A Method For Detecting Four SNPs In Folate Metabolic Pathway And The Evaluation Of Their Correlation With Congenital Heart Disease

Part one Multiplex tetra-primer amplification refractory mutation system PCR for detecting four single nucleotide polymorphisms associated with folate metabolism in a single reactionObjective:To develop a multiple tetra-primer amplification refractory mutation system PCR(M-T-ARMS-PCR)assay for detecting four single nucleotide polymorphisms(SNPs):MTHFR C677T(rs1801133),MTHFR A1298C(rs1801131),MS A2756G(rs1805087)and MTRR A66G(rs1801394)associated with folate metabolism in a single reaction tube.Methods:200 physical examination Children’s anticoagulant blood samples were collected from the Department of Clinical Laboratory,Children’s Hospital of Hebei Province from January 2016 to April 2017.Four sets of chimeric primers consisting of a universal Tag sequence and targeting rs1801133,rs1801131,rs1805087 and rs1801394 fused to the specific sequence were designed according to the T-ARMS-PCR principle and the multiplex chimeric primers strategy.A single tube PCR was then conducted after optimizing the primer concentrations and the reaction conditions.The amplified products were analyzed by QIAxcel capillary electrophoresis,and the corresponding SNP genotypes of samples were identified.Furthermore,all samples were verified by direct sequencing.Results:The M-T-ARMS-PCR combined with capillary electrophoresis can accurately verify eight different alleles of the four SNPs associated with folate metabolism at one time in 3 hours.Four SNPs of 200 samples were accurately classified and the results were completely consistent with direct sequencing.Conclusions:The proposed M-T-ARMS-PCR in this study can simply,quickly and accurately verify four SNPs associated with folate metabolism in a single reaction tube.Part two The association between four single nucleotide polymorphisms in folate metabolism way and the risk of congenital heart diseaseObjective:To investigate the association between single nucleotide polymorphisms(SNPs)of 5,10-methylenetetrahydrofolate reductase(MTHFR)C677T,A1298C,methionine synthase(MS)A2756G,methionine synthase reductase(MTRR)A66G and the risk of congenital heart disease(CHD).Methods:By restricting the corresponding conditions,a case-control study was performed.We collected 200 CHD children as case group and 200normal children as control group admitted to the Department of cardiac surgery,Children’s Hospital of Hebei Province from January 2016 to April2017.The genotypes of MTHFR C677T,A1298C,MS A2756G,and MTRR A66G polymorphisms were detected by multiplex tetra-primer amplification refractory mutation system PCR.Andχ~2 test of SPSS21.0 was used to assess the relationships between four SNPs and the risk of whole CHD and different types of CHD.Results:The mutant allete T of MTHFR C677T had contribute to the risk of developing CHD(OR=2.47,95%CI:1.86~3.29,P<0.001).Compared with the wild CC genotype,heterozygosity CT had a higher risk of CHD(OR=2.32,95%CI:1.35~3.98,P<0.05),the homozygous mutant genotype TT increased the risk of CHD by 5.37(95%CI:3.01~9.60,P<0.001).The mutant allele C of MTHFR A1298C was a protective factor for CHD(OR=0.53,95%CI:0.36~0.77,P<0.05).Compared with the wild AA genotype,heterozygosity AC had a lower risk of CHD(OR=0.41,95%CI:0.26~0.64,P<0.001).After typing,the allele frequencies and genotypes frequencies of the above two SNPs were still statistically significant(P<0.05).The combined genotype analysis of the above two SNPs showed that:compared with the CC/AA genotype,individuals with CT/AA had a higher risk of CHD(OR=4.65,95%CI:2.16~10.02),the TT/AA type increased to 7.05(95%CI:3.37~14.79).However,the polymorphisms of MS A2756G and MTRR A66G had no significant relationship with the risk of CHD(P>0.05).Conclusions:The mutant allele T of MTHFR C677T may be a risk factor for CHD and the mutant allele C of A1298C may be a protective factor for CHD.These two SNPs may have a joint effect on the occurrence of CHD.