) in a different IHH gene, further highlighting the complexity of IHH.

Grants: The Malta NGS project R&I-2012-024; Genomics of Rare Diseases I21LU04; Trialect Traineeship.

Conflict of Interest: None declared

EP02.006 NextGenerationEU — NRRP italian project “Development of the Italian Preimplantation Genetic Test (PGT) network”

Giulia Di Cola ¹, Giuseppe Castello¹, Chiara Faggionato¹, Simone Bolli², Cinzia Di Monte², Monica Mazzola², Federica Cariati³, Claudia Cartolano⁴, Jessica Parrotta⁴, Alessandro Conforti³, Alessandra Andrisani¹, Roberta Venturella⁴, Giulia Scaravelli², Carlo Alviggi³, Daniela Zuccarello¹

¹Azienda Ospedale Università, PGT Unit-UOC Genetica Clinica, Padova; ²Istituto Superiore di Sanità, Registro Nazionale PMA, Roma; ³Azienda Ospedaliera Universitaria Federico II, UOS PMA- DAI Materno-Infantile, Napoli; ⁴Università Magna Graecia, UO di Ginecologia Universitaria-Dip. Medicina Sperimentale e Clinica, Catanzaro

Background/Objectives:Preimplantation Genetic Testing (PGT) allows the analysis of embryo before its transfer into the uterus. In Italy, only 9 out of 71 public Assisted Reproduction Technology (ART) centers currently offer PGT, limiting access to patients. The NRRP project aims to enhance the accessibility and effectiveness of PGT across Italy, promoting a more widespread and equitable distribution.

Methods:In order to establish a comprehensive public network of ART-PGT centers, an HUB and SPOKE model will be set; standardized PGT protocols and key performance indicators (KPIs) will be defined; data from single PGT cycles will be collected by the Italian ART Registry (IARTR).

Results:Since the start of the project in May 2023, significant progresses have been made, including the opening of the PGT unit at Padua Hospital, the training of embryologists and molecular biologists in all 4 operative units, and the organization of embryo biopsy courses in collaboration with the Italian Society of Embryology (SIERR). PGT data collection started in January 2024, and four webinars on PGT applications and the National PGT Congress are planned for 2024.

Conclusion:The project will improve accessibility and effectiveness of PGT in Italy allowing access to ART-PGT in several public Hospitals. The identification of KPIs and protocol standardization will assess procedure quality, establishing certified guidelines. The IARTR’s single cycle data collection will provide transparent information to enable couples to make informed family planning decisions.

Grants:This work was funded by the European Union—NextGenerationEU—NRRP M6C2— Investment: 2.1, “Enhancement and strengthening of biomedical research within the NHS”, grant number PNRR-MR1-2022-12376108.

Conflict of Interest: None declared

EP02.007 Clinical applicability of next-generation sequencing in clinical exome

Michala Hrabíková ¹, Štěpán Chvojka¹, Leona Cerna¹, Jan Král¹, Filip Zembol¹, Barbora Honysová¹, Martina Bittoova¹, Monika Koudová¹

¹GNTlabs by GENNET, Molecular Genetics Laboratory, Prague 5, Czech Republic

Background/Objectives: Next generation sequencing (NGS) and Franklin Genoox (for secondary and tertiary analysis) are together very good tools for analyzing and evaluating of germline variants from multiple genes associated with HPO terms of patients.

Methods: We use the clinical exome GERDA (Gennet Exome for Rapid Diagnostic Assessment) for prenatal and postnatal diagnosis suitable especially in cases with a clear phenotype for which we assume a genetic etiology. It includes 7961 genes. We evaluate genes connected to HPO term including CNV analysis (sequencing coverage analysis and Franklin-Rainbow). Our standard is reporting variants for the reproductive risk of partners from the CarrierTest by Gennet [1], variants from the panel Czecanca (CZEchCAncerpaNelforClinicalApplication) [2] and variants from ACMG guidelines for secondary findings. Franklin integrates publicly available data from population, disease, and sequence databases and published literature, and employs machine-learning to derive a prediction aggregation score and adjust the weighting of ACMG classification criteria. It helps in order to reach a conclusion about the patogenicity of variants.

Results: The total number of so far processed samples was 670 (458 families). 61 families of them formed the prenatal clinical exome where we found genetic causality in 33%. 397 families of them formed the postnatal clinical exome where we found genetic causality in 19%.

Conclusion: The success of finding a causal variant is dependent on the precise definition of the phenotype in particular in prenatal analysis. We would like to present some interesting case history of our clinical exome.

[1] https://www.gennet.cz/en/carriertest

[2] Soukupova et al., 2018, https://doi.org/10.1371/journal.pone.0195761

Grants: none

Conflict of Interest: None declared

EP02.008 BMP15 genetic variants associated to Mayer-Rokitansky-Küster-Hauser syndrome

Nouha ABDELMOULA ¹, Balkiss Abdelmoula¹

¹Medical University of Sfax, Genomics of Signalopathies at the Service of Precision Medicine LR23ES07, Sfax, Tunisia

Background/Objectives: We reported a Tunisian female patient harboring Mayer-Rokitansky-Küster-Hauser (MRKH) syndrome and we discussed genotype-phenotype correlations related to two BMP15 genetic variants.

Methods: Clinical characteristics of the affected patient were identified. Cytogenetic and molecular analysis of the BMP15 gene were carried out. Genotype-phenotype correlation was investigated using literature and mutations databases.

Results: A 20 -year-old Tunisian female consulted for primary amenorrhea. She had a facial dysmorphism and features suggestive of Turner syndrome including short stature, low posterior hairline, neck webbing, broad chest, numerous pigmented naevi and hemangiomas, cubitus valgus and dental overcrowding. Her external genitalia and breast development appeared as completely normal feminine structures. Hormonal analysis confirmed a normogonadotropic normogonadism. US exploration revealed intra-abdominal normal ovarian anatomy with a complete aplasia of the uterus and the superior part of the vagina. Coelioscopy showed two ovaries with normal size and follicles with rudimental uterine hypoplasia and two rudimental Fallopian tubes. The two kidneys were normal. The diagnosis of a MRKH II was established. The karyotype revealed a 46,XX formula. The BMP15 sequencing revealed two heterozygous genetic variants c. 788insTCT and c. 852C > T.

Conclusion: Here, we demonstrated the presence of BMP15 genetic variants associated to Turner stigmata and Müllerian duct development disorders, while ovaries and kidneys development and functions were in the normal range. BMP15 variants were reported in many populations, but it is unproved whether these variants are unique to POF cases or present in controls as well. The variant c. 852C > T was observed in patients and controls in a recent Syrian study.

Grants: No

Conflict of Interest: None declared

EP02.009 Genotype-phenotype correlation in pure and mosaic trisomy X

Balkiss Abdelmoula ¹, Khaled Trigui¹, Wided Ltaif¹, Kays Chaker¹, Yassine Ouanes¹, Nouha ABDELMOULA¹

¹Medical University of Sfax, Genomics of Signalopathies at the Service of Precision Medicine LR23ES07, Sfax, Tunisia

Background/Objectives: Although pure 47,XXX triple X syndrome is rare, numerous mosaic trisomy X formulas exist, encompassing diverse combinations. This study sought to identify the types and frequency of trisomy X observed in women undergoing cytogenetic investigation for reproductive issues.

Methods: We conducted a retrospective analysis of patients referred to our genetic counseling at the medical university for female reproductive issues and selected women harboring 47,XXX cell lines. Clinical data were extracted from cytogenetic reports.

Results: Seven women (average age: 32.85 years) were enrolled in the present study. Trisomy X was homogeneous in only one patient, predominant in a case of 45,X/47,XXX mosaicism, while the remaining five cases exhibited low-level trisomy X mosaicism ranging from 4 to 8% with two types: 46,XX/47,XXX and 45,X/46,XX/47,XXX. The individual with pure triple X syndrome exhibited unusual physical dysmorphia and a tall stature. The individual with a 45,X[5]/47,XXX[45] karyotype displayed a short stature and several Turner’s syndrome features, excepting dysmorphic facial appearance. For patients with low-level mosaicism of 47,XXX, with and without 45,X cell line, the phenotype appeared normal, but reproductive issues varied from amenorrhea to polycystic ovaries, unexplained infertility and ART failure, to the conception of malformed children.

Conclusion: The phenotypic variability observed in patients with trisomy X poses challenges in establishing genotype-phenotype correlations. Recognizing reproductive issues as indicative clinical signs of trisomy X emphasizes the need to enhance its effective cytogenetic diagnosis in female reproductive failures. The consideration of systematic FISH analysis recommendations may be proposed to improve diagnosis accuracy.

Grants: No

Conflict of Interest: None declared

EP02.010 Genomic variants and changes in methylation level in sperm DNA of infertile brothers with oligoasthenoteratozoospermia

Marta Olszewska ¹, Marzena Kamieniczna¹, Nijole Pollock², Zuzanna Graczyk¹, Monika Fraczek¹, Katarzyna Kurek¹, Alexander N. Yatsenko², Maciej Kurpisz¹

¹Instytut Genetyki Człowieka PAN, Poznań, Poland; ²Magee-Womens Research Institute, University of Pittsburgh, Pittsburgh, United States

Background/Objectives: Male infertility represents varying degrees of severity, mostly with decreased semen parameters. Considering a fact that approx. half of the infertility cases has male background, and ~15% is related to genetic causes, it is essential to investigate both: genomic and epigenomic novel causative variants.

Our aim was to find genetic variants and altered methylation level regions causative for observed decreased sperm quality in two familial cases of infertile brothers with decreased sperm concentration and similar head and acrosome deformations.

Methods: Blood DNA samples from members of two families (F1: 3 brothers, F2: 4 brothers, father) were sequenced by Whole genome sequencing (WGS, Illumina NovaSeq6000), while sperm DNA were evaluated by whole genome methylome sequencing (WGMS, Illumina Infinium MethylationEPIC BeadChip 850k array). Immunofluorescence on spermatozoa for candidate genes was applied with proper antibodies (Leica DM5500, LASX software). Chromatin integrity tests on sperm were also performed (TUNEL, aniline blue, acridine orange).

Results: We have found genomic variants in a.o: ADCY10, DDX34, DDX3X, TCTN1, PABPC3, LENG9, USP8, while decresed methylation was documented for: SLC35F2, ATF3, GYS2, TBCD and VOPP1. Revealed mutations can clearly suggest the observed sperm phenotypes.

Conclusion: This study empasizes the strong need for genomic and epigenomic surveys in patients with severely decreased sperm parameters. Collating of WGS and WGMS data shows the complexity of genomic background for particular sperm parameters, microscopically identical.

Grants: National Science Centre in Poland: 2020/38/E/NZ2/00134 (MO), 2015/17/D/NZ5/03442 (MO), 2015/17/B/NZ2/01157 (MK)

Conflict of Interest: None declared

EP02.011 Revealing invisible chromosomal alterations involving pericentromeric regions of acrocentric chromosomes using specially designed FISH probes in patients with reproductive failure

Yusuf Bahap ¹, Meral Yirmibes Karaoguz¹, Esra Tuğ¹, Thomas Liehr², Meral Yirmibes Karaoguz¹

¹Gazi University Medical Faculty, Department of Medical Genetics, ANKARA; ²Friedrich Schiller University Jena University Hospital, Institute of Human Genetics, Jena, Germany

Background: Failure to achieve a healthy pregnancy or losing consecutive pregnancies is called a reproductive problem, and affects approximately 15% of couples. Conventional cytogenetic techniques are still crucial for uncovering microscopic chromosomal changes in these patients, but there is still unclear territory for non-microscopic ones. This project aims to identify submicroscopic changes in the pericentromeric regions of acrocentric chromosomes.

Methods: In this ongoing project, 20 couples with infertility or recurrent pregnancy loss (without evidence of non-genetic factors or on routine genetic testing), were re-examined. Fluorescent in situ hybridization (FISH) analysis was performed by means of newly designed probes that stain the pericentromeric regions of acrocentric chromosomes (13-15, 21, and 22) and their alpha-satellite regions. The brief abstract of the first results was presented at 15th National Congress of Medical Genetics in May 2022.

Results: In two couples we found aberrant patterns with the applied FISH-probe sets. An invisible reciprocal translocation emerged between Chr.15 and Chr.22. One couple never had a live birth, while the other had a dysmorphic child due to adjacent-2 segregation resulting from translocation. Additional FISH analysis of the affected child revealed three copies of the Histone Cell Cycle Regulator gene located in the 22q11 region.

Conclusion: With this study, the detection of hidden microscopic changes in acrocentric chromosomes investigates the causal cause of reproductive problems and also offers the opportunity for preimplantation and/or prenatal genetic diagnosis.

Grants: The Scientific and Technological Research Council of Turkey (TÜBİTAK) supported this project with no. 123S240 as 1002-Short-Term Support Module.

Conflict of Interest: None declared

EP02.013 Glass Syndrome Unveiled: A Unique Journey through Assisted Reproduction.

Sweta Das ^1;2, Rekha Aaron², Paramasivam g², GAUTHAM ARUNACHAL UDUPI³, Sumita Danda²

¹university of texas at austin, department of molecular biosciences, austin, United States; ²christian medical college and hospital, department of medical genetics, vellore, India; ³National Institute of Mental Health and Neuro-Sciences, department of human genetics, bengaluru, India

Glass Syndrome Unveiled: A Unique Journey through Assisted Reproduction.

Background/Objectives: Glass syndrome (OMIM #612313) is a rare genetic disorder characterized by intellectual disability and dysmorphic facial features, including down-slant of palpebral fissures, crowded teeth, cleft palate, and micrognathia. Initially described by Glass IA et al. in 1989, the syndrome was associated with an abnormal karyotype 46, XY, del(2)(q32.2q33.1). This study presents the case of a 7-year-old Indian girl diagnosed with Glass syndrome, revealing variations in clinical manifestations compared to previously reported cases. Notably, the patient was conceived through intrauterine insemination (IUI) using donor sperm.

Methods: The patient was diagnosed by clinical examination. She underwent Multiplex Ligation Probe Amplification (MLPA), identifying a 2q33.1 deletion. The finding was further confirmed through real-time polymerase chain reaction (PCR).

Results: The patient displayed classic Glass syndrome features, such as intellectual disability and dysmorphic facial characteristics. Deviations included the absence of short stature and microcephaly, alongside the presence of telecanthus and up-slanting palpebral fissures. Molecular analysis through MLPA and real-time PCR confirmed the 2q33.1 deletion, validating the Glass syndrome diagnosis.

Conclusion: This case emphasizes the clinical heterogeneity of Glass syndrome, as seen in the unique presentation of our patient. The use of IUI with donor sperm in conception added complexity in genetic counselling. The use of assisted reproductive technology, specifically IUI with donor sperm, in conception raises questions about potential genetic influences.

Grants: None

Conflict of Interest: None declared

EP02.014 LINE1-mediated epigenetic repression of androgen receptor transcription causes androgen insensitivity syndrome

Jelena Pozojevic ¹, Radhika Sivaprasad², Joshua Laß³, Franziska Haarich⁴, Joanne Trinh³, Neseebullah Kakar¹, Kristin Schulz¹, Kristian Händler¹, Annemarie Verrijn Stuart⁵, Jacques C. Giltay⁶, Koen L. I. van Gassen⁷, Almuth Caliebe², Paul-Martin Holterhus⁸, Malte Spielmann¹, Nadine Hornig²

¹Institute of Human Genetics, Lübeck, Germany; ²Institute of Human Genetics, Kiel, Germany; ³Institute of Neurogenetics, Lübeck, Germany; ⁴Institute of Cardiogenetics, Lübeck, Germany; ⁵, Department of Pediatric Endocrinology, Utrecht, Netherlands; ⁶Division Laboratories, Pharmacy and Biomedical Genetics, Utrecht, Netherlands; ⁷, Department of Genetics, Utrecht, Netherlands; ⁸, Department of Paediatrics, Division of Paediatric Endocrinology and Diabetes, Kiel, Germany

Background/Objectives: Androgen insensitivity syndrome (AIS) is a difference of sex development characterized by different degrees of undervirilized external genitalia in individuals with a 46,XY karyotype. Here, we present an undervirilized patient with a 46,XY karyotype, assigned with a female gender, with normal-for-male AMH levels at birth and normal testosterone/dihydrotestosterone ratio at age of one month following hCG stimulation.

Methods: Whole exome sequencing (WES), nanopore sequencing, quantitative PCR (qPCR), western blotting

Results: WES detected a maternally inherited L1 retrotransposon insertion in the 5´ untranslated region (5´UTR) of the androgen receptor (AR) on chromosome X. This finding coincided with reduced AR transcript and protein levels in patient-derived genital skin fibroblasts (GSF). Long-read nanopore sequencing determined the sequence and epigenetics of the L1 element and the AR promoter region where it is inserted, revealing a truncated L1 element of ≈2.7 kb. Interestingly, DNA methylation of the CpGs proximal to the L1 insertion was increased in the patient-derived GSF compared to healthy controls, suggesting that the L1 decreases AR levels by affecting its promoter methylation. To confirm this hypothesis, we compared the effects of wild-type and patient-derived AR 5´UTR, by inserting each of these sequences into a vector that contains the AR cDNA. mRNA and protein levels were decreased in the cells transfected with the L1-containing AR 5´UTR, in accordance with the results from patient-derived cells.

Conclusion: Our results show the relevance of retrotransposons in human disease. They comprise almost half of the human genome and can interfere with gene expression, especially when inserted at transcriptionally active regions.

Conflict of Interest: None declared

EP02.017 The role of gene variants for IL-6, IL-10 and TNF-α in the incidence of preterm birth

Jasenka Wagner ¹, Deni Plecko¹, Nena Arvaj¹, Kristina Kralik², Mirta Kadivnik³

¹Faculty of Medicine Osijek, Department of medical biology and genetics, Osijek, Croatia; ²Faculty of Medicine Osijek, Department of medical statistics and medical informatics, Osijek, Croatia; ³Faculty of Medicine Osijek, Department of gynecology and obstetrics, Osijek, Croatia

Background/Objectives: The association of gene variants for IL-6 (rs1800796), IL-10 (rs1800896) and TNF-alpha (rs1800629) with the occurrence of spontaneous preterm birth was examined to determine whether these genetic variants are a risk factor or not.

Methods: 175 blood samples of pregnant women who gave birth prematurely and 169 corresponding control blood samples were analyzed with the aim of determining gene variants for IL-6 (rs1800796), IL-10 (rs1800896) and TNF-alpha (rs1800629). Control samples were samples of pregnant women with term delivery. DNA isolation was performed on mini spin columns according to the manufacturer’s protocol. The quality and purity of the isolated DNA was tested with a Qubit 3 Fluorometer. Genotyping was performed on ABI PRISM 7500 SDS using Taqman SNP Genotyping Assays. Obtained genotypes were analyzed using program package 7500 Software v2.3.

Results: Carriers of the A/A genotype for rs1800629 compared to carriers of the G/G genotype, have a 5.5 times greater chance of late preterm birth. The presence of the A/A or A/G genotype for rs1800896 in the recessive model of inheritance compared to the G/G genotype represents a potentially protective factor in which the test subjects have a 6.5 times lower chance of early preterm birth. In this study, no link was established between premature birth and variants in rs1800796.

Conclusion: Rs1800629 in mothers was associated with preterm birth. Rs1800896 in this study shows a potentially protective effect for the incidence of preterm birth. No association was established between premature birth and rs1800796.

Grants: IP3-2017 Medical Faculty Osijek

Conflict of Interest: None declared

EP02.019 Establishing a quality control in PGT-A: parameters and reference values.

Andrei Kullyev ¹, Svetlana Avdeichik¹, Yaron Goikhman¹

¹Israeli Medical Center-Reproductive Medicine and Family Health, Genetics, Tashkent, Uzbekistan

Background/Objectives: While PGT-A is arguably the most complex and challenging procedure in IVF, the only relevant KPI written in the Vienna consensus is the successful biopsy rate/tubing rate. As a result, the reported efficacy of PGT-A programs varies widely. That, in turn, led to a heated discussion about whether PGT-A is a useful tool or an unnecessary add-on in IVF treatment.

Methods: We have chosen PGT-A efficiency, defined by R.J. Paulson as post–PGT-A implantation rate/(pre–PGT-A implantation rate/euploidy rate), as our main KPI, defining competency value at ≥ 75%. Other KPIs are the percentage of euploid embryos in a group of patients under 35 years of age (competency value ≥ 60%), and the clinical pregnancy rate after transferring euploid embryos (> than the clinical pregnancy rate after transferring untested embryos for all age groups).

Results: Designing our workflow from the get-go to meet defined KPIs (non-traumatic biopsy by Flicking technique, biopsied samples storage in the freezer for no longer than 6 weeks before processing, re-testing of aneuploid embryos to detect and eliminate possible false positive results) allowed us to reach PGT-A efficiency of 86.13%. The euploidy rate for patients under 35 years was kept at over 60% from the year 2022, when our laboratory was founded.

Conclusion: For the PGT-A program to be beneficial to patients, a set of quality control points and reference values should be established in the PGT-A laboratory, and efforts should be made to reach the competency value for each KPI.

Grants: none

Conflict of Interest: None declared

EP02.020 Comparative proteomics analysis of chorionic villi reveals altered complement and coagulation cascades, TCA cycle and ferroptosis in recurrent pregnancy loss

Katarina Davalieva¹, Gjorgji Bozhinovski ¹, Sanja Kiprijanovska¹, Katerina Kubelka-Sabit^2;3, Dijana Plaseska-Karanfilska¹

¹Research Centre for Genetic Engineering and Biotechnology “Georgi D Efremov”, Macedonian Academy of Sciences and Arts (MASA), 1000 Skopje, North Macedonia; ²Clinical Hospital “Acibadem Sistina”, Skopje, North Macedonia; ³Faculty of Medical Sciences, University “Goce Delcev”, Stip, North Macedonia

Background/Objectives: Recurrent pregnancy loss (RPL) represents the most common disorder during pregnancy and affect ~5% of the couples aiming at childbirth with long-term consequences on family and society. As more than half of the RPL cases do not have identified cause, uncovering the mechanisms behind the idiopathic RPL is urgently needed.

Methods: Using label-free data-independent LC-MS/MS acquisition coupled with ion mobility, we compared the proteome of 13 first trimester placental chorionic villi from RPL cases with 10 age and gestational week matched chorionic villi from normal, elective pregnancies. Transcriptional levels of selected biomarkers were determined by qPCR in extended cohort of 35 RPL and 25 controls.

Results: From 1554 proteins identified based on ≥2 peptides, statistically significant difference in abundance adjusted for multiple testing (B-H p ≤ 0.05) and fold change ≥1.5 showed 128 proteins. Bioinformatics analysis identified complement and coagulation cascades, platelet activation, TCA cycle and ferroptosis as enriched pathways with the highest significance. NEFL and NOS3 were identified with highest differential abundance between RPL and controls. The results showed that the transcription levels of NEFL, DLST, NOS3 and CP were significantly increased in RLP group, consistent with the proteomics findings.

Conclusions: While blood coagulation, complement and coagulation cascades have well established associations with the pathogenesis of RPL, our study pointed to pathways such as TCA cycle and Ferroptosis, for which at present, very limited association exists. The current study brings novel insights behind the altered pathways in RPL and opens a way for investigations regarding the clinical management.

Grants: 08-707/2023 from MASA

Conflict of Interest: None declared

EP02.021 HLA-DRB1 genotyping in Bulgarian couples with recurrent implantation failure

Mariela Hristova-Savova ¹, Petya Andreeva², Ivelina Oprova², Atanas Shterev², Ivanka Dimova³

¹SAGBAL “Dr Shterev”

, Genetic Laboratory, Sofia, Bulgaria; ²SAGBAL “Dr Shterev”

; ³Medical University Sofia

Background/objectives: The difference in spouses regarding HLA class II gene variants is one of the important conditions for successful implantation and pregnancy realization. The similarity of spouses in terms of HLA gene variants increases the probability of the appearance of an embryo with a double set of identical gene variants, i.e. HLA homozygosity, which is an unfavorable factor carrying the risk of reproductive losses. We aimed in analyzing the HLA-DRB1 genotypes in selected couples with recurrent implantation failure (RIF), i.e. more than 3 miscarriages or unsuccessful ART procedures.

Method: We used Real time PCR analysis for simultaneous detection of 13 DRB1 alleles (DNA Technology LCC), after isolation of DNA from peripheral venous blood.

Results: We analyzed eight couples for the allele variants in DRB1 locus – totally 16 genotypes and 32 alleles were detected. We established the highest frequency for DRB1*11 allele (34.4%), followed by DRB1*16 (15.6%) and DRB1*03 (12.5%). In 2 couples (25%) there was a risk for HLA-DRB1 homozygosity – the risk was 50% in one couple (genotypes of partners *03/*11 and *11/*11) and 25% in another one (genotypes *04/*11 and *04/*16, respectively).

Conclusion: Spousal HLA typing is used in diagnosing causes of reproductive failure to identify similarities in HLA gene variants in the couple. We suggest that in one fourth of couples with unexplained RIF, the contributing factor could be the HLA-II similarity.

Conflict of Interest: None declared

EP02.022 Distribution of PAI-1 5G/4G and ACE I/D polymorphism in women with recurrent pregnancy loss

Liliia Chorna ¹, Danuta Zastavna¹, Yaryna Zahanyach¹, Oksana Kolodiy², Halyna Makukh³

¹State Institution Institute of Hereditary Pathology of the National Academy of Medical Sciences of Ukraine, Diagnostics of hereditary pathology, Lviv, Ukraine; ²KNP “3rd city clinical hospital of Lviv”, Women’s consultation, Lviv, Ukraine; ³KNP “Lviv Regional Clinical Perinatal Centre”, Regional centre of newborn screening, Lviv, Ukraine

Background/Objectives: Recurrent Pregnancy Loss (RPL) defined as two or more consecutive pregnancy losses, affecting 1–5 % of reproductive-age women. Current diagnostic procedures can identify etiologic factors in approximately 50% of RPL cases. The study aimed to assess the distribution of PAI-1 (675 5G/4G) and ACE (intron16 I/D) polymorphism in women with RPL.

Methods: A study group of 55 women with a history of at least two consecutive miscarriages was compared with a control group of 57 healthy women with normal pregnancies and without miscarriages. Polymerase chain reaction (PCR-RFLP) was used to identify the polymorphism.

Results: The frequency of 4G allele of PAI gene was higher in the RPL group (67%) compared to the control group (54%). It was found that the presence of 4G allele increases the risk of RPL by 2 times (Р = 0.01). DD genotype and D allele of ACE gene were more prevalent in RPL women (31%) than in controls (19%) although the difference was not significant. The combination of DD genotype with 4G4G genotype was significantly more frequent in RPL group compared with controls (16.6% versus 2.3%). The results showed that the presence of homozygotes for two 4G alleles of the PAI-1 gene and D alleles of the ACE gene leads to an 8-fold increased risk of RPL (OR = 8.6, CI: 0.98 -75.15, P = 0.04).

Conclusion: Our study suggest that the combination of homozygosity for the PAI-1 4G and ACE D alleles could be a risk factor to RPL for Ukrainian women.

Conflict of Interest: None declared

EP02.023 Significance of the sperm DNA fragmentation index (DFI) for male fertility diagnostics - results from more than 800 cases

Michaela Blankenburg ¹, Oriane Vedrines¹, Markus Stumm¹

¹Medicover Humangenetik Berlin-Lichtenberg MVZ, Berlin, Germany

Background/Objectives: Classical sperm analysis is based on the three semen parameters concentration, morphology and motility. The current edition of the WHO Laboratory Manual for the Examination and Preparation of Human Semen also contains a chapter on advanced tests. One group of these additional analyses are DNA fragmentation tests, which are a good extension of the basic tests.

Methods: The DFI of more than 800 semen samples were analysed with the Haloperm® test. The test results were divided into three fertilisation categories (normal, impaired, severely impaired) and correlated with the patient’s age, sperm concentration, sperm morphology and sperm motility. To analyse the significance of the results, a two-tailed t-test was performed and the correlation coefficient r was calculated for each of the parameters.

Results: The data showed a significant positive correlation between the DFI and the age of the patients, but a significant negative correlation between the DFI and the basic semen parameters concentration, morphology and motility. An exception were 15 infertile patients with high sperm DNA fragmentation and normal semen parameters. All these results indicate that an elevated DFI is a good additional parameter for poor sperm quality.

Conclusion: The Haloperm® test provides additional information on sperm quality and can help to refer patients to the most appropriate assisted reproductive technology. This is especially important for infertile patients with normal sperm parameters but with high sperm DNA fragmentation.

Grants:

Conflict of Interest: None declared

EP02.024 Deciphering the role of microRNAs (miRNAs) in azoospermia: A small RNA transcriptome analysis in testicular tissue

Maria-Anna Kyrgiafini ¹, Alexia Chatziparasidou², Nikolaos Christoforidis², Zissis Mamuris¹

¹Laboratory of Genetics, Comparative and Evolutionary Biology, Department of Biochemistry & Biotechnology, University of Thessaly, Larissa, Greece; ²Embryolab IVF Unit, Kalamaria, Thessaloniki, Greece

Background/Objectives: Azoospermia, the most severe form of male infertility, affects approximately 1% of all men and 15% of those seeking infertility treatment. While genetic factors are identifiable in 25% of these cases, the vast majority remain idiopathic, posing a challenge for diagnosis and treatment. Our study aimed to identify differentially expressed microRNAs (miRNAs) in the testicular tissue of azoospermic males, aiming to reveal tissue-specific expression signatures associated with idiopathic azoospermia.

Methods: Small RNA sequencing was conducted on testicular samples from 18 IVF (in vitro fertilization) patients, organized into six groups according to the presence of spermatozoa and pregnancy outcomes. This included groups for samples with no spermatozoa, high presence of spermatozoa, and rare presence of spermatozoa with varying pregnancy results, along with specific pools for cystic fibrosis carriers and cryptozoospermic patients. Bioinformatic analysis was undertaken to identify the differentially expressed miRNAs between the different samples as well as the main pathways that are affected.

Results: Overall, approximately 20,000 miRNAs were detected and significant differences in small RNA expression (log2fold change≥2, p-value < 0.05) were observed across the analyzed pools. Bioinformatic analysis indicated that these altered miRNAs play crucial roles in metabolic processes, immune regulation, and spermatogenesis-related signaling pathways.

Conclusion: Our study investigates the complete miRNA landscape in testicular samples, revealing that variations in miRNA expression are tightly linked to sperm presence and IVF success rates, underscoring key molecular pathways that may affect male fertility. These findings suggest miRNAs as promising biomarkers and therapeutic targets for addressing male infertility.

Grants: Grant number: T1E∆K-02787

Conflict of Interest: None declared

EP02.026 Copy number variants (CNV) characterization in recurrent pregnancy losses

Evelina Dagyte ¹, Algirdas Utkus¹

¹Vilnius University, Institute of Biomedical Sciences, Faculty of Medicine, Vilnius, Lithuania

Background/Objectives: Recurrent pregnancy losses (RPL) is an important global health issue and carries psychological and financial burden for affected couples. Embryonic chromosomal abnormalities (both in the number of chromosomes and the structure) account for 50% of early pregnancy losses. Etiology of RPL in 40-60 % of couples remains idiopathic.

The aim of this study was to evaluate the role of embryonic chromosomal abnormalities and CNVs in the etiology of RPL.

Methods: SNP-genotyping was performed for patients with unexplained RPL (anatomy factors, antiphospholipid syndrome, chromosomal and endocrinological abnormalities were excluded). 32 products of conception (POCs) from miscarriage specimens were investigated using single nucleotide polymorphism array (SNP-array). Genomic DNA was extracted from all POCs using the phenol-chloroform extraction method. Genotyping was performed with Illumina HumanCytoSNP-12 v2.1 and Infinium Global Diversity Array with Cytogenetics-8 arrays according to standard protocols provided by the manufacturer.

