| Objective:To investigate the plausible role of gene PITX2 in the pathogenesis of CHD, we have selected and analysed SNPs from PITX2c gene coding regions by using bioinformatics methods and tools to ensure the SNPs which may affect the genic function. We further screened those SNPs by DNA sequencing among CHD and healthy controls to explore the relativity between SNPs and Chinese CHD. In this study, we would like to provide experimental information on prevention and reduction the morbidity of CHD, and establishment the cure measure.Methods:1. Searching all SNPs in coding region of PITX2c by 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 collected 2ml 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:through the Hardy-weinberg testing to ensure the samples in accordance with Mendelian inheritance; 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 PITX2 genes,including rs 1051887: 316G>C (Glu106Gln) and rs28936409:272G>C (Arg91Pro).2. In this research,no polymorphism was detected at the two SNPS in cases and controls.3. A new polymorphism were found by direct sequencing.We have identified 304C>G (Glu102Gln) polymorphism at codon 102 of PITX2 gene.This SNP is in the coding region exon5 of PITX2 gene,and it is a non-synonymous cSNP.4. Through the Hardy-weinberg testing,data analysis results showed that there is no difference between the cases and controls about the genotype distribution of there SNPs, that is to say the samples in accordance with Mendelian inheritance.5. In the case-control study,significant correlation were found between the PITX2 codon 102 polymorphism and CHD(χ2=7.177,P<0.05 for genotype frequencies,χ2=5.566,P<0.05 for allele gene frequencies).The odd ratio of the CG genetype was 2.667,so this haplotype is a risk factor of CHD.6. In the case-control study,significant correlation were found between the PITX2 codon 102 polymorphism and interval defect CHD(χ2=6.400,P<0.05 for genotype frequencies,χ2=5.120,P<0.05 for allele gene frequencies).The odd ratio of the CG genetype was 2.778,so this haplotype is a risk factor of interval defect CHD.7. In the case-control study,significant correlation were found between the PITX2 codon 102 polymorphism and cardiovascular anomaly CHD(χ2=11.868,P<0.05 for genotype frequencies,χ2=9.351,P<0.05 for allele gene frequencies).The odd ratio of the CG genetype was 4.000,so this haplotype is a risk factor of cardiovascular anomaly CHD.Conclusion:1. Researching the gene PITX2 through 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 PITX2 exon5 to ensure SNP:rs1051887:316G>C (Glu106Gln) and rs 28936409:272G>C (Arg91Pro), and regarded it as the SNPs of the study.2. There were no polymorphisms results on two SNPs:rs1051887:316G>C (Glu106Gln) and rs28936409:272G>C (Arg91Pro) in the cases and controls.Therefore we speculated that the two SNPs in our study have no relation to the occurrence of CHD group.3. A new SNP(304C>G) were found in this experiment. SNP CG genetype was found in gene PITX2 exon5 by direct sequencing. mRNA order 942 C→G, lead to the changing from glutamic acid (Glu, E) to glutamine (Gin, Q).4.The CG genetype of the new SNP(304C>G) may play an important pathogenic role in the occurrence of CHD, (CG gene OR=2.667), interval defect CHD (CG gene OR=2.778), and cardiovascular anomaly CHD (CG gene OR=4.000). |
PDF Full Text Request