International Journal of Rheumatic Diseases 2017; 20: 526–540

ORIGINAL ARTICLE

Lack of association between MTHFR A1298C polymorphism

and outcome of methotrexate treatment in rheumatoid
arthritis patients: evidence from a systematic review and
meta-analysis
Hongqiong FAN,1 Yanhui LI,2 Li ZHANG,3 Yuying LI1 and Wei LI1
1
Cancer Center and Departments of 2Cardiology and Echocardiography and 3Nephrology, First Hospital of Jilin University,
Changchun, China

Abstract
Objectives: The aim of this study was to evaluate the association of methylene tetrahydrofolate reductase (MTHFR)
gene polymorphism A1298C and methotrexate (MTX) outcome in rheumatoid arthritis (RA) patients.
Methods: We conducted a meta-analysis of the relevant published literature through to May 2016. Odds ratios
(ORs) and 95% confidence intervals (CIs) were calculated using fixed- and random-effect models.
Results: A total of 1325 cases (10 studies) of MTX efficacy and 2777 cases (18 studies) of MTX toxicity in RA
patients were analyzed. Pooled results showed that MTHFR gene A1298C polymorphism was not significantly
related to MTX toxicity or efficacy in RA patients. However, subgroup analysis indicated a significant association
between MTHFR gene A1298C polymorphism and decreased MTX efficacy in the South Asian population (CC
vs. CA + AA: OR = 0.45, 95% CI = 0.23–0.89, P = 0.021). Also, MTHFR gene A1298C polymorphism in the
partial folate supplementation group showed a relationship with decreased MTX efficacy (CC vs. CA + AA:
OR = 0.43, 95% CI = 0.20–0.92, P = 0.029) and toxicity (CC vs. CA + AA: OR = 0.40, 95% CI = 0.17–0.96,
P = 0.04; CC vs. AA: OR = 0.38, 95% CI = 0.16–0.94, P = 0.035).
Conclusions: Overall, our meta-analysis suggested no significant effect of MTHFR gene A1298C polymorphism
on MTX outcome in RA patients. However, due to several limitations of our meta-analysis, the results should be
interpreted cautiously and require further confirmation using high-quality studies.
Key words: meta-analysis, MTHFR, rheumatoid arthritis, SNPs.

BACKGROUND development of therapeutic interventions for RA treat-

Rheumatoid arthritis (RA) is a chronic inflammatory
joint disease that can cause cartilage and bone damage
and lead to disability.1 As one of the most prevalent
chronic inflammatory diseases, RA places a substantial
burden on both the individuals and society.2 The
Correspondence: Yuying Li MD and Wei Li MD, PhD, Cancer
Center, First Hospital of Jilin University, 71 Xinmin Street,
Changchun 10032, Jilin Province, China.
Emails: lyying1001@foxmail.com and drweili@outlook.com
Hongqiong Fan and Yanhui Li are the co-first authors.
ment in the past decade, including biological and tar-
geted synthetic disease-modifying anti-rheumatic drugs
(DMARDs), has transformed articular and systemic
outcomes.1,3 Among these therapeutics, low-dose
methotrexate (MTX) (5–25 mg/week), a conventional
synthetic DMARD, is still the ideal and most widely
used DMARD in clinical practice, due to its well-estab-
lished efficacy and low cost.4,5 However, still 40–60%
of RA patients fail to achieve a satisfactory response to
MTX and around 15–30% of them even develop
adverse drug effects.6 Thus, the MTX treatment

© 2017 Asia Pacific League of Associations for Rheumatology and John Wiley & Sons Australia, Ltd

