High-throughput functional characterization of MYH7 variants in genome-edited cardiomyocytes

Hypertrophic cardiomyopathy (HCM) is a heritable hypertrophy of the left ventricle that can present as sudden cardiac death and heart failure. Autosomal dominant missense variants in myosin heavy chain 7 (MYH7) can cause up to 40% of the familial HCM cases. While accurate interpretation of MYH7 variants can help identify patients at risk for developing HCM, there are currently ~1700 MYH7 missense variants in ClinVar that are variants of unknown significance (VUS), creating diagnostic challenges for clinicians and emotional distress for patients. Furthermore, because clinical guidelines now recommend that all HCM patients undergo genetic testing, the number of MYH7 VUS is growing rapidly. Deep mutational scanning (DMS) can help address the VUS problem by determining the functional consequence of thousands of genetic variants in a single experiment. By generating accurate variant effect maps that define the function of nearly all possible missense variants in a sequence of interest, DMS can assist with clinical variant interpretation at scale. So far, deep mutational scans have been primarily practiced in cancer-derived cell lines, which are not helpful for assessing genes such as MYH7 that are only expressed in specialized cells like cardiomyocytes. Human induced pluripotent stem cells (hiPSCs) can differentiate to many cell types, offering a solution to this problem; however, gene-editing in hiPSCs is generally low throughput. We recently developed a novel gene-editing technique called CRISPR activation On-Target Editing Retrieval (CRaTER) that can edit hiPSCs at scale, enabling the generation of large variant libraries in hiPSCs for the first time. To address the growing MYH7 VUS problem, we plan to perform a deep mutational scan of the MYH7 S2 domain, a mutational hotspot associated with HCM, in physiologically-relevant hiPSC-derived cardiomyocytes (hiPSC-CMs). In Aim 1, we propose to generate a single nucleotide variant (SNV) library encoding nearly all possible MYH7 S2 missense variants in hiPSC-CMs. We will leverage CRaTER-assisted genome-editing to be able to generate a hiPSC SNV library at this scale. In Aim 2 we will optimize and validate phenotypic assays to be able to discriminate hiPSC-CMs expressing pathogenic variants from hiPSC-CMs expressing benign variants. These phenotypic assays will inform a deep mutational scan of the MYH7 S2 domain that will determine the functional effect of nearly all possible MYH7 S2 missense variants and generate a variant effect map to assist with clinical variant interpretation. Finally, we will use the variant effect map generated to help improve the estimated ~130 MYH7 S2 VUS in the Million Veteran Program’s whole genome sequencing cohort. In summary, we will combine CRaTER, a novel gene-editing technique we developed, with hiPSCs to perform a DMS of MYH7 in genome- edited hiPSC-CMs. The results will improve the interpretation of hundreds of MYH7 VUS, enable the interpretation of thousands of possible MYH7 missense variants that may be encountered in the future, and provide a framework by which deep mutational scans can now be performed in hiPSC-derivatives for the first time, which we believe will be transformative for the field of variant interpretation. Project Number: 1I01BX006428-01A1 | Fiscal Year: 2025 | NIH Institute/Center: Veterans Affairs (VA) | Principal Investigator: Kai-Chun Yang | Institution: VA PUGET SOUND HEALTHCARE SYSTEM, SEATTLE, WA | Activity Code: I01 | Study Section: Special Emphasis Panel[ZRD1 CARA-R (01)] View on NIH RePORTER: https://reporter.nih.gov/project-details/10928069