Presence Of Pathogenic Variants Research Articles

Lipodystrophic Laminopathies: From Dunnigan Disease to Progeroid Syndromes.

Lipodystrophic laminopathies are a group of ultra-rare disorders characterised by the presence of pathogenic variants in the same gene (LMNA) and other related genes, along with an impaired adipose tissue pattern and other features that are specific of each of these disorders. The most fascinating traits include their complex genotype-phenotype associations and clinical heterogeneity, ranging from Dunnigan disease, in which the most relevant feature is precisely adipose tissue dysfunction and lipodystrophy, to the other laminopathies affecting adipose tissue, which are also characterised by the presence of signs of premature ageing (Hutchinson Gilford-progeria syndrome, LMNA-atypical progeroid syndrome, mandibuloacral dysplasia types A and B, Nestor-Guillermo progeria syndrome, LMNA-associated cardiocutaneous progeria). This raises several questions when it comes to understanding how variants in the same gene can lead to similar adipose tissue disturbances and, at the same time, to such heterogeneous phenotypes and variable degrees of metabolic abnormalities. The present review aims to gather the molecular basis of adipose tissue impairment in lipodystrophic laminopathies, their main clinical aspects and recent therapeutic strategies. In addition, it also summarises the key aspects for their differential diagnosis.

Impact of 12-SNP and 6-SNP Polygenic Scores on Predisposition to High LDL-Cholesterol Levels in Patients with Familial Hypercholesterolemia.

Familial hypercholesterolemia (FH) comprises high LDL-cholesterol (LDL-c) levels and high cardiovascular disease risk. In the absence of pathogenic variants in causative genes, a polygenic basis was hypothesized. In a population of 418 patients (excluding homozygotes) with clinical suspicion of FH, the FH-causative genes and the regions of single nucleotide polymorphisms (SNPs) included in 12-SNP and 6-SNP scores were sequenced by next-generation sequencing, allowing for the detection of pathogenic variants (V+) in 220 patients. To make a comparison, only patients without uncertain significance variants (V-/USV-) were considered (n = 162). Higher values of both scores were observed in V+ than in V-. Considering a cut-off leading to 80% of V-/USV- as score-positive, a lower prevalence of patients positive for both 12-SNP and 6-SNP scores was observed in V+ (p = 0.010 and 0.033, respectively). Mainly for the 12-SNP score, among V+ patients, higher LDL-c levels were observed in score-positive (223 mg/dL -IQR 187-279) than in negative patients (212 mg/dL -IQR 162-240; p = 0.006). Multivariate analysis confirmed the association of scores and LDL-c levels independently of age, sex, and presence of pathogenic variants and revealed a greater association in children. The 12-SNP and 6-SNP polygenic scores could explain hypercholesterolemia in patients without pathogenic variants as well as the variability of LDL-c levels among patients with FH-causative variants.

Breast density in NF1 women: a retrospective study

Neurofibromatosis type 1 (NF1) is an autosomal dominant condition caused by neurofibromin haploinsufficiency due to pathogenic variants in the NF1 gene. Tumor predisposition has long been associated with NF1, and an increased breast cancer (BC) incidence and reduced survival have been reported in recent years for women with NF1. As breast density is another known independent risk factor for BC, this study aims to evaluate the variability of breast density in patients with NF1 compared to the general population. Mammograms from 98 NF1 women affected by NF1, and enrolled onto our monocentric BC screening program, were compared with those from 300 healthy subjects to verify differences in breast density. Mammograms were independently reviewed and scored by a radiologist and using a Computer-Aided Detection (CAD) software. The comparison of breast density between NF1 patients and controls was performed through Chi-squared test and with multivariable ordinal logistic models adjusted for age, body mass index (BMI), number of pregnancies, and menopausal status.breast density was influenced by BMI and menopausal status in both NF1 patients and healthy subjects. No difference in breast density was observed between NF1 patients and the healthy female population, even after considering the potential confounding factors.Although NF1 and a highly fibroglandular breast are known risk factors of BC, in this study, NF1 patients were shown to have comparable breast density to healthy subjects. The presence of pathogenic variants in the NF1 gene does not influence the breast density value.