outcomes in RA are not uniform and indicate individ-
ual-specific variability. Therefore, it is important to
identify predictors of response in the early stage of the
disease, so that physicians can individualize treatment
plans.
MTX is an anti-folate drug with anti-inflammatory
and anti-proliferative effects.7 However, the exact mech-
anism of its actions and predictors of its treatment
response have not been fully characterized. Recently,
several of studies have attempted to identify associa-
tions between the MTX clinical response in RA and
genetic modifications involved in the MTX action mech-
anism, such as adenosine triphosphate-binding cassette
(ABC) and reduced folate carrier 1 (RFC-1) involving
MTX membrane transport pathway, and 5,10-methyle-
netetrahydrofolate reductase (MTHFR), 5-aminoimida-
zole-4-carboxamide ribonucleotide formyltransferase/
IMP cyclohydrolase (ATIC), thymidylate synthetase
(TYMS), g-folypolyglutamate synthetase (FPGS), glu-
tamyl hydrolase (GGH), dihydrofolate reductase
(DHFR) and ADORA2A involving MTX intracellular
pathways.8–11 Among these, the MTHFR gene located
on chromosome 1 (1p36.6) is one of the most critical
enzymes involved in folate metabolism and DNA syn-
thesis, which irreversibly catalyzes the conversion of
5,10-methylenetetrahydrofolate to 5-methyltetrahydro-
folate.12 As MTX indirectly inhibits MTHFR and influ-
ences the regeneration of reduced folate,10 it is
plausible that MTHFR single nucleotide polymorphisms
(SNPs) might be related to the MTX response in RA
patients.
To date, more than 80 polymorphisms have been
described in the MTHFR gene, but functional data are
available for only a few of these variants. The A1298C
polymorphism (rs1801131) is one of the most preva-
lent and extensively studied genotypes and causes a glu-
tamine to alanine replacement at codon 429 of the
MTHFR protein, subsequently decreasing the enzymatic
activity.13
Several meta-analyses have studied associations
between MTHFR A1298C polymorphism and RA out-
comes, including five meta-analyses of MTX toxicity14–18
and three of its efficacy,15,18,19 through February 2013.
However, the inconsistent results and relatively small
sample sizes of these meta-analyses, limit their power
for identifying the relationship. For instance, the meta-
analyses by Fisher et al.,14 Owen et al.15 and Song
et al.16 regarding toxicity suggested no significant rela-
tionship between MTHFR A1298C polymorphism and
MTX toxicity in RA patients. However, the meta-analysis
by Spyridopoulou et al.17 demonstrated the presence of
International Journal of Rheumatic Diseases 2017; 20: 526–540
MTHFR polymorphism and RA
a statistical association between decreased MTX toxicity
and MTHFR A1298C polymorphism in RA patients.
Regarding efficacy, three different meta-analyses15,18,19
observed similar results with relatively small numbers
of subjects (692, 743 and 1040, respectively). In the
past 3 years, seven additional studies20–26 have been
conducted to detect the association between MTHFR
A1298C polymorphism and MTX response, and thus, it
is necessary to perform a larger updated meta-analysis
to confirm this association based on all the studies out
there in the literature.
MATERIALS AND METHODS
Study selection
A systematic literature search was conducted to identify
all the clinical studies evaluating MTHFR A1298C poly-
morphism and MTX outcome in RA patients using
PubMed, Embase and Cochrane databases, up until
May 2016. The following terms were used to identify
the relevant studies: rheumatoid arthritis, RA, MTHFR
and methylenetetrahydrofolate reductase. These search
terms were combined using the Boolean operators
‘AND’ and ‘OR’ in several combinations, without
restrictions. All the eligible studies were retrieved, and
their bibliographies were checked for additional rele-
vant articles independently by two investigators.
Inclusion and exclusion criteria
The inclusion criteria for the relevant studies was as fol-
lows: (i) study should be focused on the relationship
between MTHFR A1298C polymorphism and outcome
(toxicity and/or efficacy) of MTX treatment in RA; (ii)
should be a clinical study; (iii) should use a case–con-
trol or cohort design; and (iv) should provide the avail-
able genotype data, including AA, CA, CC or CA + CC
for MTHFR A1298C polymorphism. However, studies
were excluded if they were: (i) unpublished data, case
reports, conference articles and reviews, (ii) did not
have available genotype frequencies; (iii) included
duplicate data. Disagreements about the study selection
were resolved by discussion and consensus among all
the authors.
Data extraction
Two investigators reviewed all enrolled studies and
independently extracted the data. The data extraction
form included the following items: first author, title,
date of publication, country, ethnicity, gender, mean
age or age range, disease duration, treatment protocol,
dose of MTX, MTX time, folate supplementation rate,
527
H. Fan et al.
dose of folic acid supplementation, follow-up period,
measure of MTX toxicity and/or efficacy, and genotypes
of MTHFR A1298C SNP.
Study outcomes
The two endpoints of efficacy and toxicity of MTX in RA
patients were studied. The efficacy was evaluated by
improvement in the Disease Activity Score of 28 joints
(DDAS28) based on European League Against Rheuma-
tism (EULAR) criteria, MTX dose, American College of
Rheumatology (ACR)20 at 6 months guidelines, and
other rules designed by authors from different laborato-
ries. The MTX toxicity was evaluated based on all the
reported MTX-related adverse effects or discontinuation
of MTX due to adverse effects.
Statistical analysis
The results are reported as odds ratios (ORs) with 95%
confidence intervals (CIs). Pooled ORs were calculated
for the following genetic models: dominant model
(CC + CA vs. AA); recessive model (CC vs. CA + AA);
homozygous model (CC vs. AA); heterozygous model
(CA vs. AA); and allele comparison (C vs. A). A P-value
of < 0.05 by Z-test was considered statistically
significant.
Heterogeneity between the studies was assessed using
Q test (a P-value of < 0.1 represented statistical signifi-
cance) and I2 test (values of 25%, 50% and 75% repre-
sented mild, moderate and severe heterogeneity,
respectively). If the effect appeared to be homogeneous
(P > 0.1, I2 < 50%), the fixed-effect method was used
for analysis; otherwise the random-effect model was
applied. The heterogeneity was also tested by subgroup
analyses, based on ethnicity, folate supplementation,
mono-MTX therapy, measure of efficacy and measure of
toxicity.
Sensitivity analyses were performed to identify the
potential influence of each study on the pooled OR by
sequentially omitting one individual study at a time.
Potential publication bias was assessed by the Egger’s
weighted linear regression test and Begg’s rank correla-
tion test. A P-value of < 0.05 was considered to indicate
statistically significant publication bias.
All statistical analyses were performed using Stata
12.0 (StataCorp, College Station, TX, USA) software.
RESULTS
Study characteristics
The initial search identified 116 articles. Among
these, a total of 22 studies15,20–40 encompassing 3346
528
RA patients, were found to be eligible for further
analysis according to our inclusion criteria (Fig. 1).
The main characteristics of these 22 included studies,
published between 2002 and 2015, are summarized
in Table 1.
The association between MTHFR A1298C SNP and
MTX treatment efficacy in RA was reported in four stud-
ies,22–24,36 while 12 studies20,21,26,30,32–35,37–40 reported
an association with toxicity and six studies15,25,27–29,31
provided data for both efficacy and toxicity. Overall, the
data from 10 studies22–25,27–29,31,36 (1325 RA patients)
was analyzed for determining the correlation between
A1298C polymorphism and efficacy, and 18 stud-
ies15,20,21,25–35,37–40 (2777 RA patients) were included
for the toxicity analysis.
Regarding the details of the studies, eight studies had
small sample sizes (fewer than 100
cases)9,23,24,28,30,31,34,35 whereas 14 studies had large
sample sizes (more than 100 cases).15,20–22,25–
27,29,32,33,36–38,40
In addition, the studies included
patients of different ethnicities, with 1115,20,24–
26,30,31,33,34,36,40
studying Caucasian populations,
five28,29,32,37,38 East Asian populations, two22,23 South
Asian populations, one39 a Jewish population, one35 a
Latin American population and two21,27 mixed-ethni-
city populations. Nineteen studies20–28,30,31,33–40 pro-
vided the full distribution of AA, CA and CC genotypes,
and three studies15,29,32 provided the frequencies of AA
and CA + CC genotypes. Based on the criteria used for
toxicity evaluation, 14 studies20,26–35,37–39 analyzed
adverse effects of MTX, whereas three studies21,25,40
analyzed toxicity related to MTX discontinuation. More-
over, two studies15,24 enrolled patients treated with
MTX only, whereas 16 studies20,22,25–33,36–40 enrolled
patients treated with mono-MTX or combined therapy
(MTX concomitant with DMARDs/NSAIDs/glucocorti-
coid). Among these 16 studies, two studies25,30 pro-
vided information about genotype frequency of MTHFR
A1298C polymorphism and efficacy, and one30 study
reported toxicity in mono-MTX-treated RA patients. In
terms of the frequency of patients receiving folic acid,
100% of patients in 11 studies15,20,24–27,30,33,34,36,40
received folic acid, whereas no patients received folic
acid in five studies.22,31,32,37,39 Six studies21,23,28,29,35,38
provided no information regarding folic acid supple-
mentation. Furthermore, studies were also stratified by
measure of efficacy, with two studies24,25 following
DDAS28/EULAR criteria, one29 following MTX dose,
three28,31,36 following ACR20 at 6 months, and
four15,22,23,27 following different laboratory and clinical
judgments.
International Journal of Rheumatic Diseases 2017; 20: 526–540

