Abnormalities in cardiac development are the most common form of birth defect with CHDs [12, 13]. The most important genes involved in CHDs are transcription factor genes. They are GATA4, NKX2.5, NKX2.6, T-Box, CITED2, ANKRD11, FOG2, ZIC3, and HAND2 [14]. Our laboratory’s preliminary studies revealed that two SNPs, c.608A>G and c.852G>A in the NKX2.5 gene, have an association with CHDs [15]. Earlier research from our laboratory also reported SNPs c.985 C>T in Cysteine-Rich Protein with EGF-Like Domain 1 (CRELD1) to be involved in causing CHD [16].
Echocardiography of three cases showed subaortic VSD, aortic overriding, right ventricular hypertrophy, and right ventricular outflow tract obstruction. Two TOF cases underwent intracardiac repair. The remaining one was managed medically with antibiotics because of an infected pulmonary valve, medically as infective endocarditis. The morphology of all three TOF cases was subaortic VSD in nature [17].
The homozygosity mapper tool is integrated with gene distiller to search for candidate genes and sort the candidate region’s genes. Gene distiller provided information from various data sources such as gene-phenotype associations, gene expression patterns, and protein-protein interactions [10]. Genome-wide homozygosity mapping with significant homozygous regions on chromosome 17 is marked red for both VSD and TOF cases. The presence of a homozygous region on chromosome 17 with NCOR1 and MAP2K3 genes along with its whole-exome sequence mutations aids severity towards its manifestation.
The present study identifies the presence of recessive genes NCOR1 and MAP2K3 in causing VSD and TOF through homozygosity haplotype mapping in conjunction with the WES data set where homozygous regions are identified in almost all chromosomes except 4, 8, 12, and 13. Further analysis revealed one homozygous region on chromosome 17 with genes enriched for CHD manifestation.
One of the recessive genes, NCOR1, is positioned on 17p11.2 with 54 exons with 189.31 kb length, from 16121499 to 15932191 (NCBI 37) on the reverse strand. NCOR1 is the key component of the generic corepressor complex and plays an important role in the control of heart development and differentiation [18]. The NCOR is needed for repression of myogenesis by the effect on the MYOD gene. The NCOR regulates and controls the activity of MYOD in mammalian differentiation. MYOD has a role during myogenesis, activating cell cycle, and muscle-specific gene transcription [19]. It is one of the master genes for CHD manifestation.
The other gene, MAP2K3 maps on chromosome 17 at 17q11.2, has a 30.60-kb size from 21187952 to 21218552 (NCBI 37) on the direct strand (Fig. 3). This gene is an activating transcription factor 3 (ATF3) target. ATF3 binds with the MAP2K3 promoter, recruiting histone deacetylases 1 (HDAC1), resulting in MAP2K3 gene associated histone deacetylation leading to inhibiting MAP2K3 expression. The MAP2K3 knockdown studies rescued the profibrotic/hypertrophic phenotype in ATF3KO cells. ATF3 protects against hypertensive cardiac remodeling and fibrosis, via suppressing MAP2K3 expression and p38 mitogen-activated protein kinase signaling. MAP2K3 protein is involved in P38 signaling, myogenesis, and TGF-beta signaling [17]. Hence, when the protein kinase domain in MAP2K3 is mutated, myogenesis is affected and result in CHD manifestation [20].
The physical interactors with damaging mutations affect the downstream cascade of pathways and processes, leading to disease manifestation. The NCOR1 has physically interacted with PDE9A. PDE9A as a protein regulates natriuretic peptide, stimulating and regulating cyclic guanosine monophosphate (cGMP) in heart myocytes. It is upregulated by hypertrophy and cardiac failure. When PDE9A protein expression is increased, then the cGMP-esterase activity is distinguished in left ventricular (LV) myocardium with heart failure. Therefore, the PDE9A enzyme regulates natriuretic peptide-dependent cGMP signaling in the heart, acting as a regulator of cardiac hypertrophy in myocytes [21]. Therefore, when NCOR1 protein is affected, the product of the PDE9A enzyme does not get activated. This leads to left ventricular dystrophy.
Three variants for NCOR1 present across homozygosity mapping and whole-exome sequencing are validated through Sanger sequencing. The variants of G207C and C241T are present exclusively in VSD cases. G244A variant is present in three TOF cases. These variants are not observed in any of the controls. This indicates that the variants in NCOR1 have associations with VSD and TOF phenotypes.