The developmental pathways of genes GATA4, NKx2.5, FOXH1,HAND2, 20TBX20, MEF2c, and CITED2) result in a healthy functional heart, whereas mutated genes result in a heart malfunctioning (10, 20). In China and Germany, the serine-glycerin-rich region of the CITED2 gene is reportedly the highly mutated domain (21). The common defects are VSD, ASD, AVSD, TOF, and PDA (22). The current research is based on five heart defects.
The activity of heart-associated genes (HIF-1α and TFAP2c) is influenced by the methylated and mutated transcription factor gene CITED2 in CHD patients. According to one study, CHDs can be caused by mutations in the conserved segments of the CITED2 gene (CR1, CR2, and CR3). Methylation in the promoter region of a gene likely influences transcription (15). Research studies in different populations have shown an association between the CITED2 gene and CHDs (14, 21). A primary study revealed that mice with a loss-of-function mutation in the CITED2 gene overexpress VEGF and HIF1a (Bhattacharya et al., 1999). Thus, based on these studies, the mutation in exon 2 of the CITED2 gene, SNP rs375393125, was selected for the genetic screening of 250 CHD patients for probable outcomes. The -results showed that the genotypic frequency of all samples was greater for patients with a greater number of wild-type TT genotypes, while only CHD patients were found with mutant CC genotypes. Among all CHD groups, 40% of the TOF patients had a high percentage of mutant CC genotypes (Fig. 1). A study based on CHD in China revealed a decreased interaction between CITED2 and p30°CH1 due to the presence of four mutated proteins. VEGF promoter activity could be upregulated by the significant interaction between p30°CH1 and HIF1A. This study revealed that mutations could downregulate TFAP2C in pitx2c transactivation and CITED2 (23–25). All CHD patients were found to have both T and C alleles. All CHD and control samples had a greater T allele frequency. On the other hand, the controls had no mutation C allele.
Moreover, it was found in an average of 30% of all the patients (Fig. 3A). These results showed that the high frequency of the mutant CC genotype or C allele in the respective samples may be associated with heart abnormalities. Furthermore, these results were confirmed through Sanger sequencing. The sequencing results showed that all the samples had the T > C mutation, and the PCR results were subsequently validated by cross-matching. All the samples were found to have similar PCR and Sanger sequencing results.
The chi-square test and odds ratio test were applied to determine the genetic association between the targeted CITED2 gene and CHD type and the strength of the association. The observed chi-square value of 103 was significantly greater than the tabulated value of 21.03 (Table 1). Similar results, with a greater calculated value of 43.7 than the threshold value of 1, were found by the odds ratio test (Table 2). Analysis of variance revealed significant differences between and among the CHD groups and CITED2 genotypes (Tables 3, 4). Hence, CITED2 gene mutation(s) are strongly associated with CHDs. The available data for rs13253637 in the NCBI database showed a rare and negligible mutant C allele in many different populations of normal individuals. Therefore, the outcomes of this research were compared with those from the Gnome-AD and GOE exome projects. The genome-AD project shows a 0.000004/1 distribution of the C allele. Similarly, the GOE sequencing project showed a 0.000077/1 frequency of the C allele. This research revealed a higher C allele frequency in CHD patients (Fig. 4).
An epigenetic study of congenital heart malformation revealed that environmental factors may change a gene's transcriptional activity, ultimately affecting the heart's developmental process (26, 27). Additionally, the present study focused on identifying the associations of general factors, including addiction, sex, and consanguinity, with CHDs. The exact ratios of addiction (chewing and smoking), sex, and kinship were detected in both the cases and controls. However, these factors had an insignificant effect on CHDs (Fig. 5).
It has been reported that CHDs may m caused by CITED2 gene mutation (s) in research involving varied populations. However, current research has also found similar findings. This SNP can be used as a marker for future theragnostic approaches.