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.