Research Of The Relationship Between The Genes In Folate Metabolic Pathway And Congenital Heart Disease

Objective1. We take child patients dignosed as congenital heart disease (CHD) as case group,healthy children as control. To explore the optimal reaction condition of high resolutionmelting(HRM) for detecting mutation of Cystathionine Beta Synthetase(CBS), andperform a preliminary discussion about the relationship between CBS mutation and CHD.2. Through the polymerase chain reaction and restriction fragment length polymerphi-sm analysis(PCR-RFLP), we investigate the association between methylenetetrahydrofolat-e reductase (MTHFR) C677T, A1298G, and G1793A polymorphisms(Single nucleotide p-olymorphism, SNP) and CHD susceptibility, which Provides the theoretical foundation forexploring the cause of CHD.Methods1. We conducted a case–control study including150isolated CHD cases and150controls.2. All of the DNA samples of the participants were extracted to To explore the optimalreaction condition of high resolution melting (HRM) for detecting SNPs of CystathionineBeta Synthetase(CBS).3. Take the HRM results to compared with gene sequencing to evaluate this method.4. genotyping, and draw a conclusion of the relationship of CBS and CHD bystatistical analysis.5. Detecting the methylenetetrahydrofolate reductase MTHFRC677T(rs1801133),A1298C(rs1801131), and G1793A(rs2274976) polymorphisms(SNP) by the polymerasechain reaction and restriction fragment length polymorphism analysis(PCR-RFLP).6. compare the difference of genotype and allele frequencies between the case and control by Hardy-weinberg test, x2test, t test, analysis of relative risk includingcombinations of variation of different SNPs.Discuss their separate effect and combinedeffects.Results1. The genotype distribution of control showed no statistically significance, accordwith Hardy-weinsberg genetic equilibrium.2. The optimal annealing temperature was62℃; the best reaction system ofqPCR-HRM includs primers0.2μmol/L, Mg2+2.5μmol/L, and DNA template60ng in20μl.3. Mutant type and allele distribution has been detected by HRM,how ever there is nostatistical difference in the cases and the control group.4. The frequencies of677CC, CT, TT in the case group were20.67%,52.00%,27.33%,While in the control were39.33%,44.00%,16.67%. Compared with wild CCgenotype, Heterozygosity increased the risk of CHD(OR＝2.249,95%CI:1.305-3.877,P=0.003), the homozygous mutant genotype is associated with the risk of CHDsignificantly(OR=3.121,95%CI:1.612-6.043, P=0.001). Compared with the wild allete,mutant allete increased the risk of CHD by1.813(95%CI:1.310-2.508,P=0.000)，the frequencies of1298AA, AC were76.00%,24.00%in the case group,87.34%,11.86%in thecontrol. homozygous mutant genotype was not detected. Compared with wild AA genotype,Heterozygosity increased the risk of CHD(OR＝2.177,95%CI:1.183-4.077, P=0.011).mutant allete increased the risk of CHD by2.017(95%CI:1.128-3.604, P=0.016). Thefrequencies of1793GG, GA were91.33%,8.67%in the case group,86.00%,14.00%in thecontrol. homozygous mutant genotype was not detected. The proportion of theheterozygote GA and homozygote AA had no statistical differences in the twogroups(P=0.145), also the mutant allete and wild allete(P=0.158). Joint effects were foundof MTHFR C677T and MTHFR A1298C, MTHFR A1298C and MTHFR G1793A, fortheir combined effects had increased the risk of developing CHD more significantly.Conclusion1. The technology of HRM can be used for detecting SNP of CBS gene with goodspecificity characteristics.2. There is no statistical relation between rs75616587and CHD.3. mutation of MTHFR C677T、A1298C are risk factors for CHD, while MTHFR1793GG may also be the risk factor, but the isolate function was not found.4. The genotype combinations of MTHFR677TT,1298AC and1298AC,1793GG performed joint effects.