Abstract Body: Introduction: MYBPC3 is the most prevalent gene in hypertrophic cardiomyopathy (HCM). However, the pathogenicity of most MYBPC3 variants remains unknown, and it is still challenging to interpret variants identified in patients. Although multiplexed functional assays using cancer cell lines are powerful approaches to test large number of variants in a single experiment and facilitate the interpretation of variants, there is a limitation of the assessment of genes expressing in specific cell types, such as sarcomeric genes including MYBPC3.
Hypothesis: The approach integrating base-editing mutagenesis and multiplexed functional assays using iPSC-derived cardiomyocytes (iPSC-CMs) will enable us to prospectively evaluate MYBPC3 variants in a high-throughput manner and illuminate the pathophysiological mechanism of HCM.
Methods: We focused on the intron-exon boundary including intron 11 and exon 12 in MYBPC3 and introduced variants into iPSCs by CRISPR-X, which can target multiple genomic locations and introduce diverse editing, and adenine base editor. After cardiac differentiation, we performed multiplexed functional assays. iPSC-CMs were immunostained for cMyBP-C and BNP expressions and sorted based on those expressions using fluorescence-activated cell sorting. The ubiquitin-proteasome activity associated with cellular proteostasis was analyzed by ubiquitin fusion degradation reporter system. The variant scores were calculated by comparing the frequency of each variant in each population sorted based on phenotype using next-generation sequencing.
Results: About 200 variants located at the mutagenesis target region were evaluated via in situ mutagenesis and each functional assay. The pathogenic/likely pathogenic variants from ClinVar exhibited higher variant scores compared to benign/likely benign variants in each assay. Even in variants that have not yet been reported clinically, nonsense variants and several missense and intronic variants including splice site variants showed higher variant scores compared to synonymous variants.
Conclusion: This study demonstrates that our novel approach can prospectively evaluate MYBPC3 variant effect at scale and provide clues to elucidate pathophysiological mechanism of HCM.