Figure 1 Flow chart depicting the screening and selection strategy of relevant studies.
MTHFR A1298C polymorphism and MTX
outcome in RA
Efficacy
Ten studies15,22–25,27–29,31,36 involving 1325 RA cases
addressed the association between the MTHFR A1298C
SNP and efficacy of MTX in RA patients. Eight stud-
ies22–25,27,28,31,36 provided frequencies of the full geno-
type distributions of AA, CA and CC, whereas two
studies15,29 provided frequencies of AA and CA + CC
genotypes. Thus, the pooled ORs for the dominant
model were calculated from 10 studies, while for the
other four genetic models, eight studies were analyzed.
The analysis of pooled results found no statistical asso-
ciation between A1298C SNP and MTX efficacy, under
any genetic model (Table 2). Moreover, there was no
International Journal of Rheumatic Diseases 2017; 20: 526–540
MTHFR polymorphism and RA
evidence of significant heterogeneity between these
studies.
Furthermore, subgroup analyses based on ethnicity,
showed that MTHFR A1298C SNP was significantly
associated with decreased efficacy among the South
Asian population (recessive model [CC vs. CA + AA]:
OR = 0.45, 95% CI = 0.23–0.89, P = 0.021; Fig. 2a)
and increased efficacy in the East Asian population
(heterozygous model [CA vs. AA]: OR = 3.37, 95%
CI = 1.14–9.99, P = 0.028; allele comparison [C vs. A]:
OR = 3.02, 95% CI = 1.10–8.31, P = 0.032). However,
no associations were observed in Caucasian and mixed-
ethnicity populations. In addition, there was no
evidence of significant heterogeneity between studies
performed in Caucasian and South Asian populations,
529
 Table 1 Characteristics of studies included in the meta-analysis

530
First Year Country Ethnicity No. of Female Age, years, Folate Outcome measure Therapy
author subjects (%) range supplementation, method
Toxicity Efficacy
H. Fan et al.

