Association of hyperhomocysteinemia with genetic variants in key enzymes of homocysteine metabolism and methotrexate toxicity in rheumatoid arthritis patients.

OBJECTIVES: The study investigated the association between plasma homocysteine, folate and vitamin B12 with 5,10 methylenetetrahydrofolate reductase (MTHFR C677T and A1298C), thymidylate synthase (TYMS 2R â†’ 3R) and methionine synthase (MTR A2756G) polymorphisms and methotrexate (MTX) treatment and toxicity in Tunisian Rheumatoid arthritis (RA) patients. METHODS: A total of 185 patients with RA were included. Homocysteine (Hcy) was assessed by fluorescence polarization immunoassay, and folate and vitamin B12 were measured by chemiluminescence immunoassays. The genetic polymorphisms were analyzed by PCR or PCR-RFLP. Hyperhomocysteinemia (HHC) was considered for Hcy > 15 µmol/L. RESULTS: MTHFR C677T polymorphism was associated with HHC in RA patients (multi-adjusted OR, 95% CI 2.18, [1.07-4.57]; p = 0.031). No association was detected with the remaining polymorphisms. Plasma Hcy, folate, and vitamin B12 did not differ according to each polymorphism, or with MTX treatment or toxicity. However, HHC was more prevalent in patients with than those without MTX toxicity (32.7 vs. 16.7%; p = 0.035). CONCLUSIONS: The MTHFR 677TT genotype is an independent risk factor for HHC in Tunisians RA patients. HHC could be a useful marker of MTX toxicity in RA patients.

Analysis of two susceptibility SNPs in HLA region and evidence of interaction between rs6457617 in HLA-DQB1 and HLA-DRB1*04 locus on Tunisian rheumatoid arthritis.

Previous genomewide association studies (GWAS) and meta-analyses have enumerated several genes/loci in major histocompatibility complex region, which are consistently associated with rheumatoid arthritis (RA) in different ethnic populations. Given the genetic heterogeneity of the disease, it is necessary to replicate these susceptibility loci in other populations. In this case, we investigate the analysis of two SNPs, rs13192471 and rs6457617, from the human leukocyte antigen (HLA) region with the risk of RA in Tunisian population. These SNPs were previously identified to have a strong RA association signal in several GWAS studies. A case-control sample composed of 142 RA patients and 123 healthy controls was analysed. Genotyping of rs13192471 and rs6457617 was carried out using real-time PCR methods by TaqMan allelic discrimination assay. A trend of significant association was found in rs6457617 TT genotype with susceptibility to RA (P = 0.04, pc = 0.08, OR = 1.73). Moreover, using multivariable analysis, the combination of rs6457617*TT-HLA-DRB1*04+ increased risk of RA (OR = 2.38), which suggest a gene-gene interaction event between rs6457617 located within the HLA-DQB1 and HLA-DRB1. Additionally, haplotypic analysis highlighted a significant association of rs6457617*T-HLA-DRB1*04+ haplotype with susceptibility to RA (P = 0.018, pc = 0.036, OR = 1.72). An evidence of association was shown subsequently in antiCCP+ subgroup with rs6457617 both in T allele and TT genotype (P = 0.01, pc = 0.03, OR = 1.66 and P = 0.008, pc = 0.024, OR = 1.28, respectively). However, no association was shown for rs13192471 polymorphism with susceptibility and severity to RA. This study suggests the involvement of rs6457617 locus as risk variant for susceptibility/severity to RA in Tunisian population. Secondly, it highlights the gene-gene interaction between HLA-DQB1 and HLA-DRB1.

Genetic Determinants of Methotrexate Toxicity in Tunisian Patients with Rheumatoid Arthritis: A Study of Polymorphisms Involved in the MTX Metabolic Pathway.

BACKGROUND AND OBJECTIVE: Methotrexate (MTX) is a disease-modifying anti-rheumatic drug used in the treatment of rheumatoid arthritis (RA). It is the first line drug in the treatment of this disease. However, MTX-related adverse drug reactions (ADRs) are seen in 40 % of the patients. The aim of this study was to determine the impact of six genetic polymorphisms located in five genes encoding proteins involved in the MTX metabolic pathway in Tunisian RA patients and evaluate its association with MTX toxicity. METHODS: Genotyping of 5,10 methylenetetrahydrofolate reductase (MTHFR C677T and A1298C), dihydrofolate reductase (DHFR 19-base pair deletion allele), thymidylate synthase (TYMS 2R/3R), methionine synthase (MTR A2756G) and methionine synthase reductase (MTRR A66G) was performed using PCR and PCR-RFLP method in 141 RA patients treated with MTX. Demographic and clinical characteristics were obtained and ADRs were recorded. Association analyses with regard to MTX toxicity were performed using the χ (2) test, the toxicogenetic risk index (TRI) and the Mann-Whitney U-test. RESULTS: The analysis highlighted a significant association of the T/T genotype of MTHFR C677T polymorphism with increased MTX toxicity. However, the MTHFR A1298C, DHFR 19-base pair deletion allele, MTR A2756G and MTRR A66G polymorphisms were not associated with increased MTX toxicity. The TYMS 2R/3R polymorphism had a protective effect against MTX toxicity. CONCLUSION: The results demonstrated that the C677T polymorphism in the MTHFR gene is associated with MTX toxicity in Tunisian RA patients. In contrast, the TYMS 2R/3R polymorphism is associated with a protective effect against overall MTX toxicity.