Methotrexate (MTX) continues to be the cornerstone of the treatment of rheumatoid arthritis (RA), and the major guidelines for the management of that disease recommend it as the initial therapy and as a key support for treatment with biological therapies.1–3 It is not strange that in a recent international observation study, COMORA (Comorbidities in RA), 89% of the patients received treatment with MTX or had taken it over the course of their disease.4 In Spain, the EMECAR study (Study of the Morbidity and Clinical Expression of Rheumatoid Arthritis) or in the emAR II study, conducted within the last 10 years, revealed that MTX was the initial drug of choice in 55% of the cases, and between 60% and 64% received this disease-modifying drug, either alone or in combination.5,6 Despite its wide use and the extensive research on this agent, the mechanism of action of MTX in RA is not fully established. Although the drug inhibits several metabolic pathways related to folic acid (Figure 1), the available data suggest that supplements with folic acid have a greater impact on the toxicity of MTX than on its efficacy.7 It has been shown that there is a marked individual susceptibility to its immunomodulating effect as well as to its toxicity, which makes it necessary to interrupt the treatment in up to a fourth of the patients receiving it.8 This means that, although it is the synthetic disease-modifying drug with the longest survival, less than 55% of those taking it maintain it for more than 8 years.9

Fig. 1.

Diagram of the metabolic pathways that involve folic acid and the enzymes on which methotrexate acts. The dark blue ovals indicate the enzymes studied in the present report. The red lines point to the enzymes that are inhibited by methotrexate polyglutamates. ABCB1, transporter that bonds ATP B1; ADA, adenosine deaminase; ADP, adenosine diphosphate; AMP, adenosine monophosphate; ATIC, AICAR transformylase/IMP cyclohydrolase; ATP, adenosine triphosphate; DHFR, dihydrofolate reductase; FPGS, folyl-polyglutamate synthetase; GGH, gamma-glutamyl hydrolase; IMP, inositol monophosphate; MTHFD1, 5,10-methylenetetrahydrofolate dehydrogenase 1; MTHFR, methylenetetrahydrofolate reductase; MTR, methionine reductase; MTX, methotrexate; MTXPG, methotrexate polyglutamates; RFC, reduced folate carrier; SAM, S-adenosyl-1-methionine; TYMS, thymidylate synthetase.

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Given that early treatment in RA has been shown to be essential to achieving an optimal control of the disease, it would be of great utility to have access to biomarkers that would enable us to predict which individuals will develop toxicity that could impede their taking full therapeutic doses of MTX. The study of genetic variants in the metabolic pathway enzymes in which folic acid participates has been a question of interest that has led to the performance of a number of studies that have attempted to predict the response to or toxicity of MTX, although the information obtained until now, in patients with an inflammatory rheumatic disease, is not conclusive.10 The objective of this article is to analyze the effect of genetic variants of methylenetetrahydrofolate reductase (MTHFR; rs1801131 and rs1801133), the membrane transporter that binds adenosine triphosphate (ATP) B1 (ABCB1; rs1045642), 5′-phosphoribosyl-5-aminoimidazole-4-carboxamide (AICAR) transformylase/inositol monophosphate (IMP) cyclohydrolase (ATIC; rs2372536) and folyl-polyglutamate synthetase (FPGS; rs1544105), whose function is shown in the diagram in Fig. 1, in the toxicity related to the drug in patients with early RA (ERA) exposed to MTX.

ABCB1 belongs to the ATP-binding cassette superfamily that is responsible for transport toward the exterior of the cytoplasmic membrane of a number of molecules, among them those that are cytotoxic. Specifically, the single nuclide polymorphism (SNP) rs1045642 consists in the transition of a C for a T in exon 26 of the ABCB1 transporter gene.11 This substitution provokes mRNA instability and has been associated with a decrease in the expression and, thus, in the function of ABCB1, accompanied by higher tissue levels of numerous drugs.11 In the case of MTHFR, the SNP rs1801133 involves the transition of a C for a T in exon 4, which entails the change of an alanine for a valine in position 222 of the protein, whereas the SNP rs1801131 involves the transition of an A for a C in exon 7, provoking the change of a glutamic for an alanine in position 429 of the protein. In both cases, this results in greater thermolability of the enzyme and less functional activity. With respect to rs1544105, there is a change of a G for an A in the promotor region of the FPGS gene that has been related to lower mRNA levels of this enzyme.12 The SNP rs2372536 in ATIC means a transition of a C for a G in exon 5, which brings about the change of a threonine for a serine in position 116 of the protein.13 This information on the functional impact of these genetic variants led to their being chosen for this study, which was planned in 2008.

