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

Area of Science:

• Cardiovascular Genetics
• Molecular Cardiology
• Genomic Medicine

Background:

• Hypertrophic cardiomyopathy (HCM) affects 1 in 500 people, with genetic diagnosis aiding risk identification and therapy.
• MYBPC3 gene variants are a major cause of HCM, but many variants of uncertain significance complicate clinical decisions.
• Scalable methods for interpreting genetic variants in relevant cell types are crucial for understanding disease mechanisms.

Purpose of the Study:

• To develop a scalable, multidimensional mapping strategy for evaluating the functional impact of MYBPC3 variants.
• To analyze variant effects on cardiac myosin-binding protein C (cMyBP-C) function and HCM-relevant phenotypes in patient-derived cells.
• To improve the interpretation of MYBPC3 variants and uncover novel disease mechanisms.

Main Methods:

• Developed a multidimensional mapping strategy using saturation base editing at the native MYBPC3 locus.
• Employed a long-read RNA sequencing assay to assess variant splice effects.
• Measured HCM-relevant phenotypes in human induced pluripotent stem cell-derived cardiomyocytes, including cMyBP-C abundance and hypertrophic signaling.

Main Results:

• High-resolution functional analysis of MYBPC3 variants in cardiomyocytes was achieved.
• A massively parallel splicing assay identified novel splice-disrupting variants.
• Decreased cMyBP-C abundance was identified as a key driver of HCM phenotypes, with protein degradation downregulation correlating with MYBPC3 loss of function.

Conclusions:

• The developed platform enables multiplexed assays of variant effects across diverse cellular phenotypes using genome engineering.
• This approach enhances understanding of variant pathogenicity and uncovers novel biological mechanisms for HCM.
• Findings can inform therapeutic strategies for hypertrophic cardiomyopathy.