GCK-MODY у молодых мужчин: значение поведенческих и кардиометаболических факторов риска в развитии осложнений

Aim. Present two clinical cases of Maturity-Onset Diabetes of the Young, which is based on the presence of pathogenic variants in the glucokinase (GCK) gene — GCK-MODY; to characterize behavioral and cardiometabolic risk factors for diabetes complications. Key points. In the first clinical case, the pathogenic variant p.Trp257Ter (c.770G>A, NM_000162.5) of the GCK gene, previously described in the literature, was identified in the proband and his father, in the second — the pathogenic variant p.Cys271Ter (c.1113C>A, NM_000162. 5) GCK gene. Probands are young men, 22 and 21 years old, respectively. They were first diagnosed with diabetes in the ages of 10 and 6 years during routine examinations. There were no clinical symptoms of hyperglycemia; they did not take and do not currently take hypoglycemic drugs. When examined 11 and 15 years after the diagnosis of diabetes, in each patient the level of C-peptide was within the reference values, which indicates the preservation of the secretory function of pancreatic β-cells. Antibodies were negative, the level of glycated hemoglobin (HbA1c) was 5.7 and 6.1%, respectively. No complications of diabetes were identified in either patient. The fathers of probands from both families had HbA1c levels of 6.4 and 6.5%, respectively. The father of the proband from the first clinical case does not comply with recommendations for a healthy lifestyle and nutrition, and does not deny weekly alcohol consumption. Upon examination, he was found to be overweight, arterial hypertension, dyslipidemia, steatohepatitis, and atherosclerosis of the brachiocephalic vessels, which increase the risk of cardiovascular events. The father of the proband from the second clinical case follows recommendations for a healthy lifestyle. During the examination, body weight, blood pressure, blood lipids are within the target range. Conclusion. Presenting two clinical cases and family histories of patients with GCK-MODY, the authors note that the compliance with the principles of a healthy lifestyle, assessment and correction of the main risk factors will help avoid the emergence and progression of complications in patients with GCK-MODY. When monitoring and treating patients with diabetes of any type, it is important to follow the principles of preventive medicine. Keywords: Molecular genetic research, GCK gene, monogenic diabetes mellitus, Maturity-Onset Diabetes of the Young, hyperglycemia, diabetes mellitus in young people.

Comprehensive Genomic Profiling in Predictive Testing of Cancer.

Despite the rapid progress in the field of personalized medicine and the efforts to apply specific treatment strategies to patients based on the presence of pathogenic variants in one, two, or three genes, patient response to the treatment in terms of positive benefit and overall survival remains heterogeneous. However, advances in sequencing and bioinformatics technologies have facilitated the simultaneous examination of somatic variants in tens to thousands of genes in tumor tissue, enabling the determination of personalized management based on the patient's comprehensive genomic profile (CGP). CGP has the potential to enhance clinical decision-making and personalize innovative treatments for individual patients, by providing oncologists with a more comprehensive molecular characterization of tumors. This study aimed to highlight the utility of CGP in routine clinical practice. Here we present three patient cases with various advanced cancer indicated for CGP analysis using a combination of SOPHiA Solid Tumor Solution (STS, 42 genes) for DNA and SOPHiA RNAtarget Oncology Solution (ROS, 45 genes and 17 gene fusions with any random partners) for RNA. We were able to identify actionable genomic alterations in all three cases, thereby presenting valuable information for future management of these patients. This approach has the potential to transform clinical practice and greatly improve patient outcomes in the field of oncology.

High Prevalence of a c.5979dupA Variant in the Dysferlin Gene (DYSF) in Individuals from a Semiarid Region of Brazil.

