GENETIC TESTING AND MOLECULAR BIOMARKERS ORIGINAL ARTICLES

Volume 21, Number 5, 2017

ª Mary Ann Liebert, Inc.
Pp. 275–285
DOI: 10.1089/gtmb.2016.0326

Association Between MTHFR C677T Polymorphism

and Methotrexate Treatment Outcome
in Rheumatoid Arthritis Patients:
A Systematic Review and Meta-Analysis

Wenjing Shao,1 Yi Yuan,2 and Yuying Li3

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Purpose: Methotrexate (MTX) is one of the most widely used disease-modifying antirheumatic drugs for the
treatment of rheumatoid arthritis (RA). However, its efficacy in RA patients is variable and unpredictable.
Methylene tetrahydrofolate reductase (MTHFR) is an important enzyme in the MTX pathway and is involved in
folate metabolism and DNA synthesis. Several studies have examined the association between the MTHFR
C677T polymorphism and MTX toxicity and efficacy in RA, but their conclusions remain controversial.
Materials and Methods: We conducted a comprehensive literature search of the PubMed, Embase, and
Cochrane Library databases to identify studies reporting an association between the MTHFR C677T single
nucleotide polymorphism and MTX response in RA patients.
Results: We identified 16 studies reporting MTX efficacy in 2373 RA cases, and 25 studies reporting MTX
toxicity in 4063 RA cases. The pooled data analysis indicated that the MTHFR C677T polymorphism was
associated with increased toxicity, but not efficacy, of MTX in RA patients. Further stratification based on
ethnicity revealed an association between the MTHFR 677TT genotype and overall MTX toxicity in East Asian
and Caucasian patient populations. In addition, RA patients with the MTHFR C677T polymorphism who were
supplemented with folic acid displayed significantly elevated risk for MTX toxicity.
Conclusion: Our study indicated that the MTHFR C677T polymorphism could be used as a predictor of MTX
toxicity in RA patients. However, large randomized prospective studies will be required to effectively replicate
and validate these findings.

Keywords: MTHFR, single nucleotide polymorphisms, rheumatoid arthritis, meta-analysis

Introduction 15–30% develop adverse effects (Kooloos et al., 2010). Thus,

the outcome of MTX treatment varies unpredictably between

R heumatoid arthritis (RA) is a chronic inflammatory

and autoimmune disease that affects *0.5% of the adult
population worldwide (Carmona et al., 2010). The mortality
rate for RA is higher relative to the general population (the
median and mean Standardized Mortality Ratios range from
1.5 to 1.8), with an overall reduction in survival time of be-
tween 3 and 10 years (Carmona et al., 2010).
The prognosis for RA has been improved by the devel-
opment of novel treatments, particularly disease-modifying
antirheumatic drugs (DMARDs). Low-dose methotrexate
(MTX) (5–25 mg/week) is still the most widely used
DMARD in clinical practice, due to its proven efficacy and
low cost (Kremer, 2004). However, 40–60% of RA patients
do not achieve a satisfactory response to DMARDs and about
RA patients.
MTX is an antifolate drug that displays antiproliferative
and anti-inflammatory effects by inhibiting several key en-
zymes involved in folate, methionine, adenosine, and de novo
nucleotide synthesis pathways (Lima et al., 2014). However,
its exact mechanism of action is unclear, and predictors of
MTX treatment response have not been fully characterized.
Methylene tetrahydrofolate reductase (MTHFR) is a cen-
tral regulatory enzyme in the folic acid pathway, which is
required for the regeneration of reduced folate, and is indi-
rectly inhibited by MTX. The MTHFR enzyme catalyzes the
irreversible conversion of 5,10-methylene tetrahydrofolate to
5-methyl tetrahydrofolate, which is required for purine and
thymidine synthesis, and acts as a carbon donor for the

Departments of 1Gynecology and 2Rheumatology, First Hospital of Jilin University, Changchun, China.
3
Cancer Center, First Hospital of Jilin University, Changchun, China.

275
 276 SHAO ET AL.

