Human iPSC Models for Functional Interpretation of Congenital Heart Disease Variants of Uncertain Significance

Variants of uncertain significance (VUS) complicate the genetic diagnosis and clinical interpretation of congenital heart disease (CHD). Human induced pluripotent stem cells (iPSCs) can model patient-specific genetic backgrounds, relevant cardiac cell types, and developmental processes, providing an opportunity to investigate variant effects in biologically relevant human systems. However, the observation of a cellular or molecular phenotype alone does not establish functional evidence for pathogenicity or benignity. Interpreting such phenotypes requires careful consideration of genetic background, experimental variability, developmental stage, cardiac lineage, and the biological relevance of the selected model and assay endpoint. Isogenic controls, independent iPSC clones, and reproducible phenotyping strategies can help distinguish variant-specific effects from background or assay-related variation. This review proposes a question-first framework for model selection, isogenic testing, prespecified phenotyping, replication, and calibration assay with emphasis on matching the experimental system to the established disease mechanism. Collectively, this framework defines when iPSC-based findings are sufficiently robust, reproducible, and disease-relevant to contribute to ACMG/AMP PS3 or BS3 evidence and support the functional interpretation of CHD-associated VUS.