Dysferlinopathies represent a group of limb girdle or distal muscular dystrophies with an autosomal-recessive inheritance pattern resulting from the presence of pathogenic variants in the dysferlin gene (DYSF). In this work, we describe a population from a small city in Brazil carrying the c.5979dupA pathogenic variant of DYSF responsible for limb girdle muscular dystrophy type 2R and distal muscular dystrophy. Genotyping analyses were performed by qPCR using customized probe complementary to the region with the duplication under analysis in the DYSF. A total of 104 individuals were examined. c.5979dupA was identified in 48 (46.15%) individuals. Twenty-three (22%) were homozygotes, among whom 13 (56.5%) were female. A total of 91.3% (21) of homozygous individuals had a positive family history, and seven (30.4%) reported consanguineous marriages. Twenty-five (24%) individuals were heterozygous (25.8±16 years) for the same variant, among whom 15 (60%) were female. The mean CK level was 697 IU for homozygotes, 140.5 IU for heterozygotes and 176 IU for wild-type homo-zygotes. The weakness distribution pattern showed 17.3% of individuals with a proximal pattern, 13% with a distal pattern and 69.6% with a mixed pattern. Fatigue was present in 15 homozygotes and one heterozygote. The high prevalence of this variant in individuals from this small community can be explained by a possible founder effect associated with historical, geographical and cultural aspects.

Lessons from Real Life Experience: Importance of In-House Sequencing and Smart Ratio-Based Real-Time PCR Outperform Multiplex Ligation-Dependent Probe Amplification in Prenatal Diagnosis for Spinal Muscular Atrophy: Bench to Bedside Diagnosis.

Spinal muscular atrophy (SMA) is a rare, recessively inherited neurodegenerative disorder caused by the presence of pathogenic variants in the SMN gene. As it is the leading inherited cause of infant mortality, identification of SMN gene pathogenic variant carriers is important for diagnostic purposes with effective genetic counseling. Multiple ligation probe analysis (MLPA), a probe-based method, is considered as the gold standard for SMA carrier analysis. However, MLPA might give false-negative results in cases with variations in the probe-binding regions. Here, we present a case born to consanguineous SMA carrier parents. Prenatal diagnosis with MLPA failed to detect the compound heterozygous mutant state of the proband and she was born unfortunately with SMA phenotype. Further analysis with a real-time polymerase chain reaction kit was able to detect the compound heterozygous state of the patient and was confirmed with targeted next-generation sequencing technology.

SLC26A2 Related Diastrophic Dysplasia in 42-Years Ukrainian Women.

Diastrophic dysplasia (DTD) is an uncommon pathology which falls under the group of skeletal dysplasias with its first symptoms observed from birth. The pathology is often featured by short stature and abnormally short extremities (also known as short-limbed dwarfism); the osseous structures of the body (bones and joints) are characterized through defective development in many body regions. More than 300 genes were reported to be involved in DTD etiology with autosomal recessive, autosomal dominant and X-linked manner. We describe clinical case of a 42-year-old woman from the west of Ukraine with diastrophic dysplasia and two pathogenic variants c.1020_1022del (p.Val341del) and c.1957T>A (p.Cys653Ser) identified in SLC26A2 gene. SLC26A2-related diastrophic dysplasia was confirmed based on the presence of pathogenic variants in SLC26A2, which is associated with autosomal recessive forms of skeletal dysplasia, combined with phenotypic symptoms and radiographic findings.

Description of the clinical picture and assessment of functional activity of the CFTR channel in a patient with a complex allele [S466X; R1070Q

The presence of pathogenic variants in the CFTR gene causes cystic fibrosis (CF) through various molecular mechanisms that affect the formation and functional activity of the CFTR chloride channel. An important factor affecting the phenotypic manifestations of CF and the effectiveness of targeted therapy is the presence of complex alleles with > 2 consecutive mutations per 1 allele, or in the cis position. The influence of complex alleles on the manifestations of CF has not been sufficiently studied globally due to the small number of studies.The aim of the study was to investigate the influence of the complex allele [S466X; R1070Q] on the phenotypic manifestations of CF and the effectiveness of targeted therapy in a model of intestinal organoids from a patient with [S466X; R1070Q]/CFTRdele2,3 genotype.Methods. We used medical history data, intestinal current measurement, intestinal organoid method, and forskolin test.Results. The progressive nature of the disease with a clear degradation of lung function was established. The ICM method showed absent chloride channel function. The tests on the culture of organoids obtained from the intestinal tissue indicated a complete loss of the chloride channel function. In addition, the complex allele [S466X; R1070Q] was insensitive to all targeted drugs tested.Conclusion. The complex allele [S466X; R1070Q] causes a complete loss of the functional CFTR protein and is not sensitive to any of the approved targeted drugs.

