OR01 A non-canonical splicing variant in MYBPC3 leads to cryptic splicing in Hypertrophic Cardiomyopathy

Background Hypertrophic Cardiomyopathy (HCM) is the most common genetic heart disease and a leading cause of sudden cardiac death. Despite its prevalence, approximately 50% of clinically diagnosed patients lack a genetic diagnosis (1). Traditional genetic testing primarily focuses on the detection of exonic and canonical splice site variants, potentially overlooking non-canonical splicing variants, including those located in introns, that may contribute to the disease (2). Purpose In this study, we aimed to predict and characterize the impact of intronic splicing variants identified in an HCM cohort (3), with a particular focus on MYBPC3, one of the most affected genes in HCM. Methods We used CRISPR/Cas9 gene-editing and reprogramming of patient-derived cells to generate induced pluripotent stem cells (iPSCs) harboring an intronic splicing variant identified in HCM patients. Upon differentiation into cardiomyocytes (iPSC-CMs), we used morphological and functional assays to determine pathogenicity and investigated splicing alterations triggered by this intronic variant using long-read RNA sequencing and RT-PCR. Results Mutant iPSC-CMs displayed hallmark HCM features, including increased cellular size, multinucleation and disorganized sarcomeres. Consistent with the functional phenotype of the disease, MYBPC3 mutant iPSC-CMs also exhibit prolonged calcium transients and increased relaxation times when compared with WT iPSC-CMs. Cycloheximide-mediated inhibition of nonsense-mediated decay (NMD) in mutant iPSC-CMs revealed an abnormal extension of MYBPC3 exon 14, identified by RT-PCR, caused by the creation of an intronic cryptic acceptor splice-site in the intron. Degradation of mutant transcripts by NMD led to reduced levels of MYBPC3 mRNA and myosin binding protein-C in mutant cells. Notably, partial NMD escape was observed, as stable mutant mRNAs persisted without NMD inhibition. To further investigate if this intronic variant leads to other splicing alterations, we performed long-read RNA sequencing of mutant and WT iPSC-CMs and confirmed exon 14 extension as the main aberrantly spliced MYBPC3 isoform, although other isoforms were detected. Conclusions Our findings highlight the contribution of non-canonical splicing variants to HCM and reveal the complexity of aberrant isoforms that can arise from a single nucleotide change. These results underscore the need to incorporate long-read sequencing into the studies of splicing variants to fully capture isoform diversity.