Results: Normal results were identified in 15 (46,88%) cases and abnormal results were identified in 17 (53,13%) cases. In cases with abnormal results, aneuploidy was the most common finding with 11 (64,71%) cases of autosomal trisomy and 3 (17,65%) cases of monosomy X. Triploidy was found in 3 (17,65%) cases. 15 cases with normal results were subjected to further CNV analysis. 11 CNVs were identified, including 5 deletions and 6 duplications.

Conclusion: Wider comprehension of the genetic mechanisms involved in RPL may lead to the establishment of a diagnostic panel of genetic markers for screening for individuals at risk of RPL.

Grants:

Conflict of Interest: None declared

EP02.027 Preimplantation genetic screening in partners with chromosomal polymorphisms

Radostina Raynova ^1;2, Tanya Milachich³, Petya Andreeva⁴, Tanya Timeva⁴, Maria Yunakova⁴, Atanas Shterev⁴, Alexey Savov¹, Ivanka Dimova^2;5

¹University Hospital of Obstetrics and Gynecology, National Genetic Laboratory, Sofia; ²Shterev Hospital, Genetic Laboratory, Sofia, Bulgaria; ³Shterev Hospital, Embryology, Sofia, Bulgaria; ⁴Shterev Hospital, Obstetrics and Gynecology, Sofia, Bulgaria; ⁵Medical University, Sofia, Clinical Genetics, Sofia

Background/Objectives: Chromosomal polymorphisms (CPM) are variants in chromosomes generally considered “normal” karyotypes. Studies indicate that CPM may be associated with abnormal spermatogenesis, infertility, and recurrent miscarriages. Preimplantation genetic testing (PGT) is a useful approach for reducing miscarriage and increase successful pregnancy rate in such couples. The aim of our study was to determine the frequency and spectrum of chromosomal aberrations in embryos after PGT in carriers of CPM.

Methods: Nineteen couples underwent IVF cycles, followed by PGT (Perkin Elmer protocol) due to CPM - 13 with CPM of acrocentric chromosomes (Group I – average age of 33.6 years) and 6 with chromosome 9^th CPM (Group II – 35.3 years on average).

Results:Overall, 92 embryos were subjected to trophectoderm biopsy – 4.8 embryos/cycle on average. We found 31 euploid embryos in total (44.1% in Group I and 33.3% in Group II) and lack of euploid in 7.7% and 33.3%, respectively; mosaic embryo was detected only in Group I (11%). Pregnancy was achieved in 58% and 50% of transfers in both groups. Single and complex aneuploidies were detected as follows: 17% and 12% in Group I, and 26% and 18% in Group II. The most frequently involved chromosomes were 2, 14 and 19 in Group I, and chromosomes 10, 19, 20 and 21 in Group II.

Conclusion:The polymorphism of chromosome 9 is associated with less euploid embryos than acrocentric chromosomal polymorphism. The last was associated with the presence of mosaic embryos. There is no correlation between polymorphic chromosome and aneuploidy

Grants: No grants.

Conflict of Interest: None declared

EP02.028 KIR AA1 genotype and recurrent implantation failure

Petya Andreeva ^1;1;2, Ivelina Oprova^1;2, Mariela Hristova-Savova³, Kalina Belemezova³, Ivanka Dimova⁴

¹Shterev Hospital, IVF Unit, Sofia, Bulgaria; ²South-West University Neofit Rilski, Blagoevgrad, Bulgaria; ³Shterev Hospital, Genetic Department, Sofia, Bulgaria; ⁴Medical University of Sofia, Genetic Department, Sofia, Bulgaria

Background/Objectives: The killer cell immunoglobulin-like receptors (KIRs) of a subpopulation of uterine natural killer cells (uNK cells) is made up of 16 highly polymorphic genes classified into two haplotypes: KIRAA and KIRBx. The KIRAA haplotype has predominantly inhibitory KIRs and only one activating receptor, KIR2DS4, with uncertain function. The key distinction is the balance of KIRs, which can activate or inhibit uNKcells’ ability to create components essential for embryo implantation. The study intended to explore the distribution of KIR haplotypes and genotypes in patients with recurrent implantation failure (RIF).

Methods: In total, 82 patients were genotyped for activating or inhibiting KIR genes, with 28 women with RIF serving as the study group and 32 as controls. The individual KIRAA and KIRBx haplotypes have been identified via PCR with Sequence-Specific Primer (SSP).

Results: RIF was discovered in a higher proportion in haplotype KIR AA (60% RIF vs 40% controls; P = 0.2) than in KIR Bx (37.5% RIF vs 62.5% controls; p = 0.026). Furthermore, two genotypes were identified: KIR AA1 (31.82%) and KIR AA195 (68.18%). In haplotypes KIRAA, the KIR2DS4 locus in the full-length receptor (KIR2DS4-norm) was only expressed in KIR AA1. The RIF numbers were significantly higher in KIR AA1 (75% vs 15%, p = 0.01), while there was no difference between the two observed groups in KIR AA195.

Conclusion: The KIR AA1 genotype substantially increases the risk of recurrent implantation failures. The presence of the full-length receptor KIR2DS4norm in KIRAA haplotype patients implies RIF development.

Grants: Not applicable

Conflict of Interest: None declared

EP02.029 Preimplantation Genetic Testing of Aneuploidy (PGT-A) and Structural Chromosomal Imbalances (PGT-SR) by Nanopore Sequencing – initial validation experience on archival whole genome amplification samples

Matthias Linke ¹, Charlotte Hewel¹, Bartosz Linek², Christine Skala², Susanne Gerber¹, Susann Schweiger¹

¹Institute for Human Genetics, University Medical Center of the Johannes Gutenberg University Mainz, Mainz; ²Clinic and Polyclinic for Obstetrics and Women’s Health, University Medical Center of the Johannes Gutenberg University Mainz, Mainz

Background/Objectives: Current strategies for preimplantation genetic testing for aneuploidy or structural rearrangements (PGT-A/SR) are mostly based on next-generation sequencing (NGS) and microarray platforms. Some studies have already demonstrated the feasibility of Nanopore sequencing as a PGT-A/SR screening platform for both aneuploidy and segmental imbalances.

Methods: Archival SurePlex-amplified (Whole Genome Amplification, WGA) trophectoderm biopsy samples (n = 28) previously analyzed using Vitrolife/Illumina VeriSeq PGS™ were reanalyzed using Nanopore sequencing on a PromethION P24. Libraries were prepared by using the Native Barcoding Kit 24 V14. The number of barcoded WGA products per R10.4.1 flow cell was 10 and 18 samples. Bioinformatic processing included wf-cnv based on the R package QDNAseq to call copy number aberrations.

Results: PGT-A/SR analysis based on Nanopore sequencing identified known specific aberrations in 100% of numeric chromosomal imbalances (27/27) as well as 100% (1/1) of samples with a segmental duplication (Chr.3p26.3, 2.73 Mb size). Due to the known decreasing sensitivity of NGS and nanopore-based copy number calling for imbalances smaller than 5 Mb, this copy number change was additional validated in a classical PGT-M approach. Chromosomal mosaicism previously detected by VeriSeq™, could also be validated in all of the reanalyzed samples (5/5 embryos of the PGT-A group).

Conclusion: Nanopore sequencing represents a viable alternative to current NGS-based PGT-A/SR solutions for aneuploidy and segmental imbalance screening of trophectoderm biopsy samples. Additionally, we currently investigate long-read sequencing-based SNP haplotyping for monogenic disease to replace linkage analysis and the direct readout of a classical PGT-M approach.

Grants: -

Conflict of Interest: None declared

EP02.030 Genital developement variations in a large french cohort : clinical features and genetic findings

abir talbi ¹

¹Robert Debré Hospital, paris, France

Background/Objectives: Genital development Variations (VDG) are a rare group of conditions affecting gonadal development, sexual differentiation, or chromosomal sex. The etiology of DSDs is very heterogenous and a precise diagnosis is essential for management of genetic, endocrine, surgical, reproductive, and psychosocial issues. The main goal of this present study was to characterize genetic defects in a large cohort of French VDG patients using a targeted NGS panel.

Methods: a cohort of 133 unrelated patients (109 Males/ 24females), recruited between 2022 and 2023, was studied using a panel of 59 known diagnostic genes of VDG. Gonadal dysgenesis, severe hypospadias and cryptorchidism are the most common clinical features of male patients while the primary amhenorrhea is the most frequent for the female VDG patients

Results: Pathogenic or probably pathogenic variants were identified in 28(21%)VDG patients but a molecular diagnosis is made in only 23(17%) of them. Variants in the NR5A1, WT1, SRY, CYP21A2, HSD17B3, AR, and SRD5A2 genes were the most common causes of VDG. 8(30%) of them are novel. Other variants were identified in genes associated with congenital hypogonadotropic hypogonadism (CHH), including the PROKR2 and GNRHR.

Forty five patients (33.8%) had rare variants including variants of uncertain significance (VUS) in more than one candidate gene

Conclusion: Genetic sequencing is increasingly applied to rare disease such as VDG but the achieved diagnostic rate of a targeted NGS panel is still insufficient to explain all the cases and must be completed with exome and whole genome analysis in order to unravel the complexity of this condition.

Grants:

Conflict of Interest: None declared

EP02.031 Non-invasive PGT: Risks and hopes

Daniil Ferdman ¹, Ekaterina Vasileva¹, Tutakov Maksim², Daniil Chebotarev², Svetlana Pavliuchenkova², Natalia Isaeva¹, Roman Bikanov¹

¹FIRST GENETICS JSC; ²medical genetics research center MedicaMente

Background/Objectives: Despite the high accuracy of PGT-A, it still has been remaining a significant problem associated with embryo damage risk. Non-invasive PGT (niPGT) is a technique focused on the study of extracellular DNA in a spent cultural medium (SCM). However, chromosomal DNA profile in the SCM and the real karyotype matching is questioned.

Methods: 89 SCM samples collected on the 5th or 6th day of fertilization. Whole genomic amplification (WGA) was performed using the ReproSeq™ PGS kit. WGA success revealed by agarose gel electrophoresis. Chromosome profiles were analyzed for all WGA positive SCM samples paired with invasive PGT-A using F-Genetics high-throughput sequencing system.

Results: WGA was successful for 60% SCM samples. Samples were stratified by day of SCM collection, storage duration, and chromosomal status by PGT-A (euploid vs aneuploid). The subgroup analysis demonstrated that day 6 of fertilization, less than 3 week storage and aneuploidy presence is the subgroup with minimal lack of amplification rate. Only 47% of niPGT was completely matched the PGT-A results. Contamination with cumulus or spermatozoa DNA was detected for at least 7% of niPGT samples.

Conclusion: We suggest that apoptosis in embryonic cells until the 6th day of development predominantly occurs in aneuploid cells.

The possible reason of the lack of amplification in niPGT is an euploid karyotype.

Storage SCM samples at -20°C for more than 3 weeks negatively influence on technical opportunities niPGT testing, giving higher lack of amplification rate.

Collection SCM on day 6 it better for amplification success in comparison with day 5.

Conflict of Interest: None declared

EP02.032 A successful case of preimplantation genetic testing for Hunter syndrome

Ekaterina Vasileva ¹, Nataliia Andreeva¹, Daniil Ferdman¹, Natalia Isaeva¹, Roman Bikanov¹

¹FIRST GENETICS JSC, Moscow, Russian Federation

Background/Objectives: Mucopolysaccharidosis type II (MPS II), also known as Hunter syndrome, is an X-linked recessive multisystem disorder. A couple with a female carrier of MPS II requested preimplantation genetic testing for monogenic disorder (PGT-M) in order to prevent pathogenic variant transmission to their offspring. Hunter syndrome with the hemizygous frameshift variant c.546dup (p.L183Afs*16) in the IDS gene was earlier diagnosed in proband’s brother. Preimplantation genetic testing for aneuploidies (PGT-A) was carried out before PGT-M because of advanced maternal age.

Methods: DNA of the proband, her partner and her parents was analyzed. The mother of the proband was also confirmed as heterozygous carrier of the same variant. Trophectoderm samples of each embryo undergone a whole-genome amplification (WGA). Fluorescent multiplex PCR followed by fragment analysis as indirect analysis and Sanger sequencing as direct analysis was used. Custom panel with fourteen polymorphic short tandem repeat (STR) markers was developed in order to determine the haplotype that associated with the disease. 4 out of 14 STR markers were defined as uninformative due to the homozygous status in the proband.

Results: One out of three embryos were identified by PGT-A as euploid with 46,XY karyotype. As a result of the subsequent PGT-M we revealed by both direct and indirect analyses that the pathogenic variant L183Afs*16 was not transmitted. No allele dropout was detected. This embryo was recommended for transfer.

Conclusion: We have developed and applied PGT-M panel for mucopolysaccharidosis type II with a high efficiency and accuracy, resulted in identification of healthy embryo in a high-risk couple.

Conflict of Interest: None declared

EP02.033 The role of mitochondrial dynamics genes in NOA

O. Sena AYDOS ¹, nazila farhangzad^1;2, Tulin Ozkan¹, Asuman Sunguroglu¹, Kaan Aydos³

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

Background: Mitochondria is a dynamic organelle that constantly undergoes fusion and fission. These dynamic processes work in concert to maintain functions of cell and cellular homeostasis. In process of spermatogenesis, the morphology of mitochondria undergoes significant modifications, and spermatogonia develop into highly specialized sperm cells that able to fertilize with perfectly coordinated, healthy mitochondria. Some of major molecules that mediate mitochondrial fusion and fission have been discovered in mammals, but there are not yet sufficient studies during spermatogenesis in humans.

Methods: Expression of mitochondrial fusion (OPA1) (MFN1) and mitochondrial fission (MFF) genes as biomarkers in mitochondrial dynamics was evaluated by RT-PCR in testicular cells taken from 8 infertile men with non-obstructive azoospermia (NOA) and 4 men with obstructive azoospermia (OA) as the control group.

Results: The expressions of MFN1, OPA1 genes were found to be increased in the NOA group compared to the OA group. Although the increase in OPA1 gene expression was not statistically significant, the expression of the MFN1 gene was found to be statistically significantly increased in the NOA group compared to the OA group.

Conclusion: MFN1 gene expression, which has been investigated for the first time in testicular cells of NOA patients, may play an important role in mitochondrial fusion in infertile men and may contribute to the identification of new biomarkers and the development of treatment protocols for infertile patients with mitochondrial dynamic defects. More clear results will be obtained when the study is concluded by increasing the number of patients.

Grants: This study was supported by Ankara University(BAP) (Project No:TDK-2022-2640)

Conflict of Interest: None declared

EP02.034 Ten infections before pregnancy and risk of pregnancy loss

Yevheniya Sharhorodska ^1;2;3, Anna Ulrich², Liliia Chorna¹, Ivanna Shymanska⁴, Danuta Zastavna¹, Oleg-Roman Gnateyko¹, Chiara Scapoli³, Halyna Makukh⁴, Inga Prokopenko^2;5

¹Institute of Hereditary Pathology, National Academy of Medical Science, Clinical Genetics, Lviv, Ukraine; ²University of Surrey, Department of Clinical and Experimental Medicine, Guildford, United Kingdom; ³University of Ferara, Department of Life Sciences and Biotechnology, Ferrara, Italy; ⁴Scientific Medical Genetic Center “LeoGENE”, Lviv, Ukraine; ⁵University of Surrey, People-Centred AI Institute, Guildford, United Kingdom

The loss of two or more pregnancies before 24 weeks/at any time of gestation is defined as recurrent pregnancy loss (RPL)/recurrent miscarriage (RM). Susceptibility to infections can modify individual risk of RM/RPL. We investigated the causality between ten infectious diseases – chickenpox, shingles, mononucleosis, mumps, measles, scarlet fever, bacterial meningitis, hepatitis B, hepatitis A, COVID-19 – and subsequent risk of pregnancy loss.

For outcome, we used two RPL/RM datasets: (1)LUCAR (Lviv Ukrainian Cohort for Advancing Reproductive Health) study from the Western Ukraine, including 350 women with clinically confirmed idiopathic RPL and 454 control women with at least one healthy child; (2)UK Biobank (UKBB) dataset, consisting of 556 women with RM and 2,928 control women with at least one (self-reported) healthy-born child. The LUCAR/UKBB genome-wide array datasets were QCed, imputed to TopMED/HRC reference panels density. PLINK1.9 was used for clumping (P-value = 1×10-5, r2 = 0.5, wc = 1000kb) of infection’s GWAS summary statistics. We performed two‐sample Mendelian randomization (MR, TwoSampleMR 0.5.2 package) analysis. As instruments for infections, we used summary statistics from genome‐wide association studies (GWAS; PMID:28928442, and 7th release of COVID19 Host Genetics Initiative).

The MR using 210/20 independent shingles/mononucleosis SNPs as instruments showed protective causal effect of shingles/mononucleosis on risk of RPL (OR[95%CI] = 0.40[0.32-0.50], P-value = 6.81×10-17/ OR[95%CI] = 0.34[0.14-0.80], P-value = 0.014). Then, we performed MR analyses for UKBB, meta-analysed results and detected significant (OR[95%CI] = 0.98[0.96-0.995], P-value = 0.013) protective from RPL/RM causal effect of shingles. Other infections did not show causality (P-value > 0.05) on RPL/RM risk. Our findings suggest that exposure to herpes zoster infections before pregnancy are protective from idiopathic pregnancy loss.

Grant: EMBO SLG 5423-2023

Conflict of Interest: None declared

EP02.035 Circulating anti-Müllerian hormone levels in pre-menopausal women: novel genetic insights from a GWAS meta-analysis

Natàlia Pujol Gualdo ^1;2, Minna Karjalainen^3;4, Urmo Võsa¹, Riikka K. Arffman², Reedik Mägi¹, Justiina Ronkainen³, Triin Laisk¹, Terhi Piltonen²

¹Estonian Genome Centre, Institute of Genomics, University of Tartu, Tartu, Estonia; ²Research Unit of Clinical Medicine, Department of Obstetrics and Gynecology, Oulu, Finland; ³Research Unit of Population Health, Faculty of Medicine, University of Oulu, Oulu, Finland; ⁴. Northern Finland Birth Cohorts, Arctic Biobank, Infrastructure for Population Studies, Faculty of Medicine, University of Oulu, Oulu, Finland

Background/Objectives: AMH is expressed by preantral and small antral stage ovarian follicles in women, and variation in circulating AMH levels has been associated with several conditions. However, the biological mechanisms underlying this variation are not fully understood. Previous GWAS have identified three loci associated with AMH levels in women.

Methods: We performed a GWAS meta-analysis (n = 9,668) in which we combined 2,619 AMH measurements from a founder population cohort (NFBC1966) with a previous GWAS meta-analysis that included 7,049 AMH measurements. We annotated the genetic variants, combined different data layers to prioritise potential candidate genes, described significant pathways and tissues enriched by the GWAS signals, identified plausible regulatory roles using colocalization analysis and leveraged publicly available datasets to assess genetic and phenotypic correlations.

Results: Three novel genome-wide significant loci were identified. One of these is in complete linkage disequilibrium with c.1100delC in CHEK2, which is found to be 4-fold enriched in the Finnish population compared to other European populations. We propose a plausible regulatory effect of some of the GWAS variants linked to AMH, as they colocalise with GWAS signals associated with gene expression levels of BMP4, TEX41 and EIFBP41. Gene set analysis highlighted significant enrichment in renal system vasculature morphogenesis and tissue enrichment analysis ranked the pituitary gland as the top association.

Conclusion: Our results highlight the increased power of founder populations and larger sample sizes to boost the discovery of novel trait-associated variants underlying variation in AMH levels.

Grants: EU Horizon 2020 under the MATER Marie Sklodowska-Curie grant agreement No. 813707 and Oulu university scholarship foundation.

Conflict of Interest: None declared

EP02.036 First and second trimester urine microRNA profiles in preeclampsia

Anastasia Maltseva ¹, Roman Illarionov¹, Elena Vashukova¹, Olga Pachuliia¹, Andrey Glotov¹

¹D.O. Ott Research Institute for Obstetrics, Gynecology, and Reproduction, Department of Genomic Medicine, Russian Federation

Background/Objectives: Preeclampsia (PE) is a severe complication of pregnancy, affecting 2-8 % of all pregnancies. PE is characterized by a high incidence of maternal and child morbidity and mortality. We have previously studied the microRNA profile in plasma samples from pregnant women at high risk of preterm birth. In this study, we have performed a prospective study of the miRNA profile in urine, the most non-invasive biomaterial, during the first and second trimesters in pregnant women with PE.

Methods: Total of 48 urine samples were taken from 24 women (PE = 12, CTRL = 12) in the first and second trimesters. Small RNA isolation and library preparation for sequencing was performed using miRNeasy Serum/Plasma Kit (Qiagen) and QIAseq miRNA Library Kit (Qiagen). Libraries were sequenced on a HiSeq 2500 (Illumina). Bioinformatic data analysis was performed using the GeneGlobe Data Analysis Center and DESeq2 R Package.

Results: We detected differences in the levels of 5 miRNAs (4 upregulated – hsa-miR-205-5p, hsa-miR-203a-3p, hsa-miR-223-3p, hsa-miR-184; 1 downregulated – hsa-miR-1-3p) (log₂(FC) ≥ 1.5; FDR ≤ 0.05) during the first trimester compared with the control (healthy pregnant women). During the second trimester 5 miRNAs was dysregulated (3 upregulated – hsa-miR-1-3p, hsa-miR-206, hsa-miR-9-5p; 2 downregulated – hsa-miR-205-5p, hsa-miR-223-3p). Hsa-miR-205-5p, hsa-miR-223-3p, and hsa-miR-1-3p are differentially expressed in both trimesters, but in reverse regulation.

Conclusion: Our results suggest that the miRNA profile in urine may predict a PE. Identified miRNAs in urine can be used as PE biomarkers.

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

Conflict of Interest: None declared

EP02.037 Pre-implantation Genetic Testing for Hereditary Breast and Ovarian Cancer Syndrome: The Parisian Experience

Roxana Borghese ¹, Charlotte Sonigo², Nelly Achour³, sophie monnot¹, Nadine Girarel¹, michael grynberg³, Alexandra Benachi², Sophie Frank⁴, Chrystelle Colas⁴, Dominique Stoppa-Lyonnet⁴, Julie Steffann¹

¹Necker Enfants Malades, Medecine Genomique des Maladies Rares, Paris, France; ²Antoine Béclère, Gynecologie Obstétrique, Paris, France; ³Antoine Béclère, Biologie de la reproduction, Paris, France; ⁴Institut Curie, Service de génétique, Paris, France

Background/Objectives: In France, Preimplantation Genetic Testing for Monogenic disease(PGT-M) is reserved for couples at high risk of having a child with a severe genetic disease deemed incurable, attested by a multidisciplinary prenatal diagnosis center (CPDPN). Hereditary breast and ovarian cancer syndrome is categorized as a late-onset disease for which access to PGT is disputed, in part because of the low success rate of this procedure.

Methods: Retrospective study of the 46 requests we received since 2015.

Results: A total of 27 requests (55%) were considered eligible, 10 were denied because of CPDPN refusal (4) or ovarian reserve alteration (6), and 9 couples dropped out. A total of 33 ovarian stimulation cycles were started, leading to 196 mature oocytes, 83 biopsied and 38 healthy embryos. Fresh or frozen embryos (n = 21) were transferred (n = 18), resulting in 8 pregnancies and 5 live births (23% of couples who started a PGT cycle). So far, all couples have requested to transfer embryos devoid of the pathogenic variant, regardless of gender, but none of the 42 couples who could not have a birth through PGT, requested a prenatal diagnosis.

Conclusion: PGT provides an alternative for couples who find it more “acceptable” to avoid transferring an embryo rather than opting for a pregnancy termination in the event of a predisposition to early-onset cancers. To increase the PGT pregnancy rate, the transfer of male embryos carrying the variant, assumed to have low tumor risks, should be considered.

Grants: No grants

Conflict of Interest: None declared

EP02.038 Complex chromosomal rearrangements in first trimester products of conception

Christina Katsidi^1;2, Haralambia Tsarouha², elisavet kouvidi², leandros lazaros², Periklis Makrythanasis ^3;4;5, amelia pantou², emmanouil kanavakis², ariadni mavrou²

¹National and Kapodistrian University of Athens, Medical School, Athens, Greece; ²Genesis Genoma Lab, Genetic Diagnosis, Clinical Genetics & Research, Athens, Greece; ³Biomedical Research Foundation of the Academy of Athens, Athens, Greece; ⁴National and Kapodistrian University of Athens, Laboratory of Medical Genetics, Athens, Greece; ⁵University of Geneva, Medical School, Genetic Medicine and Development, Geneva, Switzerland

Background/Objectives: Multiple or complex chromosomal abnormalities in products of conception are rare and are correlated with advanced maternal age and an early week of gestation. This study investigates their frequency in the first trimester of pregnancy.

Methods: 554 samples from first trimester aborted tissues were analyzed with conventional cytogenetic or molecular techniques. The average maternal age was 39 years (range 23-54). From each sample at least ten metaphases were analyzed using standard GTG banding. In cases where tissue samples failed to grow, molecular techniques, such as Quantitative Fluorescent Polymerase Chain Reaction (QF-PCR), were applied. Statistical analysis was performed using Pearson Chi-square exact tests to assess the association of the week of gestation and the age of the mother with single or complex chromosomal abnormalities.

Results: The success rate was 70,76%. Abnormal karyotypes were detected in 255 embryos (65%) and a normal karyotype was identified in 137 (35%). Single chromosomal abnormalities were identified in 235 cases (69,4%) with single trisomy 16 being the most frequent. Complex abnormalities were identified in 25 cases (9,8%), the most common finding was double trisomy. Polyploidies and monosomy 45,X occurred in 7,8%. Structural rearrangements and mosaicism were detected in 2,8% and 2,4%. Complex chromosomal abnormalities were more frequent among women older than 40 years old.

Conclusion: Embryos with complex chromosomal abnormalities were more frequent among women with advanced maternal age and were aborted one week earlier than embryos with single chromosomal abnormalities. Preimplantation genetic testing may be recommended in order to deliver a healthy offspring.

Grants: There were no grants

Conflict of Interest: None declared

EP02.039 Study of heterozygous carriage of genetic pathology among infertile couples in the Sverdlovsk region

Svetlana Deryabina ¹, Olga Lagutina¹, Elena Kudryavceva¹

¹Institute of medical cell technologies, Laboratory of molecular genetic researches, Yekaterinburg, Russian Federation

Background/Aims: The number of infertile patients planning to use assisted reproductive technologies (ART) is increasing significantly worldwide. However, the use of ART cannot automatically guarantee the birth of healthy children. The aim of this study was to determine the number of heterozygous carriers of severe hereditary pathology among infertile patients before in vitro fertilisation.

METHODS: In our study, we tested the first 30 married couples for carriage of fifty monogenic diseases by next generation sequencing (NGS). Informed voluntary consent was obtained from all patients.

Results: Our panel identified 28 carriers, that is, in almost every family one of the parents (and sometimes both) had an altered DNA sequence. The highest frequency of altered genetic variants was observed for congenital hearing loss (6 heterozygous carriers for GJB2 and SLC26A4 genes), cystic fibrosis and phenylketonuria (4 heterozygous carriers each). Also 3 future parents have mutations in the gene associated with biotinidase deficiency, 2 people are carriers of Wilson’s and Willebrand’s diseases. In addition, pathogenic variants in genes associated with such rare diseases for the Sverdlovsk region as isovalerian acidemia, recessive polycystic disease, metachromatic leukodystrophy, mitochondrial DNA depletion syndrome, and alpha-1-antitrypsin deficiency were identified. So far, no concordant heterozygotes have been identified in any of the pairs.

Conclusions: The findings indicate a high frequency of carrier genetic pathology among infertile couples, which makes further carrier screening appropriate.

Conflict of Interest: None declared

EP02.040 Implementation of Expanded Carrier Screening for Genetic Diseases: Report from Iran

Fatemeh Ahangari ¹, shima Dehdahsi¹, Mahsa Fadaie¹, Mehri Ashki¹, Maryam Beheshtian¹, Maryam Azad¹, Masoumeh Akbari Kelishomi¹, Negar Molaei², Parnian Alagha², Ariana Kariminejad¹, Hossein Najmabadi¹

¹Kariminejad-Najmabadi Medical Center, Tehran, Iran; ²Faculty of Social Welfare and Rehabilitation Sciences - National University, Tajrish, Iran

Background: Reproductive carrier screening (RCS) is designed to inform couples about their chances of having children with particular monogenic recessive conditions. Screening options have been revolutionized by NGS-based technologies and expanded carrier screening (ECS) is increasingly used in different populations. Here, we describe the results of the ECS test developed in Kariminejad-Najmabadi Pathology &Genetics Center, Tehran, Iran.

Methods: DNA samples of 225 Iranian cases (including 52 couples) were sequenced by NGS for 433 genes which designed for in house carrier panel considering ACMG recommended genes and genes with high carrier frequency in Iranian population. Also, deletions/duplications in DMD, SMN1 and repeats for FMR1 genes have been investigated by MLPA and TP-PCR techniques, respectively. Various routine filters applied to variants, the remaining, classified according to ACMG guideline criteria. Only variants that met the ACMG criteria for pathogenic/ likely pathogenic were reported.

Results: Of the 225 individuals, 83.11% were found to be carrier of at least one pathogenic/likely pathogenic variant. Twenty five out of the 52 couples (~48%) were carrier in same genes. The most frequent occurrence of carriers was recorded in the commonly investigated genes (CFTR, PAH, SMN1), consistent with the high carrier rates reported in the Iranian population.

Conclusion: NGS-based carrier screening offers a powerful approach for assessing carrier status to decrease the burden of genetic diseases, especially in populations with a high rate of consanguinity. The strategies for variant interpretation in a healthy population are challenging, therefore, the importance of access to studies from different populations is useful for a better interpretation of the results.

Conflict of Interest: None declared

EP02.041 Preimplantation diagnosis for mitochondrial DNA m.8344A > G (MERRF) mutation : challenge and success

sophie monnot ¹, Nadine Gigarel¹, anne mayeur², Joana Bengoa¹, Roxana Borghese¹, Benoit Funalot¹, Charlotte Sonigo², nelly achour², Julie Steffann¹

¹Necker Hospital, paris, France; ²Antoine Beclere Hospital, 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. Genetic heterogeneity of these diseases is due to the co existence of wild-type and mutated mtDNA (heteroplasmy). There is a threshold below which clinical symptoms are not likely to occur. At risk couples often ask for preimplantation genetic diagnosis (PGD) which is based on mutant load assessment in blastomeres.

Methods: Three women, asymptomatic carriers of m.8344A > G in the MT-TK gene, responsible for MERRF syndrome (Myoclonic Epilepsy with Ragged-Red Fibers), underwent PGD in our centre. Heteroplasmy level was measured on 75 cells from 40 embryos at day 3 of developpement (D3).

Results: The mutant load was stable among blastomeres from a given embryo at D3, with the exception of one embryo. We observed one mutation-free embryo. The remaining ones were all heteroplasmic (10 to 97%), suggesting random segregation of mutated DNA during maternal oogenesis. Five of them were transferred resulting in 2 pregnancies and the birth of two healthy children. Mutant load was measured at birth on cord blood showing a stable level in one case, and an increase of 18% in the other.

Conclusion: These results 1/ validate technical feasibility of PGD for the MERRF mutation on D3 embryos 2/ demonstrate clinical interest of PGD for women carrying high level of mtDNA mutations 3/ illustrate the possible variation of the mutant load during human embryo-foetal development. These data have an important impact in terms of genetic counselling for patients carrying mtDNA mutation.

