Polymorphism Of MTHFR Gene And Its Association With NSCL/P In North Of Guizhou Province

Objective: To detect the polymorphism of MTHFR gene C677 T and A1298 C in NSCL/P nuclear families and the nuclear families of control group in North of Guizhou Province.Mean while,the distribution of the alleles and genotypes and the relationship between gene polymorphism and NSCL/P was explored.Methods:1.Peripheral venous blood of 45 children without NSCL/P together with their biological parents and 60 children with NSCL/P together with their biological parents were collected.Genomic DNA was extracted from peripheral blood samples and the extraction quality of DNA was detected by agarosegel electrophoresis.2.Design of forward and reverse primers was based on C677 T gene and A1298 C polymorphism of MTHFR gene.PCR and Next-generation sequencing technology were used to detect the SNP in the core samples of the case group and control group.Online BLAST was used to test the accuracy of amplified DNA fragments.Chromas sequencing software and MEGA7 sequence analysis software were used for genotype analysis.All test data was analyzed by SPSS19.0 software,Chi square test was used to analyze the Hardy-Weinberg balance and distribution frequency of each genotype or allele were compared between these two groups.3.The HHRR analysis of nuclear family in the case group was performed based on case-control study.Parents with heterozygous genotype and their children were performed the TDT,The mutant allele polymorphism and genotypes of parents in two groups were combined to analysis in order to further explore the relationship between the polymorphisms of MTHFR gene C677 T and A1298 C and NSCL/P.Results:1.In this study,the homozygous and heterozygous mutation of MTHFR gene C677 T in the case group and control group were not detected but the mutation of A1298 C was detectable.2.Hardy-Weinberg balance test was performed in two groups based on the acture value and expected value of A1298 C genotype in MTHFR gene.While there existed no statistical difference among children,mothers and fathers in these two groups,showing that the two nuclear families were all in accordance with the Hardy-Weinberg equilibrium rule(P>0.05).3.The frequencies of A1298 C genotype and allele between the children and fathers in two groups had no statistical significance(P>0.05),while there was significant difference in the genotype and allele frequencies of the mothers in case group(P<0.05).The value of OR and 95% CI of CC genotype versus AC or AA in mother were OR=0.142(95%CI:0.027~0.745)and OR=0.135(95%CI:0.026~0.687)respectively,which indicating that the mother carrying CC genotype may reduce the risk of developing NSCL/P in offspring.The OR value of mutation isoallele C versus A was OR =0.525(95%CI: 0.289~0.953).4.In the case group,the genotype frequencies and allele frequencies of two parents were compared with those of children.There was no statistically significant difference of genotype frequencies and allele frequencies between two parents and children(P>0.05).5.In the analysis based on HHRR correlation method,transfer to the sick children in case group nuclear families parents two alleles constitute case group and not to child two alleles of a virtual control group were compared,results showed that there was no significant correlation between the NSCL/P patients and the A1298 C allele frequency(P>0.05).6.According to the transmission of parental genotype to patient children,TDT test was performed in families which parents have heterozygous allele in case group.The result indicated that there was no linkage disequilibrium between A1298 C polymorphism and NSCL/P(P>0.05).7.The total mutant allele frequency of parents in case group and control group were analyzed.The mutation allele frequencies of parents was 23.75% in control group and34.44% in case group,The difference between two mutation allele frequencies had not statistically significant(P>0.05).Conclusion:1.In this study,homozygous and heterozygous mutations of C677 T in the MTHFR gene were not detected.2.The A1298 C polymorphism of MTHFR gene may not be an important factor in the pathogenesis of NSCL/P in the north of Guizhou Province,The polymorphism of A1298 C of MTHFR gene may be a protective factor for the offspring of NSCL/P.3.There is no genetic imbalance in the MTHFR gene A1298 C site of NSCL/P patients in the north of Guizhou Province and no correlation between the polymorphism of A1298 C in MTHFR gene and the incidence of NSCL/P.