Influence of different spectra of NOTCH3 variants on the clinical phenotype of CADASIL - experience from Slovakia.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is the most common hereditary vascular disorder causing ischaemic attacks and strokes in middle-aged adults. Though the clinical spectrum includes some typical symptoms, recognition of the disease, especially at an earlier stage, is very difficult because of the highly variable manifestation and incomplete clinical picture. Characteristic brain MRI findings and the presence of pathogenic variants in the NOTCH3 gene are fundamental for CADASIL diagnosis. In this paper, we provide the first comprehensive report on CADASIL patients from Slovakia. Altogether, we identified 23 different pathogenic variants in 35 unrelated families. In our cohort of patients with clinical suspicion of CADASIL, we found a causal genetic defect and confirmed the diagnosis in 10.2% of cases. We present the case reports with up-to-date unpublished NOTCH3 variants and describe their phenotype-genotype correlation: p.(Cys65Phe), p.(Pro86Leu/Ser502Phe), p.(Arg156*), p.(Cys408Arg), p.(Tyr423Cys), p.(Asp1720His), and p.(Asp1893Thrfs*13). The most frequently described location for pathogenic variants was in exon 4, whereas the most common single variant was p.Arg1076Cys in exon 20. Based on the results of our study, we propose a re-evaluation of the criteria for the selection of patients suitable for NOTCH3 gene analysis. We hereby state that the currently used protocol of a high score requirement is not ideal for assessing molecular analysis, and it will be desirable to be less strict in criteria for genetic testing.

Novel mutations in BBS genes and clinical characterization of Chinese families with Bardet-Biedl syndrome.

Bardet-Biedl syndrome (BBS) is a rare autosomal-recessive inherited disorder characterized by multisystem anomalies. The objective of this study was to detect and analyse pathogenic variants in four Chinese families with BBS. Comprehensive clinical examinations were performed to investigate and evaluate the phenotypes of the affected individuals from four families. Genomic DNA was extracted from peripheral blood. Next-generation sequencing (NGS) was performed for four families, and the presence of pathogenic variants was confirmed via Sanger sequencing. There were two males and three females with a mean age of 16.00 years. All probands displayed the primary clinical features of BBS. Mutation screening demonstrated four novel mutations: c.613C>T; p.Q205* in the BBS5 gene, c.1391C>G; p.S464* in the BBS10 gene, and c.155delC; p.S52* and c.1584T>G; p.Y528* in the BBS12 gene. Two previously reported mutations were also identified, including c.534 + 1G>T in the BBS2 gene and c.539G>A; p.G180E in the BBS10 gene. The bioinformatic analysis revealed that all the detected mutations in BBS genes were disease causing. This study identified four novel BBS gene mutations in these Chinese families and further expanded the genotypic spectrum of BBS, thus contributing to the literature and understanding of this multisystem disease.

Challenging Occam's Razor: Dual Molecular Diagnoses Explain Entangled Clinical Pictures.

Dual molecular diagnoses are defined as the presence of pathogenic variants at two distinct and independently segregating loci that cause two different Mendelian conditions. In this study, we report the identification of double genetic disorders in a series of patients with complex clinical features. In the last 24 months, 342 syndromic patients have been recruited and clinically characterised. Whole Exome Sequencing analysis has been performed on the proband and on both parents and identified seven patients affected by a dual molecular diagnosis. Upon a detailed evaluation of both their clinical and molecular features, subjects are able to be divided into two groups: (A) five patients who present distinct phenotypes, due to each of the two different underlying genetic diseases; (B) two patients with overlapping clinical features that may be underpinned by both the identified genetic variations. Notably, only in one case a multilocus genomic variation was already suspected during the clinical evaluation. Overall, our findings highlight how dual molecular diagnoses represent a challenging model of complex inheritance that should always be considered whenever a patient shows atypical clinical features. Indeed, an accurate genetic characterisation is of the utmost importance to provide patients with a personalised and safe clinical management.

