Highlights d Integrated omics method identifies non-canonical and undocumented protein isoforms d Widespread isoform shifts during human iPSC cardiomyocyte differentiation d Unidentified protein isoforms recoverable from 80 million public mass spectra d Isoform sequences overlap with intrinsically disordered regions and PTM sites

Background: Hypertrophic cardiomyopathy (HCM) is frequently caused by mutations in myosin binding protein C3 (MYBPC3) resulting in a premature termination codon (PTC). The underlying mechanisms of how PTC mutations in MYBPC3 lead to the onset and progression of HCM are poorly understood. This study's aim was to investigate the molecular mechanisms underlying the pathogenesis of HCM associated with MYBPC3 PTC mutations by utilizing human isogenic induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). Methods: Isogenic iPSC lines were generated from patients harboring MYBPC3 PTC mutations (p.R943x; p.R1073P_Fsx4) using genome editing and then differentiated into cardiomyocytes. Comprehensive phenotypical and transcriptome analyses were performed.

-The progression toward low-cost and rapid next-generation sequencing has uncovered a multitude of variants of uncertain significance (VUS) in both patients and asymptomatic "healthy" individuals. A VUS is a rare or novel variant for which disease pathogenicity has not been conclusively demonstrated or excluded, and thus cannot be definitively annotated. VUS, therefore, pose critical clinical interpretation and risk-assessment challenges, and new methods are urgently needed to better characterize their pathogenicity. -To address this challenge and showcase the uncertainty surrounding genomic variant interpretation, we recruited a "healthy" asymptomatic individual, lacking cardiac-disease clinical history, carrying a hypertrophic cardiomyopathy (HCM)-associated genetic variant (NM_000258.2:c.170C>A, NP_000249.1:p.Ala57Asp) in the sarcomeric gene , reported by the ClinVar database to be "likely pathogenic." Humaninduced pluripotent stem cells (iPSCs) were derived from the heterozygous VUS carrier, and their genome was edited using CRISPR/Cas9 to generate 4 isogenic iPSC lines: (1) corrected "healthy" control; (2) homozygous VUS; (3) heterozygous frameshift mutation ; and (4) known heterozygous pathogenic mutation (NM_000258.2:c.170C>G), at the same nucleotide position as VUS, lines. Extensive assays including measurements of gene expression, sarcomere structure, cell size, contractility, action potentials, and calcium handling were performed on the isogenic iPSC-derived cardiomyocytes (iPSC-CMs). -The heterozygous VUS-iPSC-CMs did not show an HCM phenotype at the gene expression, morphology, or functional levels. Furthermore, genome-edited homozygous VUS- and frameshift mutation -iPSC-CMs lines were also asymptomatic, supporting a benign assessment for this particular variant. Further assessment of the pathogenic nature of a genome-edited isogenic line carrying a known pathogenic mutation,, and a carrier-specific iPSC-CMs line, carrying a HCM variant, demonstrated the ability of this combined platform to provide both pathogenic and benign assessments. -Our study illustrates the ability of clustered regularly interspaced short palindromic repeats/Cas9 genome-editing of carrier-specific iPSCs to elucidate both benign and pathogenic HCM functional phenotypes in a carrierspecific manner in a dish. As such, this platform represents a promising VUS riskassessment tool that can be used for assessing HCM-associated VUS specifically, and VUS in general, and thus significantly contribute to the arsenal of precision medicine tools available in this emerging field.