Molecular genetic study on HAND2 gene promoter in ventricular septal defect

Ventricular septal defect (VSD), as the most common type of congenital heart disease (CHD), is mainly caused by cardiac dysplasia. Heart and neural crest derivatives expressed 2 (HAND2), as a member of the basic helix-loop-Helix (bHLH) gene family, participates in the development of the right heart. And the loss of HAND2 expression in humans is closely connected with ventricular septal defects. However, no studies have hitherto examined the association between VSD and HAND2 gene promoter. We used a case-control study to analyze the genetic variations of the HAND2 gene promoter region in VSD patients and controls. Using a variety of statistical analysis methods to analyze the association of single nucleotide polymorphisms (SNPs) with VSD. The dual luciferase reporter assay was used to conduct functional analysis of genetic variations. Electrophoretic mobility shift assay (EMSA) was used to examine DNA-protein interactions. Through sequencing, we identified nine genetic variants in patients with VSD. The SNP rs2276940 G>T and rs2276941 G>A were revealed to have associations with an increased risk of VSD. The dual luciferase reporter assay showed that the SNP rs2276940 G>T and rs138531627 C>G decreased the transcriptional activity of the HADN2 gene promoter. Transcription factors (TFs) predicting suggested that all three above SNPs may change the binding of the TFs. The result of EMSA showed that rs138531627 C>G may create a new binding site for TFs while rs2276940 G>T enhanced the binding affinity for TFs. All these results indicated that genetic variants of the HAND2 gene promoter increase VSD risk by reducing HAND2 expression, and the molecular mechanism may be the change of the binding affinity of TFs.