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Fish, founders and fluorescent indicators

Boek - Dissertatie

Ondertitel:progress of inherited cardiac arrhythmia genomics in the new sequencing era
Inherited Cardiac Arrhythmia (ICA) syndromes are a group of rare genetic disorders characterised by an increased risk for potentially lethal ventricular arrhythmias, which occur in structurally (seemingly) normal myocardium. While recent technological advances in DNA sequencing have improved in the diagnostic yield in ICA, they have also led to an increased identification of variants of uncertain significance (VUS). Functional characterisation plays a crucial role in variant annotation. Nevertheless, sufficiently high-throughput ICA disease models are still lacking. We set out to fill this gap by generating a zebrafish assay for cardiac arrhythmia, with co-expression of the Ace2N-mNeon genetically encoded voltage indicator and R-GECO genetically encoded calcium indicator in the heart. Our zebrafish cardiac arrhythmia assay was able to detect electrophysiological abnormalities induced by drugs and genetic models of ICA generated at our lab. These consisted of a long QT syndrome model, induced by a knock-in mutation in the cacna1c gene and a catecholaminergic polymorphic ventricular tachycardia (CPVT) model, due to a homozygous knockout of the casq2 gene. The use of light sheet imaging also enabled us to generate three-dimensional maps of the cardiac action potential characteristics of the entire heart in zebrafish embryos. Additionally, we optimized the process of the generation of zebrafish knock-in mutants through CRISPR-Cas9 gene editing. By combining minimally invasive early genotyping with the Zebrafish Embryo Genotyper device with next-generation sequencing, we were able to achieve an almost seventeen-fold improvement in editing efficiency. Lastly, we performed clinical and functional assessments of variants in known arrhythmia genes to clarify the remaining issues in ICA genetics. We explored a novel hypothesis of autosomal dominant CPVT for a heterozygous c.738-2A>G splice site variant in CASQ2. We assessed a cohort of (c.4813+3_4813+6dupGGGT) SCN5A founder mutation carriers for clinical signs of a complex genetic architecture in Brugada syndrome. Additionally, we demonstrated the role of the disease model in the functional assessment of the allelic effect of a recurrent KCNQ1 c.1124_1127delTTCA p.(Ile375Argfs*43) variant. With increased identification of VUS and the confounding contributions of poly- and oligogenic factors, as well as variable allelic effects of established pathogenic variants, the field of ICA genetics research remains complex and challenging. Future studies in promising disease models, such as zebrafish, as well as clinical and molecular investigations in cohorts of patients carrying the same pathogenic variant, will likely aid in clarifying some of the remaining questions.
Aantal pagina's: 233
Jaar van publicatie:2023
Trefwoorden:Doctoral thesis
Toegankelijkheid:Embargoed