| Objective:To study the relationship between T-box transcription factor TBX20 gene mutation and VSD in Chinese Han children.Methods:A total of 50 children(23 males and 27 females)of Han patients with Congenital Ventricular Septal Defect were taken peripheral venous blood,three of them had Ventricular Septal Defect family history,so collect the peripheral venous blood of his brothers,sisters and parents,then extract the genomic DNA and detect the TBX20 gene.All cases were diagnosed as Ventricular Septal Defect by physical examination,echocardiography ultrasound and intraoperative diagnosis.A total of 30 healthy children(16 males and 14 females)in outpatient clinic of Shanxi children’s Hospital were taken peripheral venous blood,and extract genomic DNA according to the genomic DNA Extraction Kit introductions of Sangon Biotech,the exon of 2~6 contributing to TBX20gene T-box DNA-binding domain was amplified by Polymerase Chain Reaction of the PTC-200 automatic amplification instrument in the case group and the control group as well as in the Ventricular Septal Defect family history group.Then the PCR amplification products were purified and sequenced by ABI3700 DNA sequencer.Compare and analysis of the sequencing results with the human TBX20 genome in the gene bank through the BLAST program in the National Center for Biotechnology Information.Detection of mutation and Single Nucleotide Polymorphisms by combining the results of gene sequencing.The allele frequencies and genotype frequencies of newly discovered single nucleotide polymorphisms were analyzed and compared by statistical software SPSS20.0,so as to find out whether their genotype and allele are risk factors for Congenital Ventricular Septal Defect in Chinese Han children.Results:1.In 5 cases of Congenital Ventricular Septal Defect in Chinese Han children were detected for a synonymous mutation(T192T)and a missense mutation(G193S),the two mutations are located in the fourth exon domain,the missense mutations(c.577G/A)cause the amino acid change from glycine to serine,and these two mutations were not found in the control group,Predicted by Mutation Taster software,show that these two mutations are pathogenic.The two mutations of amino acids sites in human and animal are highly conserved through the Homologene in NCBI network.2.The new 2 Single Nucleotide Polymorphisms were found in the 5 intron of the case group and the control group,one mutation was in front of the initiation codon ATG61 base pairs,the other was in the downstream of termination codon(UGA/UAA/UGA)142bp,and these two SNPs have not been reported in the single nucleotide polymorphism database.The SNP in the downstream of termination codon 142 base pairs(A>C)was heterozygous variant in the case group and control group as well as family patients,so no statistical analysis was made.The SPSS20.0 statistical analysis found that the frequency of TC genotype were higher than the control group,the difference was statistically significant(χ~2=33.55,P<0.05),the C allele frequency in case group was higher than that in control group,with statistical significance the difference(χ~2=15.96,P<0.05)of the mutation in front of the initiation codon ATG 61 base pairs(T>C).The OR value of TC genotype(OR value is 26.83)is greater than 1,and the value of95%CI is(7.42~97.08),the lower limit is greater than 1.We believe that TC genotype may increase the risk of Congenital Ventricular Septal Defect in Chinese Han children.The OR value of allele C(OR=4.83)is greater than 1,allele T OR value(OR=0.21)is less than 1,TT genotype OR(OR=0.04)is less than 1,considering the C allele may be a pathogenic gene in Congenital Ventricular Septal Defect in Chinese Han children,the T allele may be a protective gene.3.The SNP in the downstream of termination codon 142 base pairs(A>C)was heterozygous variant in the case group and control group as well as family patients,so no statistical analysis was made.4.No new mutations and Single Nucleotide Polymorphism were found in the patients’family members,and no same mutation was found in the patients’family members and the patients.The results of this study failed to reflect the genetic relationship between the family members and the patients,and considering it’s ralated to the small number of samples collected.Conclusion:1.Synonymous mutations(T192T)and missense mutations(G193S)located in the fourth exon domain may be associated with Congenital Ventricular Septal Defect in Chinese Han children,suggesting that TBX20 may be a causative agent of Congenital Ventricular Septal Defect.2.The SNP in front of the initiation codon ATG 61 base pairs(T>C),it is analysised by the SPSS20.0 statistical found that the TC genotype may increase the risk of Congenital Ventricular Septal Defect in Chinese Han children,the C allele may be a pathogenic gene in Congenital Ventricular Septal Defect in Chinese Han children,the T allele may be a protective gene.3.The SNP in the 5 intron downstream of termination codon 142 base pairs(A>C)was heterozygous variant in the case group and control group as well as family patients,so no statistical analysis was made.This study suggesting that this variation may be irrelevant with Congenital Ventricular Septal Defect in Chinese Han children. |