Grants:

Conflict of Interest: None declared

EP02.042 Analysis of aneuploidies in products of conception: effect of maternal and gestational age

Eloisa Caviglia ¹, Micaela Galain¹, Yannina Diaz Cano¹, Mónica Fabbro¹, Sergio Papier¹, Florencia Nodar¹, Cecilia Fernandez¹

¹Novagen Genetics Laboratory. Cegyr-Eugin Group, Buenos Aires, Argentina

Background/Objectives: Between 15-20% of pregnancies result in miscarriages, of which 50% are due to embryonic chromosomal abnormalities. The aim was to describe the results of products of conception (POC) analysis and to assess the impact of maternal age and gestational age on the presence of aneuploidies.

Methods: 93 POC were studied by NGS. STRs in fetal and maternal DNA were examined to rule out contamination with maternal cells and XXX triploidies. Results were stratified according to maternal age into ≤37 (57) and >37 years (36).

Results: Results were obtained in 87% of the samples. Chromosomal abnormalities were identified in 63.4%. The most frequent aneuploidies were trisomies in chromosomes 16, 15, 21, 22, and 13. In ≤37 chromosomal abnormalities were detected in 56.1% of cases, while in >37 in 75%. Abnormalities occurred in a greater variety of chromosomes in ≤37, with trisomies predominating in chromosomes 14 and 22. Additionally, X monosomies and triploidies were identified. In >37, trisomies were more frequent in chromosomes 16, 15, and 21. The gestational age varied from 5 to 22 weeks in ≤37 and from 5 to 13 in >37. However, regardless of maternal age, 80% of abortions occurred between weeks 6-9.

Conclusion: Advanced maternal age increases the risk of aneuploidies. These abnormalities would explain the earlier pregnancy losses in women >37 years. NGS proved to be a useful tool for the study of POC. Knowing the cause of abortion allows estimating the risk of recurrence, determining the need for additional studies, and planning new reproductive strategies.

Grants:

Conflict of Interest: Eloisa Caviglia CEGYR, Micaela Galain CEGYR, Yannina Diaz Cano CEGYR, Mónica Fabbro CEGYR, Sergio Papier CEGYR, CEGYR, Florencia Nodar CEGYR, CEGYR, Cecilia Fernandez CEGYR

EP02.043 Multicenter study assessing the reproductive outcomes of mosaic embryo transfer in Argentina

Micaela Galain ¹, Eloisa Caviglia¹, Yannina Diaz Cano¹, Mónica Fabbro¹, Sebastián Menazzi¹, Valeria Paz², Idelma Serpa², Laura Sicaro³, Edgardo Young Obejero³, Andrea Dematteis⁴, Gustavo Estofan⁴, Sergio Papier¹, Florencia Nodar¹, Cecilia Fernandez¹

¹Novagen Genetics Laboratory. Cegyr-Eugin Group, Buenos Aires, Argentina; ²Grupo Gamma, Servicio de Medicina Reproductiva, Rosario, Argentina; ³IFER, Instituto de Ginecología y Fertilidad, Buenos Aires, Argentina; ⁴CIGOR, Clínica de Fertilidad, Cordoba, Argentina

Background/Objectives: Chromosomal mosaicism is defined as the presence of two or more different cell lines within an embryo. Several publications report that mosaic embryo (ME) transfer can lead to successful pregnancies and healthy deliveries. However, the impact of mosaicism on IVF outcomes remains a matter of controversy. The aim was to assess if low-level ME transfer affects reproductive outcomes.

Methods: The study included 6241 embryos from 4 fertility clinics who underwent PGT. Patients in group A (n = 894) were transferred an euploid embryo, while in group B (n = 55), a low-grade ME (<40%, 2016-2021 or <50%, 2021-2023) aneuploid cells, was transferred.

Results: 46.2% embryos were euploid, 31.3% aneuploid and 19.3% mosaic (69.5% classified as low-grade and 30.5% as high-grade) and in 3.2% no results were obtained. Fifty five low-grade ME were transferred. The positive beta-hCG rates were 50.3% and 49.1%, while the clinical pregnancy rates were 36.9% and 36.4% for Groups A and B, respectively. Regarding live births, in Group A 328 (36.7%) children were born, and in Group B,16 (29.1%) with 2 ongoing pregnancies. Conversely, pregnancy losses in Group B (9.1%) were higher compared to the euploid group (2.9%). In all cases the newborns were apparently healthy after ME transfer, with an average gestational week at birth of 39 (37-41), and an average weight of 3197 (2900-4390) grams.

Conclusion: Low-grade ME transfer would not affect reproductive outcomes, as live birth rate appears to be like those observed with euploid embryos. Therefore, mosaic embryos are a valid alternative for couples lacking euploid embryos.

Grants:

Conflict of Interest: Micaela Galain Cegyr-Eugin Group, Eloisa Caviglia Cegyr-Eugin Group, Yannina Diaz Cano Cegyr-Eugin Group, Mónica Fabbro Cegyr-Eugin Group, Sebastián Menazzi Cegyr-Eugin Group, Valeria Paz GAMMA, Idelma Serpa GAMMA, Laura Sicaro IFER, Edgardo Young Obejero IFER, IFER, Andrea Dematteis CIGOR, Gustavo Estofan CIGOR, CIGOR, Sergio Papier Cegyr, Cegyr, Florencia Nodar Cegyr, Cegyr, Cecilia Fernandez Cegyr

EP02.044 PGT-SR: effect of chromosomal rearrangement type and carrier gender on the acquisition of transferable embryos.

Yannina Diaz Cano ¹, Micaela Galain¹, Eloisa Caviglia¹, Mónica Fabbro¹, Florencia Nodar¹, Sergio Papier¹, Cecilia Fernandez¹

¹Cegyr-Eugin Group, Novagen Genetics Laboratory, Buenos Aires, Argentina

Background/Objectives: Translocation carriers have an increased risk of having unbalanced embryos. PGT-SR is a crucial tool for selecting embryos to transfer. This study aimed to explore how chromosomal rearrangement type and carrier gender affect the ability to obtain transferable embryos.

Methods: 265 embryos from 71 cycles (47-couples) were divided according to the parental rearrangement into Robertsonian translocation carrier (ROB-T, 105) and Reciprocal translocation carrier (REC-T, 160) groups. Data were stratified by maternal age (≤37 and >37) and carrier gender: male carrier (MC) and female carrier (FC). Embryos were classified as transferable if euploid/balanced or showed low-grade mosaicism (<50%). Non-transferable embryos included unbalanced, aneuploid, or displayed high-grade mosaicism.

Results: Among ROB-T embryos, 43% (45/105) were transferable, compared to only 22% (35/160) in the REC-T group (p = 0.0004612). Subgroup analysis by maternal age revealed that in the ≤37, 43% (39/91) of ROB-T embryos were transferable versus 25% (30/122) in the REC-T group (p = 0.007583), and in the >37, 43% (6/14) of ROB-T embryos were transferable compared to 13% (5/38) in the REC-T group (p = 0.04951). Within the MC, 47% (36/76) of ROB-T embryos were transferable versus 24% (22/90) in the REC-T (p = 0.003467). Conversely, in the FC, 31% (9/29) of ROB-T embryos and 19% (13/70) of REC-T embryos were transferable (p = 0.2749). Unbalanced embryos, comprising 71% of non-transferable embryos, were more prevalent in REC-T carriers across all analyzed groups.

Conclusion: The type of rearrangement is relevant to obtaining transferable embryos. Carriers of REC-T, especially female carriers, could have a higher risk of having non-transferable embryos compared to carriers of ROB-T.

Conflict of Interest: Yannina Diaz Cano Full-time employee at Novagen, Micaela Galain Full-time employee at Novagen, Eloisa Caviglia Full-time employee at Novagen, Mónica Fabbro Full-time employee at Novagen, Florencia Nodar Shareholder of Cegyr, Sergio Papier Shareholder of Cegyr, Cecilia Fernandez Full-time employee at Novagen

EP03.001 A decade of insights: prenatal cytogenetic diagnosis results of high-risk pregnancies at Atatürk University Medical Genetics laboratory (ATAGEN)

Momen Kanjee ¹, Nuran Ertargin¹, Cigdem Yuce Kahraman¹, Abdulgani Tatar¹

¹Atatürk University, Faculty of Medicine, Department of Medical Genetics, Erzurum, Türkyie

Background/Objectives: Prenatal diagnosis is the process of performing genetic testing on fetal samples to determine whether the fetus is affected by a genetic disorder. Conventional karyotype analysis is helpful in detecting large chromosomal aberrations (CA). Here, we summarize the indications and karyotype results of patients who underwent amniocentesis or cordocentesis in our clinic.

Methods: The karyotype findings of patients who underwent amniocentesis/cordocentesis in our clinic between 2014 and 2023 were evaluated retrospectively. The results were reported according to the International System for Human Cytogenomic Nomenclature (ISCN).

Results: In this study, the results from 1,330 samples were analyzed: 137(10.3%) cordocentesis and 1,193(89.7%) amniocentesis samples. Our culture success rate was 89%(1,184/1,330). The median maternal and gestational ages were 33 years(18-58) and 18 weeks(11-39), respectively. The most common indication was a positive screening result (43.1%;573/1,330), followed by fetal structural abnormalities (30.6%;407/1,330) and advanced maternal age (23.5%;313/1,330).

The prevalence of CA was 7.7%(103/1330), with aneuploidies being the most common aberrations (83.5%;86/103) followed by structural abnormalities (9.7%;10/103) and mosaicisms (6.8%;7/103). Overall, trisomy 21 was the most frequently detected alteration (n = 67; 61 standard trisomies, 5 mosaic, 1 standard trisomy with structural aberration) followed by trisomy 18 (n = 21; 20 standard trisomies, 1 standard trisomy with structural aberration), trisomy 13 (n = 3; all standard trisomies), sex chromosome aneuploidies (n = 4; 2 mosaic monosomy Xs, 1 47,XXY, 1 47,XYY), and other structural aberrations(n = 8).

Conclusion: Our study illustrated that invasive prenatal cytogenetic analysis remains a diagnostic method for detecting CA. Although additional diagnostic tests were offered to some patients, this study shows only the karyotype results.

Grants: None.

Conflict of Interest: None declared

EP03.002 A Rare Case Report: Trisomy 22 in the Second Trimester

Agnese Kalnina ^1;2, Inguna Lubaua^1;2, Ieva Grīnfelde^1;2, Klinta Lisnere¹, Maija Lubgane-Griķīte¹, Elena Zeltina¹, Gunta Kalnberza¹

¹Children’s Clinical University Hospital, Rīga, Latvia; ²Riga Stradins University, Rīga, Latvia

Background/Objectives: Trisomy 22, a chromosomal abnormality, often results in severe medical complications, typically leading to first-trimester spontaneous abortion. Live births and continued pregnancies beyond 12 weeks of gestation are exceedingly rare. We present a unique second-trimester case of trisomy 22 at our hospital.

Methods: The patient was examined at the Children’s Clinical University Hospital of Latvia’s Medical Genetics and Prenatal Diagnostics Department.

Results: At 13 weeks of gestation, the patient sought consultation at our medical genetics clinic following abnormal findings from first-trimester ultrasonography. Noninvasive prenatal testing (NIPT) indicated the possibility of trisomy 22. The fetus displayed increased nuchal translucency thickness, renal hyperechogenicity, and a complex congenital heart defect known as tetralogy of Fallot. Ultimately, the family made the decision to terminate the pregnancy at 15 weeks. Patho-anatomical examination revealed distinctive phenotypical features, such as excess nuchal skin, low-set ears, and micrognathia. The internal organs developed properly, except the prenatally diagnosed tetralogy of Fallot. Karyotype analysis from chorionic biopsy subsequently confirmed non-mosaic trisomy 22: 47,XY, + 22.

Conclusion: This case highlights the atypical presentation of trisomy 22 in later gestation, emphasizing the importance of early prenatal screening. While tetralogy of Fallot, potentially correctable postnatally, is commonly associated with genetic syndromes, its previous correlation with trisomy 22 has not been described. Early identification enables families to prepare for a child with a genetic syndrome or an elevated risk of fetal demise. Full genome NIPT played a crucial role in diagnosing this rare genetic aberration, highlighting its ability to detect conditions that might otherwise go unnoticed.

Conflict of Interest: None declared

EP03.003 Evaluation of the contribution of trio exome sequencing in targeted prenatal indications.

Manon Chretien¹, Julien Osouf², Carine Abel³, Alexandra Afenjar⁴, Tania Attie-Bitach⁵, elise brischoux-boucher⁶, lydie BURGLEN⁷, Nadège Calmels², Nicolas Chassaing⁸, Thomas Courtin⁹, Julian Delanne¹⁰, Martine Doco-Fenzy^11;12, Christele Dubourg¹³, Benjamin Durand¹, Salima EL CHEHADEH¹, Laurence Faivre^10;14, Aurore Garde¹⁰, Emmanuelle Ginglinger¹⁵, Virginie Haushalter², Damien Haye³, Solveig Heide⁹, Laurence Heidet^16;17, Delphine Heron⁹, Clemence Jacquin¹², Laetitia Lambert^18;19, Vincent Laugel²⁰, Daphné Lehalle⁹, Laurence Michel-Calemard²¹, Yohny Montoya²², Jean Muller^2;23;24, Sylvie Odent²⁵, Olivier Patat⁸, Juliette Piard^14;26, Céline Poirsier¹², Audrey Putoux³, Chloe Quelin²⁵, Nicolas Sananes^27;28, Audrey Schalk², sophie scheidecker^2;24, Christel Thauvin-Robinet^10;14, Stéphanie Valence²⁹, Anne-Sophie Weingertner^27;28, Justine Wourms¹⁸, Hélène Dollfus^1;24, Benedicte Gerard², Caroline Schluth-Bolard^2;24, Elise Schaefer ¹

¹Service de Génétique Médicale, Institut de Génétique Médicale - Hôpitaux Universitaires de Strasbourg, Strasbourg, France; ²Laboratoire de Diagnostic Génétique, Nouvel Hopital Civil - Hôpitaux Universitaires de Strasbourg, Strasbourg, France; ³Service de Génétique, Groupement Hospitalier Est, Hospices Civils de Lyon, Lyon, France; ⁴Centre de Référence Malformations et Maladies Congénitales du Cervelet, UF de génétique clinique, Hôpital Trousseau, APHP, Sorbonne Université, Paris; ⁵Service de Médecine Génomique des Maladies Rares, Hôpital Necker-Enfants Malades, Paris, France; ⁶Centre de Génétique Humaine, Centre Hospitalier Universitaire, Université de Bourgogne Franche-Comté, Besançon; ⁷Laboratoire de Neurogénétique Moléculaire, Hôpital d’Enfants Armand-Trousseau, APHP, Paris, France; ⁸Service de Génétique Médicale, Hôpital de Purpan, CHU de Toulouse, Toulouse, France; ⁹Service de Génétique Clinique et Médicale, Hôpital de la Pitié Salpêtrière, APHP, Paris, France; ¹⁰Service de Génétique Médicale, Hôpital du Bocage, CHU de Dijon, Dijon, France; ¹¹Service de Génétique, Institut de Biologie, CHU Nantes, Nantes, France; ¹²Service de Génétique, Hôpital Maison Blanche, CHU de Reims, Reims, France; ¹³Laboratoire de Génétique Moléculaire et Génomique Médicale, Hôpital Pontchaillou, CHU de Rennes, Rennes, France; ¹⁴UMR 1231 GAD, Inserm, Université de Bourgogne Franche-Comté, Dijon, France; ¹⁵Service de Génétique Médicale, Hôpital Emile Muller, GHRMSA, Mulhouse, France; ¹⁶Service de Néphrologie Pédiatrique, Centre de Référence des Maladies Rénales Héréditaires de l’Enfant et de l’Adulte (MARHEA), Hôpital Necker-Enfants Malades, APHP-centre, Université Paris Cité, Paris, France; ¹⁷Laboratoire des Maladies Rénales Héréditaires, Inserm UMR 1163, Institut Imagine, Paris; ¹⁸Service de Génétique clinique et Médecine Infantile, Hôpital d’Enfants CHU de Nancy - Hôpitaux de Brabois, VANDOEUVRE-LÈS-NANCY, France; ¹⁹INSERM-U1256 NGERE, Université de Lorraine, Vandœuvre-lès-Nancy, France; ²⁰Service de Pédiatrie 1, Hôpital de Hautepierre, CHU de Strasbourg, Strasbourg, France; ²¹Service de Biochimie et Biologie Moléculaire, Centre de Biologie et Pathologie Est, Hospices Civils de Lyon, Lyon, France; ²²Service d’Obstétrique, Hôpital du Hasenrein, GHRMSA, Mulhouse, France; ²³Unité de Bio-informatique Médicale Appliquée au Diagnostic, Hôpitaux Universitaires de Strasbourg, Strasbourg, France; ²⁴Laboratoire de Génétique Médicale, INSERM UMRS_1112, Institut de Génétique Médicale, CRBS, Université de Strasbourg, Strasbourg, France; ²⁵Service de Génétique Clinique, Hôpital Sud, CHU de Rennes, Rennes, France; ²⁶Service de Génétique Médicale, Hôpital Jean Minjoz, CHU de Besançon, Besançon, France; ²⁷Service de Gynécologie obstétrique, CMCO, CHU de Strasbourg, Schiltigheim, France; ²⁸Centre Pluridisciplinaire de Diagnostic PréNatal, Département de Médecine Fœtale, CMCO, CHU de Strasbourg, Schiltigheim, France; ²⁹Service de Neuropédiatrie, Hôpital d’Enfants Armand-Trousseau, APHP, Paris

Background/Objectives: Congenital malformations affect approximately 3% of pregnancies. The etiologies are diverse, and, in most cases, constitutional genetic abnormalities are sought.

Methods: In a multicenter, comparative, prospective study, we evaluated the yield of high-throughput sequencing analyses (in silico versus exome panel) in targeted prenatal indications strongly suggesting a monogenic pathology.

Results: Trio exome analysis of 86 fetuses (mean duration of 21 weeks of amenorrhea) led to a certain etiological diagnosis in 28% of cases (CGPA classes 4/5) and uncertain in 11.6% (VUS, class 3). The yield varied according to the type of malformation: 41.6% for vermian hypoplasia, 31% for polymalformative syndromes, 25% for hygroma colli / generalized hydrops, 23.5% for hyperechoic large kidneys, 20% for agenesis of the corpus callosum, 16.7% for holoprosencephaly, 1/2 for gyration anomalies and 1/4 for ophthalmological anomalies. The identified variants are mainly missense, in ciliopathy genes (19%) or DNA metabolism genes (16%). The mode of transmission is predominantly autosomal dominant, with 47% of variants arising de novo and 16% inherited from a pauci or asymptomatic parent. We detected a postzygotic mosaic (13%) and 4 double diagnoses. The primary in silico panel analysis provided 19% of the unambiguous molecular diagnoses; the secondary analysis of the whole exome allowed the diagnosis of a further 9% of cases, but with identification of numerous VUS.

Conclusion: This study demonstrates the added value of exome sequencing in comparison with the use of in silico panels but in targeted indications, demonstrating the value of a clear definition of the malformations justifying this approach.

Grants: No

Conflict of Interest: None declared

EP03.004 Tissue-Specific Aneuploidy in Mosaic Trisomy 18: Prenatal and Postnatal Insights

Rainer Wimmer ¹, Miriam Kinzel², Iris Dressler-Steinbach³, Holger Janke⁴, Andre Weber¹, Markus Stumm¹

¹Medicover Humangenetik Berlin Lichtenberg MVZ, Cytogenetics, Berlin, Germany; ²Medicover Humangenetik Berlin Lichtenberg MVZ, Genetic Counseling, Berlin, Germany; ³Charité Universitätsmedizin Berlin, Department of Obstetrics, Berlin, Germany; ⁴Praxis für Pränatale Diagnostik, Berlin, Germany

Background/Objectives: Edwards’ syndrome, resulting from trisomy 18, is commonly considered as incompatible with life, leading to pregnancy terminations following prenatal detection. However, mosaic trisomy 18 exhibits significant clinical heterogeneity. We report on a male pregnancy at high risk for trisomy 18, double non-invasive prenatal testing was conducted. Initial results indicated a false normal female, followed by a male result positive for trisomy 18. Fetal ultrasound revealed ventricular septal defect, thymic hypoplasia, overthrown fingers and a megacisterna magna. Subsequent amniocentesis confirmed mosaic trisomy 18. Despite the diagnosis, the parents chose to continue the pregnancy, opting for comprehensive medical care. The boy was delivered by Cesarean section and underwent heart surgery at 4 months. At 8 months, his development was evaluated as age-appropriate. This case study presents unprecedented pre- and postnatal insights into the tissue-specific distribution of his aneuploid cells.

Methods: Chromosomal and/or FISH analyses were conducted on various tissues from all germ layers.

Results: The distribution of trisomy 18 cells varied significantly: amniotic fluid (28%), heart (96%), thymus (7%), skin (5%), and buccal smear (1%). Notably, the congenital heart malformation correlated with a high percentage of aneuploid cells in the right ventricle.

Conclusion: In cases of mosaic trisomy 18, the presence of a normal cell line with unknown tissue distribution complicates predicting pregnancy outcomes. Theoretical prognostic considerations should primarily focus on confirmed fetal malformations. Clinicians should be aware of the potential for varied outcomes and consider a multidisciplinary approach when counseling parents facing similar situations.

Grants:-

Conflict of Interest: None declared

EP03.005 Expanding the allelic spectrum of the PACS1 gene: a prenatal case report with brain malformation

Marta Palucci ¹, Antonella Pizza², Allessandra Terracciano³, Valeria Orlando³, antonella spagnuolo⁴, maria assunta spina⁴, Lucia Manganaro⁵, maria cristina muzi⁶, manuela di natale⁶, barbara raso⁶, carlotta vaccari⁶, sara nuovo⁶

¹Università Cattolica del Sacro Cuore, Fondazione Policlinico Universitario A. Gemelli IRCCS, Department of Science of Life and Public Health, Rome, Italy; ²University of Campania “Luigi Vanvitelli”, Department of Precision Medicine, Naples, Italy; ³Bambino Gesù Children’s Hospital, IRCCS, Rare Diseases and Medical Genetics Unit, Rome, Italy; ⁴Centro Sant’Anna ASL Roma1, UOSD Diagnosi Prenatale, Rome, Italy; ⁵Sapienza University of Rome, Department of Radiological, Oncological and Pathological Sciences, Rome, Italy; ⁶Centro Sant’Anna ASL Roma1, UOSD Genetica Medica, Rome, Italy

Background/Objectives: Schuurs-Hoeijmakers syndrome (SHMS, OMIM #615009) is a rare autosomal dominant disorder characterized by mild-to-severe psychomotor delay/intellectual disability, abnormal craniofacial features and congenital malformations. SHMS is caused by heterozygous mutations in the PACS1 gene. Less than 90 cases have been reported worldwide (usually diagnosed postnatally), almost all sharing the p.Arg203Trp missense change.

Methods: We report a novel PACS1 variant, detected in a prenatal setting. A 32-year-old woman was referred to our prenatal diagnosis center at 20 weeks of gestation (WG) because of suspected corpus callosum (CC) hypoplasia and abnormal width of cavum septum pellucidi (CSP) appearing on ultrasound. Fetal brain MRI (at 21 WG) revealed a short, thickened and dysmorphic CC, associated with enlarged CSP and “teardrop” appearance of the frontal horns.

Results: Standard karyotype and CGH-array analysis performed on fetal DNA following amniocentesis did not reveal pathogenic alterations. Trio-based Clinical Exome Sequencing identified de novo heterozygous PACS1 variant c.2126G>A, causing the aminoacidic change p.(Arg709Gln). This variant is not described in scientific literature nor in international databases and is classified as “probably pathogenetic” (Class-IV) according to American College of Medical Genetics and Genomics (ACMG) guidelines.

Conclusion: We focused on the brain malformations observed in PACS1-mutated cases and recorded MRI findings of patients reported in the literature. Comprehensive evaluation was available only for 36 of them, mainly presenting with cerebellar hypoplasia (27%), ventricular abnormalities (19%), white matter defects (14%) and CC/CSP abnormal appearance (11%).

This work expands the allelic spectrum of PACS1, whose mutation should be considered also in cases with apparently isolated CC/CSP abnormalities.

Conflict of Interest: None declared

EP03.006 Comparisons of results from non-invasive prenatal testing (NIPT) and our cytogenetic analyses

Ilona Dietze-Armana ¹, Andreas Gerhardinger¹, Manuel Lüdeke¹, Karl Mehnert¹

¹genetikum, Neu-Ulm

Background/Objectives: Non-invasive prenatal testing (NIPT) is a well-established option for prenatal screening, primarily for the detection of trisomy 21, 18, 13 using Cell-free DNA (cfDNA) in maternal plasma. cfDNA is derived from the outer trophoblast cell layer of the placenta. NIPT tests vary in their exact methodology and there are various assays available. In a pooled meta-analysis, the detection rate across different NIPT methods was 99% (T21), 96% (T18) and 91% (T13). The cumulative false positive rate (FPR) was less than 0.1% for T21 and 0.26% for T13/18 (Gil et al. 2015). In addition to false positive detection rates, the positive and negative predictive values (PPV and NPV) of a screening test are important clinical parameters. These values depend partly on the performance characteristics of the test, but also vary with the prevalence of the tested condition in the population.

Methods: Here we report a cohort of 535 cases from 2021 to 2023 with abnormal NIPT for trisomy 13/18/21 for which we performed verification using QF-PCR and fetal karyotype.

Results: NIPT results for 418 of 535 cases could be confirmed with QF-PCR and cytogenetic analysis, whereas in 117 cases (45xT13, 43xT18, 29xT21) no chromosomal abnormality was detected.

Conclusion: The discrepancy between NIPT and cytogenetic methods is due to the fact that the cell-free fetal DNA does not always correspond to the karyotype of the fetus, which can partly be attributed to placenta mosaics.

NIPT has limitations and complexities that requesting clinicians and their patients should consider.

Conflict of Interest: None declared

EP03.007 Prenatal diagnosis and pregnancy termination in Muslims living in mixed cities versus Muslims living in isolated habitats in Israel.

Aliza Amiel ¹, Mahdi Tarabeih²

¹Tel Aviv-Yafo, School of Nursing Research, Tel Aviv-Yafo, Israel; ²School of Nursing Research The Academic College Tel Aviv-Jaffa, Tel Aviv-Jaffa, Israel

Objective: Pregnancy termination is forbidden according to the laws of Islam, thus, the need for prenatal tests is inconclusive in Muslim society. In this research, we explored whether Jewish society norms influence Muslim women living in mixed cities to undergo prenatal testing and pregnancy terminations compared to Muslims women living in their own habitats.

Methods: The sample consisted of 1081 Israeli Muslim women between 18-49 years of age. 52% living in mixed cities (i.e., Muslim and Jews living in the same city in coexistence), and 48% Muslim responders living in their own habitats.

Results: Muslim women living in mixed cities performed more prenatal tests and pregnancy terminations than in isolated Muslim-only habitats. Most pregnancy terminations, with abnormal results, were performed in the center of the country. Participants living in the center, both mixed-cities and Muslim-only habitats performed more noninvasive prenatal testing (NIPT), compared to participants living in other parts of the country. Only participants living in mixed cities and Muslim-only habitats in the center of the country did receive genetic consultancy before undergoing the different prenatal tests.

Conclusions: Muslim women living in mixed cities performed more prenatal tests and pregnancy terminations than in isolated Muslim-only habitats, due to the influence of the Jewish society living in the same city and to more genetic consultancy. Additional medical health care and genetic counselling are needed in Muslim only habitant cities in other parts than the center of the country.

Conflict of Interest: Aliza Amiel full time, Mahdi Tarabeih full time

EP03.008 A novel α1-globin gene mutation reported from north of Iran: Hb Mazandaran (HbA1 c.154 GGC > TGC)

Mohammad Reza Mahdavi¹, Atefeh Khoshaein ², Maryam Rahimi², Hossein Jalali¹, Mahan Mahdavi²

¹Thalassemia Research Center, Hemoglobinopathies Institute, Mazandaran University of Medical Sciences, Sari, Iran; ²Sinaye Mehr Research Center, Sari,Iran

Background/Objectives: Alpha thalassemia is one of the most common human genetic abnormalities and identification of all variants of Hemoglobin in different regions helps to get comprehensive knowledge about thalassemia disease and it can be used in preventive programs, and prenatal diagnosis (PND). In the present study, we describe a new α1 gene mutation (HbA1 c.154 GGC > TGC).

Methods: As a program for prevention of thalassemia a couple was referred to Fajr medical genetics laboratory (Sari, Iran) for routine hematological analysis. At first routine Cell Blood Count (CBC) and Hb capillary electrophoresis were applied. After taking written informed consent, molecular analysis was conducted on genomic DNA extracted from peripheral blood using QIAamp DNA Mini Kit (Qiagen, Germany). For identifying common Mediterranean α-Globin gene deletion multiplex Gap-PCR was performed and for detecting other mutations in the α and β-Globin genes DNA-Sequencing method was used.

Results: The results of CBC and capillary electrophoresis tests were compatible with β-thalassemia carrier in female subject. The case did not have common α-Globin gene deletions. Sequencing of α-Globin gene showed that subject carried c.315 + 1G > A and c.154G > T mutations of the β and α1-globin genes, respectively. Both of the mutations were also detected in the subject’s mother.

Conclusion: The c.154G > T variant (Hb ^Mazandaran) gives rise to a novel hemoglobin variant that was detected in two cases in combination with β-globin mutation, and it does not seem to be associated with severe hematological abnormalities in the carriers.

Grants: Not-Applicable

Conflict of Interest: None declared

EP03.010 Cell free fetal DNA at 11-13 weeks of gestation is not altered in complicated pregnancies

Zoi Koukou¹, Eleftherios Panteris², Emmanouel Manolakos³, Aristeidis Papadopoulos⁴, Ioannis Papoulidis³, Eleftheria Papadopoulou⁵, Konstantinos Relakis⁶, Stavros Sifakis ⁷

¹School of Health Sciences, International Hellenic University (IHU), Sindos Thessaloniki, Greece; ²Aristotle University of Thessaloniki, Laboratory of Forensic Medicine and Toxicology, Thessaloniki, Greece; ³, Access to Genome P.C, Clinical Laboratory Genetics, Thessaloniki, Greece; ⁴Aristotle University of Thessaloniki, School of Medicine, Thessaloniki, Greece; ⁵University Hospital of Heraklion, Department of Pediatrics, Heraklion, Crete, Greece; ⁶Department of Obstetrics & Gynecology, University Hospital of Heraklion, Heraklion, Crete, Greece; ⁷, Mitera Maternity Hospital, Heraklion, Crete, Greece

Background/Objectives: Non-invasive maternal cell-free fetal DNA (cffDNA) is a promising biomarker for screening common genetic syndromes. Alterations in the expression levels of cffDNA in the maternal circulation have been demonstrated in abnormal pregnancies. However, the results are conflicting. The present study aimed to investigate whether cffDNA levels correlate with pregnancy complications.

Methods: The study group comprised pregnant women who presented with pregnancy complications, such as preterm birth, gestational hypertension, intrauterine growth retardation, gestational diabetes, polyhydramnios, oligohydramnios, vaginal bleeding, and placental abruption. The control group comprised women who had a normal pregnancy course. Blood samples were obtained from 500 pregnant women between 11-13 weeks of gestation. cffDNA was amplified, sequenced, and analyzed using the Next-generation Aneuploidy Test of Panorama-Natera kit. Nuchal translucency (NT) thickness as well as Pregnancy Associated Plasma Protein-a (PAPP-a) and β-human chorionic gonadotropin (β-hCG) levels were also measured. Statistical analysis was performed in 494 out of the 500 samples collected with SPSS v.26 (SPSS, Inc. Chicago IL, USA) using non-parametric methods. The parameters were normalized by the Multiples of Median (MoM) method.

