Introns in action: decoding deep intronic MYBPC3 variants in hypertrophic cardiomyopathy with patient-derived iPSCs

Abstract Background Hypertrophic Cardiomyopathy (HCM) is the most common inherited cardiovascular disease and a leading cause of sudden death. Despite decades of research and advancements in sequencing technologies, the yield of genetic testing remains poor with up to 60% of individuals remaining gene elusive. This limits the clinical actionability of clinical genetic testing, vital in identifying at-risk relatives and distinguishing HCM from phenocopies. Conventional genetic tests primarily focus on the detection of exonic and canonical splice site variation. Oversighting intronic, non-canonical splicing variants potentially contributes to a proportion of HCM patients remaining genetically undiagnosed. Purpose/Aim Herein, we aimed to take advantage of iPSC technology and generate iPSC lines from HCM patients harbouring deep intronic Myosin-binding protein C3 (MYBPC3) variants (c.1224-52GA G) to determine, upon differentiation of iPSCs to cardiomyocytes (iPSC-CMs), any arising splicing abnormalities that may contribute to disease pathogenesis. Methods 4 patients harbouring deep intronic MYBPC3 variants were recruited. PBMCs obtained by venepuncture were isolated and reprogrammed into iPSCs using Sendai virus. iPSCs were characterised for pluripotency qualities and differentiated into cardiomyocytes via aggregation methodology. Spontaneously beating iPSC-CMs were cultured with cycloheximide, and RT-PCR and Sanger sequencing used to establish any splicing abnormalities. Results 4 iPSC lines (H17 A (-52) and H24 G (-23)) were generated and showed pluripotent properties. Upon differentiation, the -52 variant was shown to create a cryptic splice site upstream of the canonical 3′ splice site, resulting in the extension of exon 14 by 50 nucleotides. The resulting aberrantly-spliced transcripts were degraded by nonsense mediated decay (NMD), suggesting haploinsuffiency as a possible disease mechanism. The -23 variant was also shown to produce mis-spliced transcripts via branchpoint disruption and full retention of intron 19 leading to a PTC. However, transcripts were not flagged for NMD degradation, despite -23 cells retaining NMD machinery, suggesting a different disease mechanism. Conclusion By taking advantage of iPSC technology, we have added evidence that intronic MYBPC3 variants interfere with splicing. Whilst further work is needed to characterise morphological abnormalities arising from splicing mutations, these newly established iPSC lines provide valuable cellular models for elucidating the mechanisms by which non-canonical splicing variants cause HCM. This work may prove helpful in fulfilling the unmet clinical need to improve the clinical diagnostic yield of genetic testing.