%
Berkun 2004 Israel Jewish 93 82.8 58.7
13.7 58.8 Any adverse EULAR criteria† Mixed
effects
Caliz 2012 Spain Caucasian 468 74 49
13.4 100.0 Discontinuation NA Mixed
of MTX
Chaabane 2015 Tunisia Caucasian 141 79.4 52.8
12.48 100.0 Any adverse NA Mixed
effects
Choe 2012 Korea East Asian 167 95.2 53.9
10.4 NA Any adverse NA Mixed
effects
Davis 2014 America Mixed 319 7.96 68.75
1.89 NA Discontinuation NA NA
of MTX
Ghodke- 2015 India South 322 86 43.8
10.4 84 Any adverse ACR50 at Mixed
Puranik Asian effects† 12 months
Iqbal 2015 Pakistan South 51 86.6 42.87
13.5 NA NA ≥ 50% or NA
Asian reduction in
ESR, Richie index,
number of swollen
joints and
duration of
morning stiffness
at 6 months
Kumagai 2003 Japan East Asian 115 82.6 AE: 59.7
9.8 Non 27.8 Any adverse CRP/MTX dose Mixed
AE: 60.0
11.7 effects (6 mg/week)†
Kurzawski 2007 Poland Caucasian 174 74.1 54.8
11.1 100 NA ACR20 at Mixed
6 months
Mena 2011 Mexico Latin 70 NA NA NA MTX-related liver NA NA
American dysfunction
Owen 2013 British Caucasian 309 NA NA 100 Discontinuation CPR, ESR, and Mono
of MTX clinician MTX
judgments
Plaza-Plaza 2012 Spain Caucasian 53 81.1 54.7
14.37 100 Any adverse NA NA
effects
Salazar 2014 Spain Caucasian 124 81.5 55.62
1.297 100 Discontinuation DDAS28/EULAR Mono
of MTX† criteria MTX
Soukup 2015 Czech Caucasian 120 73.3 58.5
12.6 100 Discontinuation DDAS28/EULAR Mixed‡
of MTX criteria (mono
MTX for
efficacy)

International Journal of Rheumatic Diseases 2017; 20: 526–540

 Table 1 (continued)
First Year Country Ethnicity No. of Female Age, years, Folate Outcome measure Therapy
author subjects (%) range supplementation, method
Toxicity Efficacy
%
Stamp 2010 New Caucasian 191 72.7 60.5 (18–84) 100 Any adverse DAS28 ≤ 3.2 Mixed
Zealand effects
Swierkot 2015 Poland Caucasian 240 82 Responders 100 Any adverse DDAS28/ Mixed
54
10; effects EULAR
Non-responders: criteria†
50
11
Taniguchi 2007 Japan East Asian 156 CC: 83.3%, CC 56.1
12.2; CC 22.7; Any adverse MTX dose Mixed
CT + TT: CT + TT 55.8
12.1 CT + TT 40 effects (6 mg/week) at
87.8% 1 year and CRP

International Journal of Rheumatic Diseases 2017; 20: 526–540

Taraborelli 2009 Italy Caucasian 79 81 50
13.9 91 Any adverse ACR20 at Mixed
effects 6 months
Tasbas 2011 Turkey Caucasian 64 82.8 48.7
12.5 100 Any adverse NA Mixed‡
effects
Urano 2002 Japan East Asian 106 87.7 56.7
10.2 NA Any adverse MTX dose Mixed
effects (5 mg)
Wessels 2006 Netherlands Mixed 203 68.8 54.6
13.3 100 Any adverse DAS44 ≤ 2.4 Mixed
affects† at 6 months
Xiao 2010 China East Asian 95 74.2 49.6
14.1 NA Any adverse ACR20 Mixed
effects at 6 months
†
No genotype data. ‡Provide genotype data for mono-MTX treated RA. DDAS28, improvement in Disease Activity Score; EULAR, European League Against Rheumatism; ACR, American
College of Rheumatology; MTX, methotrexate; NA, not applicable.
MTHFR polymorphism and RA

531
H. Fan et al.

Table 2 Meta-analysis summary of pooled ORs and heterogeneity tests assessing the association of MTHFR A1298C polymor-
phism with methotrexate (MTX) efficacy in rheumatoid arthritis (RA) patients
Genetic models Sub-type No. of Test of association Test of Effect model
studies heterogeneity

OR 95% CI P Ph (%) I2 (%)