Results: The expression levels of PAPP-A, β-hCG, and the NT mean MOM values were significantly different between the study and control groups (p = 0.005, p < 0.001, and p = 0.007, respectively). However, the expression levels of cffDNA and the mean MOM values were not significantly different between these two groups (p = 0.687).

Conclusion: The results of the present study support the conclusion that cffDNA expression is not altered in a series of pregnancy complications. The prognostic value of cffDNA in predicting adverse pregnancy outcomes requires further investigation.

Conflict of Interest: None declared

EP03.011 Prenatal diagnosis of Zellweger spectrum disorder caused by novel variant in PEX6 gene

Ivana Joksic ¹, Mina Toljic¹, Andjela Stankovic¹, Zagorka Milovanovic^2;3

¹Gynaecology and Obstetric Clinic “Narodni front”, Genetic laboratory, Belgrade, Serbia; ²School of Medicine University of Belgrade, Belgrade, Serbia; ³Gynaecology and Obstetrics Clinic “Narodni front”, High risk pregnancy department, Belgrade, Serbia

Background/Objectives: Zellweger spectrum disorder (ZSD) is autosomal recesive disorder of peroxisome biogenesis caused by mutations in PEX genes. The clinical manifestations are very variable with hypotonia, facial dysmorphism, developmental delay, renal and liver dysfunction as most common features. We present a rare case of prenatally diagnosed ZSD caused by novel variant in PEX6 gene.

Methods: A 30-year old women was refered to genetic counseling in 27^th gestation week of her third pregnancy due to presence of multiple fetal anomalies. Unilateral ventriculomegaly, cystic hygroma, enlarged microcystic kidneys and bilateral club foot were detected by ultrasound. Additionally, microcephaly, pachygyria and lysencephaly were noted on fetal brain MRI. Fetal blood sampling and clinical exome sequencing (CES, TruSight One, Illumina) were done. Variants were classified according to ACMG/AMP classification system.

Results: CES revealed two variants in PEX6 gene, c.1314_1321del and c.830del (NM_000274.4), classified as pathogenic and likely pathogenic, respectfully. Compound heterozygosity for PEX6 gene variants is consistent with diagnosis of ZSD in proband.

Conclusion: Before introduction of exome sequencing (ES) in prenatal setting, ZSD was rarely diagnosed antenatally due to unspecific and late presentation of symptoms. Frameshift variant located in exon 1, c.830del, found in our patient was previously unreported in literature and databases, and is predicted to lead to nonsense mediated decay. This case confirms utility od ES in prenatal diagnosis of fatal childhood conditions and adds to mutational spectrum of PEX6 gene.

Grants: None

Conflict of Interest: None declared

EP03.013 Arthrogryposis linked to the PIEZO2 gene causing fetal birth defects

Monika Gorządek ^1;2, Hanna Moczulska^1;2, Karolina Gadzalska^1;2, Paulina Jakiel^1;2, Ewa Juścińska^1;2, Tomasz Płoszaj^1;2, Sebastian Skoczylas^1;2, Maciej Borowiec^1;2, Agnieszka Zmysłowska^1;2

¹; ²Medical University of Lodz, Department of Clinical Genetics, Lodz

Background/Objectives: Arthrogryposis (OMIM:114300, 108145; ORPHA:376, 1154) is an extremely rare multiple congenital syndrome, inherited autosomal dominantly, characterized by contractures of the hands and feet of varying severity, camptodactyly, clubfoot and, less commonly, cleft palate. A fetus with arthrogryposis carries a heterozygous mutation in the PIEZO2 gene. The PIEZO2 protein plays a role in the rapid adaptation of mechanically activated (MA) currents in somatosensory neurons.

Methods: The material for the study was DNA isolated from Sherlock AX amniotic fluid (A&A BIOTECHNOLOGY). Whole exome sequencing (WES) was performed using the Twist Human Core Exome kit (Twist Bioscience). The result will be confirmed by Sanger sequencing, also with family segregation.

Results: We present a case report conducted during fetal ultrasound in the second trimester of pregnancy. Cleft palate, intrauterine growth retardation, fetal hydrocephalus, “club hand” sign, club foot, overlapping toes of the lower limbs make up the picture of abnormal fetal growth. Whole exome sequencing revealed that the fetus carries a heterozygous variant in the PIEZO2 gene (NM_001378182.1 p.Arg2799Cys), which is the genetic cause of arthrogryposis. Bioinformatics analysis identified the PIEZO2 variant as pathogenic, according to ACMG recommendations.

Conclusions: In conclusion, we found a pathogenic variant of the PIEZO2 gene in a fetus with multiple malformations. The data obtained confirms the whole exome sequencing is a highly useful tool to identify genetic background of complex defects in the fetus.

Grants:

Conflict of Interest: None declared

EP03.014 Non-invasive prenatal testing (NIPT): combination of CNV and gene analyses using an “in-house” target enrichment NGS—solution for non-centralized NIPT laboratory?

Lucie Faldynová¹, Sylwia Walczysková ¹, Dita Černá¹, Monika Kudrejová¹, Šárka Hilscherová¹, Romana Kaniová¹, Simona Širůčková¹

¹University Hospital Ostrava, Department of Molecular and Clinical Pathology and Medical Genetics, Ostrava, Czech Republic

Background/Objectives: Recent studies have integrated copy number variant (CNV) and gene analysis using target enrichment. Here we transferred this concept to our routine genetics laboratory, which is not linked to centralized non-invasive prenatal testing facilities.

Methods: From a cohort of 100 pregnant women, 22 were selected for analysis of maternal gDNA along with fetal cfDNA. Using targeted enrichment, 135 genes were analyzed, combined with aberrations of chromosomes 21, 18, 13, X, and Y. The data were subjected to specificity and sensitivity analyses, and correlated with the results from invasive testing methods.

Results: The sensitivity/specificity was determined for the CNV analysis of chromosomes: 21 (80%/75%), 18 (-/82%), 13 (100%/67%), and Y (100%/100%). Gene detection was valid for maternal gDNA. However, for cffDNA, it was not possible to determine the boundary between an artifact and a real sequence variant.

Conclusion: The target enrichment method combining CNV and gene detection seems feasible in a regular laboratory. However, this method can only be responsibly optimized with a sufficient number of controls and further validation on a strong bioinformatic background. The present results showed that NIPT should be performed in specialized centers, and that its introduction to isolated laboratories may not provide valid data.

Grants: This work was supported by Ministry of Health, Czech Republic – conceptual development of research organization (FNOs/2019).

Conflict of Interest: None declared

EP03.015 Integration of prenatal exome sequencing with extensive clinical phenotyping to assess fetal structural anomalies in a consanguineous cohort from Egypt

Sara El-Dessouky¹, Mona Aboulghar², Reza Maroofian³, Ahmed Abdelfattah⁴, Maha Eid⁵, Wessam Sharaf-Eldin⁶, Maha Zaki⁷, Alaa Ebrashy⁸, Hassan Gaafar⁸, Ahmed Saad⁶, Mahmoud Issa⁷, Shahryar Alavi⁹, Zahra Firoozfar⁹, Mohamed Ateya⁸, Mona Fouad⁸, Rana Abdella⁸, Ahmed Ezz Elarab⁸, Ebtesam Abdalla¹⁰, Nahla Abdel-Aziz¹¹, Haitham Khaled⁴, Mohamed Elhodiby¹², Sameh Senousy⁴, Homa Tajsharghi ¹³

¹National Research Centre, Prenatal Diagnosis and Fetal Medicine Department, Human Genetics and GenomeResearch Division, Cairo, Egypt; ²Cairo University, Cairo,Egypt, Department of Obstetrics and Gynecology, Fetal Medicine Unit, Cairo, Egypt; ³, Department of Neuromuscular Disorders, london, United Kingdom; ⁴National Research Centre, Prenatal Diagnosis and Fetal Medicine Department, Human Genetics and GenomeResearch Division, Cairo, Egypt; ⁵National Research Centre, Departments of Human Cytogenetics, Human Genetics and GenomeResearch Division, Cairo, Egypt; ⁶National Research Centre, Medical Molecular Genetics Department, Human Genetics and GenomeResearch Division, Cairo, Egypt; ⁷National Research Centre, Clinical Genetics Department, HumanGenetics and Genome Research Division, Cairo, Egypt; ⁸Cairo University, Department of Obstetrics and Gynecology, Fetal Medicine Unit, Cairo, Egypt; ⁹UCL Queen Square Institute of Neurology, Department of Neuromuscular Disorders, London, United Kingdom; ¹⁰Alexandria University, Department of Human Genetics, Medical Research Institute, Cairo, Egypt; ¹¹National Research Centre, Medical Molecular Genetics Department, Human Genetics and Genome Research Division, Cairo, Egypt; ¹²M.U.S.T. University, Department of Obstetrics and Gynecology, Faculty of Medicine, Cairo, Egypt; ¹³University of Skovde, Div of Biomedicine, Skovde, Sweden

Consangeniuos unions are associated with unfavourable genetic and perinatal outcomes in the affected offspring, raising significant public health concerns due to increased rates of infant mortality and disability resulting from congenital anomalies. The additional risk of recurrent fetal structural anomalies, a major contributor to perinatal mortality, further amplifies these concerns.

The objectives of this study were to assess diagnostic outcomes in Egyptian consangeuinous couples presenting with fetal structural anomalies and to obtain insights into the clinical subtypes of these anomalies.

The cohort consisted of 250 fetuses exhibiting sonographic abnormalities. The diagnostic approach involved prenatal comprehensive clincal phenotyping, conventional karyotyping, followed by whole exome sequencing (WES) and for cases with a normal karyotype. This combined approach yielded an overall diagnostic rate of 88% (220/250). Prenatal karyotyping detected chromosomal aberrations in 20% (50/250) of fetuses, with trisomy 18 and 13 being the most frequent anomalies. For cases with a normal karyotype, WES revealed underlying molecular genetic defects in 85% (170/200) of fetuses, primarily associated with central nervous system, skeletal and renal abnormalities. Among the molecularly diagnosed cases, 74% (125/170) exhibited pathogenic or likely pathogenic variants, or variants of uncertain significance (VUS), fully correlated with the observed defects. Inconclusive diagnoses included variants partially correlated with fetal defects and VUS.

In conclusion, the integrated application of karyotyping and WES successfully identified likely causative genetic defects in 88% of consangeuinous families with fetal anomalies, underscoring the diagnostic effectiveness of these tests in the prenatal diagnosis of fetal anomalies, especially within populations characterised by high consanguinity.

Conflict of Interest: None declared

EP03.016 A rare case of Y;14 chromosome translocation in a female fetus, detected in prenatal diagnostics

Kalina Belemezova ¹, petya chaveeva², Radostina Raynova³, Petya Andreeva^2;4, Atanas Shterev², Katerina Kavaldzhieva¹, Ivanka Dimova⁵

¹Medical University of Sofia, Biology, Sofia, Bulgaria; ²Medical Complex Dr. Shterev, Sofia, Bulgaria; ³National genetic laboratory, Sofia, Bulgaria; ⁴South-West University “Neofit Rilski”, Blagoevgrad, Bulgaria; ⁵Medical University of Sofia, Medical Genetics, Sofia, Bulgaria

Background/Objectives: We present the case of a 35-year-old pregnant woman with a second spontaneous pregnancy with no previous history of fetal abnormalities. The first trimester biochemical screening test detected increased nuchal translucency and jugular lymphatic sacs bilaterally, increasing the risk for Turner and Down syndromes.

Methods: A noninvasive prenatal test was performed, followed by amniocentesis. Amniotic fluid samples were tested using QF-PCR, and chromosome analysis was performed according to standard methods on cultured cells (GTG method).

Result: The NIPT result showed gender disagreement compared to the second fetal morphology exam which was consistent for a female fetus. Following the amniocentesis, the QF-PCR showed the presence of genetic material consistent with 47, XXY karyotype. Finally, the cytogenetics showed a very rare non-balanced translocation between chromosomes Y (missing the SRY region) and chromosome 14 (karyotype: 46,XX,t(Y;14)(q10;q10)).

Discussion: Cases of Y chromosome/autosome translocation are very rare. There are only a few cases in the literature reporting a similar unbalanced translocation, usually with the presence of the SRY region, resulting in a phenotypically normal male with infertility. Moreover, cases of non-SRY Y to autosome translocation are extremely rare and are related to gonad dysgenesis and high risk of gonad malignancy.

Conclusion: The case we report shows the importance of different testing approaches whenever there is inconsistency between the results from the NIPT and the fetal morphology. There is a need for further studies of patients with Y chromosome translocation to better understand the possible complications and outcomes in those cases.

Conflict of Interest: None declared

EP03.017 Alport Syndrome and Pregnancy: Clinical and Genetic Analysis

Maria MALARSKA ¹, Paulina Pachniak¹, Hanna Moczulska¹, Paulina Jakiel¹, Karolina Gadzalska¹, Agnieszka Pukajło-Marczyk², Katarzyna Kiliś-Pstrusińska², Katarzyna Rajfur³, Alicja Majos⁴, Agnieszka Zmysłowska¹

¹Medical University of Lodz, Lodz, Poland, Department of Clinical Genetics, Łódź, Poland; ²Wroclaw Medical University, Wroclaw, Poland, Department of Pediatric Nephrology, Wrocław, Poland; ³Institute of Health Sciences, University of Opole, Poland, Opole, Poland; ⁴Medical University of Lodz, Lodz, Poland, Department of General and Transplant Surgery, Łódź, Poland

Background: Alport syndrome (AS) is a rare genetic disorder characterized by progressive damage to the basement membrane of the kidney, as well as the middle ear and retina. The purpose of this study was to analyze the impact of Alport syndrome on the course of pregnancy and to identify potential pregnancy complications associated with a genetic diagnosis of Alport syndrome.

Methods: The analysis included 12 pregnancies in 6 patients with confirmed Alport syndrome without clinical symptoms and 46 pregnancies in 23 patients with symptoms, taking into account various pregnancy parameters and molecular diagnosis of AS. Genetic testing was performed in all children using next-generation sequencing or Sanger sequencing method.

Results: Among the 12 children whose mothers were asymptomatic, 9 (75%) had genetic confirmation of Alport syndrome. Polyhydroamniosis was found in 16.67% of cases, especially in pregnancies with male fetuses and genetically confirmed Alport syndrome. In the analyzed group of 23 pregnant women with symptomatic AS, 85.29% of the children received genetic confirmation of the diagnosis. In the analyzed group, 14 pregnancies (37.84%) had polyhydroamniosis, the cause of which was not determined. Genetic testing of the children confirmed Alport syndrome in 85.29% of them.

Conclusion: The occurrence of polyhydroamniosis in pregnancies with confirmed Alport syndrome could suggest a possible influence of the syndrome on the presence of this complication. It is worth continuing research to understand the impact of Alport syndrome on pregnancy, with an emphasis on identifying potential risk factors and better managing the care of patients with this condition.

Conflict of Interest: None declared

EP03.018 Prenatal case with multiple sulfatase deficiency caused by homozygous SUMF1 mutation

Rebecca Gembicki ¹, Michael Gembicki², Jan Weichert², Moneef Shoukier³, Yorck Hellenbroich¹, Malte Spielmann^1;4

¹Institute of Human Genetics, University of Lübeck, Lübeck, Germany; ²Division of Prenatal Medicine, Department of Obstetrics and Gynecology, University Hospital of Schleswig-Holstein, Lübeck, Germany; ³Pränatal-Medizin München, München, Germany; ⁴Institute of Human Genetics, University of Kiel, Kiel, Germany

Multiple sulfatase deficiency (MSD) is a rare autosomal recessive metabolic disease caused by pathogenic homozygous or compound heterozygous variants in the sulfatase modifying factor 1 (SUMF1) gene. This gene encodes for formylglycine generating enzyme (FGE) which is involved in posttranslational modification and catalytic activation of the entire family of sulfatases.

The wide spectrum of clinical features combines symptoms of single sulfatase deficiencies whereby protein stability and residual activity influence disease severity. The most common ones are neurologic deterioration with mental retardation, skeletal anomalies, organomegaly, and ichthyosis.

Different clinical subtypes of MSD (neonatal, late infantile and juvenile) have been described based on the age of onset, the rate of progression and the severity of the disease.

The neonatal is the most severe with rapid progression and death within the first two years of life. Late infantile, the most common one, can subdivided into mild (onset beyond the second year of life) and severe (skeletal changes, progressive loss of mental/motor abilities by age 5 years, death within the first decade of life). The juvenile form is the rarest with later onset and slow progression.

Here, we report a prenatal case of MSD caused by the homozygous missense gene mutation c.739G>C,p.(Gly247Arg) in SUMF1 detected by exome analysis which was previously described with the late infantile subtype. The fetus shows multiple ultrasonographic abnormalities: decreased growth of all long bones, multiple cerebral anomalies (agenesis of the corpus callosum, colpocephaly, cerebellar vermis hypoplasia, frontal bossing), short ribs and a mild cardiomegaly.

Conflict of Interest: None declared

EP03.020 Further evidence for an attenuated phenotype of in-frame DMD deletions affecting the central rod domain of dystrophin around exon 48

Olga Bürger ¹, Angelika Humbel¹, Ivan Ivanovski¹, Alessandra Baumer¹, Anita Rauch¹

¹University of Zurich, Institute of Medical Genetics, Zürich, Switzerland

Background: Alterations in the X-linked recessive DMD gene cause dystrophinopathies with a broad clinical spectrum most commonly ranging from Duchenne muscular dystrophy (DMD) or Becker muscular dystrophy (BMD) to cardiomyopathy or intellectual disability (ID). Carrier females are commonly unaffected but may show signs of dystrophinopathies. In addition, few asymptomatic male carriers with elevated creatine kinase levels have been described possibly related to deletions around exon 48.

Results: We report an attenuated phenotype in a three-generation family with a deletion of exon 48 of the DMD-gene with clinically unaffected carrier males and females. We confirmed deep intronic breakpoints in this family by genome sequencing.

Conclusion: Predicting the phenotype in DMD-sequence alterations is challenging. The “reading frame rule” suggests that Duchenne muscular dystrophy results from DMD-mutations causing an out-of-frame transcript, whereas the milder Becker muscular dystrophy results from mutations causing an in-frame transcript. Exceptions to the “reading frame rule” are estimated at a rate below 10% [Aartsma-Rus et al 2006].

Deletions in similar regions (between exons 45 – 48 of the DMD-gene) have also been reported in patients with a severe Duchenne phenotype. This discrepancy may result from unprecise breakpoint definitions in the past that may not sufficiently discriminate between in-frame and out-of frame copy number variants, but additional modifiers may also be possible. Until more cases are characterized in more details, predicting the individual phenotype requires multiple carriers across different age groups and both genders. Cautious counselling is warranted especially regarding prenatal diagnostics.

Grants: University of Zurich clinical research priority program praeclare

Conflict of Interest: None declared

EP03.021 Paternally Inherited NFIA-Related Disorder: Prenatal and Postnatal Diagnosis at Once

Nader Khaleghi Hashemian ¹, Gioia Mastromoro¹, Daniele Guadagnolo¹, Enrica Marchionni¹, Allessandra Terracciano², Antonio Novelli², Antonio Pizzuti¹

¹Sapienza University of Rome, Department of Experimental Medicine, Rome, Italy; ²Bambino Gesù Children’s Hospital, Laboratory of Medical Genetics, Rome, Italy

Background/Objectives: We report on a male fetus exhibiting mild unilateral isolated cerebral ventriculomegaly detected through morphological ultrasonography and confirmed by magnetic resonance imaging (MRI). Upon genetic counseling, the 30-year-old father of the fetus referred a personal history of congenital hydrocephalus treated surgically at 9 months and posterior agenesis of the corpus callosum, without specific dysmorphic features or neurodevelopmental disorders.

Methods: Amniocentesis with standard karyotype and chromosomal microarray investigation yelded normal results. Exome sequencing targeting genes associated with brain anomalies was performed on the father of the fetus and his parents.

Results: A de novo heterozygous likely pathogenic variant c.82_83dup; p.(Trp30HisfsTer28) in the NFIA gene (NM_001134673) was identified in the father of the fetus. Monoallelic loss-of-function variants in NFIA are associated with “Brain malformations with or without urinary tract defects” (MIM#613735), aligning with his clinical presentation. Segregation analysis identified the variant also in the fetus. Subsequent fetal MRI at 27 weeks showed hydrocephaly-induced macrocephaly, thin cerebral cortex, agenesis of the septum pellucidum, thin corpus callosum, bilateral frontal polymicrogyria with abnormal sulcation, and reduced fetal movements. The couple opted for pregnancy termination. Fetal anatomopathological evaluation revealed hypertelorism, prominent nasal bridge, long philtrum, micrognathia, microstomia, low hairline, and low, posteriorly rotated poorly rimmed ears. Internal organ examination showed undescended testes and anomalies in pulmonary lobulation and bronchial segmentation, including an additional left lung lobe.

Conclusion: We report a hereditary NFIA-related disorder, typically arising de novo. Family history prompted exome sequencing investigation which identified a paternally trasmitted variant to the fetus.

Grants: None

Conflict of Interest: None declared

EP03.022 Whole Genome Sequencing Reveals Incidental Findings That Are Actionable In The Prenatal Setting – Case Report From a Consanguineous Population

zohra hasan ¹, MAHRUKH NASIR^1;2, Fizza Akbar¹, Salman Kirmani¹

¹Aga Khan University, Division of Women and Child Health, Karachi, Pakistan; ²International Center for Chemical and Biological Sciences (ICCBS), Dr.Panjwani Centre for Molecular Medicine and Drug Research, Karachi, Pakistan

Background: Whole Exome Sequencing (WES) and Whole Genome Sequencing (WGS) are becoming primary tools for diagnosing genetic disorders. Incidental findings can be crucial, particularly in consanguineous families, providing actionable insights for both the proband and family members. This case highlights the significance of WGS in identifying incidental pathogenic variants with immediate clinical implications.

Methods: A consanguineous couple, having experienced unexplained neonatal deaths, presented their 5-month-old with severe hypotonia and myopathic changes. WGS was employed for comprehensive genomic analysis. A homozygous pathogenic variant in TK2 was identified, confirming Mitochondrial DNA Depletion Syndrome Type 2. Incidentally, a pathogenic variant in BRCA2 was also discovered. Parental testing revealed heterozygosity for both TK2 and BRCA2 variants. Subsequent prenatal testing for both variants was offered in the next pregnancy.

Results: Prenatal testing in the subsequent pregnancy unveiled a fetus homozygous for the BRCA2 pathogenic variant, associated with Fanconi Anemia, congenital anomalies, and early cancer predisposition. Faced with this diagnosis, the family opted to terminate the pregnancy at 21 weeks. This case underscores the power of WGS in not only identifying primary genetic disorders but also revealing secondary findings with immediate clinical relevance.

Conclusion: WGS emerges as a potent diagnostic tool, capturing both primary and incidental findings. In this case, relying solely on targeted gene panels would have overlooked the BRCA2 variant. The case emphasizes the importance of comprehensive genomic analysis in offering timely interventions and appropriate genetic counseling for families facing hereditary conditions.

Conflict of Interest: None declared

EP03.023 Assessment of maternal cell contamination in all prenatal samples by quantitative fluorescent PCR

Kajsa Nilsson ¹

¹Skåne University Hospital, Clinical genetics, Lund, Sweden

Background/Objectives: The contamination of fetal samples with maternal cells, Maternal Cell Contamination (MCC), can interfere in diagnostic prenatal testing. MCC is thus recommended to be assessed in clinical practice regardless of the genetic disorder or mode of inheritance. At the Department of Clinical genetics in Lund, MCC analysis was introduced for all prenatal samples in June 2021. Here we present the outcome of this clinical approach.

Methods: Quantitative Fluorescent PCR (QF-PCR) was performed on all fetal samples obtained by amniocentesis or chorionic villus sampling and on maternal blood sample between June 2021 and November 2023 as part of clinical routine.

Results: Out of overall 1624 pregnancies analyzed, significant maternal cell contamination (MCC) was detected in 25 cases (1.5%).

Conclusion: A significant proportion of the prenatal samples were contaminated by maternal cells. To determine the pure fetal origin of fetal samples undergoing genetic analysis and to provide good laboratory practice as well as correct result interpretation, it is of high importance that MCC is assessed in all fetal samples.

Grants:

Conflict of Interest: None declared

EP03.024 From a NIPS false positive to a chromosomal rearrangement: a case report

cristina Ferreira ^1;2, Ana Rita Tarelho¹, Bárbara Marques¹, Silvia Serafim¹, Sónia Pedro¹, Cristina Alves¹, Mónica Viegas¹, Ângela Ferreira³, Hildeberto Correia¹

¹Instituto Nacional de Saúde Doutor Ricardo Jorge, I.P., Departamento de Genética Humana, Lisboa, Portugal; ²; ³Centro Hospitalar Universitário do Algarve – Hospital de Faro E.P.E., Serviço de Obstetrícia / Diagnóstico Pré-Natal, Faro, Portugal

We report a case of a pregnant woman at 21 weeks and six days of gestation who underwent cell-free fetal DNA based non-invasive prenatal screening (NIPS) without prior aneuploidy screening.

The initial NIPS result indicated a 3% fetal fraction with a high risk for chromosome X monosomy. To confirm this finding, amniocentesis was performed, and subsequent QF-PCR rapid aneuploidy test revealed a normal result for chromosome X in a female fetus, contradicting the NIPS outcome. Unusually, all polymorphic markers on chromosome X were non-informative, prompting additional investigation using multiplex-ligation dependent probe amplification (MLPA), which results revelled a terminal deletion in Xq22.3.

In order to characterize the deletion, microarray analysis was performed. The comprehensive analysis revealed a 74.51 Mb terminal deletion in Xq21.1q28 and an 85.52 Mb terminal duplication in 2p25.3p11.2, indicative of a derivative chromosome resulting from a translocation between the short arm of chromosome 2 and the long arm of chromosome X. This complex genomic rearrangement was confirmed by karyotyping. Parental karyotyping will be conducted promptly to ascertain the recurrence risk.

This case underlines the necessity of confirming NIPS results considered as high risk. While the initial suspicion of monosomy X was not substantiated, the pursuit of a deeper investigation revealed an entirely unexpected genomic anomaly with a more severe prognosis. This highlights the importance of thorough investigation following a NIPS high risk result, as QF-PCR rapid aneuploidy test, used many times as standalone method in prenatal diagnosis following NIPS high risk results, would not have solved this case.

Conflict of Interest: None declared

EP03.025 Necessity to define prenatal phenotypes to enhance diagnosis in multiple congenital anomalies.

Carla Martín-Grau¹, Carmen Orellana ², Mónica Roselló Piera², Laia Pedrola², Juan Silvestre Oltra Soler², Sandra Monfort Membrado², Marta Domínguez Martínez¹, Alfonso Caro Llopis¹, Francisco Martinez²

¹Instituto de Investigación Sanitaria La Fe (IISLAFE), Genetics Unit, Translational Genetics Research Group, Valencia; ²Hospital Universitario y Politecnico La Fe, Genetics Unit, Translational Genetics Research Group, Valencia

Background/Objectives: The Human Phenotype Ontology (HPO) provides a standardized vocabulary of phenotypic abnormalities related to human diseases. It is widely-known that HPO can be used to support differential diagnoses and translational research. HPO currently contains over 13,000 terms that mostly correspond to medically relevant postnatal phenotypes. However, HPO database only contains 262 terms directly correlated with congenital anomalies (CA). In the absence of specific fetal HPOs, comprehensive multidisciplinary knowledge about prenatal phenotypes is crucial to improve diagnostics tests.

Methods: 32 fetal samples with ultrasound and/or anatomopathological diagnosis of CA were included with normal results in the previous prenatal karyotype and 750K SNP-array performed. WES was performed using SureSelectXT HS ExonV8 (Agilent Technologies) for enrichment and NextSeq (Illumina) for sequencing. Segregation studies were confirmed by Sanger sequencing.

Results: WES diagnostic yield was 43.8% in our cohort. In 5/14 patients (35.7%), the clinical phenotype standardized by HPO terms used did not facilitate the interpretation of genetic variants detected due to the lack of genotype-phenotype correlation in fetal patients. Novel phenotype terms were described in PIGP, EP300, ROBO1, FOXP3 and ARMC9 genes.

Conclusion: Detailed fetal phenotypes in HPO terms will support filtering and prioritization of variants in order to increase diagnostic yield in CA using WES. However, a significant percentage of fetal cases may be underdiagnosed. Our results illustrate the complexity of prenatal phenotype and underline the need for further researches to better reinforce prenatal usage of HPO terms in unreported prenatal diseases.

Grants: Supported by Instituto de Salud Carlos III through “PI22/01127” and “PI22/00272” projects.

Conflict of Interest: None declared

EP03.027 A retrospective study on 162 patients that benefited of a NIPT analyze

Eusebiu Vlad Gorduza ^1;2, Violeta Martiniuc^2;3, Lavinia Caba¹, Andreea Florea¹, Ana Grigore¹, Mihaela Gramescu¹, Elena Mihalceanu^1;2

¹“Grigore T. Popa” University of Medicine and Pharmacy, Medicine of Mother and Children, Iasi, Romania; ²“Cuza Voda” Obstetrics and Gynecology Hospital, Iasi, Romania; ³SC Cytogenis SRL, Iasi, Romania

Background/Objectives: To analyze the results of NIPT test.

Methods: We made a NIPT retrospective study between August 2022 and January 2024on patients from Romania.

Results: The cohort has 162 patients, with 3 cases inconclusive (BMI ≥ 28.58). 104 patients benefited from a full test (through which full and partial aneuploidies of all chromosomes were analyzed) while 55 cases opted for the identification of only chromosomes 21, 18, 13, X and Y. We analyzed 8 twin pregnancies with normal results. We found 4 pregnancies with high risk: monosomy X, trisomy 15, microdeletion 1q21 and trisomy 18, but only the last was confirmed by chromosomal analyze after amniocentesis. No chromosomal diseases were identified in all the children born. The age of women that applied for NIPT was between 26 and 49 years: 31 (<30 years; 19.49%) 65 (30 – 35 years; 40.88%) 47 (36 – 40 years; 29.55%) 17 (>40 years; 10.69%). Between different age interval we did not found significative differences that concern medium value of free fetal DNA fraction (FFDNA) (<30 years – 13.91%; 30 – 35 years – 14.06%; 36 – 40 years – 13.20%; >40 years – 11.26%). The tests were applied at: 11 WA (18 cases; 11.32%; medium FFDNA - 13.86%); 12 WA (33 cases; 20.75%; medium FFDNA - 13.66%); 13 WA (48 cases; 30.18%; medium FFDNA - 12.96%); 14 WA (22 cases; 13.83%; medium FFDNA - 11.94%); 15 WA (19 cases; 11.94%; medium FFDNA – 12.02%); ≥16 WA (19 cases; 11.94%; medium FFDNA – 14.21%).

Conclusion: Our study confirmed the quality of NIPT as prenatal screening for chromosomal disorders.

Conflict of Interest: None declared

EP03.028 Similarities and differences in performance between two WGS-based NIPT tests – 10 years of experience of the Genomed SA laboratory

Monika Jurkowska¹, Ewa Matczyńska¹, Anna Wąsowska¹, Przemysław Łyszkiewicz¹, Magdalena Kania¹, Elżbieta Gregorczyk¹, Paweł Ogrodnik¹, Maria Jędrzejowska¹, Marek Zagulski¹, Anna Boguszewska-Chachulska ¹

¹Genomed SA, Warsaw, Poland

Non-invasive prenatal testing (NIPT) becomes a routine method of prenatal screening. Numerous testing approaches, methodologies, ranges, report options are being applied worldwide. The objective of this study was to compare data on the performance of two large series of different NIPTs provided by the Polish pan-country laboratory.

