Scaled multidimensional assays of variant effect identify sequence-function relationships in hypertrophic cardiomyopathy

Yamamoto, Y.; Chua, K.; Ferrasse, A.; Kirillova, A.; De Jong, H.; Floyd, B.; Cadisch, C.; Wiel, L.; Wang, Q.; O'Neill, M.; Tabet, D. R.; Staudt, D. W.; Gorzynski, J. E.; Huang, Y.; Wilson, R. H.; Sharma, A.; Tapales, A.; Agrawal, R.; Wheeler, M. T.; MacRae, C. A.; Roden, D. M.; Roth, F.; Glazer, A. M.; Ashley, E. A.; Parikh, V. N.

doi:10.1101/2025.05.23.655878

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bioRxiv · 10.1101/2025.05.23.655878

Scaled multidimensional assays of variant effect identify sequence-function relationships in hypertrophic cardiomyopathy

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Abstract

BackgroundAn estimated 1 in 500 people live with hypertrophic cardiomyopathy (HCM), a disease for which genetic diagnosis can identify family members at risk, and increasingly guide therapy. Mutations in the myosin binding protein C3 (MYBPC3) gene account for a significant proportion of HCM cases. However, many of these variants are classified as variants of uncertain significance (VUS), complicating clinical decision-making. Scalable methods for variant interpretation in disease-specific cell types are crucial for understanding variant impact and uncovering disease mechanisms. MethodsWe developed a scaled multidimensional mapping strategy to evaluate the functional impact of variants across a critical domain of MYBPC3. We incorporate saturation base editing at the native MYBPC3 locus, a long-read RNA sequencing-enabled assay of variant splice effects, and measurements of HCM-relevant phenotypes, including MYBPC3 abundance, hypertrophic signaling, and ubiquitin-proteasome function in human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). ResultsOur multidimensional mapping strategy enabled high-resolution functional analysis of MYBPC3 variants in iPSC-CMs. Targeted transient base editing generated a comprehensive variant library at the native locus, capturing diverse variant effects on cellular HCM-relevant phenotypes. Our massively parallel splicing assay identified novel splice-disrupting variants. Integration of functional assays revealed that decreased MYBPC3 abundance is a key driver of HCM-related phenotypes. In parallel, downregulation of protein degradation was observed as a compensatory response to MYBPC3 loss of function, and novel disease mechanisms were identified for missense variants near a critical binding domain, underscoring their contribution to pathogenesis. Bayesian estimates of variant effects enable the reclassification of clinical variants. ConclusionsThis work provides a platform for extending genome engineering in iPSCs to multiplexed assays of variant effects across diverse disease-relevant cellular phenotypes, enhancing the understanding of variant pathogenicity and uncovering novel biological mechanisms that could inform therapeutic strategies.

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Yamamoto, Y., Chua, K., Ferrasse, A., Kirillova, A., De Jong, H., Floyd, B., Cadisch, C., Wiel, L., Wang, Q., O'Neill, M., Tabet, D. R., Staudt, D. W., Gorzynski, J. E., Huang, Y., Wilson, R. H., Sharma, A., Tapales, A., Agrawal, R., Wheeler, M. T., MacRae, C. A., Roden, D. M., Roth, F., Glazer, A. M., Ashley, E. A., Parikh, V. N.. 2025-05-27. Scaled multidimensional assays of variant effect identify sequence-function relationships in hypertrophic cardiomyopathy. https://doi.org/10.1101/2025.05.23.655878

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