Abstract 769: Genomic and transcriptomic characterization of benign and malignant struma ovarii

Abstract Background: Struma Ovarii (SO) is a rare ovarian teratoma characterized by the presence of thyroid tissue in >50% of the tumor. The majority of SO are benign; however, malignant transformation occurs in up to 5% of the cases. The molecular foundations of benign and malignant SO are grossly unknown. Therefore, the goal of this study was to perform the first comprehensive genomic and transcriptomic analysis of the benign and malignant SO. Material and Method: We performed whole-exome sequencing (WES) and targeted RNA-sequencing (seq) on the DNA and RNA extracted from formalin-fixed paraffin-embedded SO tumor tissue samples. WES library was prepared using Agilent’s SureSelect XT HS2 kit, with 4 samples failing the quality assessment (QA). Variants were called from GATK processed WES data and annotated using VEP (with ClinVar and COSMIC databases). The targeted RNA-Seq library was prepared using the TruSight RNA Pan-Cancer Panel kit covering 1385 cancer genes, with all samples passing QA. The clinical characteristics of the study cohort were summarized by percentages for categorical variables and medians with 25-75% interquartile ranges for continuous variables. Results: The study included 31 tissue samples - 21 benign and 10 malignant, including 6 cases of papillary thyroid cancer (PTC), 3 of follicular variant of PTC, and 1 of follicular thyroid cancer. Patients with benign SO were characterized by the median age at diagnosis of 39 years [33-54], tumor size of 3.1 cm [2.5-5.8], while the patients with malignant SO presented at age of 45 [28-54], tumor size of 6 cm [0.85-14] and metastatic disease in 30% (3/10) - 2 patients with peritoneal metastases and 1 patient with pelvic lymph node metastases. The E1A Binding Protein P300, EP300 (6/27), and Isocitrate dehydrogenase, IDH2 (5/27) were the topmost mutated genes in the SO samples. Malignant SO samples were characterized with the presence of pathogenic variants of KRAS (pQ61L and pG12V), NRAS (pQ61R), TP53 (splice site) mutations, and Nuclear Receptor Binding SET Domain Protein 1 (NSD1) fusion as the most common molecular drivers. Among benign SO samples, the most common driver was Thyroglobulin (TG) fusion with either Guanine Nucleotide binding protein (GNAS) or Rac Family Small GTPase 1 (RAC1). Differential expression analysis showed that the member of tumor suppressor family - tumor protein 63 (TP63) was the most downregulated (Log2FC = -3), while Double-sex and Mab-3 Related Transcription Factor 1 (DMRT1), implicated in the development of germ cell tumors, was the most upregulated gene in malignant SO samples over benign (Log2FC = 2.1; padj<0.05). Conclusions: In contrast to cancer arising from the thyroid gland, characterized by BRAFV600E as the most common mutation, malignant SO belongs to RAS-like tumors. The downregulation of tumor suppressors and upregulation of DMRT1 might be implicated in the malignant transformation of SO. Citation Format: Shilpa Thakur, Darryl Nousome, Kshama Aswath, Stephanie Cardenas, Sonam Kumari, Ruth Adewale, Maria Merino, Esra Dikoglu, Padmasree Veeraraghavan, Sriram Gubbi, Joanna Klubo-Gwiezdzinska. Genomic and transcriptomic characterization of benign and malignant struma ovarii [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 769.

Clinical characteristics and long-term outcomes of patients with glycogen storage disease type 1b: a retrospective multi-center experience in Poland.

