Al Hattali, Fahad Sulaiman Badio; (2025) Evaluating trio-exome sequencing to find genetic abnormalities in familial cases of cardiomyopathies and arrhythmias in the Omani population. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Background: Inherited Cardiovascular diseases represent a significant challenge in Oman due to a lack of genetic diagnosis with standard testing panels although there have been advances in NGS technologies. Whole Exome Sequencing (WES) in trios i.e., patient and parents has proven an efficient way to identify variants in many families. In this study we performed trio-WES on 11 consanguineous Omani families with cardiomyopathies (Dilated Cardiomyopathy (DCM) and Hypertrophic Cardiomyopathy (HCM)) and arrhythmias (Long QT, Sick Sinus and early repolarization syndromes) and family history of similar phenotype presented at young age and mostly paediatric. The index patients of these families were investigated using a cardio panel that contained 174 genes (TruSight Cardio – Illumina) and no variants were found that explained their phenotype. I hypothesised that causative variants were rare and of autosomal recessive mode of inheritance. / Methods: Samples were sequenced in two batches, using library preparation kits from Nonacus Cell3 and Agilent. Prioritization for rare and exonic variants was done using Exomiser software as per the hypothesis. The prioritised variants were then confirmed and segregated using Sanger sequencing in the affected/ unaffected family members. Moreover, I cultured induced pluripotent stem cells (iPSc) and differentiated them into cardiomyocytes and used siRNA to knockdown 5 genes of interest found via trio-WES and Sanger segregation analysis. Assays such as calcium transient using Flou-4, qPCR using TaqMan and cell size using confocal imaging were used to assess the difference between controls and siRNA knockdown cultures in iPSc-CMs cells. / Results: The trio-WES and Sanger segregation analysis showed that two families (18%) with DCM/HCM showed a pathogenic variant in the ALPK3 gene. Four families (36%) with Long QT/ DCM showed no variant that followed our hypothesis. Five families (45%) had 2 or more variants and they included EML3, RAD9A, KRTAP5-7, AFF4, MAPK15, GAL3ST3, HMGA1, ADAM23, H1-1, TREM2, CPNE5, HNF4A and RBM12 and in addition two families had the same EML3 and RAD9A variants. I then selected the following genes; AFF4, RAD9A, HMGA1, EML3 and CPNE5 based on pathogenicity predictions, GWAS associations and publications for functional studies. Quantitative PCR showed successful deletions of the mRNA of the genes of interest. Calcium Transient studies showed no significant difference between controls and siRNA gene knockdown when comparing Rise Time, CaT50, CaT90, Amplitude, cycle length and conduction velocity. Cell size measurement using confocal microscopy between control cells and siRNA knockdown cells showed no significant difference. / Conclusion: Targeted gene panels for inherited cardiac conditions have become less effective over time due to the continual discovery of newly associated genes. The adoption of next-generation sequencing technologies, such as whole exome sequencing, has enabled the identification of a considerable number of novel candidate genes in a large proportion of affected families. I attempted to define abnormalities in calcium handling with a number of the genes in iPSc-CMs but there were substantial changes after knockdown. There are limitations to the use of iPSc-CMs and alternative methodologies might be needed to understand the cardiac pathogenicity of the gene variants.

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