Dominant model Overall 10 1.13 0.89–1.44 0.302 0.042 48.5 F
CC + CA versus AA Partial folate supplementation 2 0.99 0.56–1.77 0.985 0.49 0 F
100% folate supplementation 5 0.91 0.68–1.22 0.543 0.419 0 F
Combined therapy 7 1.28 0.82–2.02 0.277 0.031 56.9 R
Mono MTX 3 0.9 0.58–1.38 0.186 0.464 0 F
South Asian 2 1.39 0.71–2.72 0.343 0.258 21.9 F
Caucasian 4 1 0.66–1.51 0.995 0.469 0 F
East Asian 3 2.03 0.82–5.06 0.127 0.022 73.8 R
Mixed-ethnicity 1 0.66 0.38–1.16 0.151 NA NA R
ACR20 at 6 months 3 1.5 0.70–3.22 0.294 0.114 53.9 R
DDAS28/EULAR criteria 2 0.82 0.44–1.55 0.548 0.365 0 F
Recessive model Overall 8 0.86 0.58–1.28 0.469 0.144 35.6 F
CC versus CA + AA Partial folate supplementation 2 0.43 0.20–0.92 0.029 0.479 0 F
100% folate supplementation 4 1.33 0.78–2.27 0.297 0.154 42.9 F
Combined therapy 6 0.95 0.62–1.47 0.835 0.1 45.8 F
Mono MTX 2 1.12 0.07–18.12 0.935 0.083 66.8 R
South Asian 2 0.45 0.23–0.89 0.021 0.504 0 F
Caucasian 4 1.46 0.77–2.78 0.252 0.161 41.8 F
Mixed-ethnicity 1 1.03 0.43–2.47 0.949 NA NA R
East Asian 1 1.54 0.06–38.82 0.794 NA NA R
ACR20 at 6 months 3 1.62 0.74–3.54 0.223 0.789 0 F
DDAS28/EULAR criteria 2 1.22 0.09–16.69 0.88 0.041 76.1 R
Homozygous model Overall 8 1.04 0.66–1.64 0.868 0.263 20.9 F
CC versus AA Partial folate supplementation 2 0.58 0.24–1.40 0.227 0.777 0 R
100% folate supplementation 4 1.16 0.45–2.96 0.759 0.097 52.5 R
Combined therapy 6 1.11 0.68–1.82 0.686 0.292 18.7 F
Mono MTX 2 0.84 0.07–10.85 0.896 0.121 58.4 R
South Asian 2 0.73 0.31–1.71 0.465 0.224 32.5 F
Caucasian 4 1.46 0.73–2.94 0.288 0.129 47.1 F
Mixed-ethnicity 1 0.81 0.32–2.04 0.65 NA NA R
East Asian 1 2.12 0.08–54.08 0.649 NA NA R
ACR20 at 6 months 3 1.79 0.76–4.22 0.182 0.666 0 F
DDAS28/EULAR criteria 2 1.08 0.07–15.75 0.956 0.044 75.4 R
Heterozygous model Overall 8 1.09 0.81–1.47 0.561 0.081 44.6 F
CA versus AA Partial folate supplementation 2 1.25 0.69–2.28 0.458 0.214 35.2 F
100% folate supplementation 4 0.82 0.56–1.20 0.306 0.506 0 F
Combined therapy 6 1.07 0.79–1.46 0.657 0.081 49 F
Mono MTX 2 0.56 0.24–1.32 0.187 0.74 0 F
South Asian 2 1.96 0.97–3.96 0.062 0.346 0 F
Caucasian 4 0.94 0.60–1.45 0.764 0.739 0 F
Mixed-ethnicity 1 0.63 0.35–1.14 0.127 NA NA R
East Asian 1 3.37 1.14–9.99 0.028 NA NA R
ACR20 at 6 months 3 1.43 0.67–3.04 0.355 0.132 50.6 R
DDAS28/EULAR criteria 2 0.78 0.40–1.52 0.466 0.783 0 F
Allele comparison Overall 8 0.99 0.81–1.21 0.951 0.094 42.7 F
C versus A Partial folate supplementation 2 0.77 0.52–1.14 0.18 0.849 0 F
100% folate supplementation 4 1 0.77–1.29 0.997 0.123 48.1 F
Combined therapy 6 1.03 0.83–1.27 0.814 0.081 49.1 F

532 International Journal of Rheumatic Diseases 2017; 20: 526–540

 MTHFR polymorphism and RA

Table 2 (continued)
Genetic models Sub-type No. of Test of association Test of Effect model
studies heterogeneity

OR 95% CI P Ph (%) I2 (%)

Mono MTX 2 0.75 0.42–1.35 0.34 0.235 29 F
South Asian 2 0.83 0.56–1.23 0.36 0.382 0 F
Caucasian 4 1.09 0.80–1.47 0.589 0.194 36.4 F
Mixed-ethnicity 1 0.81 0.53–1.22 0.307 NA NA R
East Asian 1 3.02 1.10–8.31 0.032 NA NA R
ACR20 at 6 months 3 1.4 0.78–2.51 0.265 0.128 51.3 R
DDAS28/EULAR criteria 2 0.85 0.37–1.95 0.694 0.1 63.1 R
Data are highlighted in bold if P-value is significant (< 0.05). Ph, P-value of Q test for heterogeneity; NA, not applicable; DDAS28, improvement in
Disease Activity Score; EULAR, European League Against Rheumatism; ACR, American College of Rheumatology; F, fixed-effect model; R, random-
effect model.