Glycogen storage disease type 1b (GSD 1b) is an inherited metabolic defect caused by a deficiency of microsomal glucose-6-phosphate (G6P) transport protein across the endoplasmic reticulum membrane. Patients with GSD 1b have hypoglycemia episodes, lactate acidosis, hypertriglyceridemia, hypercholesterolemia, hyperuricemia, neutropenia and in imaging studies hepatomegaly and/or nephromegaly. The primary goals of treatment are to maintain proper blood glucose levels and to increase the number of properly functioning neutrophils. The aim of the study was a retrospective analysis of the clinical picture and treatment results of pediatric patients with type 1b glycogen storage disease from Poland. The study included 13 patients from 3 clinical centers, with a median age at diagnosis as 5 months. In 11/13 patients, the diagnosis was confirmed by molecular test, by the presence of pathogenic variants on both alleles of the SLC37A4 gene. Ten out of 13 patients developed the first symptoms in the form of severe infection (sepsis and/or pneumonia) already in the neonatal-infant period. A hypoglycemia episode was observed before diagnosis in 8/13 patients, of which 4/8 patients presented symptoms in the form of generalized relaxation and/or seizures. Two patients developed hypertension, and 4/13 required long-term treatment of inflammatory bowel disease.

Incidental findings from cancer next generation sequencing panels

Next-generation sequencing (NGS) technologies have facilitated multi-gene panel (MGP) testing to detect germline DNA variants in hereditary cancer patients. This sensitive technique can uncover unexpected, non-germline incidental findings indicative of mosaicism, clonal hematopoiesis (CH), or hematologic malignancies. A retrospective chart review was conducted to identify cases of incidental findings from NGS-MGP testing. Inclusion criteria included: 1) multiple pathogenic variants in the same patient; 2) pathogenic variants at a low allele fraction; and/or 3) the presence of pathogenic variants not consistent with family history. Secondary tissue analysis, complete blood count (CBC) and medical record review were conducted to further delineate the etiology of the pathogenic variants. Of 6060 NGS-MGP tests, 24 cases fulfilling our inclusion criteria were identified. Pathogenic variants were detected in TP53, ATM, CHEK2, BRCA1 and APC. 18/24 (75.0%) patients were classified as CH, 3/24 (12.5%) as mosaic, 2/24 (8.3%) related to a hematologic malignancy, and 1/24 (4.2%) as true germline. We describe a case-specific workflow to identify and interpret the nature of incidental findings on NGS-MGP. This workflow will provide oncology and genetic clinics a practical guide for the management and counselling of patients with unexpected NGS-MGP findings.

Concealed cardiomyopathy as a frequent cause of idiopathic ventricular fibrillation in a representative Czech cohort of survivors of sudden cardiac arrest (SCA)

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Ministry of Health of the Czech Republic Introduction The complex diagnostic work up in SCA survivors often does not yield a concrete cardiological diagnosis. Moreover, there is conflicting evidence whether genetic testing could support or guide clinical diagnostics. Purpose To assess the molecular architecture of idiopathic ventricular fibrillation in cases without apparent evidence of specific structural or arrhythmic cardiac disease at initial diagnostic work up in a representative Czech cohort. Patients and Methods Between 2013 - 2020 we have ascertained 100 SCA survivors (54 M / 46 F; age range at cardiac arrest 5-69 years). Genetic counselling was followed by massively parallel DNA sequencing using custom-made panels comprising 100 cardiac conditions-related genes. Subsequently, thorough cardiological screening examinations in first degree relatives were carried out. Presence of pathogenic variants was validated by Sanger DNA sequencing and through family segregation analyses. Results Highly likely or certain molecular aetiology (i.e. based on the presence of Class 4 or 5 variants) was disclosed in 20/100 (20%) in PKP2 (3x), SCN5A (4x), RYR2 (3x), TTN (2x), PLN, FLNC, PRKAG2, KCNH2, KCNQ1, SLC4A, TNNT2, and DSP. Interestingly, the KCNE1 p.Asp85Asn (LQT 5 lite) variant, was detected in further 3/100 cases, representing a recognized risk factor for ventricular arrhythmias. Conclusions Genetic testing facilitates stratification of the cause of arrhythmia in a substantial portion of SCA survivors. The utility of positive outcomes of genetic testing was substantiated in 10/20 gene positive patients, where the genetic stratification led to diagnosis of concealed arrhythmogenic cardiomyopathy, whose extent of morphological changes was under the diagnostic sensibility of imaging modalities or ECG. Our results enable individualized care in SCA survivors and assure targeted preventive approaches in their relatives.