Altogether 41064 reports, issued between 2014 and 2023, were analysed. Data have been extracted from a test-dedicated database, order forms, final reports, follow-up sheets and personal communications. Two NGS-based NIPT technologies were applied: BGI (NIFTY – 15850 samples) and Illumina (VeriSeq v1 - 5191 samples and v2 – 20020 samples, under the SANCO brand). The tests differed in the applied chemistry, sequencers, bioinformatic algorithms and range of analysis.

Despite similar populations tested, significant differences in performance were noticed between tests (Table 1, in bold).

Conflict of Interest: None declared

EP03.029 Prenatal diagnosis of Proteus Syndrome

Luana Giovannangeli¹, Virginie Magry ², Florence Jobic¹, Kahia Messaoudi², Alexis Billes¹, marianne perriere², Ségolène Delmas-Lanta³, Alice Masurel⁴, Guillaume Jedraszak², Gilles Morin¹

¹CHU Amiens-Picardie, Genetics, Amiens, France; ²CHU Amiens-Picardie, Constitutional Genetics Laboratory, Amiens, France; ³CHU Amiens-Picardie, Gynecology, Amiens, France; ⁴CHU Amiens-Picardie, Neuropediatry, Amiens, France

Background/Objectives: Proteus syndrome is a rare disorder (<1/1,000,000) characterized by progressive overgrowth which affects commonly the skeleton, skin, adipose and central nervous system. It results from a somatic activating variation in the AKT1 gene. The c.49G>A (p.Glu17Lys) variation is the most reported. Proteus syndrome has a variable severity, with a mortality rate of approximately 25% before 22 years of age. We report the second case of Proteus syndrome prenatally diagnosed with whole exome sequencing.

Clinical Report: A 34-year-old woman was referred to the prenatal diagnosis department for non-visualized anterior brain structures and suspicion of micro-penis. Subsequent ultrasonography at 21 weeks of gestation (WG) confirmed absent septum lucidum, thick corpus callosum, genital abnormalities and macrosomia. Array-CGH did not found pathogenic imbalances (46,XX). Follow-up ultrasounds and MRI at 25 and 27 WG showed persistent abnormalities and megalencephaly, leading to exome sequencing on amniocytes. A mosaic variation in AKT1 (c.49G>A) with an allele frequency of 25% was identified. Medical termination of pregnancy was performed at 30 + 1 WG.

Conclusion: Only one case of prenatal diagnosis with exome sequencing has been reported to date with similar clinical presentation (Abell et al. 2020). The diagnosis challenges lie in the clinical variability with usual postnatal beginning and the mosaic nature of the syndrome, notably in case of low mosaicism. Whole exome sequencing, with a coverage depth of 130X, was efficient in this case, offering a precise diagnosis.

Conflict of Interest: None declared

EP03.030 Prenatal diagnosis of a mosaic rearrangement of chromosome 18: the result of dicentric chromosome breakage?

Laura Feyereisen¹, Tony Yammine ¹, Leila Sahmoune Rachedi², Jean-Paul Bory², Victor Dupont-Gaudin³, Jean-Marc Dossot³, Véronique Dalstein¹, Poirsier Céline⁴, Emilie Landais¹

¹CHU de Reims, Structure de Génétique-Hématologie-Immunologie, Pôle de Biologie Territoriale, Reims, France; ²CHU de Reims, Diagnostic Prénatal - Service de Gynécologie Obstétrique, Reims, France; ³Laboratoire de Biologie Médicale Bioxa, Reims, France; ⁴CHU de Reims, Service de Génétique Clinique, Reims, France

Background: A primiparous 19-year-old patient was referred to our pluridisciplinary center for prenatal diagnosis at 26-weeks of gestation. Ultrasound examination showed severe asymmetrical intrauterine growth restriction (IUGR; with decreased abdominal and head circumferences, but spared femoral length), a single umbilical artery, and pericardial effusion.

Methods/Results: An amniocentesis was performed; rapid prenatal aneuploidy testing by fluorescence in situ hybridization (FISH) found no anomaly of chromosomes 13, 18 and 21. However, microarray-based comparative genomic hybridization (aCGH) of uncultured amniocyte DNA showed Profile A: a partial trisomy 18 with a terminal 18q monosomy (cf. table below). Conversely, the karyotype performed on short-term cell culture found 18q monosomy without partial trisomy, as confirmed by aCGH profile B. These seemingly contradictory findings made genetic counseling challenging. Due to the poor prognoses of both chromosome 18 rearrangements and the severity of IUGR, the patient elected for a therapeutic termination of pregnancy. Additional samples were collected for further testing. Array CGH of umbilical cord found Profile B. Placenta and amniocyte analyses found Profile C, a mosaic near-complete trisomy 18 with 18q monosomy.

Conclusion: We hypothesize that profile A reflects two concurrent cellular populations: one presenting a dicentric chromosome 18 (profile C), and another with a derivative resulting from dicentric chromosome breakage (profile B). This case’s complexity would not be apparent had only cultured amniocytes been analysed.

Conflict of Interest: None declared

EP03.031 ANKS6 variants underlie polycystic kidneys in prenatal and neonatal cases

Mohamed AlHamed ¹

¹King Faisal Specialist Hospital & Research Centre, Clinical Genomics, Riyadh, Saudi Arabia

Background/Objectives: Nephronophthisis (NPHP) is an autosomal recessive disorder that can result in severe renal failure. ANKS6 gene has an important role for early renal development and encodes a protein that interacts with other proteins of the cilium. Fetal ultrasound imaging and diagnosis of fetuses with ANKS6 variants still underestimated. Here, we report the detection of ANKS6 variants in consanguineous families with polycystic kidney at early stages of life.

Methods: Three unrelated Saudi Arabian patients (2 prenatal and 1 neonate) were investigated. These cases were referred due to a history of echogenic kidneys. After clinical evaluation and fetal ultrasound imaging, whole-exome sequencing (WES) was performed to identify molecular genetics causes associated with echogenic kidney phenotype.

Results: Two sequence variants were detected in ANKS6 gene. A homozygous missense novel variant ANKS6: c.1159A>C was detected in families 1 and 2. The third family harboured a homozygous loss of function known variant ANKS6: c.907+2T > A.

Conclusion: We have identified 2 ANKS6 variants in all 3 polycystic kidney families. The findings provide an expanded clinical manifestation of NPHP and emphasize the utility of WES in the phenotype of echogenic kidney at prenatal settings.

Grants: None

Conflict of Interest: None declared

EP03.032 Prenatal genetic testing in the era of next-generation sequencing: experience of a Fetal Medicine Unit in a Spanish hospital

Gema Escribano-Serrano ¹, Diana Salinas-Chaparro¹, mar borregán prats¹, Judith Armstrong^1;2;3, Delia Yubero^1;2;3, Laura Martí^1;2, Clara Xiol^1;2, Guerau Fernandez^1;2;3, Maria Edda Marimon Garcia⁴, Miriam Illa Armengol^4;5, Miriam Perez Cruz^4;5, Elisenda Eixarch Roca^4;6, Mar Bennasar Sans⁴, Joan Sabria Bach^4;5, Francesc Palau^1;2;3;7

¹Dept. of Genetic Medicine - IPER, Hospital Sant Joan de Déu, Barcelona, Spain; ²Institut de Recerca Sant Joan de Déu, Barcelona, Spain; ³CIBER de Enfermedades Raras (CIBERER), Instituto de Salud Carlos III (ISCIII), Madrid, Spain; ⁴Fetal Medicine Unit. BCNatal. Centre de Medicina Maternofetal i Neonatal, Hospital Sant Joan de Déu - Hospital Clínic, Barcelona, Spain; ⁵Maternal and Child Health and Development Network II (SAMID II), Instituto de Salud Carlos III (ISCIII), Sub-Directorate General for Research Assessment and Promotion and the European Regional Development Fund (ERDF), Madrid, Spain; ⁶Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain; ⁷Faculty of Medicine and Health Sciences, University of Barcelona, Barcelona, Spain

Background/Objectives: Foetal abnormalities affect approximately 2–3% of pregnancies. In prenatal diagnosis, clinical exome sequencing (CES) is commonly offered in complex cases with normal karyotyping and chromosomal microarray (CMA). Prenatal CES (pCES) poses inherent challenges, associated with gene filtering strategies, unrecognized foetal phenotypes, and/or genetic variant interpretation. This study aims to understand the diagnostic yield of pCES in a Women’s and Children’s Hospital.

Methods: From 2017 to 2023, 125 foetal samples obtained through amniocentesis and chorionic villus sampling, underwent pCES analysis, either singleton (74) or trio (51). Samples originated from pregnancies with foetal anomalies, where both QF-PCR and CMA studies were negative. Searching for candidate genes was performed by virtual gene panels genes and/or HPOs. We compared indications of ultrasound anomalies and virtual gene panel or pCES according to HPOs criterion. Pregnancies with identified genetic conditions received genetic counselling.

Results: Regarding the most prevalent clinical indications, 29 foetus (23%) had more than one foetal anomaly, followed by 18 (14%) which were clinically suggestive of a possible skeletal dysplasia, and 16 (13%) of cardiac alteration. Both virtual panels and HPO selection yielded similar diagnostic rates: 38% (15/39) of variants through gene panel and 33% (13/39) by HPOs.

Conclusion: The data suggest that both disease-related gene panel and HPO-addressed genetic analysis have similar diagnostic yield. Precision or deep imaging phenotyping is the main criterion to improve the diagnostic rate of pCES and pCES-trio, which would facilitate prenatal genetic counselling and informed decision-making.

Conflict of Interest: None declared

EP03.033 Rare CNVs of HBA and HBB genes identified in a group of thalassemic patients and heterozygotes of Greek origin

Panagiota Kekelou¹, Cristos Chassanidis¹, Thomai Tsantzali¹, Evangelia-Eleni Delaki¹, Effrossini Boutou¹, Eleni Yfanti¹, Maria Baou¹, Panayiotis Kyriazopoulos¹, Stamatia Theodoridou², Veroniki Komninaka³, Spiridon Kanellakis⁴, Georgia Ikonomou⁵, Dimitra Pessini⁶, Maria Dimopoulou⁷, Angeliki Balassopoulou ¹

¹Laiko Hospital, Unit of Genetics, Athens, Greece; ²Hippokration General Hospital, Thalassemia Prevention Unit, Thessaloniki, Greece; ³Laiko Hospital, Thalassemia Prevention Unit, Athina, Greece; ⁴Karamandaneio Prefecture Children Hospital of Patras, Thalassemia Prevention Unit, Patra, Greece; ⁵Larissa General Hospital Koutlimbaneio & Triantafylleio, Thalassemia Prevention Unit, Larisa, Greece; ⁶Agrinio General Hospital, Thalassemia Prevention Unit, Agrinio, Greece; ⁷Laiko Hospital, Unit for Thalassemias, Athens, Greece

Background/Objectives: Copy Number Variants (CNVs) are frequently identified in α thalassemia but they are rarely found in β thalassemia. Only some of them are routinely screened particularly in α thalassemia. This study aims to analyze the HBA and HBB CNV’s in thalassemic patients and heterozygotes whose genotypes were not defined/fully defined after routine genetic analysis

Methods: 59 DNA samples were studied. SALSA® MLPA® kits (Probemix P140, P102) MRC Holland, were used while fragment analysis was carried out in SeqStudio™ Genetic Analyzer (Thermo-Fischer Scientific). Analysis was performed using the Coffalyser.Net™ software.

Results: 9 CNV’s were identified in 16 samples. 4 deletions with sizes at least 257kb, 11,1kb, 131kb, 9,8kb, 1 duplication of the entire α globin cluster and 1 homozygous α gene triplication were identified in α globin cluster. The duplication and triplication were in compound heterozygosity with typical β thalassemia mutations giving rise to β thalassemia intermedia phenotypes. 2 deletions, 7.2kb (discontinuous) and 173kb and 1 duplication of the entire β globin cluster containing two β globin genes one carrying the wild type sequence an the other the HBB:c.93-21G > A variant, were identified in β globin cluster.

Conclusion: CNV’s analysis revealed nine rare variants three of which (deletion 9.8kb, deletion 173kb and β locus duplication) have not been previously reported. The duplication of entire globin gene clusters -particularly β- is a rare genetic phenomenon of great interest with few references so far. Characterization of these molecular lesions allows the accurate diagnosis and enables appropriate genetic counseling in case of prenatal diagnosis.

Conflict of Interest: None declared

EP03.034 Silent cis should not be silent

Neeta Lakhani ¹, Vijayalakshmi Salem Ramakumaran¹, emily craft¹

¹Leicester Royal Infirmary, Clinical Genetics, United Kingdom

Background/Objectives: Silent cis carriers of Spinal Muscular Atrophy (SMA) present a critical yet often overlooked aspect of the disease landscape. With the increased frequency of SMA carriers in our region, understanding the implications of silent cis carriers becomes imperative. Silent carriers, while phenotypically normal, harbor pathogenic heterozygous SMA alleles, which are not identified using current testing techniques. This poses significant implications for reproductive options and genetic counselling.

Methods: The estimated prevalence in our cohort is more than twice the reported prevalence of 4%. The chance of false negative parental carrier status results due to test limitations will result in erroneously concluding that the affected individual has a de novo variants and inaccurate recurrence estimation for the family. This raises ethical and emotional considerations regarding reproductive decision-making.

Results: Furthermore, the haplotyping of silent carriers are crucial adjunct to carrier testing, for accurate risk assessment and genetic counselling. Through this we can predict the likelihood of disease transmission and guide personalized reproductive strategies.

Conclusion: Here we present a case series of families with silent cis carrier status and how the family journey, though extended, significantly changes their reproductive outcomes.

Grants: None

Conflict of Interest: None declared

EP03.035 NIPT high-risk of rare autosomal aneuploidies and copy number variations are related to adverse pregnancy outcomes

Laia Pedrola ¹, Mónica Roselló Piera¹, Carla Martín-Grau², Juan Salvador Rubio Moll³, Rosa Portero Gómez³, Beatriz Marcos Puig³, Jose Vicente Cervera¹, Ramiro Quiroga³, Carmen Orellana¹

¹Hospital Universitario y Politecnico La Fe, Genetics Unit, Translational Genetics Research Group, Valencia; ²Instituto de Investigación Sanitaria La Fe (IISLAFE), Genetics Unit, Translational Genetics Research Group, Valencia; ³Hospital Universitario y Politecnico La Fe, Obstetrics and Gynaecology Unit, Valencia

Background/Objectives: Genome-wide prenatal cell-free DNA (cfDNA) screening can be used to screen for a wide range of fetal chromosomal anomalies in pregnant patients. In recent years, the scope of prenatal cfDNA screening has expanded to include optional screening for rare autosomal aneuploidies (RAAs) and copy number variations (CNVs). Recent publications have reported that these rarer chromosomal abnormalities have a clinical impact.

Methods: Blood samples were obtained from pregnant patients at 21 public maternity health centers from 1^st March 2020 to 31^st December 2022. For the cfDNA screening analysis, frozen plasma samples were processed using the VeriSeq™ NIPT Solution v2 assay (Illumina). Clinical follow-up was attempted for all high-risk cases. Confirmatory methodologies used included fluorescent in situ hybridization (FISH), karyotyping, 750 K SNP-array and quantitative fluorescent PCR (QF-PCR).

Results: A total of 6098 samples were analysed during the study period and 5880 (96.4%) had genome-wide cfDNA screening. A total of 31 RAAs and 31 CNVs cases were detected. The most common RAAs in our cohort were trisomy 7 and trisomy 16. RAA and CNV cases were related with pregnancy and perinatal adverse outcomes such as preeclampsia, gestational diabetes, spontaneous abortion, and congenital structural abnormalities.

Conclusion: Currently, professional medical societies do not recommend genome-wide cDNA screening in general pregnant population. However, our results show the importance of screening for less common chromosomal anomalies because it could identify patients at risk of perinatal complications who may require additional screening or clinical management during pregnancy.

Conflict of Interest: None declared

EP03.036 X chromosome rearrangement in familial Turner syndrome

Kristina Aleknavičienė ^1;2, Rasa Traberg^1;2, Zivile Zemeckiene^1;1, Rasa Ugenskiene^1;2

¹Lithuanian University of Health Sciences, Department of Genetics and Molecular Medicine, Kaunas; ²Hospital of Lithuanian University of Health Sciences Kauno klinikos, Department of Genetics and Molecular Medicine, Kaunas

Background/Objectives: Turner syndrome, which affects 1 in 2500 females, involves the partial or complete absence of one X chromosome, resulting in diverse developmental abnormalities.

Methods: A 34-year-old woman, with a history of five miscarriages, autoimmune thyroiditis, and V-factor mutation, underwent Non-Invasive Prenatal Testing (NIPT) during her sixth pregnancy. Despite a low risk for T13, T18 and T21, the information on sex chromosomes was not provided. Fetal polyhydramnios was diagnosed, and at the 38th week, a neonate (2380 g, <3% hypotrophic) was born. At 3-4 months, an atrial septal defect was identified. Infant molecular karyotyping based on whole-genome sequencing (3x coverage, >50 kb resolution) revealed a derivative X chromosome with a 5283 kb terminal deletion (p22.33p22.32) and a 15026 kb duplication (q27.1q28), impacting 30 and 156 genes, respectively. Mother’s FISH analysis confirmed a subtelomeric X chromosome change, identical to her daughter.

Results: X chromosomal rearrangement (46,X,der(X)t(X;X)(p22.32;q27.1)) was detected. A terminal deletion involving SHOX, CSF2RA and ARSE genes established the Turner syndrome diagnosis. This type of change could lead to growth retardation, developmental delay, hypotonia, hypogonadism, ichthyosis, Kallmann syndrome, and skeletal abnormalities. In our case, the mother presented with short stature only, and the daughter had an atrial septal defect and hypotrophy. Variable expressivity was probably due to uneven X chromosome inactivation.

Conclusion: The identified terminal deletion prompted a Turner syndrome diagnosis. With a 50% likelihood of transmitting the derivative X chromosome, this case emphasizes the importance of comprehensive genetic evaluation for informed reproductive decision-making (prenatal invasive testing, preimplantation diagnostics) in high-risk pregnancies.

Grant References: -

Conflict of Interest: None declared

EP03.037 Prenatal molecular diagnosis WES vs CMA: What is the most cost-effective approach?

Marina Martinez-Matilla ¹, Alba Velo¹, Claudia Abellán¹, Iryna Boyko¹, Gemma Cartagena¹, Carmen Fernández-Vizcaino¹, Lova Matsa², Sandra Garcia-Herrero¹

¹Igenomix. Part of Vitrolife Group, Paterna, Spain; ²Genetics Laboratory in Dubai, UAE - Igenomix, Dubai, United Arab Emirates

Background/Objectives: The chromosomal microarray analysis (CMA) testing is still more used than whole-exome sequencing (WES) as a second-line approach in pregnancies with ultrasound abnormalities. However, the prenatal diagnostic yield could be increased, and the approach could be more cost-effective by selecting WES instead of CMA, as WES allows the study of copy number variations (CNV) and single-nucleotide variations (SNV) simultaneously. Our objective was to assess which is the most cost-effective approach based on our experience.

Methods: Prenatal cases for WES from 2021 to 2023 were evaluated. WES was performed from fetal DNA using Nextera DNA Flex Pre-Enrichment Library Prep and Illumina Exome Panel and sequenced on the NovaSeq 6000 System (Illumina). Raw data were processed by the in-house bioinformatics pipeline. Detected variants were classified according to the recommendations of the American College of Medical Genetics (ACMG).

Results: A total of 53 prenatal cases were selected. Of them, 23 (43.4%) were positive, six (11.3%) non-conclusive, and 24 (45.3%) negative. Among the positive cases, in seven (30.4%) a pathogenic CNV was detected, while a pathogenic SNV was identified in 16 (69.6%) cases. All the CNV would be detected by CMA.

Conclusion: In our cohort, prenatal molecular diagnostic yield by WES technology was approximately of 43%, of which CMA would have missed approximately 70% (those related with SNV). Our results show that WES approach used as a second-line technology provides a more comprehensive diagnosis than CMA and is a more cost-effective approach, since provides CNV and SNV information in a single test.

Grants:

Conflict of Interest: None declared

EP04.001 Inherited cataracts and its genetic connection to glaucoma and other ocular phenotypes in Bulgarian patients

Kristiyana Vitanova ¹, Kunka Kamenarova¹, Nevyana Veleva², Kalina Mihova¹, Yoanna Kaneva², Reni Tzveova³, Martin Georgiev¹, Ivanka Dimova¹, Alexander Oscar², Radka Kaneva¹

¹Medical University of Sofia, Department of Medical Chemistry and Biochemistry, Sofia, Bulgaria; ²University Hospital “Aleksandrovska”, Department of Ophthalmology, Sofia, Bulgaria; ³UMHAT “Queen Joanna – ISUL”, Department of General and Clinical Pathology Department of Molecular Biology, Sofia, Bulgaria

Background/Objectives: Inherited cataracts are clinically and genetically heterogeneous cause of visual impairment at an early age. It can be associated with various ocular and systemic abnormalities. Early diagnosis and treatment are crucial for the visual prognosis. The purpose of this study was to identify the disease-causing mutations in Bulgarian patients with non-syndromic and syndromic forms of inherited cataract accompanied by glaucoma using next-generation sequencing of clinical exome (CES).

Methods: Six unrelated patients and their relatives were selected on the basis of diagnosis of cataracts. Genomic DNA of all probands was subjected to genetic analysis using clinical exome sequencing on MiSeq platform. Variants in selected genes associated with glaucoma, optic atrophy, anterior-segment dysgenesis, and inherited retinal degeneration, were extracted from the WES data and filtered using systematic bioinformatics analysis and the American College of Medical Genetics and Genomics criteria. Candidate variants were verified by Sanger sequencing and MLPA assay.

Results: Using CES, 7 potential pathogenic variants (PPVs) and 2 variants of uncertain significance were found in 9 genes, including TRPM3, MYH9, CRYAA, CLCN1, CACNA1S, COL11A1, TRPV4, CRYBB1, RP2. One patient expressing a mixed phenotype of cataracts and retinitis pigmentosa, carried a novel deletion in the RetNet-gene, RP2. Four novel variants, TRPM3- p.Val1002Met, MYH9- p.Asp25Asn, CRYAA - p.Arg116Leu, and CRYBB1- p.Arg132His, were found in non-syndromic cases presenting with congenital or nuclear cataracts, myopia, retinopathy, and glaucoma.

Conclusion: Determining the genetic cause may not only help to identify concomitant ocular and/or systemic disorders but also enable individualized genetic counselling.

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

Conflict of Interest: None declared

EP04.002 Identification of low penetrance RB1 variants and carrier screening in two families

Poh San Lai ¹, Grace Tan¹, Chunping Liu¹, thuanchong Quah¹

¹National University of Singapore, Paediatrics, Singapore, Singapore

Background: Retinoblastoma (RB) is a rare intraocular tumour caused by biallelic inactivation of RB1 gene. Although RB is a highly penetrant autosomal dominant disease, some families show low-penetrance phenotypes with reduced expressivity and incomplete penetrance of mutant RB1 alleles. In this study, we analysed two patients clinically diagnosed with unilateral and bilateral disease respectively.

Methods: Tumour and blood samples were sequenced using NGS and Sanger methods.

Results: The first proband carried a heterozygous splice mutation (NM_000321.3:c.607+1G > T) inherited from his asymptomatic father. He has a younger brother with unilateral RB harbouring the same variant. The disease-eye to carrier ratio (der) for this family is 0.66 (2/3) which is within the expected low penetrance values. The second proband inherited a germline missense variant (NM_000321.3:c.1981C>T:(p.Arg661Trp) from his unaffected father. His unaffected brother carried the same variant leading to a der = 0.66 in the family. Identification of low penetrance variants presents significance challenges in genetic counselling for both families. RB survivors are at risk of secondary malignancies while recently, there has been rare observation of asymptomatic RB1 mutation carriers developing primary osteosarcoma. In both families, highlighting the risk and implications of transmitting the low penetrance variants to future offsprings is important and life-long surveillance is indicated for asymptomatic carriers.

Conclusion: Patients with low penetrance mutations and asymptomatic members of their families remain at risk for malignancies. Genetic testing is important to identify carriers in families of patients with germline mutations as they could be asymptomatic but carry known or novel RB1 low-penetrance variants.

Grants: NMRC/0021/12

Conflict of Interest: None declared

EP04.003 the clinical and molecular Spectrum associated with isolated Wolfram Syndrome Type 1

Basamat AlMoallem ¹

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

Consortium: none

Background/Objectives Wolfram Syndrome (WS) known as DIDMOAD syndrome (Diabetes Insipidus, Diabetes Mellitus, Optic Atrophy, and Deafness) (OMIM# 222300). It is the third most common hereditary optic neuropathy that comprises three major types. WS-Type 1, is the commonest form caused due to biallelic mutations in WFS1 with neuropathy constituting one of its major diagnostic criteria. Here we aim to characterize a 45-year-old gentleman complaining of gradual loss of vision at a near distance even with glasses and intolerance to bright light that started when he was 31 years old despite corrective LASIK surgery.

Methods Detailed ophthalmogical and systemic examinations followed by Whole exome sequencing (WES)

Results Examination showed a VA 20/400 OD and 20/200 OS. He scored 1/15 on the Ishihara color vision test OU. Slit lamp examination was unremarkable except for diffuse disc pallor and cupping of 0.8-0.9 in both eyes. Visual field testing and OCT showed a significant reduction in RNFL bilaterally. WES revealed a known pathogenic biallelic frameshift variant in the WFS1 gene: NM_001145853.1:c.2673A>C, (Arg859 Pro*). Systemic evaluation including hearing assessment and neurological examination were normal. Other laboratory tests including CBC, ESR, renal, and liver function panels were within normal limits, folate = 14.8 (normal), random glucose = 6.6 (normal).

Conclusion Our case represents the first report of a patient with isolated autosomal recessive Wolfram syndrome type 1 due to confirmed biallelic pathogenic mutations in the WFS gene with isolated optic nerve atrophy and glaucomatous cupping in the absence of other systemic features and expanded its mutational spectrum.

Grants None

Conflict of Interest: None declared

EP04.004 DYRK1A syndrome presenting with familial exudative vitreoretinopathy

Siying Lin ^1;2, Ellie Hay³, Andrew Webster^1;2, Omar Mahroo^1;2, Robert Henderson^1;2;4, Gavin Arno^1;2

¹National Institute of Health Research Biomedical Research Centre at Moorfields Eye Hospital and the UCL Institute of Ophthalmology, London, United Kingdom; ²UCL Institute of Ophthalmology, London, United Kingdom; ³Great Ormond Street Hospital, Department of Clinical Genetics, London, United Kingdom; ⁴Great Ormond Street Hospital, Department of Ophthalmology, London, United Kingdom

Background/Objectives: The DYRK1A gene encodes a proline-directed kinase crucial for central nervous system development, with haploinsufficiency resulting in DYRK1A-related intellectual disability syndrome. Ocular manifestations occur in two-thirds of affected individuals, primarily involving refractive error and strabismus. Retinal involvement is rare, with only isolated cases of retinal detachment (2 patients) and peripheral retinal avascularity (1 patient) described. This study reports two patients with familial exudative vitreoretinopathy (FEVR) associated with DYRK1A disease variants.

Methods: Whole genome sequencing (WGS) was performed for 2 FEVR patients, along with comprehensive ophthalmic and systemic evaluations.

Results: Patient 1 (3 years, female) presented at 3 weeks of age with microphthalmia, microcornea and total retinal detachment in the right eye, whilst the left eye was myopic with macular dragging. There was a history of intrauterine growth retardation, and she was later noted to have short stature, microcephaly with cerebellar hypoplasia, generalised seizures and global developmental delay (GDD).

Patient 2 (19 years, female) presented at age 6 with a fixed funnel retinal detachment in the left eye; the right eye was myopic with optic atrophy, peripheral retinal ischaemia and fibrosis. Her medical history included microcephaly with cerebellar anomalies, generalised seizures, GDD, and distinctive facial features.

WGS identified heterozygous de novo DYRK1A variants in both patients; a known pathogenic variant NM_001347721.2:c.1282C>T p.(Arg428Ter) in patient 1, and a rare, novel missense variant c.857T>C p.(Leu286Pro), predicted damaging by in silico algorithms, in patient 2.

Conclusion: These cases confirm vitreoretinal involvement in DYRK1A syndrome, and highlight FEVR as a useful diagnostic handle in individuals with neurodevelopmental disorders.

Grants: none

Conflict of Interest: None declared

EP04.005 Biallelic mutations of CEP290 as a molecular background of isolated retinal dystrophy

Tadeusz Kałużewski ^1;2, Łukasz Kępczyński^1;2, Karolina Matuszczyk², Magdalena Markowska², Jordan Sałamunia², Bogdan Kałużewski², Agnieszka Gach¹

¹Polish Mother’s Memorial Hospital Research Institute, Department of Genetics, Łódź; ²Genos Sp. z o.o., Laboratory of Medical Genetics, R&D Department, Łódź

Background: The CEP290 gene encodes a centrosomal protein involved in ciliary assembly and trafficking. Homozygous or compound heterozygous mutations are well-known molecular backgrounds of ciliopathies, e.g., Joubert syndrome type 5 (OMIM: #610188), Meckel syndrome type 4 (OMIM: #611134), Senior-Loken syndrome type 6 (OMIM: #610189) and Leber congenital amaurosis 10 (OMIM: #611755). The phenotype of CEP290-related disorders ranges from isolated retinal dystrophy to severe multisystemic involvement.

Methods: This report presents two affected brothers of Polish origin, children of non-consanguineous parents. Both patients have abnormal refraction (HP:0000539) and visual field defect (HP:0001123). They do not present any syndromic features of ciliopathies. The high-throughput molecular diagnosis was carried out to investigate the molecular background of their disease.

Results: Whole exome sequencing detected compound heterozygosity for CEP290 variants: c.1992del p.(Pro665Leufs*10) and c.104T>C p.(Val35Ala) in both affected brothers. Parental testing confirmed in trans arrangement. The c.1992del variant was reported in the literature in connection with severe manifestations of ciliopathies (PMID:17345604, PMID:16909394, and PMID:23559409), while the c.104T>C was not described until now. The c.1992del was classified as pathogenic, and c.104T>C as a variant of uncertain significance according to ACMG guidelines.

Conclusion: The phenotype of the patients is consistent with the clinical presentation of isolated inherited retinal disorders caused by the CEP290 mutations. Given the shortage of data regarding genotypic-phenotypic correlations of this gene defects, clinical and molecular findings described here expand the molecular and clinical spectrum of CEP290-related disorders.

Conflict of Interest: None declared

EP04.007 Genetic spectrum of 381 patients with hearing loss diagnosed with a comprehensive approach in a clinical setting

Raquel Muguerza ^1;1, Nerea Bastida-Lertxundi¹, julien crettaz¹, Raquel Sáez-Villaverde¹, yolanda ramírez¹, jose luis eguiburu¹, laura martinez¹, irene perez¹, carla lallave¹, Zuriñe Martinez², Marta Abrego², Elena Beristain³, Blanca Gener Querol⁴

¹Donostia University Hospital, Clinical Genetics, Donostia, Spain; ²Donostia University Hospital, Otorhinolaryngology Department, Donostia, Spain; ³Araba University Hospital, Molecular Genetics Laboratory, Gasteiz, Spain; ⁴Cruces University Hospital, Clinical Genetics, Barakaldo, Spain

Background/Objectives: Deafness, the most common sensorineural impairment, is genetic in 60% of cases. It can be syndromic (30%) or non-syndromic (70%), which is genetically highly heterogeneous. Genetic diagnosis has become an essential test in the diagnosis of deafness.

Methods: 381 patients (2014-2023) from the Basque Public Health System were studied in Donostia University Hospital with a comprehensive sequential approach: 1) screening of frequent genes: GJB2 (Sanger sequencing), GJB6 (MLPA kit P163, MRC Holland), CNV of STRC (digital droplet-PCR) and mitochondrial variants (SNaPshot); 2) if negative result in the screening: Clinical Exome Sequencing TruSight One (Illumina) (65 patients) or Clinical Exome Solution-v2 (159 patients) or -v3 (Sophia Genetics) (77 patients) and virtual panel filtering (89, 91 and 109 genes, respectively).

