Analysis of rare and common variants to identify inherited and maternal genes associated with conotruncal heart defects

Congenital heart defects (CHDs) are the leading cause of infant death due to birth defects in the United States. Conotruncal defects (CTDs) are the most severe and the largest group of CHDs. Candidate gene, genome-wide gene-based analyses with meta-analyses, and pathway level-analyses of CTDs were performed, using rare and common variants, to identify novel inherited genes underlying disease occurrence. Genome-wide analyses and a meta-analysis were also performed to identify maternal genes associated with CTDs. Enrichment of genes with meta-analysis p\u3c10-3 and p\u3c0.05 was also evaluated. Both case-parent trios and case-control analyses were conducted using data from multiple cohorts. Although no gene was significantly associated with CTDs at the genome-wide level, several genes with evidence suggestive of association and several significantly enriched gene clusters were identified. Meta-analysis of inherited effects using data from trios identified 11 genes with evidence suggestive of an association (p\u3c10-3), including the Wilms tumor gene, POU6F2 (p=9.82 x10-5), and MBNL1 (p=1.50 x10-4), which is expressed in endocardial cushions and heart valves. In addition, meta-analysis of inherited effects using case-control data identified 29 genes, including: NEXN (p=1.85x10-4), which regulates cardiac differentiation; the angiogenesis regulator, PRKD2 (p=3.12x10-4); and DACT3 (p=3.18x10-4), which regulates Wnt signaling. Enrichment analysis of genes with meta-analysis p\u3c0.05 in the trio-based analyses of inherited genes identified two significantly enriched clusters, one of which included members of the solute carrier family of proteins (Enrichment Score, ES=1.78) involved in zinc transmembrane transport. Similarly, six significantly enriched clusters were identified from case-control meta-analysis of inherited genes. The top three clusters included genes that encode cadherins and gamma protocadherins (ES= 8.83, 8.74, 1.89), which are important for cell-cell adhesion, migration and cardiac differentiation. In the maternal genetic effect meta-analysis based on two case/mother-control/father comparisons, the germ cell-specific gene, GGN, was borderline significant (p=7.10x10 -6). GGN is critical for the spermatogenesis and is also involved in the development of the oocyte and preimplantation embryo. Additionally, 33 genes with suggestive evidence of association were identified, including three known CHD-related genes (SUMO1, p=1.06x10 -4; PLXND1, p=3.38x10-4; TBX20, p=8.58x10-4), and the oocyte-specific gene, H1FOO (p=7.92x10-4). Enrichment analysis of 34 genes with p\u3c10-3 from case-control meta-analysis of maternal effects identified one significantly enriched cluster of three protease inhibitor genes (ES=2.11). Similarly, for meta-analysis genes with p\u3c0.05, two significantly enriched clusters were identified, including a cluster of three hyaluronidase genes (ES=1.30) that are expressed in the ovaries and may be required for embryonic development (i.e. HYAL2). In summary, these analyses identified, several novel inherited and maternal genes with suggestive evidence of association with CTDs. The identification of enriched gene clusters among our top associations provides important clues regarding the pathogenesis of CTDs