Multiplexed functional assessments of MYH7 variants in human cardiomyocytes at scale

Background Single, autosomal-dominant missense mutations in MYH7 , which encodes a sarcomeric protein (MHC-β) in cardiac and skeletal myocytes, are a leading cause of hypertrophic cardiomyopathy and are clinically-actionable. However, ∼75% of MYH7 variants are of unknown significance (VUS), causing diagnostic challenges for clinicians and emotional distress for patients. Deep mutational scans (DMS) can determine variant effect at scale, but have only been utilized in easily-editable cell lines. While human induced pluripotent stem cells (hiPSCs) can be differentiated to numerous cell types that enable the interrogation of variant effect in a disease-relevant context, DMS have not been executed using diploid hiPSC derivates. However, CRaTER enrichment has recently enabled the pooled generation of a saturated five position MYH7 variant hiPSC library suitable for DMS for the first time. Results As a proof-of-concept, we differentiated this MYH7 variant hiPSC library to cardiomyocytes (hiPSC-CMs) for multiplexed assessment of MHC-β variant abundance by massively parallel sequencing (VAMP-seq) and hiPSC-CM survival. We confirm MHC-β protein loss occurs in a failing human heart with a pathogenic MYH7 mutation. We find the multiplexed assessment of MHC-β abundance and hiPSC-CM survival both accurately segregate all pathogenic variants from synonymous controls. Overall, functional scores of 68 amino acid substitutions across these independent assays are ∼50% consistent. Conclusions This study leverages hiPSC differentiation into disease-relevant cardiomyocytes to enable multiplexed assessments of MYH7 missense variants at scale for the first time. This proof-of-concept demonstrates the ability to DMS previously restricted, clinically-actionable genes to reduce the burden of VUS on patients and clinicians.