Biology subjects

Parikh, V. N.

Publications and source records attributed to Parikh, V. N..

5 recordsLinked to original sources

bioRxiv2026

Damaging RBM20 E-rich domain variants are not rescued by gene replacement

The promise of precision therapeutics in genetic cardiomyopathies relies on linking specific therapies to variant mechanisms. Missense variants in the cardiac splice regulator RBM20 cause a highly penetrant and arrhythmogenic dilated cardiomyopathy. Disease-causing variants in RBM20s arginine-serine rich (RS) domain act via formation of toxic gain of function cytoplasmic granules, but this is not true for a small number of clinically adjudicated pathogenic variants in its glutamate(E)-rich domain. To better define the effects of E-rich domain variants, we developed a scalable screen based on induced pluripotent stem cell (iPSC) cardiomyocyte differentiation that identified several additional damaging variants. Several of these reduced RBM20 protein abundance and stability. We therefore hypothesized that, unlike RS domain variants, these E-rich variants might be rescued by RBM20 overexpression. To test this hypothesis, we generated induced pluripotent stem cells (iPSCs) from a patient with a pathogenic E-rich domain variant (p.E913K), and confirmed reduced RBM20 protein expression in these RBM20+/p.E913K cells after differentiation to iPSC-derived cardiomyocytes (iPSC-CM, vs. engineered isogenic RBM20+/+). These iPSC-CMs also displayed aberrant transcriptional splicing, reduced contractility, increased calcium-induced calcium release, and nuclear localization of RBM20 protein, often with more than the two expected RBM20-centric splice factories. AAV-based overexpression of RBM20 reversed some, but not all of the mis-splicing events identified in RBM20+/p.E913K iPSC-CMs, and did not improve their abnormal contractility, calcium handling or supernumerary RBM20 nuclear granules. In summary, our data indicate that pathogenic E-rich domain variants reduce RBM20 protein abundance, but that their mechanism is unlikely to be explained by haploinsufficiency alone.

genetics↗

bioRxiv2026

Multimodal phenotyping defines variant-to-function maps for RBM20 in dilated cardiomyopathy

Multiplex assays of variant effects have linked thousands of genotypes to fitness effects, yet we lack profound understanding of how variants impact molecular phenotypes. Here, we introduce a deep mutational scanning framework that quantifies disease-determining molecular phenotypes in human cells, allowing readouts of protein localization and splicing regulatory function at scale. Applied to the dilated cardiomyopathy (DCM)-associated protein RBM20, we profiled [~]4,300 amino acid substitutions across disease-linked protein domains. Complemented by structure-function investigations of RBM20 bound to its nuclear import receptor TNPO3, we discover new variant hotspots affecting protein function. Finally, we systematically probed nuclear relocalization to identify variants that may be amenable to this therapeutic strategy. Together, we create comprehensive variant-to-function maps that predict variant impact, enhance clinical interpretation, and stratify RBM20-mediated DCM into mechanistically distinct therapeutic classes.

genomics↗

bioRxiv2025

Multiparametric Assessment of TNNI3 Variant Phenotypes in Human iPSC-Cardiomyocytes Correlates with Disease Severity in Patients

