Whilst in silico tools can predict whether a variant alters splicing with modest positive predicted values 9, the outcomes can only be confirmed with functional studies involving amplification and sequencing of the mRNA (Supplementary Fig. The acceptor site comprises a polypyrimidine tract around the last 20 nucleotides of the intron and includes the first three nucleotides of the exon with an almost invariant ‘AG’ dinucleotide at the end of the intron 7, 8. The donor site is defined as the last three nucleotides of an exon and the first six nucleotides of the intron, with an almost invariant ‘GT’ dinucleotide at the beginning of the intron. RNA splicing requires the recognition of the splice donor and acceptor site at the beginning and end of introns (Supplementary Fig. Assigning the correct consequence of variants at the protein level is essential for the accurate classification of pathogenicity and for understanding disease mechanisms. This includes variants previously annotated as missense, nonsense, or synonymous changes and intronic variants located far away from protein-coding regions. While genetic testing typically focuses on rare variants in protein-coding regions and the essential splice sites of genes, there is an increasing appreciation of the range and manner by which variants can disrupt RNA splicing 4, 5, 6. People with suspected inherited heart diseases should undergo genetic testing as this can clarify uncertain clinical diagnoses, guide treatment options, inform prognosis, and help stratify risk in family members 3. Inherited cardiomyopathies and arrhythmia syndromes are important causes of heart failure and sudden cardiac death 1, 2. ![]() Our study highlights that splice-disrupting variants are a significant cause of inherited heart disease, and that analysis of blood RNA confirms splicing outcomes and supports variant pathogenicity classification. Eleven variants of uncertain significance were reclassified as likely pathogenic based on functional studies and six were used for cascade genetic testing in 12 family members. Our functional studies confirmed altered splicing in six variants. ![]() Blood RNA supported the amplification of 21 out of 31 definitive disease-associated inherited heart disease genes. ![]() There was an excess burden of splice-disrupting variants in PKP2 (5.9%), FLNC (2.7%), TTN (2.8%), MYBPC3 (8.2%) and MYH7 (1.3%), in distinct cardiomyopathy subtypes, and KCNQ1 (3.6%) in long QT syndrome. We found 88 in silico-predicted splice-disrupting variants in 128 out of 1242 (10.3%) unrelated participants. Variants were functionally assessed and classified for pathogenicity. ClinGen definitively disease-associated inherited heart disease genes were amplified using RNA extracted from fresh blood, derived cardiomyocytes, and myectomy tissue. ![]() We performed burden testing of rare splice-disrupting variants in people with inherited heart disease and sudden unexplained death compared to 125,748 population controls. There is an incomplete understanding of the burden of splice-disrupting variants in definitively associated inherited heart disease genes and whether these genes can amplify from blood RNA to support functional confirmation of splicing outcomes.
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