Results: The overall diagnostic rate was 43% (163/381): 21% (80/381) in the screening and 22% (83/381) in the expanded analysis. Thirty-one different genes were involved (72.4%, (118/163) autosomal recessive, 17.2% (28/163) dominant, 9.2% (15/163) mitochondrial and 1.2% (2/163) X-linked). The more frequent autosomal recessive genes were: GJB2 (44.1%, 52/118), STRC (12.7%, 15/118), OTOF (6.8%, 8/118) and BSND (5.9%, 7/118), and among the autosomal dominant: TECTA (14.3%, 4/28), ACTG1 and WFS1 (10.7%, 3/28 each).

Conclusion: These results confirm the great genetic variability of deafness and suggest there are still many genes to be discovered. The great importance of the STRC gene and of the mitochondrial inheritance in our environment is striking, greater than that described in the literature. Moreover, the OTOF cases could be candidates for gene therapy in the near future.

Conflict of Interest: None declared

EP04.008 Genetic etiology of perrault syndrome in Iranian families: first report from Iran

Ebrahim Shokouhian ¹, Zohreh Fattahi¹, Sanaz Arzhangi¹, Kimia Kahrizi¹, Hossein Najmabadi¹, mojgan babanejad¹

¹University of Social Welfare and Rehabilitation Sciences, Genetics Research Center, Tehran, Iran

Background/Objectives: Perrault syndrome (PS) is an extremely rare autosomal recessive disorder characterized primarily by bilateral sensorineural hearing loss in both genders, and primary, or secondary ovarian failure in females. Moreover, neurological features, such as cerebral ataxia, peripheral neuropathy, epilepsy and intellectual disability are frequent manifestations of PS as well. So far, six genes have been reported to cause PS, and globally almost 100 families have been identified with this syndrome.

Methods: Here we report the first cases of Perrault syndrome from two Iranian families, with novel and reported variants in CLPP and TWNK genes, that was identified by Whole Exome Sequencing (WES).

Results: Family A, a non-consanguineous marriage, consisted of three affected individuals presented with bilateral severe to profound congenital hearing loss, cerebral ataxia, epilepsy, and intellectual disability. In Family B, with a consanguineous marriage and one affected female presented with bilateral moderate to severe hearing loss, and peripheral neuropathy. WES revealed compound heterozygous variants for the proband of Family A; a previously reported c.21delA, and a novel missense variant (c.512C>G) in the CLPP gene. In Family B, a previously reported homozygous mutation, c.874C>A, was identified in the TWNK gene.

Conclusion: This is the first report of genetic variations of CLPP and TWNK genes in affected patients with PS from Iran. We expect that our detailed clinical study will improve the genotype-phenotype interpretation of causal PS variants and the analysis of next-generation sequencing data, leading to clarification of the cause of complicated cases of rare diseases.

Grants: USWR/GRC/2020-T-2405

Conflict of Interest: None declared

EP04.009 A novel candidate gene -MACF1- associated with autosomal dominant non-syndromic hearing loss in an Iranian family

Niloofar Bazazzadegan ¹, Kimia Kahrizi¹, Hossein Najmabadi¹, Kevin TA. Booth^2;3, Sussan Banihashemi¹, Sanaz Arzhangi¹

¹Genetics Research Center, University of Social Welfare and Rehabilitation Sciences, Tehran, Iran; ²Department of Medical and Molecular Genetics, Indiana School of Medicine, Indianapolis, IN, United States; ³Department of Otolaryngology—Head and Neck Surgery, Indiana School of Medicine, Indianapolis, IN, United States

Background: Cytoskeletal dynamics, the interplay of actin and microtubules is a tightly regulated process. Defects in the proteins involved can result in a wide range of cellular consequences. Hearing loss exhibits genetic and phenotypic heterogeneity. Among 140 genes casually linked to non-syndromic hearing loss, 63 are associated with autosomal dominant inheritance. Here we add to this growing number by implicating MACF1 (reported with lissencephaly with MIM # 618325), as a novel candidate gene for autosomal dominant non-syndromic hearing loss (ADNSHL).

Methods: A large Iranian family segregating progressive ADNSHL (without any cognitive or brainstem malformation) was recruited for this study. Affected and unaffected individuals underwent clinical evaluation and after informed consent peripheral blood samples were collected. Following DNA isolation, exome sequencing (ES) was performed on proband. After read mapping and variant calling, variants were annotated with a custom bioinformatic pipeline. Variants were filtered based on quality, minor allele frequency and predicted variant impact. Subsequently, variants were further filtered and prioritized. Sanger sequencing was utilized for segregation analysis of all candidate variants in available samples.

Results: The proband had bilateral mild-moderate sensorineural hearing loss and was negative for GJB2 mutations. After applying ES on family proband, a missense mutation c.C1378T (p .H460Y) was found in a novel candidate gene, MACF1 and co-segregated with the hearing loss in the extended family.

Conclusion: We speculated that MACF1 mutations probably cause non-syndromic hearing loss inherited in an autosomal dominant manner. The potential functional impact of the identified variant will be investigated through further analysis.

Grant references: USWR: 2875

Conflict of Interest: None declared

EP04.011Missense variant in COL18A1 causing Knobloch syndrome

Laura Mauring ^1;2;3, Tiia Reimand^1;3, Ülle Murumets¹, Sander Pajusalu^1;3, Mari Petraudze², Kuldar Kaljurand^2;3, Katrin Ounap^1;3

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

Missense variant in COL18A1 causing Knobloch syndrome

Background/Objectives: Knobloch syndrome (OMIM # 267750) is a rare recessively inherited condition with significant phenotypic heterogeneity characterized by high myopia, vitreoretinal degeneration, and variable occipital skull abnormalities, with or without neurodevelopmental delay. Knobloch syndrome is caused by pathogenic variants in the COL18A1, which results in the aberrant synthesis of type XVIII collagen.

Case report: A 9-year-old boy underwent genetic consultation due to low vision, high axial myopia since early childhood, and bilateral retinal detachment. He is the second child of a healthy couple, born after uneventful pregnancy at term, with unremarkable early development and no relevant family history. The fundus demonstrates signs characteristic to high myopia, and undifferentiated fovea bilaterally, but no anterior segment defects. No occipital abnormalities are evident or palpable. He has neither cutis aplasia nor epilepsy.

Results: Next-generation sequencing of a targeted gene panel from the DNA extracted from the blood identified two heterozygous variants in trans in COL18A1: NM_001379500.1(COL18A1):c.2673dup, p.(Gly892Argfs*9) (pathogenic), and c.2443G>A, p.(Gly815Arg) (variant of uncertain clinical significance (VUS)). This variant has never been reported and is ultra-rare from the gnomAD database. Furthermore, all computational prediction tools support a deleterious effect on the gene of this variant. It is also an evolutionarily highly conserved nucleotide.

Conclusion: This case emphasizes the need for more functional data about the missense variants found in COL18A1. According to ClinVar, 1030 different missense variants have been identified, with all but one classed as VUS/benign. We are seeking collaborators to further investigate its functional effects.

Grants: PRG471, PRG2040, PSG774

Conflict of Interest: Laura Mauring: None declared, Tiia Reimand: None declared, Ülle Murumets: None declared, Sander Pajusalu PSG774, Mari Petraudze: None declared, Kuldar Kaljurand: None declared, Katrin Ounap PRG471, PRG2040

EP04.012 Genetic test results of patients clinically diagnosed with Stargardt disease: Novel ABCA4 variants from Turkey

Fulya Yaylacioglu Tuncay ¹, burak acar², murat yüksel², Gülsüm Kayhan³, mehmet ali ergün³, Sengül Ozdek²

¹University of Health Sciences, Gülhane Medical Faculty, Medical Biology, Ankara, Türkyie; ²Gazi University, School of Medicine, Ophthalmology, Ankara, Türkyie; ³Gazi University, School of Medicine, Medical Genetics, Ankara, Türkyie

Background/Objectives: This study aims to contribute to the disease-related variant data from Turkey by evaluating the genetic test results of patients clinically diagnosed with Stargardt disease.

Methods: The genetic test results of 42 patients diagnosed with Stargardt disease in the ophthalmology department of Gazi Medical Faculty were retrospectively evaluated. The genetic testing methods, reported gene variants, and variant classifications were recorded. The disease-related variants were investigated in the Human Gene Mutation Database (HGMD), ClinVar and literature to decide whether the variant was novel.

Results: Retina panel (n = 23) or clinical exome panel (n = 19) was applied as the genetic testing methods in patients. Twenty-nine ABCA4 variants were reported in 42 unrelated patients. Genetic test results supported the clinical diagnosis in 31 patients. The most frequently detected variant type was missense variants (16/29). Three variants were not available in the HGMD and ClinVar databases and were not reported in the literature: c.466_467dupAT, p.Leu157SerTer2; c.4540-1G > C; c.878delC, p.Met293SerfsTer7. Although seven variants were in the ClinVAR database, their association with a retinal disease was not mentioned in the literature and databases: c.4529C>T, p.Pro1510Leu; c.6121G>A, p.Gly2041Ser; c.6568C>T, p.Gln2190Ter; c.1916A>G, p.Tyr639Cys; c.6568C>T, p.Gln2190Ter; c.5018+81C>T; c.303-181G>A. c.5882G>A, p. Gly1961Glu was detected in 7 patients in this study and was the most frequently recurring variant similar to different patient populations.

Conclusion: This study is the most extensive series providing variant data on Stargardt disease from Turkey. Newly detected variants contribute to the variant spectrum of ABCA4 in Stargardt disease.

Conflict of Interest: None declared

EP04.013 Whole-exome sequencing revealed a novel candidate gene, ARHGAP22, as potential cause of non-syndromic hearing loss in an Iranian family

Raziye Rezvani Rezvandeh ¹, negar kazemi¹, Farzane Zare Ashrafi¹, Masoud Edizadeh², fatemeh ghodratpour¹, Fatemeh Keshavarzi¹, Khadijeh Jalalvand¹, Sanaz Arzhangi¹, Kimia Kahrizi¹, Hossein Najmabadi¹, Marzieh Mohseni¹

¹Genetics Research Center, University of Social Welfare and Rehabilitation Sciences, Tehran, Iran; ²Genoks Genetic Diagnosis Center, Department of Bioinformatics, Ankara, Türkyie

Background: Non-syndromic hearing loss (NSHL) affects one out of every 500 newborns worldwide. In Iran, it is the second most frequent disability due to the high rate of consanguineous marriages.

Methods: Whole exome sequencing (WES) was performed to identify the disease-causing gene in a consanguineous family with hereditary bilateral severe sensorineural NSHL in three affected members.

Results: WES revealed a homozygous missense variant c.1873G>A in the ARHGAP22 gene (NM_021226.4) for the patients. Co-segregation analysis revealed that all affected individuals were homozygous for this variant with the heterozygous carrier parents.

Conclusion: The c.1873G>A variant located in the Coiled Coiled structure at position 625 that substitutes lysine for glutamic acid. The protein that is expressed by ARHGAP22 is found in the Rho GTPase protein cluster and interacts with proteins that lead to hearing loss; such as CDC42, JAG1, ROCK2, and DIAPH3. The bioinformatics databases indicate that ARHGAP22 is expressed in the ear. We propose that the ARHGAP22 gene could be a candidate as a new hereditary hearing loss gene. Further studies need to confirm the effect of ARHGAP22 on the auditory system.

Grant number: USWR/GRC/2024 /3072

Institutional ethical approval number: IR.USWR.REC.1402.187

Conflict of Interest: None declared

EP04.014 DBX2: A novel candidate gene for non-syndromic hearing loss identified in an iranian family

negar kazemi ¹, Raziye Rezvani Rezvandeh¹, Farzane Zare Ashrafi¹, fatemeh ghodratpour¹, fatemeh keshavarzi¹, Khadijeh Jalalvand¹, Sanaz Arzhangi¹, Masoud Edizadeh², Kimia Kahrizi¹, Hossein Najmabadi¹, Marzieh Mohseni¹

¹Genetics Research Center, University of Social Welfare and Rehabilitation Sciences, Tehran, Iran; ²Genoks Genetic Diagnosis Center, Department of Bioinformatics, Ankara, Türkyie

Background: More than 70 percent of pathogenic hearing loss genes are involved in autosomal recessive non-syndromic hearing loss (ARNSHL). In Iran, due to the high rate of consanguineous marriage, hearing loss is the second most common neurosensory disorder, affecting, approximately 1 in 166 individuals.

Methods: In this study, after excluding GJB2 and OtoSCOPE mutations, we performed Whole Exome Sequencing (WES) of two Iranian siblings born to a consanguineous family, with late-onset moderate to severe sensorineural hearing loss.

Results: We identified a homozygous missense variant (c.28G>A (p.Ala10Thr)) in the DBX2 gene (NM_001004329.3) as a novel candidate gene.

Conclusion: DBX2, known as developing Brain Homeobox 2, possesses DNA-binding transcription factor activity, and RNA polymerase II-specific. This gene interacts with LS1, LMX1A, and LMX1B in auditory pathways. Additionally, it is a crucial component of the superior olivary complex, responsible for sound localization. The c.28G>A (p.Ala10Thr) variant leads to the formation of a larger and more hydrophobic amino acid potentially altering the protein structure and its hydrophobic interactions. Further investigation of the DBX2 gene may contribute to our understanding of the genetic basis of auditory function.

Grant number: USWR/GRC/2024 /3072

Institutional ethical approval number: IR.USWR.REC.1402.187

Conflict of Interest: None declared

EP04.015 Comprehensive Molecular Diagnosis of Inherited Retinal Diseases: Use of an Extended Virtual NGS Panel for Enhanced Detection of Genetic Variants

Ornella Rondinone ¹, Giada Moresco¹, Carola Conca Dioguardi², Martina Miceli², Rita Gorgoglione², Chiara Pesenti², Giovanni Marfia^3;4, Stefania Navone³, Gianluca Tolva², Leonardo Colombo⁵, Luca Rossetti^5;6, Monica Miozzo^1;2, Laura Fontana^1;2

¹University of Milan, Medical Genetics, Department of Health Sciences, Milan, Italy; ²ASST Santi Paolo e Carlo Hospital, Medical Genetics Unit, Milan, Italy; ³Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Laboratory of Experimental Neurosurgery and Cell Therapy, Neurosurgery Unit, Milan, Italy; ⁴Aeronautica Militare, Clinical Pathology Unit, Istituto di Medicina Aerospaziale “A. Mosso”, Milan, Italy; ⁵ASST Santi Paolo e Carlo Hospital, Department of Ophthalmology, Milan, Italy; ⁶University of Milan, Department of Health Sciences, Milan, Italy

Background/Objectives: Inherited retinal diseases (IRDs) are a clinically and genetically heterogeneous group of disorders characterized by photoreceptor degeneration. Currently, over 300 genes have been identified to cause both non-syndromic and syndromic retinal degeneration, and this number is increasing. Molecular diagnosis is fundamental for clinical management, prognostic assessment, and treatment prospects, as gene therapy is available for some types of IRDs.

Methods: We developed a comprehensive in-house extended virtual NGS panel that includes both SNV/indels and CNV in coding and specific non-coding regions of 332 IRDs-genes. Using eVai software (enGenome), we analyzed a cohort of 52 patients affected by rod-cone and other retinal dystrophies, suspected of having a genetic etiology. Some of these patients has previously undergone genetic testing, yielding either negative or inconclusive results.

Results: Our extended approach enabled conclusive diagnoses in 31 patients, achieving a diagnostic rate of 60%. We identified 35 variants in total: 2 CNVs (deletions), 17 missense, 10 frameshift/nonsense, 7 splicing, and 2 intronic variants. Of these, 23 variants are classified as pathogenic/likely pathogenic (P/LP), 2 as VUS/LP, and 10 as VUS strongly correlating with phenotype. The most frequently mutated genes were USH2A (16% of cases) and EYS (10% of cases). In addition, this in-house panel facilitated the diagnosis of tricky cases, including the identification of a deep intronic variant in USH2A and the re-diagnosis of Pseudoxanthoma elasticum in a patient with biallelic ABCC6 variants.

Conclusion: Our study supports the use of a comprehensive diagnostic approach, extending also to non-coding regions, to achieve an accurate molecular diagnosis of IRDs.

Grants: None.

Conflict of Interest: None declared

EP04.016 A novel frameshift variant in compound heterozygous state in the USH2A gene associated with Usher syndrome

Mert Coşkun ¹, Mehmet Erkan Dogan², Alp Peker¹, Aslı Toylu¹, Özden Altıok Clark¹

¹Akdeniz Üniversitesi Hastanesi, Medical Genetics, Antalya, Türkyie; ²Akdeniz Üniversitesi Hastanesi, Ophthalmology Department, Antalya, Türkyie

Background/Objectives: Usher syndrome (USH) is an autosomal recessive disorder characterized by bilateral sensorineural hearing loss and progressive retinitis pigmentosa. USH2A, one of the causative genes of this syndrome, codes a protein named Usherin which is important for the functions of photoreceptor cells and cochlear hair cells. In this study, we present a patient with clinical findings of USH. She had congenital hearing loss and experienced progressive visual impairment beginning in the second decade. We aimed to evaluate the sequence variants of the USH associated genes in order to confirm the patient’s diagnosis.

Methods: Genomic DNA was extracted from the patient’s peripheral blood sample. Exons and exon-intron transition regions of the USH associated genes were investigated by next generation sequencing. Variants were analyzed using Sophia DDM program. Variant information servers dbSNP, ClinVar, Ensembl and ACMG 2015 criteria were used for evaluations.

Results: In USH2A gene, c.2845dup (p.Leu949Profs*12) frameshift and c.4628-2A > T splice site variants were detected in compound heterozygous state. The splice site variant was reported as a “pathogenic” change in ClinVar database. The frameshift variant which is predicted to cause premature termination of protein translation was not reported in population or disease-specific databases.

Conclusion: The detected novel frameshift mutation is evaluated as a candidate variant that may be associated with USH, due to its predicted functional effects and presence with a known pathogenic variant. To better understand genotype-phenotype correlation, segregation of the variant in the patient’s family and larger population data will be examined.

Grants: None

Conflict of Interest: None declared

EP04.017 Diagnosis of Smith-Magenis syndrome in an adult patient with rapidly progressive profound hearing loss

Vanessa Hochheimer ¹, Zafer Yüksel¹, Hanno Bolz¹

¹Bioscientia Institute for Medical Diagnostics GmbH, Bioscientia Human Genetics, Ingelheim, Germany

Background/Objectives: Smith-Magenis syndrome (SMS) is an autosomal dominant multisystem disorder initially found to be caused by microdeletions on chromosome 17p11.2. Since then, whole-exome sequencing (WES) has revealed an increasing number of point mutations in RAI1, the disease-causing gene contained in the SMS deletion locus. Symptoms include typical facial features, intellectual disability, sleep disturbances and behavioral problems; diagnosis is usually given in childhood. Hearing loss (HL) occurs in about 80%, as conductive HL in childhood and sensorineural HL in adulthood. It is progressive and usually mild.

We report a 27-year-old male for whom parents reported lip reading since early infancy, and hearing aids were provided in the 1^st decade for his mild-to-moderate HL. Progression to profound HL required bilateral cochlear implantation (CI) at 20 years. Previous genetic testing for Usher syndrome, optic atrophy and hearing loss because of night blindness and visual field constriction revealed a heterozygous pathogenic USH2A variant.

Methods: Trio-WES on DNA from peripheral blood samples (patient, parents) was conducted on an Illumina NovaSeq 6000 system.

Results: Medical history and physical examination revealed moderate developmental delay, hypotonia, bilateral iris coloboma, brachycephaly, brachydactyly, and facial dysmorphism. The cause of the visual complaints remained elusive (ophthalmological assessment did not confirm retinal dystrophy). We identified a novel heterozygous truncating de novo c.106_116del p.(Ala36Leufs*13) variant in RAI1. Reverse phenotyping revealed onychotillomania and heavy sleep disturbance.

Conclusion: SMS may atypically present with predominant progressive HL requiring bilateral CI and should thus be considered in genetic analysis for profound sensorineural HL.

Conflict of Interest: None declared

EP04.019 Double trouble, incidental finding or clinical irrelevant – the interpretation challenge

Sinje Geuer ¹, Cord-Christian Becker¹

¹MVZ genetikum GmbH, Neu-Ulm, Germany

Background: We report on a 4-year-old female child with hearing impairment without any signs of syndromic involvement. The family history is empty for early onset hearing impairment.

Methods: In-silico panel analysis for syndromic and non-syndromic hearing impairment based on whole exome sequencing (TWES, Twist Human Core Exome with additional customized probes and NGS with NovaSeq Illumina and deep sequencing of mitochondrial DNA) was performed.

Results: Panel-analysis revealed potentially relevant variants in three genes. A pathogenic variant and a variant of unclear significance (VUS) in MYO15A might be causative for autosomal recessive non-syndromic hearing loss if present in compound- heterozygosity. A heterozygous VUS in WFS1 could be causative for autosomal dominant non-classical Wolfram syndrome if arisen de-novo. A pathogenic variant in mt-TL1 with low heteroplasmy of 7% and known to cause MELAS-Syndrome might cause syndromic or (rarely) non-syndromic hearing impairment. Noteworthy, we cannot exclude that MELAS will never manifest in our patient. The relevance of the variants detected might be partly resolvable by segregation analysis. Nevertheless, the MELAS-variant presents an interpretation challenge due to its extremely low heteroplasmy and tissue specific variability.

Conclusion: This case report highlights the challenge to interpret multiple variants of which each might explain the patient phenotype while certain syndromic characteristics are not present (yet), and leaves us with the question: Is this double trouble or an incidental finding? Technological improvement leads to a third option: A known pathogenic variant might be clinical irrelevant due to its low allele frequency.

Conflict of Interest: None declared

EP04.020 De novo variants in PDE6B retinitis pigmentosa

Luigi Monti ^1;2, Elena Luppi^1;2, Nicole Balducci³, Giovanni Innella^1;2, Francesca Montanari¹, Cesare Rossi¹, Daniela Turchetti^1;2, Marco Seri^1;2

¹Medical Genetics Unit, IRCCS Azienda Ospedaliero-Universitaria di Bologna, Bologna, Italy, Bologna, Italy; ²Alma Mater Studiorum University of Bologna, Bologna, Italy, Department of Medical and Surgical Sciences, Bologna, Italy, Bologna, Italy; ³Ophtalmology Unit, S.Orsola-Malpighi Hospital, University of Bologna, Department of Medical and Surgical Sciences, Bologna, Italy, Bologna, Italy

Background/Objectives: PDE6B pathogenic variants are associated with autosomal recessive Retinitis Pigmentosa (RP) and occur in a small proportion of affected patients (2-5%). In this study, we aim to expand the clinical and mutational spectrum of PDE6B-associated RP by presenting two new unrelated cases and investigating the occurrence of novel variants in the gene.

Methods: Patients’ histories were recorded by medical charts. Both patients underwent ophthalmic examinations and a hereditary eye disease NGS panel analysis. Variant validation and segregation analysis were performed through Sanger-sequencing.

Results: Both patients, a 42-year-old woman and a 13 years-old boy, reported the onset of symptoms in childhood/adolescence (narrow visual field and hemeralopia) and share preserved visual acuity, extrafoveal bone spicules at the ocular fundus, atrophy of the bilateral extrafoveal photoreceptors and intraretinal cysts at OCT. Both resulted carriers of a known pathogenic variant inherited from a parent and a novel variant (c.928-1G > A;p.? and c.340C>T;p.Gln114Ter) of de novo origin respectively on the maternal and the paternal allele (false paternity was excluded).

Conclusion: PDE6B-associated disease is a classic RP with relatively preserved central vision at older ages. Intraretinic cysts are common findings. Our identification of two novel mutations in PDE6B increases the spectrum of genetic variations. Their de novo origin may not be coincidental, suggesting a more common finding than previously thought, and could explain the high prevalence of novel variants in PDE6B (up to 43% in some studies). Further research will be needed to understand the mechanism underlying this phenomenon.

Grants:

Conflict of Interest: None declared

EP04.021 Natural history of DFNA9 (COCH) in a six-generation family from North America segregating the Belgian founder mutation (c.151C>T; p.P51P/S)

Tammy Benteau ¹, Maxime Maheu², David McComiskey³, Anne Griffin¹, Kim Furlong⁴, Susan Stanton⁵, Terry-Lynn Young¹

¹Memorial University, Faculty of Medicine, St. John’s, Canada; ²Universite’ de Montreal, Ecole d’orthophonie d’audiologie, Montreal, Canada; ³University of Ottawa, Neuroradiology, Ottawa, Canada; ⁴NL Balance and Dizziness Centre, St. John’s, Canada; ⁵Western University, National Centre of Audiology, London, Canada

Background/Objectives: DFNA9 hearing loss (HL) due to COCH (c.151C>T; p.P51P/S) also causes severe vestibular dysfunction and an increased risk of dementia. Potential treatment targets anti-sense oligonucleotides (AONs) to silence the mutant allele. Success, in part, depends on the identification of SNPs that increase efficacy and a deeper understanding of the full natural history to identify the precise therapeutic window. This study describes the DFNA9 haplotype and natural history in a multigenerational North American family.

Methods: We identified a six-generation family from the genetic isolate of Newfoundland, Canada with late onset SNHL segregating as an AD trait due to COCH (c.151C>T). To determine if this is the Belgian founder allele, microsatellite markers spanning DFNA9 were compared with 3 Dutch mutation carriers. Phenotypic investigations on mutation carriers will be presented, including nature of HL and vestibular disturbance.

Results: Genotyping revealed this family segregates the Belgian founder mutation, based on a shared 1.2 MB haplotype. The proband experienced bilateral HL (sloping to moderate loss at high frequencies) that worsened over a 15-year period and was treated with a cochlear implant (CI). Although CT scan at age 40 detected no vestibular abnormalities, by age 56, the proband required supports for daily activities.

Conclusion: Extended families with founder mutations are particularly useful for capturing the natural history of adult-onset HL and are a valuable source of pre-symptomatic carriers for prospective studies required for targeted gene therapies.

Grants: ACOA (Atlantic Canada Opportunities Agency); CIHR (Canadian Institutes for Health Research); CFI (Canadian Foundation for Innovation).

Conflict of Interest: Tammy Benteau I am employed as a Research Assistant with the senior author, Maxime Maheu: None declared, David McComiskey: None declared, Anne Griffin: None declared, Kim Furlong: None declared, Susan Stanton: None declared, Terry-Lynn Young: None declared

EP04.022 GLRA2-Associated X-linked dominant high myopia in a multigenerational family

Neta Feinstein-Goren ^1;2, Dalia Eli³, Daphna Prat^2;4;5, Ortal Barel Barel⁶, Odelia Chorin^2;7, Shelly Lev-Hochberg^2;7, Abraham Spierer^2;4, Guy Ben-Simon^2;4;5, annick REIN ROTHSCHILD^2;7, Ben Pode-Shakked^2;5;7

¹Sheba Medical center, Tel-Hashomer, The Danek Gertner Institute of Human Genetics, Ramat Gan, Israel; ²Tel Aviv University, Faculty of Medicine, Tel Aviv, Israel; ³Sheba Medical center, Tel-Hashomer, The Opthalmo-Genetics Clinic, Department of Ophthalmology, Goldschleger Eye Institute, Ramat Gan, Israel; ⁴Sheba Medical Center, Tel-Hashomer, Department of Ophthalmology, Goldschleger Eye Institute, Ramat Gan, Israel; ⁵Sheba Medical center, Tel-Hashomer, The Talpiot Medical Leadership Program, Ramat Gan, Israel; ⁶Sheba Medical center, Tel-Hashomer, The Genomic Unit, Sheba Cancer Research Center, Ramat Gan, Israel; ⁷Edmond and Lily Safra Children’s Hospital, Sheba Medical center, Tel-Hashomer, The Institute for Rare Diseases, Ramat Gan, Israel

Background: High myopia is a complex ocular condition influenced by both genetic and environmental factors. The global rise in myopia has positioned it as a significant public health concern. Understanding its genetic basis is crucial for unraveling its etiology. To date, over 100 genes have been associated with myopia (with only 17 considered causal). We sought to investigate the molecular basis of isolated high myopia affecting multiple individuals across four generations of a non-consanguineous Moroccan-Jewish family, with an X-linked dominant inheritance pattern.

Methods: Targeted gene panel sequencing was conducted on a multigenerational family with isolated high myopia. Comprehensive clinical ophthalmic examinations were performed to characterize the myopic phenotype.

Results: Our investigation revealed a pathogenic variant in the GLRA2 gene, implicating its involvement with isolated high myopia within the family. Notably, GLRA2 was previously associated with a neurodevelopmental phenotype. In addition to our findings, only recently (February 2023) was GLRA2 identified as a player in the genetic landscape of isolated high myopia in two large Chinese families and a simplex case, collectively emphasizing the significance of this gene in the manifestation of high myopia across diverse populations.

Conclusion: This study advances our understanding of GLRA2-related high myopia, underscoring its distinctiveness from the syndromic neurodevelopmental phenotype previously associated with this gene. Our findings advocate for the inclusion of GLRA2 in the differential diagnosis of isolated high myopia cases, marking a step toward unravelling the complex genetic landscape of high myopia, enhancing diagnostic precision, prognosis accuracy, and holding promise for targeted therapeutic interventions.

Conflict of Interest: None declared

EP04.023 Characterization of sensorineural hearing loss in Finland reveals enrichment of pathogenic founder variants

Tyrni Pykäläinen^1;2, Minna Kraatari-Tiri^1;2, Pia Pohjola³, Sanna Häkli⁴, Elisa Rahikkala ^1;2

¹University of Oulu, Research Unit of Clinical Medicine and Medical Research Center, Oulu University Hospital and University of Oulu, Oulu, Finland; ²Oulu University Hospital, Dept of Clinical Genetics, Oulu, Finland; ³Turku University Hospital, Dept of Genomics, Turku, Finland; ⁴Oulu University Hospital, Dept of Otorhinolaryngology and Phoniatrics, Oulu, Finland

Background/Objectives: To examine the clinical and genetic characteristics of childhood-onset bilateral sensorineural hearing loss (SNHL) in Finland.

Methods: A retrospective cohort study was conducted in the Oulu University Hospital from 2017 to 2022. Inclusion criteria were children younger than 18 years with diagnosed bilateral SNHL (ICD-10: H90.3).

Results: The cohort consisted of 249 children. Pathogenic or likely pathogenic sequence variants explaining the SNHL were identified in 34 % (N = 84/249) of children, including 60 non-syndromic SNHL patients (71 %, N = 60/84) and 24 syndromic (29 % N = 24/84) SNHL patients. Biallelic pathogenic variants in GJB2 were the most common etiology, explaining 16 % of SNHL in this cohort (N = 40/249). The most common hearing loss syndrome in this study was Usher type 3 (N = 5). Seventeen (N = 17/249, 7 %) children had chromosome abnormalities or copy number variants explaining their SNHL, including deletion of STRC-CATSPER2 genes (N = 4), other deletion (N = < 3), Down syndrome (N = 6), Turner syndrome (N = 4), or duplication (N = < 3). Pathogenic or likely pathogenic variants enriched in the Finnish population were identified in TMC1, CABP2, MYO7A, TWNK, SUCLA2, and CLRN1 genes.

Conclusion: SNHL is genetically and clinically heterogeneous. The most common causative gene was GJB2. Hearing loss syndromes explained 29 % of all SNHL caused by pathogenic or likely pathogenic sequence variants. The Finnish-enriched variants comprised of 23 % (N = 19/84) of all likely causative variants.

