High-Throughput Studies of MYH7 Variant Cardiomyocytes to Identify Pathogenic Variants and Factors Governing Variable Expressivity of Hypertrophic Cardiomyopathy

PROJECT SUMMARY / ABSTRACT Hypertrophic cardiomyopathy (HCM), characterized by unexplained left ventricular hypertrophy, affects 1:200- 500 people. HCM can cause heart failure and sudden cardiac death. Pathogenic single nucleotide variants (SNVs) causing missense substitutions in myosin heavy chain 7 (MYH7; encoding β-MHC) account for 35% of inherited HCM cases. Pathogenic MYH7 variants are clinically actionable because they identify patients at risk of developing HCM, prompting early interventions that can prevent sudden cardiac death. However, 75% of MYH7 missense variants are of unknown significance (VUS) because there is insufficient evidence to determine pathogenicity. Furthermore, variable expressivity, or symptomatic severity, in patients with pathogenic MYH7 variants limits their prognostic value. Identification of HCM-modifying factors would enable clinicians to personalize patient counselling. The goal of this proposal is to establish a new system to accelerate MYH7 VUS reclassification and determine the role of putative HCM-modifiers using high-throughput in vitro models to provide clinically actionable information for patients with MYH7 variants. Human induced pluripotent stem cells (hiPSCs) retain their donor’s genetic background, can be differentiated to cardiomyocytes (hiPSC- CMs), and are an ideal model for MYH7 variant effect studies. hiPSC-CMs with pathogenic MYH7 variants have shown decreased survival, decreased variant-to-wildtype β-MHC protein, and increased atrial natriuretic peptide (ANP) expression, which correlate with measures of HCM severity. During my postdoctoral training, I developed a novel gene-editing tool to enable pooled generation of MYH7 variant hiPSC libraries. High-throughput assessments of a pilot MYH7 variant library in hiPSC-CMs correctly segregated all pathogenic and benign variants, showing that my approach can be scaled to determine the functional effect of thousands of MYH7 SNVs in hiPSC-CMs (Aim 1). These functional data will aid in the reclassification of up to 140 VUS, yet HCM expressivity is variable and the roles of putative HCM-modifiers remain largely untested. Genome-wide association studies (GWAS) have linked many single nucleotide polymorphisms (SNPs) with HCM. I hypothesize that these GWAS SNPs act as genetic modifiers that drive the expressivity of pathogenic MYH7 HCM variants. To test this hypothesis, I propose to measure the phenotypic variability of pathogenic MYH7 variants in hiPSC-CMs generated in an isogenic hiPSC background with three different HCM-associated SNPs separately introduced (Aim 2). Moreover, males manifest HCM earlier than females and have increased expressivity after puberty, however, the source of this sex difference remains unknown. I hypothesize that both sex chromosomes and sex hormones contribute to HCM sexual dimorphism. To test this hypothesis, I propose to establish a co-culture ‘village’ of pooled MYH7 variant hiPSC-CMs generated on healthy XX and XY hiPSC backgrounds and examine phenotypic variability with and without sex hormones (Aim 3).