Our data suggest that functional genetic variants of proteins ABCB1, FPGS and MTHFR are associated with the presence of greater toxicity and the use of smaller doses of MTX in patients with ERA. These data are congruent with previous reports, especially in the case of MTHFR, concerning which we have greater evidence in the field of rheumatology,18,19 including the Spanish population.20–22 In patients with inflammatory rheumatic diseases, the roles of ABCB1 and FPGS have been less extensively studied, as the focus has been mostly on the efficacy of MTX and less on its toxicity.10

A noticeable aspect of our results is the differential effect of the genetic variants studied here, with SNP rs1045642 in ABCB1 transporter and rs1801133 in MTHFR mainly affecting liver toxicity, whereas SNP rs1544105 in FPGS has been associated with hematologic toxicity. There are no previous studies that investigate the differential toxicity of these genetic variants in the field of rheumatology, since the majority of the studies we cite here were based on dichotomous variables involving the discontinuation of the drug due to toxicity, and the cause was not specified.18–22

In the case of ABCB1, the cells of the patients who were carriers of the minor allele rs1045642 would not easily eliminate MTX and, consequently, there would be a higher bioavailability, which could explain the greater toxicity observed. In fact, our data confirm the findings of Gregers et al., who reported a greater liver toxicity in children with acute lymphoblastic leukemia treated with MTX when they were homozygous carriers of CC genotype of ABCB1 rs1045642, rather than carriers of CT or TT genotype.23

In the case of SNP rs1801133 in MTHFR, the infrequent presence of the T allele is associated with decreased functional activity, which is estimated to be around 30% for TT homozygotes and 70% for CT heterozygotes.24 Moreover, this genetic variant has been associated with an elevation of homocysteine levels and the corresponding increase in cardiovascular risk.25 The elevation of homocysteine in patients with low levels of MTHFR activity occurs because the catalysis of the conversion of homocysteine to methionine requires 5-CH3-THF, which is generated from 5,10-CH2-THF by MTHFR (Fig. 1). The relationship with liver toxicity would occur because these patients have a lower production of methionine and of S-adenosyl-methionine (Fig. 1), which is of great importance in hepatic metabolism.26 Although our data only show a trend toward higher levels of GPT in carriers of CT and TT genotypes of rs1801133, these findings are consistent with previous studies that associate these genotypes with an elevation of transaminases.27,28

With respect to FPGS, as we mentioned above, the infrequent presence of the rs1544105 A allele has been associated with lower levels of the enzyme12 and differences in the efficacy of MTX in both leukemia and RA.29 In relation to our findings, the presence of the ancestral G allele is associated with greater hematologic toxicity and the need for lower doses of MTX. It is probably related to a greater efficacy in MTX polyglutamylation and a higher activity of the drug on purine and pyrimidine synthesis pathways (Fig. 1), and thus greater toxicity in the 3 hematologic series.

The major limitation to this study lies in the fact that it was performed in a single population of ERA, and that local practices in the use of MTX may have biased the results. For example, the attending physician could have decided not to initiate the drug in patients with a profile that predisposed them a priori to toxicity, such as elderly patients or alcoholic individuals or those with liver disease, which could lead to the underestimation of the effect observed. However, the reproduction of factors associated with greater MTX toxicity, such as obesity in hepatotoxicity,30 or the association of genetic factors related to changes in levels of GPT, Hb, leukocytes and platelets recorded in visits in which the patients had been receiving MTX, but not in those in which this drug was not being administered, confers credibility to the approach proposed in our article.

As another limitation, it is intriguing that we cannot identify the reason why genetic variants of proteins like ABCB1 and FPGS, which act on the same levels in the management of MTX and folic acid, have different impacts in terms of MTX toxicity, involving hepatotoxicity in the former and hematologic toxicity in the latter. Finally, as a result of the organization in the collection of samples in PEARL, it was not possible to determine intracellular MTX polyglutamates, which could have provided to a better understanding of the effect of the SNP studied in FPGS.

Finally, the effect observed may have less clinical relevance than expected, since the variants of ABCB1 and FPGS that we studied, although associated with MTX toxicity, have also been related to a greater efficacy of the drug.10 However, the combination of genotypes that could best limit the MTX dose used, according to our study, would be being homozygote for the G allele of rs1544105 in FPGS and CT or TT genotype of rs1801133 in MTHFR, which was found in 22% of our patient population. In short, the percentage of carriers of the SNP that we studied is considerable, and the determination of genetic variants of ABCB1, MTHFR and FPGS could help to identify the ERA patients who should take a lower dose of MTX or replace it with another disease-modifying drug.

This study was performed with funds from projects RD12/0009/0017, PI11/00551, PI14/00442 and PIE13/00041 of the Spanish Ministry of Economy and Competiveness (Instituto de Salud Carlos III, Madrid, Spain), co-financed by the European Regional Development Fund (ERDF).

IG-A has received funding for the last 5 years for research projects from BMS, Roche and UCB, as well as fees as a consultant and for scientific presentations from Abbvie, BMS, Lilly, Pfizer, Roche and UCB. IG-A and AMO are co-inventors of the patent entitled “Prognostic method for autoimmune disorders through genotyping vasoactive intestinal peptide variants”, code PCT/ES2015/070182.

LS, AL, EG-L and PM-F declare they have no conflicts of interest regarding this report.