Reanalysis of Next-generation Sequencing Data in Patients With Hypertrophic Cardiomyopathy: Contribution of Spliceogenic MYBPC3 Variants in an Italian Cohort

Abstract

Hypertrophic cardiomyopathy (HCM) is a genetic cardiac muscle disease characterized by clinical and genetic heterogeneity. Genetic testing can reveal the presence of disease-causing variants in genes encoding sarcomere proteins. However, it yields inconclusive or negative results in 40-60% of HCM cases, owing to, among other causes, technical limitations such as the inability to detect pathogenic intronic variants. Therefore, we aimed to increase the diagnostic yield of molecular analysis for HCM by improving the in-silico detection of intronic variants in MYBPC3 that may escape detection by algorithms normally used with tagged diagnostic panels. We included 142 HCM probands with negative results in Illumina TruSight Cardio panel analysis, including exonic regions of 174 cardiomyopathy genes. Raw data were re-analyzed using existing bioinformatics tools. The spliceogenic variant c.1224-80G>A was detected in three patients (2.1%), leading us to reconsider their molecular diagnosis. These patients showed late onset and mild symptoms, although no peculiar phenotypic characteristics were shared. Collectively, rare spliceogenic MYBPC3 variants may play a role in causing HCM, and their systematic detection should be performed to provide more comprehensive solutions in genetic testing using multigenic panels.

Keywords: High-throughput nucleotide sequencing; Hypertrophic cardiomyopathy; Introns; Loss of function mutation; RNA splicing.

Fig. 1. Parallel multistep workflow for diagnostic setting. Left side (first approach): development and application of an in-silico hotspot for previously reported PVs; right side (second approach): in-silico filtering and in-vitro analysis of unknown variants.

Abbreviations: PVs, pathogenic variants; IGV, Integrative Genomics Viewer; ACMG, American College of Medical Genetics and Genomics.

Fig. 2. MYBPC3 exon 14 splicing. (A) Schematic representation of the nucleotide sequences of exon 13, intron 13, and exon 14. Canonical splicing of intron 13 with the juxtaposition of the 3′-exon 13/5′-exon 14 junction is shown above (green lines). The variant c.1224-80G>A (in red) creates a cryptic splice acceptor site in intron 13 (dinucleotide “AG”), which leads to the juxtaposition of 3′-exon 13 with a locus upstream of 5′-exon 14 (bottom, red lines), inserting 78 intronic nucleotides into the transcript. (B) Representation of aligned NGS reads (top) and Sanger electropherogram (bottom) corresponding to the heterozygous variant c.1224-80G>A (arrow).

Abbreviation: NGS, next-generation sequencing.