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Rare Variants Linked to Congenital Heart Defects in Humans

NEW YORK (GenomeWeb News) — Through exome sequencing and functional assays, researchers led by the Wellcome Trust Sanger Institute's Matthew Hurles uncovered rare variants linked to congenital heart defects in humans.

As the researchers reported in the American Journal of Human Genetics today, they performed whole-exome sequencing of a dozen parent-offspring trios as well as of more than 110 unrelated individuals. From this, they identified a number of rare variants within the highly conserved NR2F2 gene associated with certain heart defects called atrioventricular septal defects. NR2F2, they further reported, is expressed in the developing heart, and the variants affect NR2F2 activity.

"Taken together, these data support our hypothesis that rare and private variants in NR2F2 probably contribute to AVSDs and other CHDs during human development," Hurles and his colleagues write.

According to the researchers, atrioventricular septal defects account for about 4 percent to 5 percent of all congenital heart defects, and occur in 0.3 to 0.4 of 1,000 live births.

To search for de novo coding mutations linked to this heart defect, Hurles and colleagues performed exome sequencing on 13 parent-offspring trios using Agilent SureSelect Target Enrichment V3 followed by sequencing on the Illumina HiSeq platform. After variant calling, they identified 13 de novo coding mutations, subsequently validated by capillary sequencing, in nine genes.

Only two of those genes — ZMYND8 and NR2F2 — were known to be expressed in heart tissue, and after testing the burden of rare coding mutations in those nine genes, the researchers found that only NR2F2, which encodes a transcriptional regulator, had a significant enrichment of rare missense mutations.

This analysis uncovered four additional missense mutations in AVSD-affected individuals, but also found four in control subjects, which the researchers suggested could be due to either incomplete penetrance or benign variants.

Hurles and his colleagues also examined whether NR2F2 variants were present in people with heart defects other than AVSDs, and uncovered three additional families affected by other types of heart defects that had NR2F2 variants.

Using whole-mount in situ hybridization and optical projection tomography, the researchers mapped the pattern of NR2F2 mRNA expression in developing mouse embryos. It's expressed, they found, in the atria, branchial arches, somites, and olfactory placode.

Meanwhile, in developing human hearts, they added, NR2F2 is expressed in the atria, coronary vessels, and aorta, as indicated by immunofluorescent analysis of fixed human fetal heart tissue.

These missense mutations, the researchers noted, affect spots throughout the NR2F2 protein, though three are within the ligand-binding domain. To test how these variants might affect the transcriptional activity of NR2F2, Hurles and his colleagues used luciferase assays coupling wild-type and variant NR2F2 to a promoter and luciferase reporter.

When coupled to the NGFI-A promoter, two NR2F2 variants showed significantly lower activity, while two others had increased activity, the researchers said. And when coupled to the alternative APOB promoter, four variants had reduction in activity, compared to the wild type, while one now had increased activity.

"That individual mutations have promoter-specific effects on gene function probably reflects the complexity of the protein-protein interactions NR2F2 engages in depending on tissue, stage, and genomic context," the researchers said.

They further noted that NR2F2 could act as a sort of an environmentally responsive factor that mediates the effects of non-genetic heart defect risk factors like high glucose or retinoic acid levels. In hepatocyte and pancreatic cells, the researchers noted, insulin and glucose levels are known to negatively control NR2F2 expression, and, during development, NR2F2 is known to play a role in retinoic acid signaling.

Still, further investigation of how glucose or retinoic acid levels affect NR2F2 expression in the developing heart is needed, Hurles and his colleagues added.