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

Area of Science:

• Genetics
• Cardiology
• Molecular Biology

Background:

• Hypertrophic cardiomyopathy (HCM) affects 1 in 500 people, with genetic diagnosis aiding risk identification and therapy.
• Mutations in the myosin binding protein C3 (MYBPC3) gene are a common cause of HCM.
• Many MYBPC3 variants are of uncertain significance (VUS), hindering clinical decisions and disease mechanism understanding.

Purpose of the Study:

• To develop a scalable, multidimensional mapping strategy for evaluating the functional impact of MYBPC3 variants.
• To analyze variant effects on HCM-relevant phenotypes in human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs).
• To improve variant interpretation and uncover novel disease mechanisms for potential therapeutic strategies.

Main Methods:

• Developed a scaled multidimensional mapping strategy using saturation base editing at the native MYBPC3 locus.
• Employed long-read RNA sequencing to assess variant splice effects.
• Measured HCM-relevant phenotypes including MYBPC3 abundance, hypertrophic signaling, and ubiquitin-proteasome function in iPSC-CMs.

Main Results:

• High-resolution functional analysis of MYBPC3 variants in iPSC-CMs was achieved.
• Identified novel splice-disrupting variants and revealed decreased MYBPC3 abundance as a key driver of HCM phenotypes.
• Observed compensatory downregulation of protein degradation and identified novel disease mechanisms for missense variants.

Conclusions:

• The developed platform enables genome engineering in iPSCs for multiplexed variant effect assays.
• Enhanced understanding of variant pathogenicity and uncovered novel biological mechanisms.
• Provides a foundation for informing therapeutic strategies for HCM.