Contribution and Functional Characterisation of Splice-Disrupting Variants in Inherited Heart Disease and Sudden Cardiac Death

Splice-disrupting variants are increasingly recognised as causing inherited heart disease. We sought to determine the contribution of these variants in cardiac disease aetiology and functionally validate the effects on splicing using RNA analysis. We selected patients with inherited heart disease in which gene panel, exome or genome sequencing identified variants in-silico predicted to disrupt splicing of established cardiac disease genes. Candidate variants were functionally characterised with Sanger sequencing of patient’s reverse-transcribed RNA. Variants were classified for pathogenicity using standard guidelines. Ninety in-silico predicted splice-disrupting variants were found in 138 out of 1,459 (9.5%) unrelated probands. Splice-disrupting variants were mostly found in MYBPC3 (n=35; 38.9%) and TTN (n=15; 16.7%). Exonic missense and synonymous variants contributed 32 (35.6%) variants, followed by 22 (24.4%) deep intronic splice-gain variants. There were 23 (25.6%) variants located in the canonical ± 1,2 splice site regions, whilst variants in the extended splice regions (-3 to +6 in the donor site; -20 to +3 in the acceptor site) contributed 8 (8.9%) variants. Variants located in the extended donor region mostly occurred at the +5 position (n=3; 3.3%). Fifty-three variants were selected for RNA analysis and disrupted splicing was successfully confirmed for 24 variants in ELN, KCNQ1, MYBPC3, and TTN. Functional assessment led to a clinically meaningful reclassification of 12 variants. Splice-disrupting variants are a significant cause of inherited heart diseases. Analyses of patient’s RNA extracted from blood confirms splicing outcomes and can provide additional evidence for pathogenicity.