Genetic polymorphisms of enzyme proteins and transporters related to methotrexate response and pharmacokinetics in a Japanese population

Methotrexate (MTX) is currently the anchor drug widely used worldwide in the treatment of rheumatoid arthritis (RA). However, the therapeutic response to MTX has been shown to vary widely among individuals, genders and ethnic groups. The reason for this has been not clarified but it is considered to be partially due to several mechanisms in the cellular pathway of MTX including single-nucleotide polymorphisms (SNPs). The purpose of this study was to investigate the allelic frequencies in different ethnic and/or population groups in the 10 polymorphisms of enzyme proteins and transporters related to the MTX response and pharmacokinetics including MTHFR, TYMS, RFC1, FPGS, GGH, ABCB1, ABCC2 and ABCG2 in unrelated healthy Japanese adults and patients with RA. Ten polymorphisms, methylenetetrahydrofolate reductase (MTHFR) 1298, thymidylate synthase (TYMS) 3'-UTR, reduced folate carrier 1 (RFC1) 80 and−43, folypolyglutamyl synthase (FPGS) 1994, γ-glutamyl hydrolase (GGH) 452 and−401, the ABC transporters (ABCB1 3435, ABCC2 IVS23 + 56, ABCG2 914) of enzyme proteins and transporters related to MTX response and pharmacokinetics in 299 unrelated healthy Japanese adults and 159 Japanese patients with RA were investigated to clarify their contributions to individual variations in response and safety to MTX and establish personalized MTX therapy. SNPs were evaluated using real-time polymerase chain reaction (PCR). Comparison of allelic frequencies in our study with other ethnic/population groups of healthy adults and RA patients showed significant differences in 10 polymorphisms among healthy adults and 7 among RA patients. Allelic frequencies of MTHFR 1298 C, FPGS 1994A and ABCB1 3435 T were lower in Japanese than in Caucasian populations and those of ABCC2 IVS23 + 56 C and ABCG2 914A were higher in Japanese than in Caucasian/European populations in both healthy adults and RA patients. Allelic frequencies of MTHFR 1298 C, GGH−401 T, ABCB1 3435 T, and ABCG2 914A were higher in healthy Japanese adults than in an African population, and those of RFC1 80A, RFC1−43C and ABCC2 IVS23 + 56 C in healthy Japanese adults were lower than in Africans. However, no significant differences were seen in the distribution of allelic frequencies between healthy Japanese adults and RA patients. The variations in allelic frequencies in different ethnic and/or population groups in healthy adults and RA patients may contribute to individual variations in MTX response and toxicity.


Background
Rheumatoid arthritis (RA) is a systemic autoimmune inflammatory disease and its pathogenesis remains unclear. Although no curative therapy for RA has been established, the therapeutic goal is to delay symptom progression using disease-modifying anti-rheumatic drugs, gold preparations and biologics and achieve pain relief using nonsteroidal anti-inflammatory drugs (DMARDs). Among the therapeutic options, methotrexate (MTX) is currently the anchor drug widely used worldwide in the treatment of RA because it is inexpensive, effective and safe. In the 2014 Japanese therapeutic guidelines for RA treatment published by the Japan College of Rheumatology (JCR) [1], MTX was also cited as a first-line drug. However, the therapeutic response to MTX has been shown to vary widely among individuals, genders and ethnic groups [2,3]. The reason for this has been not clarified but it is considered to be partially due to several mechanisms in the cellular pathway of MTX including single-nucleotide polymorphisms (SNPs) in transporters, glutamation, the folate pathway and adenosine pathway [2] such as reduced folate carrier 1 (RFC1), folypolyglutamyl synthase (FPGS), γ-glutamyl hydrolase (GGH), the ABC transporters ABCB1, ABCC2 and ABCG2, methylenetetrahydrofolate reductase (MTHFR), and thymidylate synthase (TYMS). We previously reported that the distribution of MTHFR C677T between black and Japanese populations, of TYMS 5'-UTR alleles between Caucasian or black and Japanese populations, and of TYMS 3'-UTR alleles between Caucasian and Japanese populations showed significant differences, as well as gender differences in TYMS 3'-UTR allelic frequency in Japanese [3].
Up to the present, a clear genetic difference has not been confirmed between healthy individuals and RA patients which would indicate susceptibility to RA, as such genetic variations do for many other diseases. There are few data on the possible genetic differences in RA, and therefore, it is important to clarify them to obtain useful information from the viewpoint of human genetics and/ or population genetics, leading to the establishment of personalized medicine for those susceptible to the development of RA.
The purpose of this study was to investigate the allelic frequencies in different ethnic and/or population groups in the 10 polymorphisms of enzyme proteins and transporters related to MTX response and pharmacokinetics including MTHFR, TYMS, RFC1, FPGS, GGH, ABCB1, ABCC2 and ABCG2 in unrelated healthy Japanese adults and patients with RA.