Comparison of variant detection rate in genes between two cohorts of Czech living patients versus victims of sudden cardiac death with clinical / post mortem diagnosis of non-ischemic cardiomyopathy

Abstract Introduction Hereditary cardiomyopathy is associated with an increased risk of ventricular arrhythmia and sudden cardiac death (SCD). Genetic stratification substantiates risk assessment and enables the primary prevention of SCD in relatives at risk. We have analyzed the genetic aetiology of SCD in a representative Czech cohort with post mortem diagnosis of various forms of cardiomyopathy and compared it to living cases with these cardiac disorders. Patients and methods Between 2018 and 2019, altogether 47 victims of SCD with post mortem diagnosis of hypertrophic- (HCM; 18/47), arrhythmogenic- (ACM; 19/47) and dilated cardiomyopathy (DCM; 10/47) were identified. Concurrently, genetic testing was performed in 114 living patients (HCM 54/114, ACM 22/114, DCM 38/114). Genetic counselling and cardiologic examination had been carried out in first-degree relatives in all patients/SCD victims. Massively parallel sequencing (MiSeq platform; Illumina.com) was utilized for a custom-made panel comprising 100 candidate genes (Sophia Genetics, Switzerland). The presence of pathogenic variants was validated by Sanger DNA sequencing and through family segregation analyses. Results The causative detection rate (according to ACMG.net classes 4 or 5) in SCD victims with DCM was 60% (6/10) and in living patients with DCM 47.4% (18/38). Variants in TTN, RBM20, DES and FLNC (mainly truncating variants) prevailed in both groups. The detection rate in ACM was 5% (1/19 in SCN5A gene) in SCD victims and 31.8% (7/22) in living patients. Interestingly, the most prevalent mutated gene PKP2 in living patients was not detected in SCD victims. The detection rate in SCD victims with post mortem diagnosis of HCM was 16% (3/18) and in living patients 35% (19/54). The most prevalent gene was MYBPC3 in both groups, while PRKAG2 was detected in one SCD victim and in one living case who survived cardiac arrest. Conclusion Post-mortem genetic analysis in DCM yields a high detection rate and allows potentially effective primary prevention of SCD in relatives at risk. In contrast, the molecular autopsy of HCM and ACM renders a much lower yield which is below the mutation detection rate in living phenotype positive individuals. The results help to improve the genetic counselling in affected families in Czech Republic. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): Ministry of Health of the Czech Republic

Relevance and diagnostic performance of genes involved in arrhythmogenic cardiomyopathy

Arrhythmogenic cardiomyopathy (ACM) is an inherited myocardial disease, affecting preferentially people under 40 years old with ventricular arrhythmia to sudden death. To date, the rate of successful genotyping is about 50%. Desmosomal genes (PKP2, DSC2, DSG2, DSP, JUP) are the most frequently mutated, the implication of the other genes associated with other cardiomyopathies is still debated. The pathogenicity of variants among these genes should be assessed not only based on the rarity because of the presence of rare variants in individuals in the general population. Assess the pathogenicity of variants and the relevance of genes involved in ACM. DNA from 179 patients with ACM were sequenced on a panel of 71 heart disease genes including 11 genes most often described in ACM. The pathogenicity of variants is assessed according to international guidelines (ACMG score) with 2 bioinformatic tools. We performed burden (CAST) and dispersion tests (SKAT, SKAT-O) on the 71 genes between patients and a control population. Among the 179 patients, 34.6% have a pathogenic variant with PKP2 in 42 patients. Furthermore, 17.3% of patients have uncertain significance variants in genes involved in ACM. Burden tests showed a significant enrichment in pathogenic variants in PKP2, DSP, DSC2 and DSG2 ( P < 0.001) and in uncertain significance variants in the PKP2, DSG2 and DSC2 genes ( P < 0.01). No enrichment was observed among other genes. Patients with a variant in genes involved in ACM are diagnosed earlier than patients without variant ( P < 0.05). Only desmosomal genes count a significate proportion of pathogenic variants in patients with ACM compared to control population. Uncertain significance variants in PKP2, DSC2, DSG2 genes seem to contribute to ACM and should be investigated. The age of symptom onset is correlated with the presence of pathogenic variant, thus justifying early family screening for better clinical management.

