NKX2-5 congenital heart disease mutations show diverse loss and gain of epigenomic, biochemical and chromatin search functions underpinning pathogenicity

Congenital heart defects (CHD) occur in [~]1% of live births, with inherited and acquired mutations and environmental factors contributing to causation. However, network perturbations in CHD remain poorly understood. We report an integrated functional-epigenomics approach to CHD, focusing on the cardiac homeodomain (HD) transcription factor NKX2-5, mutations which cause diverse heart structural and conduction defects. We selected twelve NKX2-5 CHD variants affecting different residue classes - homeodomain DNA base-contacting, backbone-contacting and helix-stabilizing, and those affecting other conserved protein:protein interaction (PPI) domains. In HL-1 cardiomyocytes, we profiled genome-wide DNA targets of NKX2-5 wild type (WT) and variants, their DNA binding affinity and specificity, PPI with known NKX2-5 cofactors and chromatin search dynamics. Variants showed diverse yet class-specific behaviours. All variants failed to bind many WT targets but retained binding to a subset of core cardiomyocyte-related targets, and bound hundreds of unique "off-targets" via changes to DNA binding site specificity, homodimerization, cofactor interactions and chromatin search functions. Our data suggest that complex residue-by-residue scale epigenomic, biochemical and chromatin search defects involving both loss-and gain-of-function contribute to CHD. These findings may inform precision molecular therapeutic approaches in patients with CHD.