mutations in hiPSCs inform mechanisms for maldevelopment of the heart, pancreas, and diaphragm.

Damaging variants cause cardiac outflow tract defects, sometimes with pancreatic and diaphragmic malformations. To define molecular mechanisms for these diverse developmental defects, we studied transcriptional and epigenetic responses to loss of function (LoF) and missense variants during cardiomyocyte differentiation of isogenic human induced pluripotent stem cells. We show that GATA6 is a pioneer factor in cardiac development, regulating that activates and that with orchestrates outflow tract formation. LoF variants perturbed cardiac genes and also endoderm lineage genes that direct expression and pancreatic development. Remarkably, an exon 4 missense variant, highly associated with extra-cardiac malformations, caused ectopic pioneer activities, profoundly diminishing , and expression and increasing normal retinoic acid signaling that promotes diaphragm development. These aberrant epigenetic and transcriptional signatures illuminate the molecular mechanisms for cardiovascular malformations, pancreas and diaphragm dysgenesis that arise in patients with distinct variants.