Investigating genetic and mechanistic interactors in familial cardoimyopathy through advanced disease modeling

Abstract

Hypertrophic cardiomyopathy (HCM) is the most common inherited cardiac disease and a frequent cause of heart failure and sudden cardiac death. HCM is a highly complex condition defined by clinical and genetic heterogeneity. During last decades, our understanding of the diverse genetic landscape and the pathological molecular mechanisms underlying HCM has increased significantly. However, studying the effect of genetic modifiers of cardiomyopathies is limited by their complex genetic aetiology. A better understanding of the complex genetic mechanisms underlying cardiac diseases is an imperative hallmark for precision medicine. With this aim, we sought to investigate the differing molecular and genetic mechanisms of two siblings with an extensive family history of HCM but divergent clinical manifestations using patient-specific induced pluripotent stem cells (hiPSCs).

For this purpose, we generated patient-specific iPSC from the male, diagnosed with a severe hypertrophic phenotype, and from the female, with mild hypertrophy, whose genetic testing revealed a common pathogenic mutation in the MYBPC3 gene (K600Nfs*2). Morphological characterization of iPSC-derived cardiomyocytes from mutant carriers revealed that sarcomeric alignment and structure was not compromised. However, MYBPC3 deficient iPSC-CMs showed reduced contractile force generation without cell shape remodelling. We then took advantage of the CRISPR/Cas9 gene-editing technology to generate MYBPC3-corrected isogenic controls in order to better ascribe genotype-phenotype correlations. Functional evaluation of mutant and isogenic iPSC-CMs revealed that cardiomyocytes from the symptomatic patient presented a hypercontractile phenotype as well as faster calcium transients. Further analysis on the mitochondrial bioenergetics indicated an inefficient ATP consumption in sarcomeres from both mutant carriers.

In order to explore whether additional genetic variants were modifying the pathological outcomes in the symptomatic carrier, we performed a whole- exome sequencing of the mutant carriers. We identified a variant of unknown significance (VUS) in the MYH7 gene (I1927F), the second most common mutated gene in HCM, uniquely present in the severe HCM individual. Although the identified VUS has been previously described in HCM patients, there is not sufficient clinical and functional evidence to ascertain pathogenicity. To precisely evaluate the effect of the VUS, we generated a MYH7 I1927F corrected isogenic iPSC line using CRISPR/Cas9. Functional evaluation of double and single mutant iPSC-CMs revealed that the additional presence of the MYH7 variant was responsible for the faster cardiac contraction, strongly supporting a severe pathogenic contribution. Our study provides a unique platform to functionally assess the effect of genetic modifiers.