Grants: Research Council of Finland (grant number 338446), Uolevi Mäki Foundation, State Subsidy of Oulu University Hospital.

Conflict of Interest: None declared

EP04.024 Relationship between physical and psychological functioning and health-related quality of life in congenital aniridia

Erlend Christoffer Sommer Landsend¹, Charlotte von der Lippe ², Line Merete Mediå³, Jeanette Ullmann Miller³, Knut Erik Berge⁴, Solrun Sigurdardottir³

¹Oslo University Hospital, Department of Ophthalmology, Oslo, Norway; ²Telemark Hospital Trust, Department of Medical Genetics, Skien, Norway; ³Oslo University Hospital, Centre for Rare Disorders, Oslo, Norway; ⁴Oslo University Hospital, Department of Medical Genetics, Oslo, Norway

Background: Congenital aniridia is a serious eye disease characterized by absence of iris to various degrees. Genetic causes may be pathogenic variants in PAX6 or in regulatory regions of PAX6. We will describe health-related quality of life (HRQoL) in adults with PAX6-related aniridia and assess the relationships between HRQoL, psychological status, ocular health and obesity.

Methods: Twenty-nine adults with PAX6-related aniridia (48% male, aged 18-79 years) participated. HRQoL was measured with SF-36 and the EQ visual analogue scale (VAS). Symptoms of anxiety and depression were measured using the Hospital Anxiety and Depression Scale (HADS). Obesity was assessed with the Patient-Reported Outcomes in Obesity (PROS). Sociodemographic characteristics, genetic variants and ocular and medical health variables were also analysed.

Results: The participants scored significantly lower in the general health domain of the SF-36 than the general population (65.2 vs. 75.3, p = 0.017). The EQ VAS score was also lower in the aniridia group (64.9 vs. 77.9, p = 0.021). Low PCS score was correlated with presence of ocular pain (p = 0.019), high HADS score (p = 0.017) and high PROS score (p = 0.009). High HADS and PROS scores were both related to low EQ VAS scores.

Conclusion: Adults with PAX6-related aniridia scored worse on certain measures of HRQoL than the general population. Poorer HRQoL was associated with increased symptoms of anxiety, depression and obesity and with presence of ocular pain.

Reference: Published in PMID: 38131258 https://doi.org/10.1111/aos.16615

Conflict of Interest: Erlend Christoffer Sommer Landsend Landsend is member of the scientific committee of the patient organization Aniridia Norway., The work was funded by the National Advisory Unit on Rare Disorders in Norway. The funding organization had no role in the design or conduct of this research., Charlotte von der Lippe: None declared, Line Merete Mediå: None declared, Jeanette Ullmann Miller: None declared, Knut Erik Berge: None declared, Solrun Sigurdardottir: None declared

EP04.025 Optimisation of the custom IRD NGS panel - a further step in the improvement of a diagnostic tool

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

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

We present data on the genetic diagnosis of inherited retinal dystrophies (IRD), including the diagnostic yield, resulting from the panel design improvement, as well as bioinformatic pipeline development.

NGS analysis based on an annualy optimised and expanded Twist Bioscience custom panel (v3), targeting coding regions of 360 IRD genes, along with over 400 selected deep intronic variants and mtDNA sequence, was applied to identify genetic causes of IRD for 378 Polish patients. Bioinformatics involved the GATK(v4) best practice pipeline. CNV analysis was conducted using GATK GermlineCNVCaller. The variant classification was performed according to ACMG/AMP guidelines with the help of the in-house interpretation tool BroVar(v3). mtDNA analysis was routinely performed and included into the standard diagnostic pipeline.

Positive results were provided for 243 patients (64.29%). In 8 cases, this was possible due to the analysis of deep intronic regions. An increase in mean coverage (247x, SD 37) and uniformity improved CNV analysis resulting in 13 patients with the diagnosis confirmation based on CNVs. Coverage in the difficult RPGR ORF15 region was further improved with additional probe tiling. Pathogenic variants in the RPGR gene were identified in 25(6.6%) patients, 13 of them were uncovered in the ORF15 region.

The described changes in panel construction, in particular the inclusion of an updated list of deep intronic variants, together with improvements in the sensitivity of CNV analysis, should provide a higher diagnostic yield than current IRD diagnostics based on standard exome enrichments.

Partially funded by Ministry of Education and Science, applied PhD project PCTT/1874/2019.

Conflict of Interest: Ewa Matczyńska Full time employment in Genomed S.A., Marta Beć-Gajowniczek Full time employment in Genomed S.A., Larysa Sivitskaya Full time employment in Genomed S.A., Elżbieta Gregorczyk Full time employment in Genomed S.A., Ewa Suchecka Full time employment in Genomed S.A., Przemysław Łyszkiewicz Part time employment in Genomed S.A., Robert Szymańczak Full time employment in Genomed S.A., Maria Jędrzejowska Part time employment in Genomed S.A., Sławomir Teper: None declared, Maciej Krawczyński Employment in Diagnostyka GENESIS Sp. z o.o, Anna Boguszewska-Chachulska Ownership of Genomed S.A. stocks, Full time employment in Genomed S.A.

EP04.026 Variable expression of novel FGF10 splice-site mutation in large kindred with aplasia of lacrimal and salivary glands (ALSG).

ofek freund ¹, Baker Alsana², Nadav Agam¹, Matan M. Jean¹, Amit Safran¹, Tomer Poleg¹, libe gradstein², Erez Tsumi², Ohad Shmuel Birk^1;3

¹ben gurion university, Faculty of Health Sciences, beer sheva, Israel; ²soroka medical center, Department of Ophthalmology, beer sheva, Israel; ³soroka medical center, genetic institute, beer sheva, Israel

Purpose: Twelve individuals from 6 generations of single Bedouin kindred presented with epiphora from an early age. We aimed to delineate the disease’s clinical phenotype and identify the underlying genetic defect.

Methods: Clinical phenotyping was determined by senior ophthalmologists and geneticists following informed consent and IRB approval. Genetic studies combined linkage analysis and whole exome sequencing, followed by filtration of candidate variants and segregation analysis. Aberrant splicing of mutant FGF10 exon 2 was assayed in HEK-293 cells transfected with plasmids harboring the wild-type or mutant sequences, followed by RT-PCR using primers from exon 1 and 3.

Results: Ophthalmologic examination of the affected individuals revealed tear deficiency and congenital punctal atresia. Multiple caries with no concomitant abnormalities of the ears or digits were also noted, determining a diagnosis of aplasia of the lacrimal and salivary glands (ALSG). Variable expression and penetrance were evident between affected individuals and bilaterally within individuals. Genetic analysis identified a disease-causing novel heterozygous splice-site mutation in intron 2 of FGF10, segregating through the kindred as expected for dominant heredity with incomplete penetrance. Transfection of HEK-293 cells with plasmids expressing wildtype or mutant FGF10 sequences proved that the mutation eliminates exon 2 in the mutant RNA.

Conclusions: We report a novel dominant splice-site mutation in FGF10, abrogating transcription of exon 2 and causing ALSG with epiphora from a young age, with variable expression and incomplete penetrance.

Conflict of Interest: None declared

EP04.027 Recurrent MYO6 variants as a result of a small genome cohort study for hearing loss

Michaela AH Hofrichter ¹, Christian W Remmele^1;2;3, Asuman Koparir¹, Daniel Bengl¹, Tabea Böttcher¹, Tobias Müller⁴, Marcus Dittrich^1;4, Wafaa Shehata-Dieler⁵, Sophie Flandin⁵, Thomas Haaf¹

¹Institute of Human Genetics at the University of Würzburg, Würzburg, Germany; ²Center for Rare Diseases, University Hospital Würzburg, Würzburg, Germany; ³Bavarian Genomes Network for Rare Diseases, Germany; ⁴Department of Bioinformatics, Würzburg, Germany; ⁵Comprehensive Hearing Center, Department of Otorhinolaryngology, University Hospitals, Würzburg, Germany

Background/Objectives: Hearing loss (HL) is one of the most common sensory disorders, affecting around 5% of the world’s population. A genetic predisposition is suspected in 60% of cases. Because of the considerable heterogeneity in HL, pinpointing the genetic source poses a challenge. The implementation of advanced technologies such as whole genome sequencing (WGS) consequently enhances the efficacy of elucidating the root cause of HL.

The scope of this genome cohort study with 15 patients is to find the causative HL variants.

Methods: DNA samples from each index patients were prepared according to the Illumina WGS protocol and sequenced on the NovaSeq6000. Most patients were already pre-tested at least for GJB2. Analyses for SNVs, INDELs and structural variations were performed using in-house genome analysis pipeline. Disease causing variants were validated by Sanger sequencing and segregation analysis.

Results: Overall, a causing variant was discovered in 5/15 patients (33%). However, the analysis is still ongoing. Interestingly, we also detected a heterozygous variant c.884_893del in MYO6 (NM_004999.4) in two index patients with expected autosomal dominant trait for HL. Pre-analysis of the haplotype suggest a hotspot or founder effect.

Conclusion: Overall, a genetic diagnosis was contributed for 1/3 of our patients. In half of these patients, this was only possible using WGS analysis after preliminary exome/panel analysis failed. This allowed the detection of the MYO6 variant and determined the recurrent character, which expand the genetic landscape of the gene MYO6 as well as hereditary HL.

Grants:

Conflict of Interest: None declared

EP04.029 20 years of aniridia testing in Udine: more than meets the eye

Catia Mio¹, Jessica Zucco², Carla Reale³, Concetta Ambrosino³, Federica Baldan¹, Lorenzo Allegri¹, Alessandra Franzoni², Angela Valentina D’Elia², Flavio Faletra⁴, Giuseppe Damante ¹

¹Università degli Studi di Udine, Dipartimento di Medicina, Udine, Italy; ²Azienda Sanitaria Universitaria Friuli Centrale (ASUFC), Istituto di Genetica Medica, Udine; ³Biogem Scarl, Istituto di Ricerche Genetiche, Ariano Irpino, Italy; ⁴Azienda Sanitaria Universitaria Friuli Centrale (ASUFC), Istituto di Genetica Medica, Udine, Italy

Background/Objectives: Anterior segment dysgenesis (ASD) refers to a spectrum of congenital disorders characterized by a wide range of heterogeneity that affect the development of the cornea, the iris and the lens. The most well-known anterior segment abnormality affecting the iris is aniridia, which is associated with heterozygous loss of function variants in PAX6. Genomic research has indeed boosted our understanding in the molecular basis of ASD. Here we describe the molecular characterization of a cohort of 162 patients displaying isolated or syndromic congenital ASD (i.e., isolated or syndromic aniridia, Peters anomaly, Axenfeld-Rieger and Gillespie syndromes).

Methods: Sanger sequencing and multiplex ligation-dependent probe amplification were used to analyse the 11p13 locus. Whole exome sequencing (WES) was performed on PAX6-negative samples.

Results: Our data reiterate the notion that PAX6 alterations are primarily associated with ASD since the majority of the cohort (66.7%) has a pathogenic or likely pathogenic variant in the 11p13 locus. WES allowed us to assess variants in known genes (i.e., CYP1B1, ITPR1, MAB21L1, PXDN and PITX2) and to identify rarer phenotypes (i.e., MIDAS and OGIN syndromes). Besides, a heterozygous MAF deletion was identified in a patient with bilateral aniridia. Morpholino treatments in Danio Rerio suggested a putative involvement of MAF in lens dysgenesis.

Conclusion: Our data clearly suggest that WES allows expanding the analytical portfolio of ocular dysgenesis, both isolated and syndromic, and that is pivotal for the differential diagnosis of those conditions in which there may be phenotypic overlaps and in general in ASD.

Grants: n.a.

Conflict of Interest: None declared

EP04.030 Genetic Profile of Retinopathies Due to Disease-Causing Variants in Leber Congenital Amaurosis (LCA)-Associated Genes in a Greek Cohort

Smaragda Kamakari ¹, Stavrenia Koukoula²

¹OMMA Ophthalmological Institute of Athens, Ophthalmic Genetics, Athens, Greece; ²Ophthalmica Institute of Ophthalmology and Microsurgery, Electrophysiology, Thessaloniki, Greece

Background/Objectives: Leber congenital amaurosis (LCA) is the most frequent cause of congenital blindness in children and most severe form of inherited retinal dystrophies. To date, more than 25 genes have been implicated in the pathogenesis of LCA and Early-Onset Severe Retinal Dystrophy (EOSRD). The aim of the study was to report the molecular basis of LCA/EOSRD in 51 Greek families.

Methods: DNA microarrays and/or Next Generation Sequencing panel for LCA genes were performed to identify potentially pathogenic variants. Sanger sequencing was used for identification of the 2nd allele (2 cases) and segregation analysis to confirm biallelism within the families.

Results: The molecular background was established in 51 independent patients. 48 distinct variants were identified in 12 genes: CEP290, CRB1, GUCY2D, RDH12, RPGRIP1, AIPL1, RPE65, NMNAT1, TULP1, SPATA7, LCA5 and IQCB1. Almost 1/5 of the patients were affected with LCA10 due to CEP290 gene variants with its intronic mutation c.2991+1655A > G being the most frequent. CEP290 was the most prevalent gene in our cohort (17.6%), followed by CRB1 and GUCY2D (11.8% each), RDH12 and RPGRIP1 (9.8% each), AIPL1 and RPE65 (7.8% each), NMNAT1, TULP1 and SPATA7 (5.9% each), LCA5 (3.9% each) and IQCB1 (2%).

Conclusion: This study provides the first molecular genetic characteristics of patients with LCA from the previously unexplored Greek population. Our study expands the mutational spectrum as we report novel variants in LCA genes and allows for the formation of a cohort for target therapy of the disorder.

Grant: EU FP6, Integrated Project ‘EVI-GENORET’ (LSHG-CT-2005-512036; S.K)

Conflict of Interest: None declared

EP04.031 A novel disease-causing TUBB4B variant in an Italian family diagnosed with progressive cone dystrophy and early onset sensorineural hearing loss

Margherita Scarpato ¹, Francesco Testa², Roberta Zeuli¹, Sandro Banfi^1;3, Francesca Simonelli², Marianthi Karali^1;2

¹Università degli Studi della Campania ‘Luigi Vanvitelli’, Medical Genetics, Department of Precision Medicine, Naples, Italy; ²Università degli Studi della Campania ‘Luigi Vanvitelli’, Multidisciplinary Department of Medical, Surgical and Dental Sciences, Eye Clinic, Naples, Italy; ³Telethon Institute of Genetics and Medicine, Naples, Italy

Background: Leber congenital amaurosis with early-onset deafness (LCAEOD) is a rare autosomal dominant syndrome manifesting retinal degeneration and sensorineural hearing loss (SHL). LCAEOD was first described in 2017 in four families segregating heterozygous missense mutations in TUBB4B, a gene encoding a β-tubulin isotype. To date, only three other families with TUBB4B-associated LCAEOD have been reported. All described cases harboured missense variants affecting the same amino acid (Arg391).

Methods: Whole Exome Sequencing (WES) was performed on a 45-year-old female and her 9-year-old son, both diagnosed with progressive cone dystrophy and early onset bilateral SHL.

Results: Ophthalmological assessment of the proband showed macular dystrophy with osteoblast-type pigmentation at the posterior pole, reduced visual acuity, nonrecordable photopic and subnormal scotopic electroretinography (ERG) responses. Her son presented diffuse dystrophy of the retinal pigment epithelium, thinning of the macular area and markedly reduced photopic ERG responses. WES of the proband and her son identified a shared c.1049A>C point mutation in TUBB4B (NM_006088) which causes the substitution of a highly conserved Lysine with Threonine at amino acid position 350. The variant is novel, absent from population databases, and classified as Likely Pathogenic according to the ACMG criteria.

Conclusion: Here, we report a novel, disease-causing, missense variant in TUBB4B in two affected members of an Italian family segregating with progressive cone dystrophy and early onset SHL. Our findings corroborate the recently reported association of TUBB4B variants with sensorineural syndromic forms, expand the list of pathogenic TUBB4B variants, and demonstrate that TUBB4B mutations can also cause cone-dominated retinal phenotypes.

Conflict of Interest: None declared

EP04.032 Unveiling Genetic Insights: NGS Analysis of 57 Subjects with Inherited Retinal Disorders

Federico Rondot¹, Amedeo Primerano², Patrizia Dentelli¹, Enrico Grosso², Alessandra Pelle ², Barbara Pasini^1;2

¹University of Turin, Department of Medical Sciences, Turin, Italy; ²AOU Città della Salute e della Scienza di Torino, Turin, Italy

Background/Objectives: Inherited Retinal Disorders (IRD) constitute a heterogeneous group of genetic conditions marked by retinal dystrophy, resulting in progressive visual field impairment, whether presented in a syndromic or isolated clinical context. This study presents a cohort of 57 subjects clinically diagnosed with IRD, exhibiting a spectrum of clinical presentations, including retinitis pigmentosa, Stargardt disease, macular dystrophy, and less specific retinal dystrophy. Employing a Clinical Exome platform and a virtual panel approach in Singleton, Duo, and Trio settings, subjects underwent Next-Generation Sequencing (NGS) analysis, enhancing the comprehensive genetic evaluation of IRD phenotypes.

Methods: SureSelect-Agilent Custom-Constitutional-Panel-17Mb encompassing 5219 genes. Virtual panel “Inherited Retinal Disorder” (157 genes).

Results: A definitive molecular diagnosis was successfully established in 30 out of 57 patients. However, in 27 patients, conclusive molecular diagnosis remained elusive, attributed to uncertain significance variants (6/27), heterozygous pathogenic variants in autosomal recessive genes (7/27), and the absence of detectable candidate variants (14/27). Despite the cohort’s heterogeneity, a mutation detection rate of 53% was achieved, aligning with published data on genetic diagnoses in Inherited Retinal Disorders (IRD).

Conclusion: This study deepens our understanding of Inherited Retinal Disorders (IRD), underscoring the necessity for integrated clinical and genetic approaches in both diagnosis and management. The identification of unreported pathogenic variants broadens insights into the pathological molecular mechanisms of these disorders, laying the groundwork for further functional and diagnostic investigations in unresolved cases.

Grants:

Conflict of Interest: None declared

EP04.033 Our experience in analysing stereocilin (STRC) gene alterations detected by next-generation sequencing

Sara Álvaro Sánchez ¹, mar borregán prats¹, Judith Armstrong^1;2;3, Daniel Castillo¹, Sara Cardelus Vidal⁴, Águeda Díaz Anadon⁴, Maurizio Levorato⁴, Francesc Palau^1;5;6, Loreto Martorell Sampol¹

¹Sant Joan de Déu Barcelona Hospital, Molecular and Genetic Medicine Department, Esplugues de Llobregat, Spain; ²Institut de Recerca Sant Joan de Déu (IRSJD), Genómica para el diagnóstico de enfermedades raras, Barcelona, Spain; ³CIBER - Center for Biomedical Research Network, CB06/07/0061, Madrid, Spain; ⁴Sant Joan de Déu Barcelona Hospital, Otorhinolaryngology Department, Esplugues de Llobregat, Spain; ⁵Institut de Recerca Sant Joan de Déu (IRSJD), Neurogenética y Medicina Molecular, Barcelona, Spain; ⁶CIBER - Center for Biomedical Research Network, CB06/07/0001, Madrid, Spain

Background/Objectives: Approximately 60% of hearing loss cases have a genetic basis, identifying it is crucial for prognosis and treatment.

Biallelic mutations in STRC gene result in DFNB16. STRC is located in a complex chromosomal region, which comprises its pseudogene (STRCP1).

Acknowledging NGS limitations in detecting Copy-Number-Variants (CNVs), we aim to elucidate STRC alterations identified through short-read exome sequencing by comparing them with multiplex ligation-dependent probe amplification (MLPA) technique.

The goal is to provide an accurate genetic diagnosis for patients with DFNB16.

Methods: Exomes were analysed using a custom Hearing~Loss panel. Cases without STRC alterations were excluded.~The remaining were grouped and CNVs were validated using MLPA.

Results: DFNB16 patients were categorized into six groups:

Results were not always coincident; for solving challenging cases, MLPA and familial segregation studies were necessary. The main cause of DFNB16 was large homozygous CNVdeletion of 48.5kb, encompassing STRC and the adjacent CASPER2 gene.

Conclusion: While NGS has limitations in detecting CNVs at STRC, complementing with MLPA allows for precise genetic diagnoses. Careful interpretation of the results by experienced professionals is required to provide accurate information and, consequently, appropriate genetic counselling for these families.

Grants: Not aplicable.

Conflict of Interest: None declared

EP04.034 Blended phenotypes in rare Inherited Retinal Dystrophies; Coinheritance of CNGB3-associated Achromatopsia and RLBP1-related Retinitis Punctata Albescens

Ayse Gurel ¹, Omer Yildiz²

¹Abant Izzet Baysal University Izzet Baysal Training and Research Hospital, Department of Medical Genetics, Bolu, Türkyie; ²Abant Izzet Baysal University Izzet Baysal Training and Research Hospital, Department of Ophthalmology, Bolu, Türkyie

Background/Objectives: Achromatopsia (ACHM, MIM #262300) is a congenital cone dystrophy characterized by poor color discrimination, low visual acuity, nystagmus and photoaversion. It is caused by biallelic mutations in CNGB3, a gene encoding cone photoreceptor channel subunits. Concurrently, Retinitis Punctata Albescens (RPA, MIM #136880) is a fleck retina disease presenting with yellow-white deposits throughout the retina and night blindness, caused by biallelic mutations in the RLBP1 gene. This study reports the coinheritance of homozygous pathogenic mutations in CNGB3 and RLBP1 genes within the proband, clarifying the notable clinical variability observed among affected siblings.

Methods and results: The proband, 34-year-old female born to consanguineous parents, displayed poor visual acuity and absent color discrimination since birth, along with photosensitivity. Ophtalmological evaluation revealed myopia, nystagmus and flecks over the fundus. The proband’s brother presented with photosensitivity, absent color vision and reduced visual acuity while the affected sister, clinically diagnosed with retinitis pigmentosa, exhibited progressive vision impairment and night blindness. Clinical exome sequencing of the proband revealed a homozygous splice acceptor mutation in CNGB3 c.1579-1G > A and a homozygous missense mutation located in the last nucleotide of exon 5 of RLBP1 c.346G>C (p.Gly116Arg), presumably affecting the normal splicing mechanism.

Conclusion: To the best of our knowledge, this study demonstrates the coinheritance of ACHM and RPA for the first time in the literature, accentuating mutually deleterious outcome in the proband. The blended phenotypes observed in Inherited Retinal Dystrophies, which are clinically and genetically heterogeneous, highlight the importance of comprehensive gene panels and detailed evaluation of intrafamilial phenotypic variability.

Grants:

Conflict of Interest: None declared

EP04.035 Prevalence of STRC variants among mild-moderate hearing loss or presumptive autosomal recessive inheritance in the Brazilian population

Stella Diogo-Cavassana¹, Danillo Alencar-Coutinho¹, Allan Anjos-Monteiro¹, Thais Pailo-Carvalho², Emi Z. Murano³, Jeanne Oiticica^1;3, Ricardo F. Bento^1;3, Regina Mingroni-Netto², Ana Carla Batissoco¹, Karina LEZIROVITZ ¹

¹Hospital das Clínicas HCFMUSP, Faculdade de Medicina, Universidade de São Paulo, Laboratório de Otorrinolaringologia/LIM32, São Paulo, Brazil; ²Instituto de Biociências, Universidade de São Paulo, Departamento de Genética e Biologia Evolutiva, Centro de Pesquisas sobre o Genoma Humano e Células-Tronco, São Paulo, Brazil; ³Hospital das Clínicas HCFMUSP, Faculdade de Medicina da Universidade de São Paulo, ENT Department, São Paulo, Brazil

Background/Objectives: Hereditary non-syndromic hearing loss has 80% of its inheritance transmitted recessively. Regarding mild to moderate hearing loss, studies have suggested that pathogenic variants in STRC (DFNB16), especially large deletions, should be considered as the second major cause of this phenotype, with an estimated frequency between 1% to 5%. Due to the high prevalence of STRC causative variants, STRC screening is recommended in laboratory routines around the world. A genome sequencing study of 2097 normal-hearing Brazilian individuals revealed a heterozygous ~57.9kb STRC microdeletion in 38 individuals; which suggested it as the most recurrent CNV (AF = 0.91%) in Brazilians. The present study aims to determine the frequency of STRC deletions by investigating selected Brazilian subjects with mild-moderate hearing loss or presumptive autosomal recessive inheritance.

Methods: The study subjects were selected for STRC deletion screening based on mild-moderate hearing loss or presumptive autosomal recessive inheritance. A TaqMan-based qPCR assay was used to identify STRC CNVs. In cases with heterozygous CNVs, a long-range PCR and sequencing assay screened the hemizygous allele.

Results: Our cohort comprised 66 mild-moderate and 70 presumptive autosomal recessive cases. To date, three Brazilian families showed homozygosity for STRC deletions, all exhibiting familial recurrence of moderate deafness.

Conclusion: Given the high prevalence of STRC deletions among subjects with moderate hearing loss, we suggest screening as a routine investigation. Further studies will aim to find if it is the same deletion found in the general Brazilian population.

Grants: The São Paulo Research Foundation FAPESP funded this research (numbers 23/07188-7 and CEPID 2013/08028-1).

Conflict of Interest: None declared

EP04.036Frequency of STRC-related hearing loss in GJB2 negative individuals

Silvia Borecka ¹, Klaudia Cipkova¹, Lukas Varga^1;2, Dimitrios Paouris³, Martina Skopkova¹, Miroslava Huckova¹, Dominika Hromnikova¹, Milan Profant², Daniela Gasperikova¹

¹Institute of Experimental Endocrinology, Biomedical Research Center SAS, Bratislava, Slovakia; ²Medical Faculty of Comenius University and University Hospital, Department of Otorhinolaryngology – Head and Neck Surgery, Bratislava, Slovakia; ³Medical Faculty of Comenius University and National Institute of Children’s Diseases, Clinic of Pediatric Otorhinolaryngology, Bratislava, Slovakia

Background/Objectives: Deletions and mutations in the STRC gene are the second most common cause of autosomal recessive, mild to moderate, nonsyndromic sensorineural hearing loss (SNHL). Larger deletions overlapping into the CATSPER2 gene may lead to a syndromic hearing disorder associated with male infertility (DIS syndrome). Exploring these changes is methodologically complicated due to a high homology between the STRC gene and its pseudogene. The aim of our work was to identify the copy number variations and pathogenic variants in the STRC gene in a selected population of Slovak patients.

Methods: 371 GJB2 negative probands with mild to moderate SNHL onset up to 15 years were selected out of the 1 200 probands registered in the SNHL biobank. For determination of the genetic etiology, MLPA, long-range PCR, nested PCR, Sanger sequencing and KASP genotyping assay were used.

Results: We identified a homozygous deletion of the STRC, CKMT1B and CATSPER2 region in 8 probands. Five of these probands are male, suggesting the DIS syndrome. Homozygous deletions of separate STRC exons of different lengths were observed in another 8 probands. Eight probands were compound heterozygotes for deletion of the STRC gene and pathogenic variants on the second allele. The variant R1073* was the most frequent one and was detected in five probands.

Conclusion: The genetic etiology of hearing impairment was determined in 24 out of 371 probands (6,5%). Our results confirmed that changes in the STRC gene are the second most common cause of hearing loss in Slovakia.

Grants: APVV-20-0236, VEGA 1/0572/21

Conflict of Interest: None declared

EP04.038 Genetics of eye disorders in Cypriot patients

Andri Miltiadous ¹, Paul Costeas¹, maria kanezou¹, Petroula Gerasimou¹, rafaella metaxa², Emily Athanasiou³, George A. Tanteles⁴, Violetta Anastasiadou²

¹Karaiskakio Foundation, Molecular Diagnostic Laboratory; ²Karaiskakio Foundation, Genetics Clinic; ³Makario Children’s Hospital, Genetics Department, Cyprus; ⁴The Cyprus Institute of Neurology and Genetics, Egkomi Lefkosias, Cyprus

Background/Objectives: Inherited eye diseases are the leading cause of blindness in adults and in children. Diagnosis of these disorders has witnessed significant progress the last decade in the advent of next generation sequencing.

Methods: Patients affected with eye diseases including but not limited to retinitis pigmentosa, glaucoma, cone rod dystrophy, leber congenital amaurosis, and ocular albinism were referred to our laboratory and NGS sequencing was performed with Agilent’s Exome V8 panel and data were analysed using Franklin software.

Results: Diagnostic rate in eye disease genetic investigation was >80% and causative or likely causative variants were reported in the following genes:ABCA4, CRB1, TYR, MAK, FOXC1, POC1B, NR2E3, GRK1, CFH, EYS, GPR143, RPE65. In addition syndromic cases with eye abnormalities were reported to harbour variants in BBS7 and CEP41.

Conclusion: Eye disease is a significant cause of vision loss and therefore it’s crucial that is is diagnosed early on the course of disease for the correct type of treatment and/or surveillance. Non syndromic eye diseases with known monogenic causes have a relatively high diagnostic rate due to the very specific phenotype that is investigated compared to broader or more complex phenotypes.

Grants: No grants were received for this study

Conflict of Interest: None declared

EP04.039 Oculocutaneous albinism

Elena Colastra Ugena ¹, Ana Peña Cabia¹, Irene Pereira Gonzalez¹, Raquel Gomez Molina¹, Maria Perez Gomez¹, Maria Concepcion Calderon Alva¹

¹Hospital Virgen de la Luz, Análisis Clínicos, Cuenca, Spain

Background/Objectives: 19-year-old woman with a personal history of hypermetropia and astigmatism, with a suspected diagnosis of macular dystrophy.

Methods: A clinical exome was performed to test for genes related to hereditary retinal dystrophies, and no variant was detected that could explain its phenotype.

It was decided to extend the study to genes related to albinism, since it presented flat macules with absence of fovea.

Results: In this reanalysis, three genetic variants were identified in the TYR gene:

• c.405del(p.Phe135Leufs*6) in heterozygosis, classified as probably pathogenic.

• c.575C>A(p.Ser192Tyr) in heterozygosity and c.1205G>A(p.Arg402Gln) in homozygosis, both classified as of uncertain clinical significance.

The detection of these variants is compatible with the patient’s clinical features.

A family segregation study is requested from the parents to determine the phase of the variants identified, since pathogenic variants in the TYR gene are associated with oculocutaneous albinism type IA and IB (autosomal recessive pattern).

After analysis, the p.Arg402Gln variant is in cis phase with the p.Ser192Tyr variant, and both are in trans phase with the p.Phe135Leufs variant. The p.Arg402Gln variant is considered a hypomorphic allele and when combined with the p.Ser192Tyr variant in cis, it is estimated to have a loss of function effect equal to or greater than a pathogenic variant in the TYR gene.

Conclusion: The patient is a carrier in compound heterozygosis for variants probably pathogenic in the TYR gene, diagnosis of the disease.

Family segregation study plays a fundamental role in the diagnosis of this disease, thus it is essential to provide adequate genetic counseling.

Grants: None

Conflict of Interest: None declared

EP04.042 A patient with ATOH7 variants with Delayed Sleep-Wake Phase Disorder and Optic Nerve Hypoplasia

Jennifer Brzezynski ¹, Caroline Johnson¹, Sandra Smieszek¹, Changfu Xiao¹, Christos Polymeropoulos¹, Mihael Polymeropoulos¹

¹Vanda Pharmaceuticals Inc., Washington, DC, United States

Background/Objectives: We are conducting a study to evaluate the effects of tasimelteon in Delayed Sleep-Wake Phase Disorder (DSWPD) patients. We report our first completed patient from the Open-Label Extension (OLE), a 24-year-old female diagnosed with DSWPD and Optic Nerve Hypoplasia (ONH), with delayed Dim Light Melatonin Onset.

