Genetic Variants And Functional Analyses Of The GATA4 Gene Promoter In Dilated Cardiomyopathy

Objective: To study the sequence variation of the GATA4 gene promoter region in patients with DCM and healthy individuals,and to explore the role of the GATA4 gene promoter in the occurrence and development of DCM.Method:1.This study was divided into a dilated cardiomyopathy group and a healthy control group,with 110 subjects in the dilated cardiomyopathy group and 203 subjects in the healthy control group.Clinical data was collected from two groups for statistical analysis,and peripheral blood samples were collected for DNA extraction.2.A primer was designed for the GATA4 gene promoter,and the target fragment was amplified by PCR.The PCR product was then directly sequenced,and the GATA4 mutation site was identified.3.The wild-type target fragment and the GATA4 gene promoter fragment,containing a mutation site,were constructed on a p GL3-basic vector.They were then co-transfected with the internal reference plasmid PRL-TK through transient transfection into HEK293 cells and neonatal rat cardiomyocytes(NRCMs).The transcriptional activity of the GATA4 gene promoter was observed.Result:1.A total of six DNA mutation sites(DSVs)were detected through gene sequencing.Two SNPs were detected in the DCM group,g.31487C>G(rs1053351749)and g.31601G>A(rs73537869),but none were detected in the control group.Four DSVs were detected in the control group,g.31715C>A,g.31566G>C,g.31492T>A,and g.31403G>T,all of which were not detected in the experimental group.2.The internal reference plasmid and p GL3-basic vector containing the target gene were co transfected into NRCMs and HEK293 cells,and then the transcriptional activity was detected using luciferase.It was found that only two SNPs,g.31487C>G(rs1053351749)and g.31601G>A(rs73537869),which were present in the DCM group,significantly increased the transcriptional activity of the GATA4 gene promoter;The four DSVs present in the control group,g.31715C>A,g.31566G>C,g.31492T>A,and g.31403G>T,had no significant effect on the transcriptional activity of the GATA4 gene promoter.3.Through prediction using the online database TRANSFAC,it was found that g.31487C>G(rs1053351749)could abolish the binding site of KLF4 and weaken the binding of the transcription factor KLF6.g.31601G>A(rs73537869)may have created a binding site for CIZ,weakening Churchill’s binding site.Conclusion: This study detected two gene mutation sites that significantly alter the transcriptional activity of the GATA4 gene promoter in DCM patients(P<0.01).These two aberrant sites may alter the expression of the GATA4 gene and have an important impact on the occurrence and development of DCM,providing a theoretical basis for the future development of prevention and treatment of DCM.