DNA extraction
For genetic analysis, a 5-mL peripheral blood sample was obtained from each study participant using the standard venipuncture technique. The samples were placed in tubes containing ethylenediaminetetraacetic disodium salt (EDTA 2Na) and stored at−20°C until DNA extraction. DNA was extracted using the Wizard Genomic DNA Purification Kit (Promega Corporation, WI, USA) following the manufacturer's instructions. Total genomic DNA was quantified and its purity and integrity were analyzed using the NanoDrop 1000 spectrophotometer v3.7 (Thermo Scientific, Wilmington, DE, USA).

Assessment of ethnic or population and gender differences
We  [4] on the assumption that it contained healthy adult data. The search terms were MTHFR, methylenetetrahydrofolate reductase, TS, TYMS, thymidylate synthase, dihydrofolate reductase, RFC1, reduced folate carrier 1, Solute carrier family 19 (folate transporter) member 1, SLC19A1, folylpolyglutamate synthase, FPGS, gamma-glutamyl hydrolase, GGH, ATP-binding Cassette Sub-family B Member 1, ABCB1, ATP-binding cassette sub-family C member 2, ABCC2, ATP-binding cassette transporter G2 and ABCG2, in combination with genotype and polymorphism. All languages were included. Additionally, a manual search of reference listings from all of the articles retrieved from the electronic databases was performed. Selection criteria were case-control, crosssectional or prospective cohort studies, which assessed any allelic frequencies of MTHFR 1298 A > C, TYMS 3'-UTR 6-bp deletion, RFC1 80 G > A, RFC−43 C > T, FPGS 1994 G > A, GGH 452 C > T, GGH−401C > T, ABCB 3435 C > T, ABCC2 IVS23 + 56 T > C, and ABCG2 914 C > A in healthy adults and RA patients and in which ethnic group was clearly identified. In case-control studies, only the allelic frequencies of the healthy adult control group were extracted. If we could not identify the ethnic group in the literature, population groups were assumed to be from the country of residence of the lead author.

Statistical analysis
Comparisons of ethnic and gender differences in the distribution of allelic frequencies among genotypes, and/ or gender differences and tests for Hardy-Weinberg equilibrium were carried out using the chi-square test. A p-value of <0.05 was considered to represent a significant difference in all statistical analyses. All statistical analyses were performed using JMP 12.0.1 software (SAS Institute Inc., Cary, NC, USA).
Statistically significant differences were found in the distributions of the MTHFR 1298A > C, RFC1 80 G > A, RFC−43 T > C, FPGS 1994 G > A, GGH−401 C > T, ABCB1 3435 C > T, ABCC2 IVS23 + 56 T > C, and ABCG 914 C > A alleles in healthy adults between our Japanese population and Caucasians and Africans. Statistically significant differences were found in the distributions of the RFC1 80 G > A and ABCC2 IVS23 + 56 T > C alleles in healthy adults between our Japanese population and Asians (Table 1). Statistically significant differences were found in the distributions of the MTHFR 1298A > C, TYMS 3'-UTR −6 > +6, RFC1 80 G > A, FPGS 1994 G > A, GGH 452 C > T, ABCB1 3435 C > T, ABCC2 IVS23 + 56 T > C, and ABCG 914 C > A alleles in RA patients between our Japanese population and Caucasians (Table 2).