whereas the heterogeneity was not assessed for studies
on other ethnic groups due to the small number of
studies. Next, subgroup analyses based on folate sup-
plementation showed that MTHFR A1298C SNP was
significantly associated with decreased efficacy in the
partial folate supplementation group (recessive model
[CC vs. CA + AA]: OR = 0.43, 95% CI = 0.20–0.92,
P = 0.029), but not in the 100% folate supplementa-
tion group (Fig. 2b). Again, no significant heterogeneity
was observed between these studies. Furthermore, sub-
group analyses based on therapy method (mono-MTX
and combined therapy) and measure of efficacy
(DDAS28/EULAR criteria, ACR20 at 6 months) showed
possible alteration in efficacy, but the data were not sig-
nificant in any genetic model. The findings of these dif-
ferent meta-analysis and heterogeneity tests are
summarized in Table 2.
Toxicity
There were 18 studies analyzed to assess the relation-
ship between MTHFR A1298C SNP and MTX treatment
toxicity. Fifteen studies20,21,25–28,30,31,33–35,37–40 pro-
vided frequencies of the full genotype distributions of
AA, CA and CC, whereas three studies15,29,32 provided
frequencies of AA and CA + CC. The pooled ORs calcu-
lated for the dominant model were from 18 studies,
and the other four genetic models were analyzed based
on 15 studies.
The pooled results showed that MTHFR A1298C SNP
was not significantly associated with MTX toxicity in
RA, and there was significant heterogeneity between the
studies under dominant and heterozygous models and
allele comparison (CC + CA vs. AA: I2 = 53.4%,
P = 0.004; CA vs. AA: I2 = 53.8%, P = 0.007; C vs. A:
I2 = 65.2%, P < 0.001). Next, subgroup analyses based
on folate supplementation revealed that A1298C SNP
was statistically associated with decreased MTX toxicity
in the partial folate supplementation group (recessive
model [CC vs. CA + AA]: OR = 0.40, 95% CI = 0.17–
0.96, P = 0.04; homozygous model [CC vs. AA]:
OR = 0.38, 95% CI = 0.16–0.94, P = 0.035), but not
in the 100% folate supplementation group. Also, we
did not observe any evidence of significant heterogene-
ity between the studies (CC vs. CA + AA: I2 = 39.2%,
P = 0.193; CA vs. AA: I2 = 25.3%, P = 0.262). How-
ever, stratification based on the treatment method indi-
cated that MTHFR A1298C SNP had a trend of
association with increased MTX toxicity in the mono-
MTX group but decreased MTX toxicity in the combined
therapy method group. However, these results did not
reach significance. Also, there was no evidence of signif-
icant heterogeneity between the studies in these two
subgroups.
Furthermore, subgroup analyses based on ethnicity
revealed that A1298C SNP was significantly associated
with increased toxicity among the Latin American pop-
ulation (allele comparison [C vs. A]: OR = 2.75, 95%
CI = 1.12–6.76, P = 0.027) and mixed ethnicity popu-
lation (dominant model [CC + CA vs. AA]: OR = 2.29,
95% CI = 1.46–3.61, P < 0.001; heterozygous model
[CA vs. AA]: OR = 2.24, 95% CI = 1.39–3.60,
P = 0.001). In contrast, A1298C SNP was associated
with decreased toxicity among the Jewish population
(recessive model [CC vs. CA + AA]: OR = 0.20, 95%
CI = 0.05–0.74, P = 0.015; homozygous model [CC vs.
AA]: OR = 0.19, 95% CI = 0.05–0.73, P = 0.015; allele
comparison [C vs. A]: OR = 0.35, 95% CI = 0.18–0.70,
P = 0.003). There was no evidence of significant hetero-
geneity between these studies. Further stratification by

International Journal of Rheumatic Diseases 2017; 20: 526–540 533

H. Fan et al.

Figure 2 Forest plot of association between the MTHFR A1298C polymorphism and methotrexate (MTX) efficacy in RA patients:
odds ratios (ORs) with 95% confidence intervals (CIs). (a) Subgroup analysis based on different ethnicities: recessive model, CC
versus CA + AA; (b) subgroup analysis based on folate supplementation: recessive model, CC versus CA + AA.

534 International Journal of Rheumatic Diseases 2017; 20: 526–540


measure of toxicity indicated that the MTHFR 1298 CC
genotype was associated with increased discontinuation
of MTX in RA patients previously treated with MTX (re-
cessive model [CC vs. CA + AA]: OR = 1.8, 95%
CI = 1.07–3.04, P = 0.027). However, MTHFR A1298C
was associated with decreased adverse effects, but this
did not reach significance. All the results of these meta-
analyses and heterogeneity tests are summarized in
Table 3.
Sensitivity analysis and publication bias
Sensitivity analysis was performed in all genetic models
for efficacy and toxicity. These results indicated that
pooled ORs were not significantly affected by the omis-
sion of the individual studies. Therefore, the present
results appeared to be statistically reliable. In addition,
the Begg’s rank correlation test and Egger’s linear regres-
sion test did not provide any statistical evidence of pub-
lication bias for either the efficacy or toxicity analyses
(Fig. 3a,b).
DISCUSSION
We analyzed the association between MTHFR
A1298C SNP and clinical response to MTX in RA
patients in our meta-analysis. The results from five
previously published meta-analyses were inconsistent,
likely due to the relatively small sample sizes, which
limited the power to identify this relationship. Thus,
in our meta-analysis, we included additional recently
published studies, including four studies22–25 (448
RA cases) of MTX efficacy and four studies20,21,25,26
(749 RA cases) of MTX toxicity, to undertake the
comprehensive meta-analysis of the association, if
any, and draw a more concrete conclusion. The
pooled results of the current meta-analysis showed
that MTHFR gene A1298C polymorphism was not
significantly related to the toxicity and efficacy of
MTX in RA patients. Our efficacy results were consis-
tent with the results published by Owen et al.,15 Lee
et al.18 and Morgan et al.19 and our toxicity results
were consistent with the results published by Owen
et al.,15, Lee et al.18 and Fisher et al.14
The MTHFR A1298C polymorphism, first discovered
in 1998, leads to a decrease in MTHFR enzyme activity,
which can be further indirectly inhibited by MTX. The
homozygous variant carries about 60% of the wild-type
function.13 The influence of MTHFR A1298C polymor-
phism on MTX efficacy and toxicity has been explored
in other diseases, such as acute lymphoblastic leuke-
mia.41,42 However, our meta-analysis revealed that
International Journal of Rheumatic Diseases 2017; 20: 526–540
MTHFR polymorphism and RA
MTHFR A1298C polymorphism was not associated
with the efficacy or toxicity of MTX in RA patients.
The main reason for this discrepancy between biolog-
ical and epidemiological findings remains unclear but
may partially be explained as follows. First, several
additional genetic mutations, such as MTHFR C677T,17
ATIC C347G,27 RFC-1 G80A43 and TYMS (50 untrans-
lated region repeat element),44 in the coding sequences
of key enzymes in the MTX cellular pathway have been
identified, and they likely all affect the efficacy or toxic-
ity of MTX. Specifically, Ongaro et al. pointed toward
the existence of linkage disequilibrium between the
MTHFR 677T and MTHFR 1298C alleles.45 Soukup
et al. identified a synergistic effect of 677CT and
1298AC genotypes on the MTX efficacy in Czech RA
patients.25 Choe et al. suggested that the 677C/1298A
haplotype was associated with overall MTX toxicity in
Korean RA patients.32 In summary, it appears that link-
age disequilibrium and other genetic variations can
modify the relationship between MTHFR A1298C SNP
and the outcome of MTX treatment in RA patients. In
addition, MTX response in RA is not only determined
by genetic factors, but also by clinical factors. Recently
published studies have identified several clinical factors
as predictors of MTX outcome in RA, including gender,
age, smoking, disease duration, history of failed
DMARDs and baseline disease activity.46–49 These non-
genetic factors may have some influence on the rela-
tionship between the genetic factors and MTX clinical
outcome in RA patients. We wanted to address these
issues by subgroup analyses but were unable to conduct
a fully stratified analysis with these variables of non-
genetic or genetic factors due to a lack of sufficient data
from the available studies. Therefore, further investiga-
tion of the polymorphism in the context of specific
non-genetic or genetic factors is required to elucidate
the effect of MTHFR A1298C SNP on clinical outcome
following MTX treatment in RA patients.
Additionally, our results from the subgroup analyses
based on ethnicity showed that MTHFR A1298C poly-
morphism was related with MTX outcome among dif-
ferent populations. However, the power of the
subgroup analysis to produce statistically significant
results was limited due to the low number of studies
enrolled in the analysis (less than three). Furthermore,
the A1298C polymorphism varied between ethnic
groups, as the observed frequency of the 1298C allele
was 34% in Caucasian patients, 21% in Japanese
patients and 9% in African patients.50 Thus, these
results emphasize the need for future studies to confirm
the relationship among different populations.
535
H. Fan et al.

