Functional annotation of non-coding variants identifies a novel enhancer with activity in neural crest cell-derived...

Congenital heart disease (CHD) is one of the most prevalent and severe structural birth defects, affecting approximately 1% of live births. Because a substantial proportion of CHD patients do not harbor pathogenic mutations in protein-coding regions, non-coding variants are thought to underlie many unexplained cases. However, predicting the functional consequences of non-coding variation from sequence alone remains challenging due to our limited understanding of how regulatory information is encoded in the genome. Here, we used the Survey of Regulatory Elements (SuRE) assay to functionally screen 4.7 million non-coding variants from six individuals with an unexplained genetic predisposition to CHD in cultured AC16 human cardiomyocytes, identifying 18,201 regulatory quantitative trait loci (raQTLs). Systematic prioritization highlighted rs74330989, leading to the discovery of a novel cardiac enhancer, hs3112. Using a LacZ transgenic mouse assay, we demonstrate that hs3112 functions as a robust cardiac enhancer at embryonic day (E) 11.5, with predominant activity in neural crest cell (NCC)-derived populations, as demonstrated by co-staining with an anti-Sox10 antibody. In particularly, enhancer activity was detected in the outflow tract (OFT). Notably, introduction of the alternate rs74330989 allele completely abolished enhancer activity. Collectively, these results provide functional validation of the non-coding variant rs74330989 and demonstrate its requirement for hs3112 enhancer activity. As hs3112 is predicted to regulate HES1, which encodes a transcription factor essential for cardiogenesis, our findings suggest a direct mechanistic link between non-coding variation and CHD pathogenesis.