Abstract Body: Introduction: MYBPC3 is the most common genetic cause of hypertrophic cardiomyopathy (HCM). However, many variants identified in patients remain classified as variants of uncertain significance (VUS), limiting genetic diagnosis and clinical interpretation. Multiplex assays of variant effect (MAVEs) provide a scalable strategy to functionally interpret disease-associated variants, but most prior MAVEs have been performed in non-cardiac immortalized cells, which do not fully recapitulate the biological context of sarcomeric gene variants.
Hypothesis: Scaled multidimensional functional assays in iPSC-derived cardiomyocytes (iPSC-CMs) will enable systematic interpretation of MYBPC3 variants and elucidate the underlying mechanisms of HCM.
Methods: We developed multidimensional functional assays in iPSC-CMs to evaluate MYBPC3 variant effects across a critical region of the gene. To evaluate variant effect on splicing, we performed a minigene splice reporter assay combined with long-read sequencing. In parallel, MYBPC3 variants were introduced by base editing at the native locus in iPSCs and HCM-relevant phenotypes including MYBPC3 protein abundance, hypertrophy signaling, and ubiquitin-proteasome activity were evaluated in iPSC-CMs.
Results: Our multidimensional functional assays provided a high-resolution functional map of over 400 MYBPC3 variants. Splicing reporter assay identified 21 splicing-disrupting variants across intronic and exonic regions. Targeted in situ mutagenesis using base editors generated a native-locus variant library and high-throughput phenotyping captured diverse functional variant effects on HCM-relevant phenotypes. Integration of functional datasets indicated that reduction of MYBPC3 abundance is a primary pathogenic mechanism for MYBPC3 variants. In addition, several missense variants located in critical interacting domain with actin/calmodulin may contribute to the pathogenesis via a novel mechanism.
Conclusion: Multidimensional functional assays in iPSC-CMs enable scalable interpretation of MYBPC3 variants and provide new mechanistic insights into HCM.