Multiplexed functional assessments of MYH7 variants in human cardiomyocytes at scale

Single, autosomal-dominant missense mutations in MYH7, which encodes a sarcomeric protein (MHC-b) in cardiac and skeletal myocytes, are a leading cause of hypertrophic cardiomyopathy and are clinically-actionable. However, approximately 75% of MYH7 variants are of unknown significance (VUS). Deep mutational scans (DMS) can determine variant functional 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 to enable the interrogation of variant effect in a disease-relevant context, DMS have not been executed using diploid hiPSC derivates, presumably due to low hiPSC gene-editing efficiency. To overcome this, CRaTER enrichment has enabled the pooled generation of a saturated five amino acid MYH7 variant library in hiPSCs suitable for DMS for the first time. As a proof-of-concept, we differentiated Ahmed, this MYH7 variant hiPSC library to cardiomyocytes (hiPSC-CMs) for multiplexed assessment of MHC-b variant abundance by massively parallel sequencing (VAMP-seq) and hiPSC-CM survival. We confirm MHC-b protein loss occurs in a failing human heart with a pathogenic MYH7 mutation. We find the multiplexed assessment of MHC-b abundance and hiPSC-CM survival both accurately segregate all pathogenic from synonymous variants. Additionally, these assays provide functional data for another 62 MYH7 missense variants, including four VUS. 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.