ROBO2 Variants Associated With Atrial Septal Defect Define a Novel Regulatory Element

BACKGROUND: Atrial septal defects (ASDs) are a prevalent type of congenital heart disease. Previous GWAS (Genome-Wide Association Studies) have identified common variants associated with ASDs, though their mechanisms remain unknown. We aimed to expand insights into the architecture of common variants associated with ASD risk and elucidate functional mechanisms. METHODS: We conducted a GWAS using isolated ASD cases and healthy controls and replicated findings in an independent cohort. We examined epigenetic marks within this ASD locus in human induced pluripotent stem cell-derived cardiomyocytes and fetal human hearts. We characterized the consequences of deletions introduced by CRISPR-Cas9 mutagenesis of human induced pluripotent stem cells to assess the effect on downstream gene expression. In addition, we investigated the 3-dimensional genome architecture of the locus using chromosome conformation capture sequencing. RESULTS: We identified a novel ASD locus on chromosome 3p12.3 encompassing the ROBO2 gene, which encodes the Roundabout guidance receptor 2 for Slit ligands. This locus includes 15 common single nucleotide polymorphisms, an enhancer, and a CCCTC-binding factor (CTCF)-binding site. Deletions of varying lengths within the ASD-associated locus in human induced pluripotent stem cell-derived cardiomyocytes reduced ROBO2 expression and dysregulated the expression of extracellular matrix genes. Chromosome conformation capture sequencing indicated that this region physically interacts with the ROBO2 promoter and demonstrates that the CTCF-binding site is essential for this contact. CONCLUSIONS: Novel common single nucleotide polymorphisms in regulatory elements controlling ROBO2 transcription contribute to risk for ASDs. These data infer key roles for the Roundabout guidance receptor 2 and Slit ligands in embryogenic development and maturation of the atrial septa.