remethylation of homocysteine to methionine (Malik and Statistical analyses

Ranganathan, 2013; Zhang et al., 2014).
Thus, the MTHFR gene, located on chromosome 1
(1p36.6), appears to play a key role in maintaining the bal-
ance between DNA synthesis, repair, and methylation, and
polymorphisms in this gene could impact MTX responses in
RA patients. Several MTHFR polymorphisms have been
described, of which the C677T polymorphism (rs1801133) is
the most prevalent and extensively studied. This mutation
results in the replacement of the amino acid alanine with
valine at codon 222 of the MTHFR protein that in turn re-
duces its enzyme activity and subsequently results in elevated
plasma homocysteine levels and altered folate distribution
(Kang et al., 1988; Hider et al., 2007).
To date, seven meta-analysis studies (Fisher and Cron-
stein, 2009; Lee and Song, 2010; Spyridopoulou et al., 2012;
Davila-Fajardo et al., 2013; Owen et al., 2013; Morgan et al.,
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All statistical analyses were performed using Review
Manager 5.3 (The Nordic Cochrane Center, The Cochrane
Collaboration, Copenhagen, Denmark) and Stata 12.0 (Stata-
Corp, College Station, TX) software. The results were re-
ported as odds ratios (ORs) with 95% confidence intervals
(CIs). Pooled ORs were calculated for the following ge-
netic models: dominant model (TT + TC vs. CC); recessive
model (TT vs. TC + CC); homozygous model (TT vs. CC),
and heterozygous model (TC vs. CC). Z-test p values <0.05
were considered statistically significant.
Subgroup analyses were performed based on ethnicity,
folate supplementation, mono-MTX therapy, measure of ef-
ficacy, and measure of toxicity. Heterogeneity was assessed
using the Q test and I2 test (Higgins and Thompson, 2002). A
random effects model was applied to assess the outcome if
significant heterogeneity (Q test: p £ 0.1, I2 test: I2 ‡ 50%)

2014; Song et al., 2014) have reported an association between
the MTHFR C677T polymorphism and clinical response to
MTX in RA patients. However, the results of these studies
were not consistent, and their relatively small sample sizes
further limited their power to characterize this relationship. In
the last 3 years, another 10 studies were published that con-
firmed this association. Thus, to have a conclusive result
about this association, we performed a new meta-analysis
that includes all recent publications to review and summarize
the association between the MTHFR C677T polymorphism
and MTX response in RA patients.
Materials and Methods
Study selection
Two authors independently searched the PubMed, Em-
base, and Cochrane Library databases from their inception up
to September 2016 using the following key words: methylene
tetrahydrofolate reductase (MTHFR), and RA. Additional
information was procured by manually searching the refer-
ence lists of relevant articles and reviews.
was observed, whereas a fixed effects model was used to
assess outcomes with low heterogeneity (Q test: p > 0.1, I2
test: I2 < 50%).
To assess the influence of each study on the pooled OR,
sensitivity analyses were performed by sequentially omitting
one individual study at a time.
Results
Study characteristics
The search criteria identified 116 articles. Among these, 32
studies, representing data from 5496 RA patients, met the
inclusion criteria (Fig. 1). The main characteristics of the 32
included studies are summarized in Table 1. Briefly, all studies
were published between 2001 and 2015. Seven studies re-
ported the relationship between the MTHFR C677T poly-
morphism and MTX treatment efficacy in RA, 16 studies
reported toxicity, and nine studies reported both. Overall, ef-
ficacy analyses included data from 2373 RA patients, whereas
toxicity analysis was based on data from 4063 patients.

Inclusion and exclusion criteria

Articles conforming to the following criteria were included
for analysis: (1) evaluated an association between the MTHFR
C677T polymorphism and MTX treatment outcome (efficacy
and/or toxicity) in RA patients; and (2) provided sufficient
information about the frequency of the MTHFR C677T poly-
morphism (CC, CT, TT, or CT + TT). Articles were excluded if
they: (1) did not report genotype frequencies; (2) included
duplicate data; (3) analyzed only the association between the
MTHFR C677T polymorphism and risk of RA development;
or (4) were only case reports, conference abstracts, or review
articles. Any disagreements about the study selection were
resolved by discussion until all authors reached a consensus.

Risk of bias assessment

Potential publication bias was assessed using an Egger’s
test (Egger et al., 1997) and Begg’s test (Begg and Ma-
zumdar, 1994). p values <0.05 were considered to indicate
statistically significant publication bias. In addition, we also
performed the Duval and Tweedie nonparametric trim and fill FIG. 1. Flowchart depicting the screening and selection
procedure to further assess the possible effect of publication criteria of relevant studies. Thirty-two studies were selected
bias (Higgins and Thompson, 2002). for the meta-analysis.
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Table 1. Characteristics of the Studies Included in the Meta-Analysis

Folate Outcome measure
Subject Female Age years supplementation Therapy
First author Year Country Ethnicity number (%) (range) (%) Toxicity Efficacy method
Aggarwal et al. 2006 India South Asian 150 86.6 42.9 – 11.1 100 Any adverse DDAS28/EULAR Mixed
affects criteria
Berkun 2004 Israel Jewish 93 82.8 58.7 – 13.7 58.8 Any adverse EULAR criteriaa Mixed
affects
Bohanec 2008 Slovenia Caucasian 150 88.3 61 (51–69) 62.0 Any adverse DDAS28/EULAR Mixed
Grabar et al. affects criteriaa
Caliz 2012 Spain Caucasian 468 74 49 – 13.4 100 Discontinuation NA Mixed
of MTX
Chaabane 2015 Tunisia Caucasian 141 79.4 52.8 – 12.48 100 Any adverse NA Mixed
affects
Choe 2012 Korea East Asian 167 95.2 53.9 – 10.4 NA Any adverse NA Mixed
affects
Davis 2014 America Mixed 319 7.96 68.75 – 1.89 NA Discontinuation NA NA
of MTX
Ghodke-Puranik 2015 India South Asian 322 86 43.8 – 10.4 84 Any adverse ACR50 at 12 Mixed
affectsa months
Iqbal 2015 Pakistan South Asian 51 86.6 42.87 – 13.5 NA NA ‡50% reduction NA
in ESR, Ritchie
index, number

