Image-based, pooled phenotyping reveals multidimensional, disease-specific variant effects

Genetic variants produce complex phenotypic effects that confound current assays and predictive models. We developed variant in situ sequencing (VIS-seq), a pooled, image-based method measuring variant effects on molecular and cellular phenotypes in diverse cell types. Applying VIS-seq to ∼3,000 LMNA and PTEN variants yielded high-dimensional morphological profiles capturing changes in protein abundance, localization, activity, and cell architecture. VIS-seq identified a subset of linker-subdomain LMNA variants that increase nuclear circularity, in contrast to aggregating or low-abundance rod-subdomain variants that decrease circularity. VIS-seq also identified autism-associated PTEN variants that mislocalize and accurately distinguished autism-linked from tumor syndrome-linked and gnomAD control variants. Most variants impacted a multidimensional phenotypic continuum not recapitulated by any single functional readout. By linking variants to cell images at scale, VIS-seq illuminates how variant effects cascade from molecules to subcellular structures to cells, providing a framework for resolving the complexity of variant function.