Natural Missplicing Events Amplified by an Elusive Deep-Intronic MYBPC3 Variant Cause Hypertrophic Cardiomyopathy

Disruption of MYBPC3 precursor mRNA splicing is a frequent genetic cause of hypertrophic cardiomyopathy (HCM). Most often, it reflects changes at canonical sites or the creation of novel splice sites. Prediction tools usually prioritize splice variants with lower efficiency when they are distant from canonical sites. These elusive variants may explain HCM in patients considered genotype-negative after DNA testing.

Massively parallel sequencing identified the previously unreported deep-intronic variant MYBPC3 c.2308+227G>A in a cohort of genotype-negative patients with HCM from Salamanca, Spain. SpliceAI predicted a benign effect (Δ score, 0.03). However, its recurrent detection prompted us to conduct extensive analyses to investigate its pathogenicity, including genetic testing, clinical assessment, family studies, splicing assays, and haplotype reconstruction.

We identified 35 unrelated HCM probands carrying c.2308+227G>A, plus 46 relatives from 27 families, yielding 81 confirmed carriers. Of these, 56 (69%) met diagnostic criteria for HCM (66% male; mean age, 53±15 years). By age 60 years, cumulative penetrance was estimated to be 81% in women and 96% in men. The combined logarithm of the odds score of 5.51 across 20 informative families provided strong evidence of cosegregation. During follow-up of probands, no significant differences were observed in heart failure, arrhythmic events, atrial fibrillation, or mortality when compared with cohorts carrying other pathogenic MYBPC3 variants. Blood splicing assays, confirmed in cardiac tissue, showed that c.2308+227G>A disrupts splicing by the use of pre-existing cryptic donor (c.2308+299) and acceptor (c.2309-580) splice sites, generating 2 misspliced mRNAs with partial intron retention or cryptic exon inclusion, which encode truncated proteins. These cryptic sites are the leading natural, unannotated missplicing events in intron 23, and some of the most frequent across the whole MYBPC3 precursor mRNA, as revealed by SpliceVault analysis of RNA sequencing databases. This deep-intronic variant does not alter essential splice motifs but promotes the use of pre-existing cryptic sites, likely through gain of splicing regulatory enhancer elements. The identification of a shared haplotype among HCM probands carrying this variant explains its recurrent detection, consistent with an ancient founder effect.

MYBPC3 c.2308+227G>A is a pathogenic splice-altering variant that causes HCM by amplifying natural missplicing events. These background splicing errors help explain how this elusive variant disrupts splicing without creating novel essential splice sites, thereby expanding the known mechanisms by which deep-intronic variants alter MYBPC3 splicing and ultimately contribute to HCM pathogenesis. Elusive MYBPC3 splice-altering variants should be considered in HCM patients with an unresolved genetic origin of disease.