Table 3 Meta-analysis summary of pooled ORs and heterogeneity tests assessing the association of MTHFR A1298C polymor-
phism and methotrexate (MTX) toxicity in rheumatoid arthritis (RA) patients
Genetic models Sub-type No. of Test of association Test of Effect
studies heterogeneity model

OR 95% CI P Ph (%) I2 (%)

Dominant model Overall 18 0.94 0.72–1.22 0.64 0.004 53.4 R
CC + CA versus AA Not 100% folate supplement 4 0.75 0.50–1.14 0.182 0.39 0.4 F
100% folate supplement 8 0.95 0.77–1.19 0.677 0.248 21.9 F
Not mono MTX 14 0.85 0.70–1.03 0.089 0.297 14.3 F
Mono MTX 2 1.7 0.57–5.10 0.341 0.153 51 R
Jewish 1 0.44 0.18–1.06 0.057 NA NA F
Caucasian 9 0.87 0.69–1.09 0.221 0.411 2.9 F
East Asian 5 0.76 0.53–1.08 0.13 0.53 0 F
Mixed-ethnicity 2 2.29 1.46–3.61 <0.001 0.167 47.6 F
Latin American 1 3.58 0.98–13.04 0.053 NA NA F
Any adverse affects 14 0.9 0.73–1.10 0.296 0.097 34.8 F
Discontinuation of MTX 4 1.12 0.54–2.33 0.758 0.002 79.7 R
Recessive model Overall 15 1.14 0.82–1.60 0.436 0.128 30.2 F
CC versus CA + AA Partial folate supplementation 3 0.4 0.17–0.96 0.04 0.193 39.2 F
100% folate supplement 8 1.16 0.73–1.83 0.538 0.565 0 F
Not mono MTX 12 0.93 0.63–1.37 0.701 0.287 16 F
Mono MTX 1 2.57 0.24–28.09 0.439 NA NA F
Jewish 1 0.2 0.05–0.74 0.015 NA NA F
Caucasian 8 1.34 0.80–2.22 0.265 0.656 0 F
East Asian 3 0.89 0.27–2.97 0.856 0.492 0 F
Mixed-ethnicity 2 1.39 0.46–4.22 0.561 0.066 70.4 R
Latin American 1 5 0.63–39.39 0.126 NA NA F
Any adverse affects 12 0.84 0.54–1.31 0.452 0.286 16.1 F
Discontinuation of MTX 3 1.8 1.07–3.04 0.027 0.316 13.1 F
Homozygous model Overall 15 1.21 0.85–1.73 0.282 0.023 46.9 F
CC versus AA Partial folate supplementation 3 0.38 0.16–0.94 0.035 0.262 25.3 F
100% folate supplement 8 1.16 0.72–1.87 0.544 0.52 0 F
Not mono MTX 12 0.91 0.61–1.37 0.658 0.328 11.9 F
Mono MTX 1 4.8 0.38–59.89 0.223 NA NA R
Jewish 1 0.19 0.05–0.73 0.015 NA NA F
Caucasian 8 1.18 0.70–1.98 0.528 0.518 0 F
East Asian 3 0.8 0.23–2.73 0.716 0.589 0 F
Mixed-ethnicity 2 2.2 0.59–8.24 0.242 0.051 73.7 R
Latin American 1 8.75 0.95–80.26 0.055 NA NA F
Any adverse affects 12 0.88 0.55–1.39 0.572 0.19 25.8 F
Discontinuation of MTX 3 1.64 0.54–5.02 0.386 0.037 69.8 R
Heterozygous model Overall 15 0.92 0.68–1.25 0.601 0.007 53.8 R
CA versus AA Partial folate supplementation 3 0.82 0.47–1.42 0.469 0.469 0 F
100% folate supplement 8 0.9 0.71–1.15 0.4 0.197 29 F
Not mono MTX 12 0.84 0.68–1.03 0.097 0.218 23 F
Mono MTX 1 3.6 0.71–18.25 0.122 NA NA R
Jewish 1 0.85 0.30–2.44 0.76 NA NA F
Caucasian 8 0.79 0.61–1.01 0.063 0.506 0 F
East Asian 3 0.65 0.41–1.03 0.064 0.208 36.3 F
Mixed-ethnicity 2 2.24 1.39–3.60 0.001 0.357 0 F
Latin American 1 3.06 0.80–11.76 0.103 NA NA F
Any adverse affects 12 0.91 0.73–1.14 0.409 0.109 35.1 F
Discontinuation of MTX 3 1 0.34–2.96 0.995 0.001 84.7 R

