Multiplexed functional assessments ofMYH7variants in human cardiomyocytes at scale

Abstract

AbstractBackgroundSingle, autosomal-dominant missense mutations inMYH7, 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% ofMYH7variants 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 positionMYH7variant hiPSC library suitable for DMS for the first time.ResultsAs a proof-of-concept, we differentiated thisMYH7variant 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 pathogenicMYH7mutation. 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.ConclusionsThis study leverages hiPSC differentiation into disease-relevant cardiomyocytes to enable multiplexed assessments ofMYH7missense 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.