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

Area of Science:

• Genetics
• Cardiology
• Developmental Biology

Background:

• Atrial septal defects (ASDs) are common congenital heart diseases.
• Previous Genome-Wide Association Studies (GWAS) identified common variants linked to ASDs, but their functional roles are unclear.
• Further investigation is needed to understand the genetic architecture and functional mechanisms of ASD risk variants.

Purpose of the Study:

• To identify novel common variants associated with ASD risk.
• To elucidate the functional mechanisms underlying ASD development.
• To expand insights into the genetic architecture of ASDs.

Main Methods:

• Conducted a Genome-Wide Association Study (GWAS) using isolated ASD cases and healthy controls, with replication in an independent cohort.
• Examined epigenetic marks in the identified ASD locus using human induced pluripotent stem cell-derived cardiomyocytes and fetal human hearts.
• Utilized CRISPR-Cas9 mutagenesis to assess the functional consequences of deletions within the locus and chromosome conformation capture sequencing to investigate 3D genome architecture.

Main Results:

• Identified a novel ASD locus on chromosome 3p12.3 containing the ROBO2 gene, an enhancer, and a CTCF-binding site.
• Deletions within the locus reduced ROBO2 expression and dysregulated extracellular matrix genes in cardiomyocytes.
• Chromosome conformation capture sequencing revealed physical interactions between the locus and the ROBO2 promoter, mediated by the CTCF-binding site.

Conclusions:

• Novel common single nucleotide polymorphisms in regulatory elements controlling ROBO2 transcription contribute to ASD risk.
• These findings highlight critical roles for Roundabout guidance receptor 2 and Slit ligands in atrial septum development and maturation.