Molecular and Cellular Characterisation of Non-coding Variants in Inherited Heart Disease

Abstract

Genetic variants outside of protein-coding regions of genes, i.e non-coding variants, are increasingly identified to cause inherited diseases. However, the clinical relevance of these variants is challenging to determine from sequence context alone, leaving many classified as variants of uncertain significance. This thesis aimed to study the functional consequences and clinical relevance of non-coding variants in people with inherited heart disease and sudden cardiac death (SCD) through genetic and cellular studies. A rare variant burden analysis in Chapter 2, found an enrichment of RNA splice-disrupting variants in six cardiac disease genes in over 1000 people with inherited heart disease or SCD compared to population controls, supporting their relevance to disease causation. RNA extracted from blood amplified two-thirds of cardiac disease genes, highlighting the suitability of blood RNA for functional analyses of splicing variants. Chapter 3 presents functional analysis reports confirming the pathogenicity of eight putative splice-disrupting variants following blood RNA analyses. RNA and protein studies using primary heart tissue samples presented in Chapter 4 confirmed that a 5’ untranslated region deletion in TAFAZZIN abrogated protein expression from the gene, leading to haploinsufficiency and causing Barth syndrome. Eight MYBPC3 midigenes were developed in Chapter 5 to functionally assess the impact of putative splicing variants without requiring patient-derived RNA samples. In Chapter 6, RNA sequencing of patient-specific induced pluripotent stem cell cardiomyocytes confirmed the pathogenicity of a minor intron variant in SCN5A. CRISPR-Cas9 correction of this variant in Chapter 7, reversed cryptic splicing to wild-type. Collectively, data presented in this thesis led to a clinically meaningful reclassification of 13/21 (62%) non-coding variants and demonstrated how incorporating RNA analyses in genetic testing shortens the diagnostic odyssey for families.