Methods: The study consists of screening and treatment, followed by an OLE to explore the safety and efficacy of daily dosing with tasimelteon over 11 months. During the OLE, patients answer sleep diaries and take one dose of tasimelteon 1 hour before their desired bedtime.

Results: The patient’s average sleep onset was 01:27 during screening and 00:42 during the OLE, an improvement of 45 minutes. At screening, the patient reported their symptoms as moderate on the Patient Global Impression of Severity (PGI-S). On average during the OLE, the patient reported their symptoms as mild on the PGI-S.

The patient’s history of ONH could indicate lack of proper optic response to light. They carry two variants in the ATOH7 gene, rs61854782 and rs7916697, known to be associated with ONH. They have a VNTR PER3^5/5 genotype, associated with normal sleep patterns, and no identified predicted loss-of-function mutations within other circadian genes.

Conclusion: This case illustrates the positive effect of tasimelteon on a patient diagnosed with DSWPD, based on an earlier sleep onset shift and improvement in PGI-S responses. It also provides the opportunity for research into ONH and ATOH7 variants, and the relationship with DSWPD and sighted Non-24.

Grants: Not Applicable.

Conflict of Interest: Jennifer Brzezynski Employee and stock owner., Employee and stock owner., Caroline Johnson Employee and stock owner., Employee and stock owner., Sandra Smieszek Employee and stock owner., Employee and stock owner., Changfu Xiao Employee and stock owner., Employee and stock owner., Christos Polymeropoulos Employee and stock owner., Employee and stock owner., Mihael Polymeropoulos CEO and stock owner., CEO and stock owner.

EP04.043 Optical Genome Mapping facilitates rapid characterisation of structural variants in families with developmental eye anomalies

Solomon Merepa ¹, Karthikah Jeganathan¹, Bertrand Chesneau^1;2;3, Richard Holt¹, Lidiya Talbot¹, Fabiola Ceroni¹, Dorine Bax¹, Fiona Watkins¹, Mira Kharbanda⁴, Katherine Lachlan⁴, Patrick Calvas^3;5, Julie Plaisancié^3;5, Nicolas Chassaing^3;5, Elfride De Baere^6;7, Nicola Ragge^1;8

¹Oxford Brookes University, Faculty of Health and Life Sciences, Oxford, United Kingdom; ²Université de Toulouse, Molecular, Cellular and Developmental Biology Unit (MCD), Centre de Biologie Intégrative (CBI), Toulouse, France; ³CHU de Toulouse, Centre de Référence pour les Affections Rares en Génétique Ophtalmologique (CARGO), Toulouse, France; ⁴University Hospital Southampton NHS Foundation Trust, Wessex Clinical Genetics Service, Southampton; ⁵Laboratoire National de Référence (LBMR), Génétique des anomalies malformatives de l’œil, Toulouse, France; ⁶Ghent University, Department of Biomolecular Medicine, Ghent, Belgium; ⁷Ghent University Hospital, Center for Medical Genetics Ghent (CMGG), Ghent, Belgium; ⁸Birmingham Women’s and Children’s NHS Foundation Trust, West Midlands Regional Clinical Genetics Service and Birmingham Health Partners, Birmingham, United Kingdom

Introduction: Developmental eye anomalies, including anophthalmia, microphthalmia and coloboma (AMC), are a genetically heterogeneous group of disorders. Structural variants (SVs) affecting coding and non-coding regions of the genome can alter expression, function and regulation of genes implicated in eye development. Conventional diagnostic methods, including array CGH and WGS, can limit resolution and/or exact nature of rearrangements. Here, we use Optical Genome Mapping (OGM) to identify and characterise SVs in a cohort of individuals with AMC.

Methods: Using ultra-high molecular weight DNA, we screened 53 AMC families for SVs using OGM (Bionano Genomics, USA).

Results: We identified heterozygous deletions involving known eye development genes in the retinoic acid pathway (ALDH1A3 and STRA6) in two independent families with bilateral anophthalmia. These SVs were inherited in trans with corresponding rare SNVs, providing diagnosis. In three further families, OGM characterised complex rearrangements involving genes part of pathways relevant to eye development; i) a complex SV with two deletions flanking an inversion affecting CYP26A1 and CYP26C1, genes encoding enzymes involved in retinol metabolism, affecting two unrelated families; ii) an individual with 3 separate SVs, including a chromosome 11q13 duplication containing SIMO and other cis regulatory elements of PAX6; and iii) an insertional translocation involving chromosomes 3 and 5 which may impact important regulatory elements.

Conclusion: We demonstrate that OGM can easily identify clinically relevant SVs, including complex rearrangements, in individuals with AMC. This technology has the potential to complement clinical genetic testing, and support research into the mechanisms underlying such genetic disorders.

Funding: Baillie Gifford

Conflict of Interest: None declared

EP05.001 Long read genome sequencing facilitates CNV mapping at highly homologous loci

Nina Bögershausen ¹, Alexander Wolff¹, Yun Li¹, Tassilo Wollenweber², Julia Schmidt¹, Silke Kaulfuß¹, Karl Koehrer², Arne Zibat¹, Gökhan Yigit^1;3, Bernd Wollnik^1;3;4

¹Institute of Human Genetics, University Medical Center Göttingen, Göttingen, Germany; ²Genomics and Transcriptomics Laboratory, Biologisch-Medizinisches-Forschungszentrum (BMFZ), Düsseldorf, Germany; ³German Center for Cardiovascular Research (DZHK), partner site Göttingen, Göttingen, Germany; ⁴Cluster of Excellence “Multiscale Bioimaging: from Molecular Machines to Networks of Excitable Cells” (MBExC), University of Göttingen, Göttingen, Germany

Background/Objectives: High sequence similarity in segmental duplications can facilitate copy number variation (CNV). Within such regions, sequence identity may hinder exact short read mapping and consequently, the detection of CNV boundaries by short read sequencing (SRS) approaches. Here, we present a family with two siblings, affected by juvenile onset exocrine pancreatic insufficiency (EPI), in whom long read genome sequencing (LRGS) was successfully applied.

Methods: We initially employed short read exome sequencing and/or qPCR in both siblings and their parents. Additionally, we performed HiFi LRGS in one sibling, using the PacBio Sequel II system, and analyzed the data with our in-house pipeline.

Results: Exome sequencing identified a homozygous deletion at the CELA3A / CELA3B locus in both affected siblings. CELA3A and CELA3B are highly homologous genes, likely owing to gene duplication. The boundaries of the deletion could not be determined with SRS due to misalignment. LRGS, with an average read length of ~20.000 bp, allowed us to map the breakpoints more precisely. The deletion creates a CELA3A/CELA3B fusion gene, possibly disrupting the function of both genes. CELA3A and CELA3B code for digestive enzymes that are detected clinically as stool elastase. The fact that the deletion affects both genes, likely explains the observation of extremely low stool elastase levels in both siblings.

Conclusion: We present a biallelic deletion at the CELA3A/B locus as a potential novel cause for EPI and demonstrate the efficiency of LRGS for breakpoint mapping in highly homologous regions. We aim to identify further individuals with CELA3A/B associated EPI.

Grants: Deutsche Forschungsgemeinschaft (Project 417959134)

Conflict of Interest: None declared

EP05.002 Outcomes of patients with Juvenile Polyposis - Hereditary Haemorrhagic Telangiectasia caused by pathogenic SMAD4 variants in a pan-Scotland cohort.

Madeline Pearson ¹, Ruth McGowan², Philip Greene³, Zosia Miedzybrodzka⁴, jonathan berg¹

¹University of Dundee, School of Medicine, United Kingdom; ²West of Scotland Centre for Genomic Medicine, United Kingdom; ³South East of Scotland Clinical Genetics Services, United Kingdom; ⁴University of Aberdeen, School of Medicine, United Kingdom

Background/Objectives: SMAD4 is integral to mediating signal transduction for serine threonine kinase receptors. Germline loss of SMAD4 function (LOF) results in Juvenile Polyposis-Hereditary Haemorrhagic Telangiectasia Overlap Syndrome (JP-HHT). JP-HHT is associated with life-threatening complications including gastrointestinal cancers and pulmonary arteriovenous malformations (PAVMs). We investigated the JP-HHT phenotype in a Scottish cohort.

Methods: A retrospective multi-centre case-note review identified 28 patients with a pathogenic LOF SMAD4 variant from 13 families across all Scottish Clinical Genetics Centres, providing a complete clinical picture of the Scottish JP-HHT cohort.

Results: Colonic polyps were identified in 87%(20/23) of screened patients. 43%(10/23) of patients with polyps required a colectomy at a median age of 24years (IQR20.0–47.0). Colorectal cancer occurred in 25%(7/28) of patients at a median age of 33years (IQR29.5–45.0).

Gastric polyps were identified in 67%(12/18) of screened patients. 42%(5/12) of patients with gastric polyps required a gastrectomy at a median age of 47years (IQR37.0–59.0). 1 patient was diagnosed with gastric cancer, at age 66 years.

PAVMs were identified in 47%(9/19) of screened patients. When investigated for, epistaxis and telangiectasia were present in 75%(15/20) and 29%(4/14) of patients respectively.

88%(23/26) and 81%(17/21) of patients exhibited JP and HHT features respectively, with 70%(14/20) demonstrating features of both conditions.

Conclusion: SMAD4 variant carriers are at very high risk of life-threatening gastrointestinal disease, including early onset bowel cancer and gastric obstruction caused by non-malignant polyposis. PAVM incidence was at least equivalent to that seen in HHT type 1, if not higher. All individuals with pathogenic LOF SMAD4 variants require comprehensive screening.

Grants:

Conflict of Interest: None declared

EP05.003 Defining PKD1 variants in patients with cystic kidney

Deimante Brazdziunaite ¹, Gabija Mazur², Algirdas Utkus¹

¹Institute of Biomedical Sciences, Faculty of Medicine, Vilnius University, Vilnius, Lithuania; ²Vilnius University Hospital Santaros Klinikos, Vilnius, Lithuania

Background: PKD1 gene pathogenic variants are the most common cause of autosomal dominant polycystic kidney disease (ADPKD; OMIM #173900; ORPHA:730), which is the most common in the group of cystic kidney diseases. The ADPKD database counts more than 1200 pathogenic/likely pathogenic variants in PKD1, while more than 200 remain as variants of uncertain significance (VUS).

Aim: To identify and classify genetic variations within PKD1 gene among patients exhibiting multiple renal cysts.

Methods: 62 patients with multiple renal cysts were included in the study. The study group consisted of 35 women and 27 men; 57 adults and 5 children. Patients were tested with a kidney-focused next-generation sequencing panel of 498 genes, including the main polycystic kidney disease genes PKD1, PKD2, PKHD1, ALG5, DZIP1L, DNAJB11, GANAB. Pathogenicity of detected variants was assessed using the American College of Medical Genetics and Genomics guidelines.

Results: Variants in PKD1 gene were identified in 34 patients, of which 18 were not described in the literature. Family history was negative for 11 PKD1-patients. After evaluating pathogenicity of the variants, 26 were classified as pathogenic/likely pathogenic, and 8 as VUS. In 12 patients clinically significant variants were not identified in the analyzed genes. However, some of those patients were not clinically diagnosed with polycystic kidney disease or had an atypical form of the disease.

Conclusion: A genetic diagnosis was established for the majority of patients in the conducted study. All PKD1 variants identified in the study are unique and did not repeat between families.

Conflict of Interest: None declared

EP05.004 PERCC1-associated congenital enteropathy: Delineating the natural history of a new disorder of enteroendocrine cell function

lev dorfman^1;2;3, Mansa Krishnamurthy^4;5, Yael Haberman^1;3;6;7, emily stenke⁸, Anat Guz Mark^2;3, Hengameh Abdollahpour⁹, seba saleh nadeef¹⁰, Khaled Warasnhe¹¹, Figen Ozcay¹², vivien a. nguyen¹³, Martin G Martin¹⁴, stanley f. nelson^15;16, Julian Martinez-Agosto^14;15, james m. wells^4;5;17, Fowzan Alkuraya^10;18, Billy Bourke^8;19, Yair Anikster^3;20;21, Ben Pode-Shakked ^3;20

¹Cincinnati Children’s Hospital Medical Center, Division of Gastroenterology, Hepatology and Nutrition, Cincinnati, United States; ²Schneider’s Children’s Medical Center, Institute of Gastroenterology, Nutrition and Liver Diseases, Petach Tikva, Israel; ³Tel-Aviv University, Faculty of Medicine, Tel Aviv, Israel; ⁴Cincinnati Children’s Hospital Medical Center, Division of Endocrinology, Cincinnati, United States; ⁵Cincinnati Children’s Hospital Medical Center, Center for Stem Cell and Organoid Medicine (CuSTOM), Cincinnati, United States; ⁶Edmond and Lily Safra Children’s Hospital, Sheba Medical Center, Pediatric Gasteroenterology Unit, Ramat Gan, Israel; ⁷University of Cincinnati College of Medicine, Department of Pediatrics, Cincinnati, United States; ⁸Children’s Health Ireland-Crumlin, National Centre for Paediatric Gastroenterology, Dublin, Ireland; ⁹University Medical Center Hamburg-Eppendorf, Institute of Human Genetics, Hamburg, Germany; ¹⁰King Faisal Specialist Hospital and Research Center, Department of Translational Genomics, Center for Genomic Medicine, Riyadh, Saudi Arabia; ¹¹Başkent University Faculty of Medicine, Ankara, Turkey, Department of Pediatrics, Ankara, Türkyie; ¹²Başkent University Faculty of Medicine, Department of Pediatric Gastroenterology and Hepatology, Ankara, Türkyie; ¹³UCSF Benioff Children’s Hospital, Division of Pediatric Gastroenterology, Hepatology and Nutrition, Oakland, United States; ¹⁴University of California Los Angeles, Department of Pediatrics, Los Angeles, United States; ¹⁵University of California Los Angeles, Department of Human Genetics, David Geffen School of Medicine, Los Angeles, United States; ¹⁶University of California Los Angeles, Department of Neurology, David Geffen School of Medicine, Los Angeles, United States; ¹⁷Cincinnati Children’s Hospital Medical Center, Division of Developmental Biology, Cincinnati, United States; ¹⁸College of Medicine, Alfaisal University, Department of Anatomy and Cell Biology, Riyadh, Saudi Arabia; ¹⁹University College Dublin, School of Medicine, Dublin, Ireland; ²⁰Sheba Medical Center, Metabolic Disease Unit, Edmond and Lily Safra Children’s Hospital, Ramat Gan, Israel; ²¹Sheba Medical Center, The Wohl Institute for Translational Medicine, Ramat Gan, Israel

Consortium: Undiagnosed Diseases Network

Abstract

Background: Congenital diarrheas and enteropathies (CODEs) constitute a heterogeneous group of individually rare disorders, characterized by infantile-onset chronic diarrhea. Recently, biallelic deletions encompassing an unannotated open reading frame termed PERCC1, were implicated in an autosomal-recessive CODE among Iraqi-Jewish families. Disruption of PERCC1 was further shown to hamper enteroendocrine cell (EEC) function in organoids and in vivo models. We sought to elucidate the natural history of this newly-recognized disorder.

Methods: Clinical data was obtained directly from the patient and/or their parents, via a virtual semi-structured medical interview. When a direct interview was not possible, clinical data was collected by the physician caring for the patient, based on their medical records.

Results: An international cohort of n = 11 patients was recruited, at an average age of 18.5 years (range, 2-37). Molecular diagnosis was reached at an average age of 14.8 years (range, 1.5m-36y). TPN was required in all patients, and was successfully weaned off in 9/11, at an average age of 8 years (range, 10m-20y). The PERCC1-related genotype underlying their disease varied between biallelic chromosomal deletions (6/11 patients; 54%); a recently identified stopgain mutation (c.390C>G) shared by unrelated Irish patients (3/11; 27%); a novel c.555C>G point mutation (1/11; 9%); or maternal UPD of chromosome 16 encompassing PERCC1 harboring a novel c.348C>G truncating variant (1/11; 9%).

Conclusions: PERCC1-associated congenital enteropathy is an ultra-rare, underdiagnosed disorder, typically manifesting at early infancy with persistent watery diarrhea and hypernatremic dehydration. Our findings expand the current knowledge regarding both the molecular basis and phenotypic spectrum of this disorder.

Conflict of Interest: None declared

EP05.005 Broadening the phenotype of MAFB disease spectrum- kidney, eye, ear and nervous system involvement

aviva eliyahu ^1;2;3, Danit Atias-Varon⁴, Ortal Barel Barel⁵, Yulia Khavin⁵, Elon Pras³, haike reznik wolf⁶, Lior Greenbaum^3;6, Pazit Beckerman^3;7, Nabil Abu amer^3;7, Tamara Vignanski-Jaffe^3;8, Asaf Vivante^3;9;10

¹Sheba, The Danek Gertner Institute of Human Genetics, Ramat Gan, Israel; ²Bar Ilan university, The Goodman faculty of life science, Ramat Gan, Israel; ³Tel-Aviv University, Faculty of Medicine, Tel aviv, Israel; ⁴Edmond and Lily Safra Children’s Hospital, Sheba Medical Center, Tel-Hashomer, Pediatric Nephrology Unit, Ramat Gan, Israel; ⁵Sheba Medical Center, Tel-Hashomer, The Genomic Unit, Sheba Cancer Research Center, Ramat Gan, Israel; ⁶Sheba Medical Center, Tel-Hashomer, The Danek Gertner Institute of Human Genetics, Ramat Gan, Israel; ⁷Sheba Medical Center, Tel-Hashomer, Institute of Nephrology and Hypertention, Ramat Gan, Israel; ⁸Sheba Medical Center, Tel-Hashomer, Goldschlager Eye Institute, Ramat Gan, Israel; ⁹Sheba Medical Center, Tel-Hashomer, Department of Pediatrics B, Edmond and Lily Safra Children’s Hospital, Ramat Gan, Israel; ¹⁰Sheba Medical Center, Tel-Hashomer, Pediatric Nephrology Unit, Edmond and Lily Safra Children’s Hospital, Ramat Gan, Israel

Background/Objectives: Focal segmental glomerulosclerosis (FSGS) is a leading cause of steroid resistant nephrotic syndrome (SRNS) and chronic kidney disease (CKD). Monogenic etiologies are detected with increasing frequency for FSGS. Many of the disease-causing genes are involved in the structure of the slit diaphragm and podocyte maintenance.

Missense variants in the MAF BZIP Transcription Factor B (MAFB) gene have been recently reported as a cause of a dominantly inherited syndrome with variable expressivity, ranging from syndromic FSGS to isolated renal or ocular disease.

Methods: We report herein five affected individuals, members of an extended family, presenting with proteinuria and variable renal and extra-renal phenotype.

Results: A novel heterozygous missense variant c.797T>C p(Leu266Pro) in MAFB was made in the proband and in a sibling by Exome Sequencing (ES) and subsequent variant segregation by Sanger sequencing in the mother.

In addition to CKD and FSGS, the proband presented with congenital auricular malformations, hearing loss and neurodevelopmental delay. Her male sibling had Duane retraction syndrome (DRS) and neurodevelopmental involvement, while an additional family member presented with an isolated renal phenotype. Some of these clinical features have not been previously determined as part of the MAFB phenotypic spectrum.

Conclusion: Taken together, MAFB-related disease spectrum is broader than previously described. Awareness of this may promote accurate diagnosis and improve medical treatment.

Grants: N/A

Conflict of Interest: None declared

EP05.006 Multi-locus imprinting disturbances in patients with PHP1B/iPPSD3

Yerai Vado¹, Africa Manero Ruiz de Azua¹, Arrate Pereda¹, Guiomar Perez de Nanclares Leal ¹

¹Bioaraba Health Research Institute, Genetics, Epigenetics and Cellular Biology, Gasteiz, Spain

Background/Objectives: iPPSD3 (previously known as pseudohypoparathyroidism 1B) is a rare epigenetic disorder characterized by PTH resistance, hypocalcemia, hyperphosphatemia and, sometimes, TSH resistance. It is caused by epigenetic variants occurring in one or more GNAS-DMR, with hypomethylation at GNAS A/B:TSS-DMR always present. In some imprinting disorders, methylation disturbances at multiple loci (MLID) have been described and genetic defects at genes involved in methylation establishment/maintenance reported. Our interest was to determine if MLID is present in iPPSD3 and its genetic causes.

Methods: 90 patients with epigenetically confirmed iPPSD3 were checked for MLID by MS-MLPA (kit ME034-C1). 49 of these individuals were also studied with a custom targeted methylation sequencing panel (ImprintSeq). Samples of 30 trio or, at least duo (index and mother) were analysed by NGS with a custom panel of imprinting regulator genes, specifically looking for maternal effect genes.

Results: From the 90 patients who presented an epigenetic defect, the genetic cause was identified in 24 (18 with STX16 deletions and 6 with upd(20)pat). MLID was not observed by ME034-C1, but ImprintSeq detected it in 18 patients affecting different DMRs with (mainly) no known clinical effect. Preliminary analyses of maternal effect genes have allowed the identification of some VUS variants.

Conclusion: In MLID-iPPSD3 cases (36.7% of our series) the involved DMRs are not included in currently available MS-MLPA kits and their clinical significance is unknown. Further clinical studies are needed to evaluate their impact.

Grants: Institute of Health Carlos III (PI20/00950); Basque Department of Health (GV2021/111056).

Conflict of Interest: None declared

EP05.007 Growth Hormone deficiency: spectrum of causal genetic variants in Portuguese patients

Ana C. Ribeiro ¹, Najeeb Syed², Luís R. Saraiva³, Manuel C. Lemos¹

¹CICS-UBI - Health Sciences Research Centre, University of Beira Interior, Covilhã, Portugal; ²Laboratory of Genomic Medicine, Sidra Medicine, Doha P.O. Box 26999, Qatar; ³Translational Medicine Division, Sidra Medicine, Doha P.O. Box 26999, Qatar

Background/Objectives: Growth Hormone (GH) deficiency is a rare disorder which in the absence of treatment can result in severe short stature. Combined (CPHD) or isolated (IGHD) GH deficiency can have a genetic origin, due to variants in genes affecting the hypothalamic-pituitary development and/or function. We aimed to characterize the causal variants of GH deficiency in a cohort of 205 Portuguese patients.

Methods: This study included 81 IGHD and 124 CPHD patients. The GH1 and GHRHR or PROP1 genes were analysed by Sanger sequencing in these groups, respectively. In patients without causal variants, and in 324 controls, whole exome sequencing (WES) was performed. Putatively pathogenic variants were filtered in 46 known GH deficiency-related genes.

Results: Causal variants were identified in 20% of GH deficiency patients. These variants were distributed across the PROP1, GLI2, PROK2, PROKR2, CDON, CHD7, GHRHR, and HESX1 genes. Regarding controls, only 2% had variants that would potentially originate this disorder. Comparing the frequency of individuals with variants of uncertain significance (VUS), the difference between patients and controls was not statistically significant. However, VUS in the RBM28 and GLI2 genes were significantly more frequent in GH deficiency patients.

Conclusion: A genetic diagnosis was established in 20% of the studied patients. At least for the RBM28 and GLI2 genes, identified VUS may also be involved in the pathogenesis of GH deficiency. Further studies are needed to confirm this hypothesis.

Grants: Portuguese Foundation for Science and Technology (PTDC/SAU-GMG/098419/2008, UIDB/00709/2020, and UI/BD/151021/2021), and Sidra Medicine (SDR200059).

Conflict of Interest: None declared

EP05.008 Dent disease is effectively identified in clinical practice and is well confirmed by whole-exome sequencing: report of seven cases

Anastasiia Buianova ¹, Anastasiia Ryzhova², Mariia Proskura², Edita Petrosyan², Valeriia Gavrilova², Anna Shmitko¹, Alina Samitova¹, Oleg Suchalko¹, Zhanna Repinskaia¹, Galit Ilyina¹, Anna Kuznetsova¹, Iuliia Vasiliadis¹, Natalia Ponikarovskaya¹, Vera Belova¹, Dmitriy Korostin¹

¹Pirogov Russian National Research Medical University, Center for Precision Genome Editing and Genetic Technologies for Biomedicine, Moscow, Russian Federation; ²Pirogov Russian National Research Medical University, Russian Children’s Clinical Hospital, Moscow, Russian Federation

Background/Objectives: Dent disease is a rare renal tubular disorder characterized by X-linked recessive inheritance. It primarily affects the proximal tubules of the kidneys and is divided into two types (associated with CLCN5 and OCRL genes, respectively). The accessibility and high effectiveness of whole-exome sequencing (WES) in patients provide the opportunity for early diagnosis and more timely treatment of such children, delaying end-stage renal disease.

Methods: WES analysis paired with comprehensive clinical evaluation was assessed in seven pediatric male patients with suspected Dent disease.

Results: The patient group is characterized by minor clinical heterogeneity (see table). Variants of patients 1-3 were previously undescribed.

Conclusion: In this study, the preliminary diagnosis was confirmed in all patients, so we ensured that biochemical analysis of blood, urine tests, kidney ultrasound, as well as a physical examination combined with history-taking are exhaustive differential diagnostic methods for Dent disease.

Grants: №075-15-2019-1789.

Conflict of Interest: None declared

EP05.009 Novel targets for unravelling renal tubular injury in diabetes and hypertension: Effect of increased exosomal levels of miR-200a-3p

Olga Martinez-Arroyo ¹, Ana Flores-Chova¹, Marta Mendez-Debaets¹, Laia Garcia-Ferran¹, Lorena Adan¹, Lesley Escriva¹, Sara Vela¹, Carlos Bea^1;2, Josep Redon^1;2;3;4, Maria J Forner^1;2;3, Raquel Cortés¹, Ana Ortega^1;5

¹Incliva Biomedical Research Institute, Cardiometabolic and Renal Risk Research Group, Valencia; ²Clinic Hospital of Valencia, Internal Medicine Unit, Valencia; ³Medicine’s Faculty, Department of Medicine, Valencia; ⁴Carlos III Health Institute, CIBER OBN; ⁵Carlos III Health Institute, CIBER CV

Background/Objectives: Hypertension and diabetes mellitus (DM) are major risk factors of renal damage. Evidence places the renal tubule as a driving force in kidney disease. Exosomes are disease biomarkers mediating specific cell communication by transferring molecules such as microRNAs. Previously, we found that miR-200a-3p has promising roles in renal injury. We aimed to analyse the levels of miR-200a-3p in exosomes from patient’s urine and deepen into its role in tubular damage.

Methods: Urinary exosomal levels of miR-200a-3p were measured by RT-qPCR in hypertensive patients with and without DM (n = 69) and increased UAE (n = 42). We investigated the exosomal and pellet levels of miR-200a-3p and their targets at mRNA and protein levels (Western blot) in tubular cells (RPTECs) subjected to treatments and observed their influence on apoptosis (flow cytometry), injury and RPTEC markers. Furthermore, we performed mimic and inhibitor miRNAs transfection in treated RPTECs.

Results: We found increased levels of miR-200a-3p in urinary exosomes from patients with increased UAE. Further, this miRNA was able to discriminate UAE with an AUC of 0.75 (p = 0.003). In in vitro experiments, miR-200a-3p and renal injury markers are increased and RPTEC markers and apoptosis decreased, under glucose and angiotensin-II conditions. miR-200a-3p mimic and inhibitor experiments showed an important influence on its targets involved in tubular damage (SIRT1 and CLDN1) and in injury markers.

Conclusion: Increased exosomal levels of miR-200a-3p emerge as a potential disease marker and treatment target to decrease renal tubular injury associated to hypertension and diabetes.

Grants: PI19/01796; PI21/00249; PI23/01179; FI20/00096; FI22/00032; CD00069; IJC2020-045308-I. ERDF.

Conflict of Interest: None declared

EP05.010 Involvement of TBC1D8B novel variant as a cause of x-linked corticosteroid resistant nephrotic syndrome.

Anney Yelena Rosario Vargas ¹, María Jesus Izquierdo Ortiz¹, Irene Oñate Alonso¹, Badawi Hijazi Prieto¹, María Isabel Sáez Calero¹, Marta Boya Fernández¹, Raquel Toro Casado¹, Vanesa Camarero Temiño¹, Basilia González Diez¹, María Luisa Carrasco Prado¹, Alejandra Martin Rosique¹, Sheyla Cristal Álvarez Parra¹

¹Burgos University Hospital, Burgos, Spain

Background/Objectives: Steroid-resistant nephrotic syndrome (SRNS) is a glomerular disease characterized by massive proteinuria, most often associated with a focal and segmental glomerulosclerosis^.1 This histological pattern has variety of potential causes, including rare variants of podocyte-related genes. TBC1D8B plays a regulatory role by inhibiting endogenous Rab11, a key protein in vesicular reycling cells, that interacts with nephrin dynamics in the glomerular podocyte.³

Variants in TBC1D8B gene on the X chromosome can lead to SRNS type 20 and its progression to kidney failure in individuals under 25 years-old.¹ Its most commonly in boys ², women should not express severe manifestations unless the phenomenon of lyonization is present.

We identified novel TBC1D8B variant in a 32 years-old, spanish woman on hemodialysis for unrelated chronic kidney disease since 18 years-old. Additionally, she carries a pathogenic variant in KMT2D, associated with congenital malformations of the kidney-urinary tract and the Kabuki phenotypic spectrum not present in the patient.

Methods: We carried out a genetic study with a global panel of nephropathy (529 genes).

Results: We found that the patient is a heterozygous carrier of the possibly pathogenic variant c.1426C>T:p.Arg476 in the geneTBC1D8B (NM_017752.2).

Conclusion: Pathogenic variants in the TBC1D8B gene are associated with the development of nephrotic syndrome type 20, a kidney disorder with an X-linked inheritance pattern, of variable expression, especially in women in whom the severity of the disease is assumed by the pattern of chromosome X inactivation.

Grants: No grants to declare.

Conflict of Interest: None declared

EP05.012 Exploring genetic variants for the diagnosis of chronic liver disease in the pediatric population

Olga Parshina ¹, Anastasiia Buianova¹, Ekaterina Filimonova², Mariia Venediktova², Alexandra Shagiazdanova², Elena Dobronravova², Stella Temurian², Anna Shmitko¹, Alina Samitova¹, Oleg Suchalko¹, Zhanna Repinskaia¹, Galit Ilyina¹, Iuliia Vasiliadis¹, Anna Kuznetsova¹, Mariia Sidorchuk¹, Natalia Ponikarovskaya¹, Vera Belova¹, Dmitriy Korostin¹

¹Pirogov Russian National Research Medical University, Center for Precision Genome Editing and Genetic Technologies for Biomedicine, Moscow, Russian Federation; ²Pirogov Russian National Research Medical University, Russian Children’s Clinical Hospital, Moscow, Russian Federation

Background/Objectives: Genetic factors contribute to nearly half of the chronic liver diseases diagnosed in childhood [1]. Genetic testing is vital in the diagnosis process, significantly influencing the disease’s progression and improving the overall quality of life for affected children. Approximately 30% of cases employing whole-exome sequencing (WES) have proven beneficial in reaching a diagnosis [2,3].

Methods: We conducted WES on pediatric patients afflicted with chronic liver diseases.

Results: The examined patient group comprised 45 children (median 5 years, age range 1 month−17 years), consisting of 25 boys and 20 girls. Family history of liver diseases was present in only 5 children, and 13 patients had comorbidities. Liver cirrhosis was diagnosed in 4 children, while idiopathic cholestasis was identified in 5 cases. Additionally, 2 children were found to have alpha-antitrypsin deficiency. Among the remaining 34 patients, unclassified hepatitis was predominant. A genetic cause of the disease was identified in 12 cases, with 6 out of 45 patients (13,3%) exhibiting at least one heterozygous genetic variation. The study revealed 19 distinct pathogenic variants, including 8 novel ones, distributed across 11 different genes (JAG1, ATP7B, ABCC2, ATP8B1, KI12, PKD1, SBDS, ABCB11, ABCB4, HADHA, IFIH1) were identified.