277
of swollen
joints and
duration of
morning
stiffness at 6
months
Kim 2006 Korea East Asian 385 92.2 50.4 – 11.0 100 Any adverse NA Mixed
affects
Kumagai 2003 Japan East Asian 115 82.6 AE: 59.7 – 9.8; 27.8 Any adverse CRP/MTX dose Mixed
Non-AE: affects (6 mg/week) a
60.0 – 11.7
Kurzawski 2007 Poland Caucasian 174 74.1 54.8 – 11.1 100 NA ACR20 at 6 Mixed
months
Lee 2009 America Caucasian 120 85.8 61.5 – 12.6 NA NA DAS28 £ 3.2 at 6 Mixedb
months
Lima et al. 2014 Portugal Caucasian 233 84.1 52 – 11.9 50.6 NA DSA28 <3.2 at 6 Mixed
months
Mena 2011 Mexico Latin American 70 NA NA NA MTX-related NA NA
liver
dysfunction
Owen et al. 2013 British Caucasian 309 NA NA 100 Discontinuation CRP, ESR, Mono
of MTX clinician MTX
judgment

(continued)
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Table 1. (Continued)
Folate Outcome measure
Subject Female Age years supplementation Therapy
First author Year Country Ethnicity number (%) (range) (%) Toxicity Efficacy method
Plaza-Plaza 2012 Spain Caucasian 53 81.1 54.7 – 14.37 100 Any adverse NA NA
effects
Ranganathan 2008 America Caucasian 52 NA NA 71.1–85.2 Any adverse NA Mixed
affects
Salazar et al., 2014 Spain Caucasian 124 81.5 55.62 – 1.297 100 Discontinuation DDAS28/EULAR Mono
of MTXa criteria MTX
Saleh 2015 Jordan Caucasian 120 85 52.00 – 13.08 29.2 Any adverse DAS28a Mixed
affects
Soukup 2015 Czech Caucasian 120 73.3 58.5 – 12.6 100 Discontinuation DDAS28/EULAR Mixedb
of MTX criteria
Stamp 2010 New Zealand Caucasian 191 72.7 60.5 (18–84) 100 Any adverse DAS28 £3.2 Mixed
effects
Swierkot 2015 Poland Caucasian 240 82 Responders: 100 Any adverse DDAS28/EULAR Mixed
54 – 10; effects criteriaa
Non-responders:
50 – 11
Taniguchi et al. 2007 Japan East Asian 156 CC: 83.3; CC: 56.1 – 12.2; CC: 22.7; Any adverse MTX dose (6 mg/ Mixed