Carriers of cystic fibrosis among sperm donors: complete CFTR gene analysis versus CFTR genotyping

To determine the frequency of cystic fibrosis (CF) carriers among sperm donors in Spain studied through a complete analysis of the CFTR gene and to compare the results with those that would have been obtained by the 4 genotyping panels of the CFTR gene most commonly used as a carrier test in the context of assisted reproduction in our country. Descriptive observational study. Private center. Nine hundred thirty-five sperm donors, from January 2014 to June2019. None. Presence of pathogenic variants in the CFTR gene. 17% of the donors were carriers of at least 1 pathogenic variant in CFTR, with 39 different pathogenic variants detected. Only 4 of these 39 variants (10.27%) would have been detected by the 4 genotyping tests considered, and 22 variants (56.41%) would not have been detected by any of the genotyping tests. The pathogenic variants of the CFTR gene included in the different genotyping tests analyzed vary widely, and <50% are common to all of them. Although the was not based in the general population, these results show that the use of genotyping tests is associated with a high reproductive risk, because the rate of detection of CF carriers was lower when these panels were applied, in comparison with the complete study of the CFTR gene. We recommend that complete sequencing of the CFTR gene by next-generation sequencing be performed as a screening method for CF in sperm donors.

P1107First results of molecular autopsy examinations in sudden cardiac death drawn from nationwide multidisciplinary and multicentric collaboration in the Czech Republic

Abstract Funding Acknowledgements Supported by Ministry of Health of the Czech Republic, grant nr. NV18-02-00237. All rights reserved." Introduction "Molecular autopsy" in sudden cardiac death (SCD) is an important diagnostic tool for primary prevention of cardiac arrest in victim´s relatives and requires multicentric and multidisciplinary collaboration. Purpose To establish a national network for SCD diagnostics, to assess the acceptance of genetic testing in at risk relatives and to elucidate the genetic etiology of SCD in a representative Czech cohort aged below 45 years. Patients and Methods Between 2016 and 2019 we ascertained 70 SCD (50 M / 20 F), with positive family history for cardiac arrest in 8/70 cases. Only one family did not agree with molecular autopsy. Genetic counselling and cardiological screening examination was carried out in first degree relatives of SCD survivors accompanied by custom-made targeted panel massively parallel DNA sequencing comprising 100 cardiac conditions-related genes. Presence of pathogenic variants was validated by Sanger DNA sequencing and through family segregation analyses. Results According to post-mortem diagnosis most victims died of HCM (17/70), ACM (16/70), no structural heart disease was found in 15/70 cases, DCM/LVNC in 9/70, 6/70 were SIDS cases, other rare diagnoses comprised aortic dissection, or myocarditis. Most of victims died at sleep, only 9/70 victims died during strenuous activities. About 50% of SCD victims did not have cardiac complaints before death. Very likely or certain molecular etiology (i.e. based on presence of ACMG.net Class 4 to 5 variants) was disclosed in 12/70 (17%) in RYR2, FLNC, TTN, KCNH2 and KCNQ1 genes, whilst potentially causative DNA variants (Classes 3-4) were observed in 13/70 (18%) cases. In SIDS we did not find any pathogenic variants. Interestingly, the KCNE1 p.Asp85Asn (LQT 5 lite) variant, was detected in 3/70 cases as a recognized risk factor for ventricular arrhythmias. Conclusion The multidisciplinary cooperation in Czech Republic has been established, family survivors are willing to receive genetic and cardiological care, while centralised molecular genetic analysis enables reliable results which are in accordance with other multicentric studies. The molecular autopsy should, especially in SIDS, be expanded to whole exome sequencing.