Abstract 18022: Multiplexed Generation and Functional Annotation of Hypertrophic Cardiomyopathy-Associated MYH7 Missense Variants in Isogenic Gene-Edited Stem Cell-Derived Cardiomyocytes

Hypertrophic cardiomyopathy (HCM) is an inheritable cardiac condition affecting 1:200-500 people and is characterized by left ventricular hypertrophy that can cause heart failure. Autosomal-dominant, missense mutations in MYH7 (encoding MHC-β) cause 33% of genotype ⁺ HCM cases. Despite this, 75% of MYH7 missense variants are of unknown clinical significance (VUS). Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) can elucidate the effect(s) of VUS, however, new variant identification by genetic testing outpaces methods to gene edit hiPSCs. Thus, we developed a method called

ISPR On- arget diting etrieval (CRaTER) which increased successful gene-editing 25-fold over current methods. We leveraged CRaTER to enrich for edited hiPSCs, generating a library of 113 MYH7 missense variants. As mutations can reduce protein stability and abundance, we flow sorted MYH7 variant hiPSC-CMs into bins based on differing MHC-β protein abundance followed by next generation sequencing to calculate variant abundance scores. This multiplexed assay identified 31 residue substitutions as functionally abnormal, including all tested pathogenic variants and VUS. MHC-β depletion was validated in clonal hiPSC-CMs and by Western blotting of patient myocardium with a MYH7 mutation. Additionally, we found that severe MHC-β depletion is associated with increased cell size, a hallmark pathological phenotype of HCM. Finally, single-cell contractility analyses of hiPSC-CMs with a pathogenic MYH7 variant revealed hyperdynamic physiology relative to isogenic controls, suggesting an association between MHC-β loss and systolic dysfunction. Together, CRaTER enables the generation of isogenic hiPSC variant libraries at unprecedented scale for differentiation to disease-relevant cell types. Functional analyses of MYH7 variant hiPSC-CMs have revealed a novel correlation between MHC-β protein depletion and pathogenicity that accurately predicts MYH7 variant effect and has implications for future MYH7 variant effect interpretations.