278
CT + TT: CT + TT: CT + TT: 40 effects week) at 1 year
87.8 55.8 – 12.1 and CRP
Taraborelli 2009 Italy Caucasian 79 81 50 – 13.9 91 Any adverse ACR20 at 6 Mixed
effects months
Tasbas 2011 Turkey Caucasian 64 82.8 48.7 – 12.5 100 Any adverse NA Mixedb
effects
Urano et al. 2002 Japan East Asian 106 87.7 56.7 – 10.2 NA Any adverse MTX dose (5 mg) Mixed
effects
Uribarri 2015 Italy and Hungry Caucasian 197 79.2 59.3 – 12.5 NA Discontinuation Continuation of Mixedb
of MTX MTX
Van Ede 2001 The Netherlands Caucasian 236 72.4 56 – 12 66.5 Discontinuation DASa Mono
of MTX MTX
Wessels 2006 The Netherlands Mixed 203 68.8 54.6 – 13.3 100 Any adverse DAS44 <2.4 at 6 Mixed
affectsa months
Xiao 2010 China East Asian 95 74.2 49.6 – 14.1 NA Any adverse ACR20 at 6 Mixed
effects months
Zeng et al. 2008 China East Asian 193 89.7 40 – 14.8 NA Any adverse NA Mixed
effects
a
No genotype data.
b
Provide genotype data for mono-MTX-treated RA.
DDAS28, improvement in Disease Activity Score; CRP, C-reaction protein; ESR, erythrocyte sedimentation rate; MTX, methotrexate; EULAR, European League Against Rheumatism; ACR,
American College of Rheumatology; AE, adverse events; NA, not applicable.
 MTHFR POLYMORPHISMS AND RHEUMATOID ARTHRITIS
MTHFR C677T polymorphism and MTX
outcome in RA
Efficacy. Sixteen studies involving 2373 RA cases de-
scribed the association between the C677T polymorphism
and MTX efficacy in RA patients. Twelve studies reported
the frequency of CC, CT, and TT genotypes, whereas four
studies (Urano et al., 2002; Aggarwal et al., 2006; Taniguchi
et al., 2007; Owen et al., 2013) provided only the frequency
of CC and CT + TT genotypes. Thus, the pooled OR for the
dominant model (TT + TC vs. CC) was analyzed based on 16
studies, whereas analysis of pooled ORs for the other three
genetic models was based on only 12 studies.
Analysis of the pooled results indicated no significant as-
sociation between the C677T polymorphism and MTX
treatment efficacy in RA under any genetic model (Fig. 2).
Moreover, there was no evidence of significant heterogeneity
between these studies. However, subgroup analyses based on
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ethnicity (Caucasian, South Asian, East Asian, or mixed
ethnicity populations), folate supplementation (100% folate
supplementation or partial folate supplementation), mono-
MTX therapy, and measure of efficacy (improvement in
Disease Activity Score [DDAS28]/European League Against
Rheumatism [EULAR], the American College of Rheuma-
tology [ACR]20 at 6 months, or DAS28 £3.2 at 6 months)
revealed nonsignificant trends in efficacy. Efficacy meta-
analysis and heterogeneity tests are summarized in Table 2.
Toxicity. Twenty-five studies involving 4063 RA patients
assessed the relationship between the C677T polymorphism
and MTX toxicity. Eighteen studies provided the frequency
of the full genotype distributions of CC, CT, and TT, whereas
seven studies (van Ede et al., 2001a; Urano et al., 2002;
Aggarwal et al., 2006; Taniguchi et al., 2007; Bohanec
Grabar et al., 2008; Zeng et al., 2008; Owen et al., 2013)
provided the frequency of only the CC and CT + TT geno-
types. Thus, the pooled OR for the dominant model (TT + TC
vs. CC) was calculated based on 25 studies, whereas in the
other three genetic models, the pooled ORs were derived
from only 18 studies.
FIG. 2. Forest plot of the MTHFR C677T polymorphism and MTX efficacy in RA patients; ORs with 95% CIs for
dominant model, TT + TC versus CC. MTX, methotrexate; RA, rheumatoid arthritis; OR, odds ratio; CI, confidence interval.
279
The pooled results revealed that the C677T polymorphism
was significantly associated with increased overall adverse
affects in RA patients treated with MTX (TT vs. CC:
OR = 1.61, 95% CI = 1.03–2.50, p = 0.037; TT + TC vs. CC:
OR = 1.36, 95% CI = 1.02–1.80, p = 0.033, Fig. 3; TT vs.
TC + CC: OR = 1.49, 95% CI = 1.18–1.87, p = 0.001). How-
ever, we observed significant heterogeneity between studies
under the homozygous and dominant model (TT vs. CC:
I2 = 56.7%, p = 0.002; TT + TC vs. CC: I2 = 69.4%, p < 0.001),
but not the recessive model (TT vs. TC + CC: I2 = 6.3%,
p = 0.38).
Further ethnicity subgroup analyses revealed that the
C677T polymorphism was significantly associated with
increased toxicity among Caucasians (TT vs. TC + CC:
OR = 1.55, 95% CI = 1.14–2.10, p = 0.006; TT vs. CC:
OR = 1.77, 95% CI = 1.26–2.47, p = 0.001), and East Asians
(TT vs. TC + CC: OR = 1.66, 95% CI = 1.12–2.47, p = 0.012),
but not in Jewish, or Latin American populations.
Stratification based on treatment method indicated a sig-
nificantly increased association between the MTHFR C677T
polymorphism and overall adverse effects in RA patients
receiving mono-MTX therapy (TT + TC vs. CC: OR = 1.50,
95% CI = 1.01–2.23, p = 0.044). The subgroup analyses based
on folate supplementation revealed that the C677T poly-
morphism was significantly associated with increased tox-
icity in RA patients receiving MTX concomitant with folic
acid (TT vs. CC: OR = 2.54, 95% CI = 1.36–4.75, p = 0.003;
TT + TC vs. CC: OR = 1.57, 95% CI = 1.05–2.36, p = 0.03;
TT vs. TC + CC: OR = 1.82, 95% CI = 1.33–2.48, p < 0.001;
TC vs. CC: OR = 1.66, 95% CI = 1.01–2.71, p = 0.044). In
this study, we observed evidence of significant heteroge-
neity between studies investigating TT versus CC, TT + TC
versus CC, and TC versus CC (I2 = 56.4%, p = 0.025; I2 =
70.4%, p < 0.001; and I2 = 71.6%, p = 0.001, respectively).
The stratification based on measure of toxicity indicated that
increased discontinuation of MTX was associated with the
C677T polymorphism, but the differences did not reach
significance. The toxicity meta-analysis and heterogeneity
tests are summarized in Table 3.
 280 SHAO ET AL.

