Scaled Multidimensional Assays of Variant Effect Identify Sequence-Function Relationships in Hypertrophic Cardiomyopathy
Abstract
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.
Article Details
Authors (27)
Yuta Yamamoto
Institute of Materials and Systems for Sustainability
Kaiser Chua
Stanford 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.
David Staudt
Alexis Ferrasse
Stanford 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.
Anna Kirillova
Medical Scientist Training Program, University of Pittsburgh, PA (A.K.).
Hannah N. De Jong
Department of Genetics, Stanford School of Medicine, Palo Alto, CA. (H.N.D.J., J.E.G.)
Brendan J. Floyd
Stanford 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.
Christian Cadisch
Stanford 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.
Laurens Wiel
Qianru Wang
Dalian Institute of Chemical Physics
Matthew J. O’Neill
Brigham and Women’s Hospital, HMS, Boston, MA (M.J.O., C.A.M.).
Daniel Tabet
Donnelly Centre and Department of Molecular Genetics, University of Toronto, ON, Canada (D.T., F.P.R.).
John E. Goryznski
Stanford 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.
Yong Huang
National Laboratory of Solid State Microstructures, School of Physics
Fang Bai
Rachel H. Wilson
Stanford 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.
Arman Sharma
Stanford 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.
Althea Tapales
Stanford 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.
Rani Agrawal
Stanford 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.
Matthew T. Wheeler
Mark Mercola
Stanford Cardiovascular Institute, Department of Medicine, and Department of Chemical and Systems Biology
Calum A. MacRae
Dan M. Roden
Frederick P. Roth
Andrew M. Glazer
Euan A. Ashley
Victoria N. Parikh
Stanford 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.