Global Evaluation of Congenital Heart Disease-Associated Non-Coding Variants

Abstract (Summary) Genome-wide association studies (GWAS) have mapped thousands of congenital heart disease (CHD)-associated variants within non-coding regions of the genome. Non-coding variants can alter regulatory mechanisms, such as transcription factor (TF) binding control of gene expression, potentially contributing human diseases. However, with the increasing number of disease-associated variants, comprehensive functional validation remains a significant challenge. In this work, we developed a novel method called SNP Bind-n-Seq to evaluate >3,000 CHD-risk variants for allelic binding for the cardiac TFs NKX2-5, GATA4, and TBX5 in a high-throughput manner. These binding affinity data sets were coupled with a massively parallel reporter assay (MPRA) to screen CHD-risk variant genotype-dependent regulatory activity. We identified 170 variants that exhibit allelic TF binding and 187 that modulate gene expression. Combining both approaches revealed three high-confidence variants with genotype-dependent TF binding, genotype-dependent transcriptional activity, and eQTL behavior in cardiac cells. Collectively, this study provides the first combined high-throughput biochemical and functional genomic evaluation of thousands of CHD-risk variants. Highlights: Allelic binding affinity measurements of ∼9,600 variants for NKX2-5, GATA4, and TBX5 EvaluaFon of >3,000 CHD-risk variants for genotype-dependent regulatory acFvity InteracFon networks idenFfy funcFonal variants and genes involving cardiac eQTLs Competing Interest Statement The authors have declared no competing interest. Footnotes ↵† Joint supervised work This version of the manuscript has been revised to update the following: Edwin G. Pena-Martinez and Shreya Sharma are now listed as equally-contributing authors; Devesh Bhimsaria is now listed as corresponding author.