Table 2. Summary of the Pooled Odds Ratios and Heterogeneity Tests for the MTHFR C677T
Polymorphism and Methotrexate Efficacy in Rheumatoid Arthritis Patients
Test of association Test of heterogeneity
No. of
Genetic models Subtype studies OR 95% CI p ph I2 (%)
Dominant model Overall 16 0.91 0.76–1.09 0.300 0.679 0
TT + TC vs. CC Partial folate supplement 4 0.79 0.56–1.12 0.187 0.935 0
100% folate supplement 8 0.94 0.74–1.20 0.627 0.317 14.1
Mixed therapy method 10 0.89 0.71–1.10 0.286 0.232 22.9
Mono MTX 5 0.85 0.59–1.23 0.390 0.746 0
South Asian 3 0.94 0.56–1.55 0.799 0.962 0
Caucasian 9 0.91 0.73–1.14 0.406 0.323 13.3
East Asian 3 0.82 0.52–1.29 0.393 0.321 12
Mixed ethnicity 1 1.04 0.60–1.81 0.894 NA NA
DDAS28/EULAR criteria 4 0.87 0.59–1.30 0.509 0.811 0
ACR20 at 6 months 3 1.61 0.99–2.63 0.056 0.652 0
DAS28 £3.2 at 6 months 3 0.74 0.52–1.06 0.101 0.529 0
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Recessive model Overall 12 1.03 0.75–1.42 0.841 0.266 18.1

TT vs. TC + CC Partial folate supplement 3 0.90 0.24–3.30 0.868 0.061 64.3
100% folate supplement 6 1.29 0.79–2.13 0.308 0.891 0
Mixed therapy method 7 0.95 0.64–1.41 0.789 0.210 28.6
Mono MTX 4 1.58 0.33–7.46 0.565 0.041 63.6
South Asian 2 1.28 0.25–6.67 0.766 0.407 0
Caucasian 8 0.96 0.67–1.37 0.826 0.113 39.9
East Asian 1 1.12 0.35–3.57 0.846 NA NA
Mixed ethnicity 1 1.57 0.58–4.26 0.373 NA NA
DDAS28/EULAR criteria 2 1.73 0.54–5.50 0.354 0.593 0
ACR20 at 6 months 3 1.42 0.69–2.92 0.335 0.832 0
DAS28 £3.2 at 6 months 3 0.97 0.28–3.37 0.956 0.022 73.9
Homozygous model Overall 12 1.00 0.71–1.41 0.998 0.261 18.6
TT vs. CC Partial folate supplement 3 0.79 0.21–2.92 0.723 0.115 53.8
100% folate supplement 6 1.19 0.71–2.02 0.506 0.673 0
Mixed therapy method 7 0.88 0.57–1.36 0.574 0.158 35.5
Mono MTX 4 1.51 0.64–3.54 0.346 0.150 43.6
South Asian 2 1.32 0.25–6.95 0.745 0.434 0
Caucasian 8 0.89 0.60–1.32 0.568 0.130 37.5
East Asian 1 1.52 0.39–5.91 0.543 NA NA
Mixed ethnicity 1 1.56 0.55–4.40 0.402 NA NA
DDAS28/EULAR criteria 2 1.63 0.49–5.40 0.426 0.493 0
ACR20 at 6 months 3 1.67 0.71–3.92 0.237 0.816 0
DAS28 £3.2 at 6 months 3 0.81 0.24–2.74 0.737 0.038 69.4
Heterozygous model Overall 12 0.99 0.79–1.22 0.896 0.711 0
TC vs. CC Partial folate supplement 3 0.88 0.57–1.34 0.538 0.998 0
100% folate supplement 6 0.99 0.73–1.34 0.928 0.263 22.7
Mixed therapy method 7 0.98 0.75–1.28 0.883 0.327 13.5
Mono MTX 4 0.89 0.52–1.51 0.659 0.718 0
South Asian 2 0.94 0.48–1.83 0.845 0.692 0
Caucasian 8 0.97 0.75–1.25 0.796 0.421 1.1
East Asian 1 1.55 0.56–4.31 0.403 NA NA
Mixed ethnicity 1 0.98 0.55–1.76 0.951 NA NA
DDAS28/EULAR criteria 2 0.88 0.45–1.70 0.699 0.530 0
ACR20 at 6 months 3 1.55 0.94–2.56 0.089 0.583 0
DAS28 £3.2 at 6 months 3 0.76 0.52–1.10 0.151 0.619 0
ph, p-value of Q-test for heterogeneity; NA, not applicable; OR, odds ratio; CI, confidence interval; MTHFR, methylene tetrahydrofolate
reductase.

