The Experimental Research Of TBX₂₀ Gene Ploymorphism Associated With Neonatal Congenital Heart Disease

Objective:To explore the relativity between T-box20(TBX20) gene SNPs (rs6950175、 rs3999941) and Chinese CHD of newborn. We have selected and analysed SNPs from TBX20gene coding regions which may affect the genic function. In this study, we would like to provide experimental information on prevention and treatment measures of perinatal CHD.Methods:1. Searching all SNPs in coding region of TBX20by using NBCI website SNPs database; meanwhile searching the non-synonymous SNPs by online bioinformatics software, SNPper. Finally settled the SNPs based on comparation the results and literatures.2.We collected and organized clinical data and samples. Through the method Case-control study, we have collected2ml blood from each CHD patients and controls peripheral blood, anticoagulated by sodium citrate, and preserved in the condition of-80℃.3.Gene sequencing and evaluate the SNP and CHD relativity. DNA extraction�'primer preparation�'PCR amplified target fragment�'identification of PCR product�'sequencing of PCR product�'data analysis. To analyse related data by software SPSS17.0:compared allelic gene frequency and genetype frequency between cases and controls, the associations between haplotype and the risk of CHD.Results:1.Two coding region SNPs were picked in TBX20genes,including rs6950175:c.765C>A(Ile255Ile)and rs3999941:c.766T>C (Phe256Leu).2.1n this research, rs6950175sites are all performing for C/C type homozygous genes in neonatal cases and controls. There was not statistical analysis between allele frequency and genotype frequency. Significant association was found between the TBX20gene SNP site rs3999941and HD(x2=9.93,P<0.05for genotype frequencies, x2=7.29,P<0.05for allele gene frequencies). The odd ratio of the TC genetype was7.11,so this haplotype is a risk factor of CHD.3.A new polymorphism were found by direct sequencing. We have identified657A>C (Ile219Ile) polymorphism at codon219of TBX20gene. This SNP is in the coding region exon5of TBX20gene, and it is a non-synonymous cSNP.4.In the case-control study, significant correlation were found between the TBX20codon219polymorphism and CHD(x2=6.52,P<0.05for genotype frequencies, x2=13.46,P<0.05for allele gene frequencies). The odd ratio of the AC genetype was4.57, so this haplotype is a risk factor of CHD.Conclusion:1. Researching the gene TBX20through Biological information analysis, combining the searching result from dbSNP database and SNPper software, and refering to related literature, we selected the common SNPs which in gene TBX20exon5to ensure SNP(rs6950175and rs3999941) and regarded it as the SNPs of the study.2.The selected SNPs site rs6950175(c.765c> A) in the cases and in the control group, both present C/C homozygous type gene, presumably the site with the occurrence of CHD group may have nothing to do. There was polymorphisms results on rs3999941:(c.766T>C) in the cases and controls(TC genetype OR=7.11).Therefore we speculated that the SNPs in our study have relation to the occurrence of CHD group.3.Anew SNP c.657A>C (Ile219Ile) were found in this experiment. SNP AC genetype was found in gene exon5by direct sequencing. mRNA order657A�'C.4.The AC genetype of the new SNP657A>C may play an important pathogenic role in the occurrence of CHD,(AC genetype OR=4.57).