Genetic Variants And Functional Analyses Of The TBX20 Gene Promoter In Dilated Cardiomyopathy

Background:Dilated cardiomyopathy(DCM)is a disease characterized by ventricular remodeling and cardiac fibrosis.Its main clinical manifestations are ventricular enlargement and impaired heart function.DCM is one of the leading causes of death in the world and an important cause of sudden cardiac death and heart transplantation.By 2015,the global prevalence of cardiomyopathy was approximately 2.5 million,an increase of 27%from 2005.DCM is the production of the interplay of multiple factors.In fact,both innate heredity and acquired environment are involved in the occurrence and development of DCM.As a genetically heterogeneous disease,genetic variation accounts for approximately 25%to 40%of DCM cases.To date,genes involved in DCM have been successively discovered,but this does not explain all cases.Cardiac transcription factors mainly regulate chamber differentiation and myocardial proliferation during heart development,and affect the expression of cardiac development related genes through multiple pathways.As a result,mutations in cardiac transcription factors not only cause congenital heart defects,but also alter heart function,trigger heart diseases and arrhythmias.TBX20 is one of the most important transcription factors related to the normal development and morphological regulation of heart,and is mainly involved in the regulation of heart structure and function.Therefore,TBX20 is now known to be linked to a variety of heart diseases,including congenital heart disease,arrhythmias and other cardiovascular diseases.Although abnormal expression of TBX20 in DCM has been reported,there are no studies on TBX20 promoter region variation in the occurrence and development of DCM.Therefore,we hypothesized that mutations in the promoter region of TBX20 gene might alter the expression of TBX20 gene and thus participate in the pathogenesis of DCM.Objective:Based on the genetic and functional studies of TBX20 gene promoter in DCM patients,DSVs of TBX20 gene promoter region was determined by sequencing.The effect of DSVs on promoter function of TBX20 gene was analyzed by vector construction and cell transfection.Using DNA-protein binding technology to explore the possible regulatory mechanism of DSVs affecting promoter function of TBX20 gene,which is aimed at provide the genetic basis for occurrence and development of DCM.Methods:1.136 DCM patients and 210 control group were included in the case-control study.3-4ml of fasting blood samples were taken from participants for DNA extraction.2.Into the GenBank database of NCBI to search for TBX20 gene promoter DNA sequences,design and synthesize TBX20 gene promoter PCR primers.promoter were amplified and sequenced.3.DSVs in the TBX20 gene promoter region were determined by comparing the sequencing results with the wild type TBX20 gene promoter DNA sequences.4.The TRANSFAC database was used to predict whether DSVs affected the binding site of the TBX20 gene promoter.5.The sequences of wild type and variant TBX20 gene promoter were cloned into pGL3-basic expression vector to construct recombinant plasmids,and the constructed pGL3 recombinant plasmid and internal reference plasmid pRL-TK were co-transfected into HEK-293 and neonatal rat cardiomyocytes(NRCMs).The relative luciferase activity of the transfected cells was determined and the results were analyzed to determine the effect of DSVs on the transcriptional function of TBX20 gene promoter.6.Electrophoretic mobility shift assay(EMSA)was used to determine whether DSVs affected the binding of TBX20 gene promoter to transcription factors.Results:A total of ten DSVs were identified in TBX20 promoter of all subjects by sequencing.Among them,one new DSV(g.4275G>T)and four single nucleotide polymorph isms(SNPs)[g.4169G>A(rs1263874255),g.4949C>T(rs119174599),g.5114G>A(rs 112076877),g.5252C>T(rs1356932911)]only existed in DCM group.One SNP[g.4028T>C(r s186936022)]and one new DSV(g.4075G>T)only in the control group.Three SNPs[g.5198G>A(rsl39651523),g.4740T>C(rs336284),g.5295T>C(rs73099190)]was present in both the DCM group and the control group,The frequency of SNPs[g.4740T>C(rs 336284)and g.5295 T>C(rs73099190)]were higher in DCM group than in control gro up(P=0.041,P=0.005).But the frequency of SNP[g.5198G>A(rs 139651523)]was not statistically significant between the two groups(P>0.05).TRANSFAC prediction results showed that the SNP[g.4169G>A(rsl263874255)]may eliminate the binding site of ZXDA and ZXDB,create the binding site of CSX.The DSV(g.4275G>T)may abolish the binding site of TFIIB and SMAD1,and produced the binding sites of SMAD5.The SNP[g.4949C>T(rs1191745927)]may generate the binding sites of ELF2,ERG and GABP-alpha,modified the binding sites of CPBP.The SNP[g.5114G>A(rs 112076877)]may disrupt the binding sites of GKLF and PU.1,create the binding sites of ZNF35.The SNP[g.5252C>T(rs1356932911)]may disrupt the binding sites of CSX and produced the binding sites of NFAT and EHF.Transfection results showed that expression vectors[pGL3-4275T、pGL3-4740C+4949T+5295C、pGL3-5114A、pGL3-5252T]significantly enhanced the transcriptional activity of TBX20 gene promoter in HEK-293 cell and NRCMs(P<0.05).But,expression vectors[pGL3-4028C,pGL3-4075T,pGL3-4169A]don’t alter the transcriptional activity of TBX20 gene promoter in HEK-293 cell and NRCMs(P>0.05).EMSA results showed that the DSV(g.4275G>T)abolished the binding of unknown transcription factor to the TBX20 gene promoter and create the binding of unknown transcription factor to the TBX20 gene promoter.The SNP[g.4949C>T(rs1191745927)]created the binding of unknown transcription factors the TBX20 gene promoter to.The SNP[g.5114G>A(rs112076877)]enhanced the binding of unknown transcription factor to the TBX20 gene promoter.The SNP[g.5252C>T(rs1356932911)]disrupted the binding of unknown transcription factors to the TBX20 gene promoter.While SNPs[g.4169G>A(rs1263874255)]did not affect the binding of transcription factors to the TBX20 gene promoter.Conclusion:In this study,four DSVs[g.4275G>T,g.4949C>T(rs119174599),g.5114G>A(rs112076877),g.5252C>T(rs1356932911)]were found significantly increased the transcriptional activity of TBX20 gene promoter in HEK-293 cells and NRCMs by affecting the binding of transcription factors.This may alter the expression level of TBX20 and play a role in the genesis and development of DCM.Our findings will improve the diagnosis of pathogenic promoter variants,and it also helps us to understand the role of transcriptional regulation in the development of DCM.