Multiplexed Functional Assessments of Variants in Human Cardiomyocytes.

Background: Pathogenic autosomal-dominant missense variants in (), which encodes the sarcomeric protein (β-MHC [beta myosin heavy chain]) expressed in cardiac and skeletal myocytes, are a leading cause of hypertrophic cardiomyopathy and are clinically actionable. However, ≈75% of missense variants are of unknown significance. While human-induced pluripotent stem cells (hiPSCs) can be differentiated into cardiomyocytes to enable the interrogation of variant effect in a disease-relevant context, deep mutational scanning has not been executed using diploid hiPSC derivates due to low hiPSC gene-editing efficiency. Moreover, multiplexable phenotypes enabling deep mutational scanning of variant hiPSC-derived cardiomyocytes are unknown.

Methods: To overcome these obstacles, we used CRISPRa On-Target Editing Retrieval enrichment to generate an hiPSC library containing 113 codon variants suitable for deep mutational scanning. We first established that β-MHC protein loss occurs in a hypertrophic cardiomyopathy human heart with a pathogenic variant. We then differentiated the missense variant hiPSC library to cardiomyocytes for multiplexed assessment of β-MHC variant abundance by massively parallel sequencing and hiPSC-derived cardiomyocyte survival.

Results: Both the multiplexed assessment of β-MHC abundance and hiPSC-derived cardiomyocyte survival accurately segregated all known pathogenic variants from synonymous variants. Functional data were generated for 4 variants of unknown significance and 58 additional missense variants not yet detected in patients.

Conclusions: This study leveraged hiPSC differentiation into disease-relevant cardiomyocytes to enable multiplexed assessments of missense variants for the first time. Phenotyping strategies used here enable the application of deep mutational scanning to clinically actionable genes, which should reduce the burden of variants of unknown significance on patients and clinicians.