Discovery of a cryptic founder variant in MYBPC3 associated with hypertrophic cardiomyopathy by promoting the most frequent natural missplicing events
Authors: Gallego Delgado M , Torrado M , Perez Barbeito M , Lorenzo Hernandez S L , Rodriguez Diego S , Garcia Berrocal B , Garcia Hernandez S , Garcia Monsalvo M , Garcia Salgado M J , Maneiro Pampin E , Alvarez Martinez C , Ochoa J P , Sanchez P L , Isidoro Garcia M , Villacorta E 
Published: October 2024

Abstract

Introduction
MYBPC3 splicing errors are a common cause of hypertrophic cardiomyopathy (HCM). Most known actionable intronic variants are located near exons. However, genetic variants in deep intronic regions are also emerging as casuals factors of HCM. The previously undescribed variant MYBPC3 (NM_000256.3):c.2308+227G&gt;A is not reported in gnomAD or dbSNP but is overrepresented in a cohort of probands with HCM from the same geographical area. This led us to suspect its clinical relevance despite the very low probability of being splice-altering as assigned by SpliceAI (Δ score = 0.03).

Purpose
To conduct a phenotypic and clinical description of the cohort, ascertain the pathogenicity of the variant, and investigate the existence of a common ancestor.

Methods
(1) Observational analysis of clinical data and familial evaluation results, (2) Functional study involving mRNA expression analysis in peripheral blood, along with in silico analysis of the splicing disruption mechanism, and (3) Haplotype analysis to evaluate the founder effect and assess the age of the mutation.

Results
MYBPC3:c.2308+227G&gt;A was identified in 26 probands with HCM (69% male; mean age at diagnosis 53±10 years). Eighty relatives from 20 families were screened resulting in 17 cases diagnosed (mean age 49±19 years; 53% males), 19 positive genotype/negative phenotype and 38 non-carriers. The greatest cosegregation involved three cases carrying the variant, confirmed in four different families. Males were diagnosed at a younger age but without significant differences in clinical features or events during follow-up (table). RT-PCR expression analysis in patients' blood revealed that this variant disrupts pre-mRNA splicing by promoting the use of cryptic donor and acceptor splice sites, located downstream and upstream of the variant, respectively, which are already present in the intron 23 reference sequence. Strikingly, these cryptic splice sites represent one of the most frequent natural unannotated missplicing events in MYBPC3, as evidenced by SpliceVault analysis of high-throughput RNA sequencing databases. The direct primary event induced by the variant is expected to disrupt a regulatory RNA-protein binding site, thereby unblocking the use of preexisting cryptic splice sites and amplifying natural exonification missplicing events within intron 23 (figure). Finally, the haplotype analysis, based on the copy number of nine STRs covering a total of 29.2 Mb surrounding MYBPC3:c.2308+227G&gt;A, revealed that 17 unrelated carriers shared a DNA segment of 10 Mb (10,067,150 bp). The origin of this mutation is estimated to be at least 82 generations ago and roughly 1649 years in the past (IC95%:1180-2300).

Conclusions
MYBPC3:c.2308+227G&gt;A is an elusive novel deep intronic variant causing HCM by a molecular mechanism not previously described in this context. The founder effect of this variant is confirmed in our geographical area.Phenotypic and clinical characteristics