Sensitivity analysis and publication bias
For sensitivity analysis, the omission of any single study
had no significant effect on the overall results for efficacy and
toxicity among all the genotype models, which indicated the
stability of our meta-analysis.
In addition, Begg’s test and Egger’s test provided no sta-
tistical evidence of publication bias for toxicity analysis un-
der any model, except the recessive model (Begg’s test
p = 0.038 and Egger’s test p = 0.022). The trim and fill method
was used to evaluate the influence of possible missing stud-
ies. However, no trimming was performed and the results
 MTHFR POLYMORPHISMS AND RHEUMATOID ARTHRITIS
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FIG. 3. Forest plot of the MTHFR C677T polymorphism and MTX toxicity in RA patients; ORs with 95% CIs for a
dominant model, TT + TC versus CC.
were unchanged. Similarly, the assessment of publication
bias for efficacy analysis showed no evidence of bias based
on the Begg’s test in any genetic model. However, Egger’s
test suggested significant publication bias with a recessive
model ( p = 0.024) and homozygous model ( p = 0.012). Using
the trim and fill method the imputed studies produced a
symmetrical funnel plot. The pooled analysis incorporating
the hypothetical studies did not significantly alter the asso-
ciation between the MTHFR C677T polymorphism and
MTX efficiency in RA patients (OR = 0.883, 95% CI =
0.643–1.212, p = 0.440; OR = 0.748, 95% CI = 0.540–1.035,
p = 0.08; Fig. 4).
Discussion
In this study we investigated the association between the
MTHFR C677T polymorphism and MTX clinical response in
RA patients. Only seven previous meta-analysis studies have
attempted to analyze this association, and among them four
analyzed the relationship between the MTHFR polymorphism
and MTX toxicity (Fisher and Cronstein, 2009; Spyridopoulou
et al., 2012; Davila-Fajardo et al., 2013; Song et al., 2014), one
analyzed efficacy (Morgan et al., 2014), and the other two
(Lee and Song, 2010; Owen et al., 2013) analyzed both. Since
the results of these different studies were inconsistent, and
several additional recent studies also reported this association,
we performed an enhanced power analysis by including the
data from all studies published to date.
Overall, our analysis revealed no association between the
MTHFR C677T polymorphism and MTX treatment efficacy
in RA patients. This observation was consistent with previous
reports from Owen et al. (2013) and Lee and Song (2010).
However, we did observe a statistically significant associa-
tion between the MTHFR C677T polymorphism and MTX
281
toxicity, which was consistent with previous meta-analyses
from Song et al. (2014), Fisher and Cronstein (2009), and
Spyridopoulou et al. (2012).
The MTHFR C677T polymorphism has been reported to
reduce MTHFR enzyme activity. The enzymatic activity of
the homozygous and heterozygous variants is reduced by
70% and 40%, respectively (Ranganathan and McLeod,
2006; Hider et al., 2007). Despite these differences, we did
not find any association between the MTHFR C677T poly-
morphism and MTX efficacy, and instead observed an as-
sociation with an increased risk of toxicity.
This discrepancy may be partially attributed to linkage
disequilibrium or individual patient characteristics. Several
other genetic modifications, such as MTHFR A1298C (Salazar
et al., 2014), ATIC C347G (Grabar et al., 2010), RFC-1 G80A
(Hayashi et al., 2013), ABCB1 C3435T (Drozdzik et al., 2006),
TYMS (5’-UTR repeat element), and TYSM (3¢-UTR, 6 bp
deletion) (Szekanecz et al., 2013), were found to have the
potential to affect the efficacy or toxicity of MTX. Specifically,
Ongaro et al. highlighted the known linkage disequilibrium
between the MTHFR 677T and MTHFR 1298C alleles (On-
garo et al., 2009). These genetic variations can modify the
relationship between the MTHFR C677T polymorphism and
MTX outcome in RA patients. However, due to a lack of rel-
evant data, we could not address this issue in greater detail.
Second, clinical factors, including patient gender, age, and
smoking behavior, along with disease-specific factors, such as
disease duration and disease activity at baseline, can influence
the efficacy or toxicity of drugs used to treat RA patients
(Anderson et al., 2000; van Ede et al., 2001b; Hoekstra et al.,
2003; Hider et al., 2009). These nongenetic factors may affect
the relationship between genetic factors and clinical outcome.
We tried to address this issue through a subgroup analysis, but
were unable to conduct a fully stratified analysis based on
 282 SHAO ET AL.