BackgroundThe routine genetic testing of cardiomyopathy patients has significantly accelerated the identification of causative cardiomyopathy variants. However, translating these genetic insights into effective patient management poses significant challenges, since the impact of gene variants on physiological function and clinical outcomes is not yet fully understood. Therefore, there is an urgent need for large-scale methods to assess the effects of genetic variants on cardiomyocyte physiology and to establish correlations between functional phenotypes and clinical severity. MethodsWe developed a high throughput imaging platform to measure force generation and calcium handling throughout the cardiac cycle of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). By expressing variants of a sarcomeric protein [cardiac Troponin-I (TNNI3)] in a healthy genetic background, we were able to assess sarcomeric calcium sensitivity as well as systolic and diastolic function. Analysis of these parameters distinguished subgroups of variants, and permitted the correlation of in vitro physiological effects with a measure of disease severity in a single-center cardiomyopathy cohort. ResultsCombining contractile force and calcium cycling measurements accurately distinguished known pathogenic from non-pathogenic TNNI3 variants and also revealed pathogenicity of two variants of unknown significance (VUS) that occurred in two families, suggesting the ability to prospectively discern pathogenicity. Clustering of TNNI3 variants based on quantitative physiological phenotypes identified subgroups that correlated with age of disease onset across a well-characterized cardiomyopathy patient cohort, showing clinical relevance of the in vitro phenotypes. Interestingly, normalized measures of in vitro diastolic function correlated with age of onset (R2 = 0.6), but calcium sensitivity, which accurately predicted pathogenicity, did not translate into disease severity. ConclusionsA high throughput in vitro platform that measures multidimensional cardiomyocyte function can link subgroups of human genetic variants in TNNI3 with differential patient outcomes. Comprehensive determination of variant effects on disease-relevant cardiomyocyte function will help classify variants into different pathogenic mechanisms leading to variable disease severity, and potentially lead to class-targeted ameliorative strategies.

genetics↗

bioRxiv2025

Spatial genomics of the cardiac sarcomere

Structure-function mapping of proteins has improved our understanding of disease mechanisms and protein domains while human population genetics has provided a window into variational tolerance that can be modeled at a structural level. Here, to reveal novel insight into the cardiac sarcomere, the motor unit of the heart, we develop a novel analytical framework to integrate data from over 17,000 patients with hypertrophic cardiomyopathy (HCM) and combine it with two population-scale genomic databases that incorporate data from more than 800,000 individuals. We integrate in silico genetic predictions of gene variant pathogenicity with 3 dimensional integrative spatial scanning across multiple structural models of cardiac motor proteins. Results reveal both recognized and novel regions of structural variant intolerance across the critical genes of the cardiac sarcomere including novel insights into protein function. We discuss the structural relevance of variant enrichment in the context of sarcomere organization and destabilization of sequestered myosin leading to hypercontractility seen in HCM, by incorporating the recently defined high-resolution structure of human cardiac myosin filament. We extend and validate these findings through pathogenic variant class enrichment and reveal novel associations with the earlier onset of disease in a large clinical cohort. In summary, our study provides a multi-dimensional framework for integrating structural, genomic, and modeling data to reveal novel insight into the cardiac sarcomere.

genomics↗

bioRxiv2025

Scaled multidimensional assays of variant effect identify sequence-function relationships in hypertrophic cardiomyopathy

BackgroundAn estimated 1 in 500 people live with hypertrophic cardiomyopathy (HCM), a disease for which genetic diagnosis can identify family members at risk, and increasingly guide therapy. Mutations in the myosin binding protein C3 (MYBPC3) gene account for a significant proportion of HCM cases. However, many of these variants are classified as variants of uncertain significance (VUS), complicating clinical decision-making. Scalable methods for variant interpretation in disease-specific cell types are crucial for understanding variant impact and uncovering disease mechanisms. MethodsWe developed a scaled multidimensional mapping strategy to evaluate the functional impact of variants across a critical domain of MYBPC3. 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 MYBPC3 abundance, hypertrophic signaling, and ubiquitin-proteasome function in human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). ResultsOur multidimensional mapping strategy enabled high-resolution functional analysis of MYBPC3 variants in iPSC-CMs. Targeted transient base editing generated a comprehensive variant library at the native locus, capturing diverse variant effects on cellular HCM-relevant phenotypes. Our massively parallel splicing assay identified novel splice-disrupting variants. Integration of functional assays revealed that decreased MYBPC3 abundance is a key driver of HCM-related phenotypes. In parallel, downregulation of protein degradation was observed as a compensatory response to MYBPC3 loss of function, and novel disease mechanisms were identified for missense variants near a critical binding domain, underscoring their contribution to pathogenesis. Bayesian estimates of variant effects enable the reclassification of clinical variants. ConclusionsThis work provides a platform for extending genome engineering in iPSCs to multiplexed assays of variant effects across diverse disease-relevant cellular phenotypes, enhancing the understanding of variant pathogenicity and uncovering novel biological mechanisms that could inform therapeutic strategies.

genetics↗