Reduced folate carrier 1 gene expression levels are correlated with methotrexate efficacy in Japanese patients with rheumatoid arthritis.

Responsiveness to methotrexate (MTX), the "anchor drug" for treating rheumatoid arthritis (RA), varies among individual patients. In this study we investigated the effects of folate transporter gene expression levels on disease activity among 56 Japanese patients with RA who were undergoing MTX therapy. We also assessed gene expression levels for 15 healthy control subjects. The mRNA expression levels of reduced folate carrier 1 (RFC1) and proton-coupled folate transporter (PCFT) in PBMCs from these patients and controls were determined using real-time quantitative polymerase chain reaction (PCR). Compared with PCFT, there were large individual differences in RFC1 mRNA expression levels in both RA patients and healthy controls. RFC1 mRNA expression levels and RA disease activity scores were significantly negatively correlated, as disease activity scores were lower for patients with higher RFC1 mRNA expression levels. However, RFC1 mRNA levels were not correlated with MTX doses. Thus, the clinical efficacy of MTX for Japanese RA patients was associated with the expression level of a folate transporter gene. Increased RFC1 expression may increase MTX uptake by immune cells, such as lymphocytes, and as a result, RA disease activity would be reduced.

A single nucleotide polymorphism of reduced folate carrier 1 predicts methotrexate efficacy in Japanese patients with rheumatoid arthritis.

  Methotrexate (MTX) exhibits large inter-individual and inter-ethnic differences in the dose required for its anti-rheumatic effect. To maintain low disease activity, patients may require increased MTX doses or co-administration of biologic disease-modifying anti-rheumatic drugs (bDMARDs). The availability of a marker predicting the effect of MTX will make it possible to increase the MTX dose and prescribe bDMARDs to patients at an early stage. To establish individualized medication for rheumatoid arthritis (RA), we investigated genetic polymorphisms of the folate pathway in Japanese RA patients. Eighty-nine patients were treated with MTX alone (MTX group). MTX and bDMARDs were co-administered to 81 patients because of insufficient MTX efficacy (MTX + bDMARDs group); an equally stable therapeutic effect was achieved in both groups. Polymorphism analyses using bDMARD co-treatment as the objective variable revealed a significant association between age and the G80A polymorphism of the reduced folate carrier 1 gene (RFC1) as an explanatory variable. Compared to patients with the A allele, patients with the G allele may have less intracellular MTX uptake and, therefore, poor efficacy; a greater number of them were found to be bDMARD concomitant cases. The results of this study suggest that the RFC1 G80A polymorphism may be a useful marker for predicting MTX efficacy in Japanese patients with RA.

An artifact derived from a pseudogene led to the discovery of microRNA binding site polymorphism in the 3'-untranslated region of the human dihydrofolate reductase gene.

A novel single-nucleotide polymorphism (SNP) in the 3'-untranslated region of the human dihydrofolate reductase (DHFR) gene with enhanced expression was identified in 2001. In 2007, it was reported that this SNP, DHFR C829T, was located close to a microRNA binding site and contributed to the stability of mRNA. Many researchers have analyzed this SNP in several races including Asians and Caucasians. However, the mutation allele is not yet confirmed in most populations. In this study, we reinvestigated the frequency of this SNP using three methods. First, this SNP in genomic DNA was analyzed by a PCR-restriction fragment length polymorphism method. Second, this SNP in mRNA was analyzed by a single nucleotide extension method following a reverse transcription reaction. Third, the mRNA expression level was analyzed by a real-time PCR method. The findings in our study, regarding the discovery of this SNP, suggest that the SNP is an artifact caused by contamination by the genomic DNA of the pseudogene DHFRP1. This study is a reinvestigation of a newly discovered genetic polymorphism.

Dihydrofolate reductase gene intronic 19-bp deletion polymorphisms in a Japanese population.

Dihydrofolate reductase gene (DHFR) 19-bp deletion polymorphisms result in varied DHFR enzymatic activity affecting the risk for preterm delivery, spina bifida, and the efficacy of methotrexate (MTX). Ethnic differences in DHFR 19-bp polymorphisms may be responsible for the divergent findings in previous genetic studies. We compared genotype and allele frequency of DHFR intronic 19-bp deletion polymorphisms in ethnically homogenous East Asians (from Japan) and others by polymerase chain reaction assay conducted on 277 healthy Japanese individuals. The genotype distribution was as follows: wild/wild, 11.9% (n=33); wild/deletion, 40.1% (n=111); deletion/deletion, 48.0% (n=133). The frequencies of wild type and deletion alleles were 0.32 and 0.68, respectively. The obtained genotype distribution was consistent with those calculated by Hardy-Weinberg equilibrium. The genotype distribution and allele frequencies in the Japanese population were significantly different from those previously reported for other ethnic populations. Determination of intronic 19-bp deletion polymorphisms of DHFR may be useful for monitoring the efficacy and side effects of MTX for the treatment of diseases such as rheumatoid arthritis and childhood acute leukemia in the Japanese population because the frequency of the deletion allele is higher.