Table 3. Summary of the Pooled Odds Ratios and Heterogeneity Tests for the MTHFR C677T
Polymorphism and Methotrexate Toxicity in Rheumatoid Arthritis Patients
Test of association Test of heterogeneity
No. of
Genetic models Subtype studies OR 95% CI p ph I2 (%)
Dominant model Overall 25 1.36 1.02–1.80 0.033 <0.001 69.4
TT + TC vs. CC Partial folate supplement 8 1.35 0.75–2.40 0.316 0.002 69.1
100% folate supplement 10 1.57 1.05–2.36 0.030 <0.001 70.4
Mixed therapy method 17 1.38 0.96–1.96 0.079 <0.001 81.1
Mono MTX 5 1.50 1.01–2.23 0.044 0.490 43.8
South Asian 1 0.76 0.33–1.73 0.509 NA NA
Jewish 1 1.20 0.51–2.81 0.675 NA NA
Caucasian 14 1.35 0.99–1.84 0.058 0.009 53.8
East Asian 7 1.76 0.84–3.65 0.132 <0.001 84.2
Latin American 1 0.88 0.24–3.26 0.846 NA NA
Mixed ethnicity 1 0.65 0.37–1.16 0.144 NA NA
Discontinuation of MTX 6 1.16 0.90–1.50 0.246 0.085 48.3
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Recessive model Overall 18 1.49 1.18–1.87 0.001 0.380 6.3

TT vs. TC + CC Partial folate supplement 5 1.03 0.59–1.79 0.927 0.373 5.9
100% folate supplement 8 1.82 1.33–2.48 <0.001 0.384 6.0
Mixed therapy method 12 1.57 1.21–2.05 0.001 0.204 24.4
Mono MTX 3 2.59 0.98–6.87 0.056 0.665 0.0
Jewish 1 1.24 0.32–4.76 0.752 NA NA
Caucasian 11 1.55 1.14–2.10 0.006 0.230 22.4
East Asian 4 1.66 1.12–2.47 0.012 0.485 0.0
Latin American 1 0.85 0.16–4.47 0.852 NA NA
Mixed ethnicity 1 0.69 0.25–1.92 0.480 NA NA
Discontinuation of MTX 4 1.48 0.98–2.23 0.060 0.326 13.3
Homozygous model Overall 18 1.61 1.03–2.50 0.037 0.002 56.7
TT vs. CC Partial folate supplement 5 0.86 0.44–1.69 0.663 0.377 5.2
100% folate supplement 8 2.54 1.36–4.75 0.003 0.025 56.4
Mixed therapy method 12 1.70 0.98–2.92 0.057 0.003 61.7
Mono MTX 3 2.50 0.85–7.36 0.095 0.656 0.0
Jewish 1 1.33 0.33–5.45 0.689 NA NA
Caucasian 11 1.77 1.26–2.47 0.001 0.059 43.8
East Asian 4 2.11 0.68–6.53 0.197 0.002 79.6
Latin American 1 0.80 0.12–5.20 0.815 NA NA
Mixed ethnicity 1 0.59 0.21–1.68 0.324 NA NA
Discontinuation of MTX 4 1.44 0.93–2.24 0.106 0.170 40.3
Heterozygous model Overall 18 1.31 0.92–1.86 0.135 <0.001 68.5
TC vs. CC Partial folate supplement 5 1.19 0.56–2.53 0.648 0.048 58.2
100% folate supplement 8 1.66 1.01–2.71 0.044 0.001 71.6
Mixed therapy method 12 1.37 0.89–2.11 0.150 <0.001 71.5
Mono MTX 3 1.26 0.60–2.64 0.537 0.482 0.0
Jewish 1 1.17 0.47–2.88 0.738 NA NA
Caucasian 11 1.34 0.93–1.94 0.120 0.027 50.6
East Asian 4 1.47 0.46–4.67 0.516 <0.001 88.1
Latin American 1 0.90 0.23–3.55 0.884 NA NA
Mixed ethnicity 1 0.67 0.36–1.24 0.199 NA NA
Discontinuation of MTX 4 0.96 0.70–1.32 0.798 0.286 20.6
Data are highlighted in bold if p-value is significant (<0.05).

