Germline hereditary, somatic mutations and microRNAs targeting-SNPs in congenital heart defects

Abstract Somatic mutations and dysregulation by microRNAs (miRNAs) may have a pivotal role in the Congenital Heart Defects (CHDs). The purpose of the study was to assess both somatic and germline mutations in the GATA4 and NKX2.5 genes as well as to identify 3′UTR single nucleotide polymorphisms (SNPs) in the miRNA target sites. We enrolled 30 patients (13 males; 13.4 ± 8.3 years) with non-syndromic CHD. GATA4 and NKX2.5 genes were screened in cardiac tissue of sporadic and in blood samples of familial cases. Computational methods were used to detect putative miRNAs in the 3′UTR region and to assess the Minimum Free Energy of hybridization (MFE, kcal/mol). Difference of MFEs (ΔMFE) ≥ 4 kcal/mol between alleles was considered biologically relevant on miRNA binding. The sum of all ΔMFEs (|ΔMFEtot | = ∑|ΔMFE|) was calculated in order to predict the biological importance of SNPs binding more miRNAs. No evidence of novel GATA4 and NKX2.5 mutations was found both in sporadic and familial patients. Bioinformatic analysis revealed 27 putative miRNAs binding to identified SNPs in the 3′UTR of GATA4. ΔMFE ≥ 4 kcal/mol between alleles was obtained for the + 354A > C (miR-4299), + 587A > G (miR-604), + 1355G > A (miR-548v, miR-139-5p) and + 1521C > G (miR-583, miR-3125, miR-3928) SNPs. The + 1521C > G SNP showed the highest ΔMFEtot (21.66 kcal/mol). Luciferase reporter assays indicated that miR-583 was dose-dependently effective in regulating + 1521 C allele compared with + 1521 G allele. Based on the analysis of 100 CHD cases and 204 healthy newborns, the + 1521 G allele was also associated with a lower risk of CHD (OR = 0.5, 95% CI 0.3–0.9, p = 0.03), likely due to the relatively low binding of the miRNA and high levels of protein. These results suggest that common SNPs in the 3′UTR of GATA4 alter miRNA gene regulation contributing to the pathogenesis of CHDs.