Genetic screening for hypertrophic cardiomyopathy (HCM) has traditionally comprised of sequencing the coding regions of genes and looking for single nucleotide (SNVs) and copy number variants (CNVs) that could disrupt gene function. These methods typically expand ~10 base pairs into the non-coding intronic regions that flank the coding exons and are therefore unable to detect variants that may lie deeper within the introns. The yield in genetic testing in HCM is only ~50%, this may be increased by analysing intronic regions of HCM genes.
The aim of this study was to analyse the complete sequences of the MYBPC3 and MYH7 genes to identify potentially pathogenic rare deep-intronic SNVs and CNVs.
This study included 281 patients who had been clinically diagnosed with HCM and had previously undergone routine diagnostic genetic testing. Targeted sequencing of MYBPC3 and MYH7 coding and non-coding regions had been previously performed. Rare deep-intronic variants predicted to affect splicing were selected for further analysis and characterisation at the RNA level. Splicing outcome was analysed by sizing cDNA amplification products and Sanger sequencing. CNV analysis was carried out using an in-house CNV caller utilising read depth analysis.
Analysis of deep-intronic SNVs in MYBPC3 enabled molecular diagnoses in four HCM patients and uncovered potentially pathogenic variants in a number of others that could be tested at the RNA level in future. CNV analysis in MYBPC3 revealed a single molecular HCM diagnosis. Analysis of MYH7 did not reveal any new variants.
There is an increasing awareness of the importance of the non-coding regions in the genome in clinical diagnostics. Although only two HCM genes were looked at here, this study has implications for other cardiomyopathy genes, as well as other diseases, where pathogenic variants in deep-intronic regions may be missed due to the lack of sequencing and analysis of these regions.