Allelic frequencies and gender differences in frequencies in healthy Japanese adults
The distribution of MTHFR 1298A > C, and TYMS 3'-UTR (−6/+6), RFC1 80G > A, RFC1−43 T > C, FPGS 1994G > A, GGH 452C > T, GGH−401C > T, ABCB1 3435C > T, ABCC2 IVS 23 + 56 T > C, and ABCG2 914C > A polymorphisms in 299 healthy Japanese adults are summarized in Table 3. The proportions of genotypes at each site for their transporter carrier proteins and enzymes were generally in agreement with Hardy-Weinberg equilibrium. Therefore, the proportions at each site followed Mendelian principles. Only three genotype combination patterns were found for RFC1 80G > A and RFC1−43 T > C, consisting of 80G/G,−43 T/ T genotype, 80G/A,−43 T/C genotype, and 80A/A,−43C/ C genotype, respectively and a strong linkage disequilibrium was observed in healthy Japanese adults. Gender differences in the allelic frequencies are also summarized in Table 3. Allelic frequencies of RFC1 80G > A and RFC1−43 T > C were 61% in men and 52% in women for the A and C alleles and those of ABCC2 IVS23 + 56 T > C were 80% in men and 69% in women for the C allele. These differences in distribution were statistically significant (p = 0.0226 for RFC1 80G > A and RFC1−43 T > C, p = 0.0009 for ABCC2 IVS23 + 56 T > C, chi-square test). No gender differences in the distribution of other genotypes were observed (Table 3), although a tendency for a difference by gender was noted in the distribution of FPGS 1994 G > A (p = 0.0775, chi-square test) and GGH 452 C > T (p = 0.097, chi-square test).

Allelic frequencies and gender differences in frequencies in Japanese RA patients
The distributions of MTHFR 1298A > C, and TYMS 3'-UTR (−6/+6), RFC1 80G > A, RFC1−43 T > C, FPGS 1994G > A, GGH 452C > T, GGH−401C > T, ABCB1 3435C > T, ABCC2 IVS 23 + 56 T > C, and ABCG2 914C > A polymorphisms in 159 Japanese RA patients are summarized in Table 3. The proportions of genotypes at each site for their transporter carrier proteins and enzymes were generally in agreement with Hardy-Weinberg equilibrium, following Mendelian principles. Only three genotype combination patterns occurred for RFC1 80G > A and RFC1−43 T > C, consisting of the 80G/G,−43 T/T genotype, 80G/A,−43 T/C genotype, and 80A/A,−43C/C genotype, respectively and a strong linkage disequilibrium was observed in Japanese RA patients. Allelic frequencies of TYMS 3'-UTR−6 > +6 were 38% in men and 32% in women for the +6 allele, and a significant gender difference in TYMS 3'-UTR −6 > +6 (p = 0.0064, chi-square test) was seen, but the proportions of genotypes in men were not in agreement with Hardy-Weinberg equilibrium (p = 0.003, chi-square test). Gender differences in the distribution of other genotypes were not observed (Table 3), although a tendency toward gender differences was seen in the distribution of MTHFR 1298 A > C (p = 0.0736, chisquare test).