these nongenetic factors due to the limited data available and
different stratification standards used in each study. There-
fore, the MTHFR C677T polymorphism should be further
studied in the context of specific nongenetic factors.
In addition, folate supplementation in clinical practice is
thought to reduce the risk of adverse effects of MTX (Harten,
2005). However, in our study, we found that overall toxicity
related to MTX was directly associated with the MTHFR
C677T polymorphism in RA patients taking folic acid. This
unexpected observation could be explained based on the as-
sumption that patients who received folic acid were likely to
have more severe disease, and thus were more susceptible to
either direct or indirect adverse effects of MTX (van Ede
et al., 1998). Furthermore, folic acid might not affect MTX-
related adverse events aside from hepatotoxicity (van Ede
et al., 2001b). Thus, patients with folate supplementation
were more likely to exhibit specific toxicity, which may in-
fluence the results. However, due to the limited number of
studies providing data about individual MTX-related side
effects, we could not address the association between the
C677T polymorphism and individual adverse effects asso-
ciated with folate supplementation.
 MTHFR POLYMORPHISMS AND RHEUMATOID ARTHRITIS
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FIG. 4. Funnel plot analysis of the MTHFR C677T
polymorphism and MTX efficacy in RA patients with and
without trim and fill method. (A) Begg’s funnel plot in a
recessive model, TT versus TC + CC; (B) Filled funnel plot
in a recessive model, TT versus TC + CC.
In clinical practice, MTX is generally administered as a
monotherapy or in combination with DMARDs/nonsteroidal
antiinflammatory drugs (NSAIDs)/glucocorticoids to im-
prove its efficacy. These drugs can also induce several ad-
verse effects associated with MTX. Thus, the enrollment of
patients receiving combined therapy may have confounded
the results and masked an association between MTHFR
polymorphisms and clinical outcome. To specifically address
the issue, we performed subtype analysis based on mono-
MTX therapy. Toxicity was observed to be significantly as-
sociated with the C677T polymorphism in RA patients re-
ceiving mono-MTX therapy. In addition, we also observed a
nonsignificant increase in efficacy in RA patients with the TT
genotype who received mono-MTX therapy. As the number
of studies included in this meta-analysis is small, further in-
vestigations will be required to confirm the relationship be-
tween the MTHFR C677T polymorphism and RA outcome in
patients treated with mono-MTX therapy.
Previous studies indicated that the frequency of the
MTHFR 677T gene variant differs among ethnic groups. The
frequency of the 677 T allele is 24–40% in Europeans, 40% in
Koreans, 26–37% in Japanese, and <11% in Africans, as well
as 49% and 44.7% in Jewish and Latin American populations,
respectively (Schneider et al., 1998; Efrati et al., 2013; Wang
et al., 2013). Furthermore, another study suggested that the
283
MTHFR C677T polymorphism is involved in the genetic
susceptibility of RA in Asian populations (Cen et al., 2016).
Thus, we also performed subgroup analysis based on eth-
nicity. Our study indicated a greater association between the
MTHFR 677TT genotype and overall toxicity of MTX in East
Asian and Caucasian patients. Studies by Owen et al. (2013)
and Song et al. (2014) also suggested a similar statistical
relationship in non-Caucasian and East Asian patients, re-
spectively, whereas in Caucasians they observed elevated,
but nonsignificant, toxicity. This inconsistency could be at-
tributed to the limited number of studies analyzed in the
previous meta-analysis. Further ethnicity-based investiga-
tions will be required to confirm the relationship between
C677T polymorphism and RA outcome.
Another important challenge in undertaking this meta-
analysis was the variability in the definitions and measures of
efficacy and toxicity. Ideally, definitions of outcome should
be standardized between studies; however, lack of stan-
dardization has been an issue, especially in retrospective RA
studies. In the present meta-analysis, several measures were
used to assess efficacy, including DAS28 score, DAS44
score, DDAS28/EULAR, ACR50, ACR20, continuation of
MTX, and MTX dose. Definitions of toxicity also varied,
including discontinuation of MTX and adverse effects cate-
gorized according to different toxicity criteria. Furthermore,
the time point of evaluation ranged from 4 weeks to 12
months after treatment, which may have influenced the tox-
icity and efficacy data. Taken together, these different out-
come criteria made it difficult to compare results between
studies. To address this issue, we repeated the meta-analysis,
including only the studies that used the same definitions of
efficacy and toxicity. The results of this subgroup analysis for
toxicity (discontinuation of MTX) and efficacy (DDAS28/
EULAR, ACR20 at 6 months, and DAS28 £3.2) were similar
to the overall meta-analysis results. However, as the number
of studies employing each outcome was small, further in-
vestigations are required to validate these results.
In summary, our meta-analysis suggests that the MTHFR
C677T polymorphism could be considered as a reliable predictor
of low-dose MTX toxicity, but not efficacy, in RA patients.
These findings may be helpful in clinical practice and could
improve the safety profile of MTX in RA patients. However, we
have to acknowledge the limited power of our analysis for
identification of genetic biomarkers for MTX clinical responses.
Larger, well-designed clinical studies that describe genetic and
nongenetic factors will be important to further improve our
understanding of the relationship between the MTHFR C677T
polymorphism and MTX outcome in RA patients.
Author Disclosure Statement
No competing financial interests exist.
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Address correspondence to:
Yuying Li, MD
Cancer Center
First Hospital of Jilin University
71 Xinmin Street
Changchun, Jilin 10032
China
E-mail: lyying1001@foxmail.com