Detection And Significance Study Of Genetic Polymorphisms In Methylenetetrahydrofolate Reductase And Thymidylate Synthase

Objective: To investigate the distribution of the 677C>T and 1298A>C polymorphisms of the methylenetetrahydrofolate reductase (MTHFR) and the triple or double 28bp tandem repeats (3R/2R) in 5'-untranslated region (UTR) and the 6bp deletion/insertion (6bp-/6bp+) in the 3'-UTR polymorphisms of the thymidylate synthase (TS) in healthy Han population of Chaoshan area.Subjects and methods: A total of one hundred healthy individuals (64 men and 36 women) from Chaoshan area were studied. DNA samples were isolated from peripheral blood samples. MTHFR 677C>T and 1298A>C and TS 3'-UTR 6bp-/6bp+ polymorphisms were genotyped by polymorphism chain reaction (PCR) -restriction fragment length polymorphism (RFLP). TS 5'-UTR 3R/2R polymorphism was determined by PCR. In order to further investigate the geographical and ethnic variation of four polymorphisms in China, we compared our results with published data from the others Han and minority population.Results: For MTHFR 677C>T polymorphism, the frequency of CC homozygous was 65.0%; CT heterozygous was 30.0%; and TT homozygous was 5.0%. For 1298A>C, AA homozygous was 45.0%; AC heterozygous was 50.0%; and CC homozygous was 5.0%. The allelic frequency of 677T and 1298C was 20% and 30% respectively. Genotypes distribution for each polymorphism was in agreement with Hardy-Weinberg equilibrium. Three haplotypes of tow loci were constructed by EH program. The frequency of 677C-1298A, 677C-1298C and 677T-1298A was 50.0%, 30.0% and 20.0%. Two SNPs were strong in linkage disequilibrium (LD),︱D'︱=1.0. The frequency of the 677T allele in Chaoshan Han population was closed to most southern Han and minority ethnic groups, but lower than the northern populations (p<0.05). The 1298C frequency of Chaoshan Han was similar with southern populations, but higher than the northern populations (p<0.05) and lower than the southern minority ethnic groups. The distribution of TS 5'-UTR 3R/3R, 3R/2R and 2R/2R was 61.0%, 36.0% and 3.0%. For 3'-UTR 6bp-/6bp+, 6bp-/6bp- was 47.0%; 6bp-/6bp+ was 42.0%; 6bp+/6bp+ was 11%. The frequency of 5'-UTR 2R and 3'-UTR 6bp+ was 21% and 31% respectively. Total of four haplotyeps were constructed. And︱D'︱value was 0.7, significant LD was also found. Two loci mutation frequencies were similar with the others Chinese populations.Conclusion: Our preliminary data indicated the distribution of four SNPs of MTHFR and TS genes in Chaoshan Han population had geographic and genetic feature. Objective: In order to investigate whether the genetic polymorphisms in enzymes involved in folate and homocysteine metabolism pathway—methylenetetrahydrofolate reductase (MTHFR 677C>T and 1298A>C) and thymidylate synthase (TS 5'-UTR triple or double 28-bp tandem repeats 3R/2R and 3'-UTR 6bp deletion or insertion 6bp-/6bp+) are related with risk of Chaoshan Han patients with AS and RA.Subjects and methods: A total of 114 AS patients, 100 RA patients and 100 controls were recruited in this hospital-based, case-control study. DNA samples were isolated from peripheral blood samples. MTHFR 677C>T and 1298A>C and TS 3'-UTR 6bp-/6bp+ polymorphisms were genotyped by polymorphism chain reaction (PCR) -restriction fragment length polymorphism (RFLP). TS 5'-UTR 3R/2R polymorphism was determined by PCR.Results: Two loci of MTHFR 677C>T and 1298A>C were strong in linkage disequilibrium (LD) in AS and RA patients, D value was 0.9 and 0.8 respectively. D value of TS 5'-UTR 3R/2R and 3'-UTR 6bp-/6bp+ was 0.8 and 0.7 in AS and RA patients respectively, LD was also found. It was found that in female AS patients, odd ratio (ORs; 95% confidence interval) for TS 3'-UTR 6bp-/6bp+ compared with 6bp-/6bp- was 2.6(1.03～6.58), for allele 6bp+ and haplotype 3R-6bp+ were 7.8 (0.93~66.33) and 3.0 (1.0～9.0) respectively (compared with allele 6bp- and haplotype 3R-6bp-). However, there were only 10 women with AS in our study, the statistical power was limited. Neither genotype nor allele frequencies for MTHFR were found to be significantly different between AS patients and normal controls. In RA patients, we observed a significant association between 677C>T polymorphism and cases. Subjects with the 677CT genotype had higher risk for developing the RA, with the OR of 2.69 (95%CI,1.48～4.89). The frequency of 677T allele was more popular in RA than normal control (33.5% vs. 20.0%,p<0.05). After stratified analysis by sex variable, this statistical tendency was still obvious. For the MTHFR 1298A>C, although there were more 1298AC, CC genotypes and C allele carriers in normal controls than RA patients, but no statistical difference was found between two groups. Analysis of haplotype showed that the 677T-1298A was associated with RA development, compared with 677C-1298A, the OR 95%CI was 1.83 (95%CI 1.12~2.97). We observed a significantly lower risk of RA associated with the TS 5'-UTR 3R/2R genotype (OR 0.5, 95%CI 0.26~0.93) and 2R-6bp+ haplotype (OR 0.55, 95%CI 0.31~0.99). No association was found between TS 3'-UTR 6bp-/6bp+ polymorphism and risk of RA in the study.Conclusion: Our preliminary findings indicate that genetic polymorphisms in MTHFR and TS enzymes may contribute to risk of the development of RA. Objective: To examine the association between MTHFR 677C>T, 1298A>C and TS 5'-UTR 3R/2R,3'-UTR 6bp-/6bp genetic polymorphisms and MTX related efficacy and toxicity in RA and AS patients.Methods: Total of 58 RA and 62 AS patients were involved in efficacy assessment, and 85 RA and 98 AS patients were recruited in toxicity assessment. In RA patients, DAS28 and modified ACR20 were used to evaluate the MTX-response. For AS patients, the efficacy was assessed by the ASAS20, 50, 70. Drug related side effects were judged referred to the adverse drug react association evaluation recommended by State Food and Drug Administration.Results: Forty-three, forty and thirty-eight patients responded to MTX treatment judged by the DAS28 (ESR), DAS28 (CRP) and ACR20 criteria respectively. In AS group, there were 12, 17 and 22 patients fulfilled the ASAS70, ASAS50 and ASAS20 respectively. We could not find a significant association between the efficacy and genetic polymorphisms in RA and AS patients. Sixty seven patients presented one and more MTX-related toxicity. Neither genotypes nor haplotypes were associated with MTX-related toxicity in RA and AS patients.Conclusion: Our preliminary study does not demonstrate that four polymorphisms in MTHFR and TS are associated with MTX efficacy and toxicity. To test our results, the larger, multicentre, polyethnic studies should be performed.