Discussion
We comprehensively investigated the allelic frequencies in the gene polymorphisms of enzymes and transporter proteins including MTHFR, TYMS, RFC1, FPGS, GGH, ABCB1, ABCC2 and ABCG2, which affect    MTX pharmacokinetics and the therapeutic response to it, between a healthy Japanese population and Japanese RA patients to obtain the fundamental data for the personalized MTX therapy for RA. Characteristics of MTX-related enzyme gene and transporter polymorphisms are summarized in Table 4. In the comparison of allelic frequencies in ethnic and/or population groups in healthy adults and RA patients, the frequency of the MTHFR1298 C allele in our Japanese study was significantly lower than in Caucasian [4,7,8], but higher than in Africans [4]. The MTHFR gene is associated with the generation of 5-methyl THF, the MTHFR 1298A > C polymorphism decreases MTHFR activity, and is thereby associated with MTX efficacy [41,42] and toxicity [26,43]. In terms of the effect of each SNP on MTX efficacy and toxicity, it is considered to be the greatest in Caucasians [4,7,8], followed by Japanese and Africans [4]. However, currently there are few data comparing Japanese with Africans in relation to the efficacy and toxicity of MTX treatment. As such one example, Hughes et al. [26] reported that there was an association between scores of MTX toxicity and the rs4846051 C allele, that is, a higher mean toxicity score among African-Americans than among Caucasians, and haplotypes containing this allele in African-Americans, but not in Caucasians.
The frequency of RFC1 80 A and RFC1−43C alleles in the present Japanese study was found to be higher than in Caucasian healthy adults [4,7,8,11] and RA patients [24,[34][35][36]. The present study found it to be lower in Japanese than in African healthy adults [4]. RA patients with the RFC1 80 A/A genotype had increased MTX-PG concentrations in RBCs [9,41], and the mean MTX-PG concentration in RBCs in RA patients with that genotype was reported to be 3.4-fold greater than in other genotypes [44]. Those patients showed a good clinical response to MTX treatment [35]. On the other hand, the−43 T > C change decreased the expression of RFC1 protein in patients with RA [34]. The directions in MTX-PG influx between RFC1 80 G > A and−43 T > C are completely opposite. To clarify the contributions of these two genotypes to MTX-PG concentrations in RBCs, it will be necessary to measure the concentrations in RFC1 80G > A and RFC1−43 T > C RA patients.
The frequency of the FPGS1994 A allele in our Japanese study was significantly lower than reported in Caucasian [4] and Africans [4]. FPGS 1994 A > G polymorphism reported to have no effect on MTX efficacy or toxicity [45], although there was an association between FPGS mRNA expression in peripheral blood mononuclear cells and a poor response to MTX in RA patients with that genotype [46]. Therefore, it is unknown whether the FPGS 1994 A > G polymorphism is associated with the inter-individual difference in MTX efficacy or toxicity.
For the frequency of the GGH 452 T allele, our Japanese study found it to be significantly lower than reported in Caucasian healthy individuals [4] and was the almost same as in Caucasian RA patients [27,37,38]. The GGH−401 T allelic frequency in our study was also lower than in HapMap-JPT [4] and in Caucasians [4], but it was higher than in Africans [4]. The GGH gene is  [47]. The−401C > T mutation resulting in greater GGH promotor activity increases the hydrolytic activity of MTX-PGs and decreases polyglutamation in the−401TT genotype compared with the−401CC or CT genotype [44]. Thus, MTX might be more effective in Caucasian than in Japanese RA patients at the same dose, when we speculate on the intracellular MTX-PG concentration.
The frequency of the ABCB1 3435 T allele in our study was lower than reported in Caucasian [4] but higher than in African healthy adults [4]. Conversely, the ABCC2 IVS23 + 56 C allelic frequency was higher in our study than reported in Caucasians [4] but lower than in Africans [4]. We also found a higher frequency of the ABCG2 914 A allele in our study than reported in Caucasians [4] and Africans [4]. The ABCB1 3435C > T mutation resulting in decreased stability and expression of mRNA reduces the activity of efflux transporters, may affect P-glycoprotein function and MTX efflux from cells [48], and is associated with MTX efficacy. It was reported that the length of time before it became necessary to reduce the MTX dose or discontinue administration due to the occurrence of toxicity was 2 months in patients with the T/T genotype, 23 months in those with the T/C genotype, and 29 months in those with the C/C genotype, and the time period was significantly correlated with genotype [40]. Therefore, it is considered that the ABCC2 IVS23 + 56 T > C mutation may affect ABCC2 enzyme activity involved in MTX efflux from cells [40]. The ABCG2 914C > A mutation was reported to increase MTX-PG 1 and MTX-PG 2 concentrations in RA patients [27]. It is considered that MTX efficacy is the highest in Caucasians, followed by Japanese and Africans, from the viewpoint of ABCB1 3435C > T; conversely, it is the highest in Caucasians, followed by Japanese and Africans, from the viewpoint of ABCC2 IVS 23 + 56 T > C; and the highest in Japanese, followed by Caucasians and then Africans, from the viewpoint of they included different ss numbers. 3) Several papers used for comparison with our results did not report the rs numbers for ethnic/race and/or population groups. Therefore, our comparisons may not be completely accurate in using the same rs numbers of SNPs. 4) All SNPs studied here could not be compared among ethnic/race and/or population groups due to a lack of data. This study may also include mixed ethnicities/races in a population group that we treated as a single national population, because that was assumed based on the lead author's country of residence. 5) The distributions of several SNPs in the ethnic/population group in healthy adults and RA patients were also not in agreement with Hardy-Weinberg equilibrium. 6) We did not evaluate allelic frequencies using combinations of SNPs such as diplotypes or haplotypes in our Japanese population. 7) There are few studies of MTX pharmacokinetics among ethnic/population groups. We also did not investigate MTX-PGs pharmacokinetics among ethnic/population groups by comparing the genotypes of MTX-related SNPs to clarify interethnic variations in MTX pharmacokinetics and therapeutic response. This is the greatest limitation in this study.

Conclusion
This study identified allelic frequencies in ethnic and/or population groups compared with healthy Japanese and RA patients. The differences in frequencies may be variables in the interethnic variations in MTX response. Further study is needed to confirm the association of these genetic/phenotypic factors and clinical outcomes and whether these SNPs may be useful in determining personalized MTX therapy for RA.