LAUSR

Congenital heart disease (CHD) affects ~1% of live births and remains the leading cause of mortality in infants. While large-scale genetic studies have uncovered genes associated with CHD, distinguishing variants that confer risk from the background noise of inconsequential variants remains a challenge. Causative mutations in transcription factors (TF) essential for cardiovascular development, such as NKX2-5, GATA4 and TBX5, have been identified in familial cases of CHD, however they are rare. To expand our understanding of their molecular function and to test whether their interacting proteins may be enriched for variants associated with CHD. We defined the protein-protein interaction (PPI) network of NKX2-5, GATA4 and TBX5 using unbiased mass spectrometry in human iPSC-derived cardiac progenitors (iPS-CPs). This approach yielded a network of 172 proteins. An interdependent gene-regulatory role has been reported at the DNA-binding level for these 3 TF during cardiac development, and we also found interdependent protein interactomes where loss of one TF affected the interactome of the others. Interactomes for each were enriched in proteins involved in similar biological processes, such as chromatin remodeling and gene regulation, or previously unrelated processes such as splicing and mRNA transport. Integration of the iPS-CP-PPI network with the CHD-associated damaging variants found in the Pediatric Cardiac Genomics Consortium whole-exome sequencing cohort revealed statistically significant enrichment in the GATA4 interactome for de novo missense variants. In contrast, neither the TBX5 or NKX2-5 PPIs were enriched for either de novo missense or rare damaging variants. Finally, we developed a framework to rank PPIs with reported damaging variants for functional validation studies. Overall, this work identified novel protein interactors of TFs essential for cardiac development, offering new insights regarding their regulatory roles and the mechanisms through which they may cause CHD.