Scaled Multidimensional Assays of Variant Effect Identify Sequence-Function Relationships in Hypertrophic Cardiomyopathy.

BACKGROUND

An estimated 1 in 500 people lives with hypertrophic cardiomyopathy (HCM), a disease for which genetic diagnosis can identify family members at risk and increasingly guide therapy. Variants in the MYBPC3 gene, which encodes cardiac myosin-binding protein C (cMyBP-C), account for a significant proportion of HCM cases. However, many of these are classified as variants of uncertain significance, complicating clinical decision-making. Scalable methods for variant interpretation in disease-specific cell types are crucial for understanding variant impact and uncovering disease mechanisms.

METHODS

We developed a scaled multidimensional mapping strategy to evaluate the functional impact of variants across a critical domain of cMyBP-C. 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 cMyBP-C abundance, hypertrophic signaling, and ubiquitin-proteasome function in human induced pluripotent stem cell-derived cardiomyocytes.

RESULTS

Our multidimensional mapping strategy enabled high-resolution functional analysis of MYBPC3 variants in induced pluripotent stem cell-derived cardiomyocytes. Our massively parallel splicing assay identified novel splice-disrupting variants. Targeted transient base editing generated a comprehensive variant library at the native locus, capturing diverse variant effects on cellular HCM-relevant phenotypes. Integration of functional assays revealed that decreased cMyBP-C abundance is a key driver of HCM-related phenotypes. In parallel, downregulation of protein degradation was observed to correlate with MYBPC3 loss of function, and novel potential disease mechanisms were identified for missense variants near a critical binding domain. Bayesian estimates of variant effects enable the reclassification of clinical variants.

CONCLUSIONS

This work provides a platform for extending genome engineering in induced pluripotent stem cells to multiplexed assays of variant effects across diverse disease-relevant cellular phenotypes, enhancing our understanding of variant pathogenicity and uncovering novel biological mechanisms that could inform therapeutic strategies.