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Published on: August 8, 2022
Scaled Multidimensional Assays of Variant Effect Identify Sequence-Function Relationships in Hypertrophic
Yuta Yamamoto1, Kaiser Chua1, David Staudt1,2
1Stanford Center for Inherited Cardiovascular Disease, Department of Medicine, Division of Cardiovascular Medicine, (Y.Y., K.C., D.S., A.F., B.J.F., C.C., L.W., Q.W., J.E.G., Y.H., F.B., R.H.W., A.S., A.T., R.A., M.T.W., M.M., E.A.A., V.N.P.), Stanford School of Medicine, Palo Alto, CA.
Insights
Genetic variants in MYBPC3 cause hypertrophic cardiomyopathy (HCM). This study developed a new method to interpret these variants, revealing decreased cardiac myosin-binding protein C (cMyBP-C) abundance as a key driver of HCM and identifying new disease mechanisms.
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.
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.
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