536 International Journal of Rheumatic Diseases 2017; 20: 526–540

 MTHFR polymorphism and RA

Table 3 (continued)
Genetic models Sub-type No. of Test of association Test of Effect
studies heterogeneity model

OR 95% CI P Ph (%) I2 (%)

Allele comparison Overall 15 0.94 0.72–1.21 0.613 <0.001 65.2 R
C versus A Partial folate supplementation 3 0.62 0.34–1.13 0.119 0.124 52.1 R
100% folate supplement 8 0.98 0.81–1.17 0.805 0.32 14.1 F
Not mono MTX 12 0.88 0.75–1.03 0.124 0.124 33.3 F
Mono MTX 1 2.63 0.84–8.20 0.095 NA NA R
Jewish 1 0.35 0.18–0.70 0.003 NA NA F
Caucasian 8 0.92 0.76–1.12 0.434 0.368 8 F
East Asian 3 0.73 0.50–1.07 0.108 0.679 0 F
Mixed-ethnicity 2 1.62 0.88–3.00 0.123 0.041 76.1 R
Latin American 1 2.75 1.12–6.76 0.027 NA NA F
Any adverse affects 12 0.88 0.68–1.15 0.347 0.019 51.8 R
Discontinuation of MTX 3 1.12 0.55–2.31 0.752 0.001 84.8 R
Data are highlighted in bold if P-value is significant (< 0.05). Ph, P-value of Q test for heterogeneity; NA, not applicable; F, fixed-effect model; R,
random-effect model.

Another challenge in undertaking this meta-analysis

Figure 3 Assessment of publication bias using funnel plot for
the association between the MTHFR A1298C polymorphism
and (a) methotrexate (MTX) efficacy (recessive model, CC vs.
CA + AA) and (b) MTX toxicity (recessive model, CC vs.
CA + AA).
was the use of inconsistent criteria for the evaluation of
MTX response and toxicity among the enrolled studies.
In the current meta-analysis, there were numbers of
measures for efficacy, including DDAS28/EULAR crite-
ria, ACR20 and MTX dose. For MTX toxicity, different
measures, including discontinuation of MTX and types
of adverse effects categorized according to different toxi-
city criteria, were used in different studies. Furthermore,
the time point of evaluation ranged from 4 weeks to
12 months in the enrolled studies, which could also
influence the evaluation results. Taken together, these
unstandardized outcome criteria can variably impact
the results. Subgroup analysis indicated the altered rela-
tionship between MTHFR A1298C SNP and MTX out-
come in RA, based on measures of toxicity and efficacy,
but the small number of studies for each outcome made
it difficult to confirm the validity of these results.
In addition, different treatment protocols were used
between studies, including mono-MTX, MTX in combi-
nation with DMARDs/NSAIDs/glucocorticoids, and
MTX in combination with folic acid. We know that the
combination of MTX with DMARDs/NSAIDs/glucocor-
ticoids improves the response to treatment, but it also
results in induction of more adverse effects than mono-
MTX treatment. Similarly, folate supplementation has
also been suggested in clinical practice to reduce the risk
of MTX adverse effects.51 This suggests that the combi-
nation of drugs may confound the results for the associ-
ation between MTHFR SNP and clinical outcome. To
get a detailed understanding of this issue, we tried to
perform a subtype analysis based on treatment

International Journal of Rheumatic Diseases 2017; 20: 526–540 537

H. Fan et al.
protocol. However, the results were inconclusive for
multiple reasons. First, patients were treated with
mono-MTX treatment in fewer than three studies. Sec-
ond, the dose and time of folate supplementation were
different between different studies. Also, there were dif-
ferences in dietary folate intake between studies per-
formed in different populations.52 Thus, further
research is needed to confirm the association between
A1298C SNP and outcome of RA patients with mono-
MTX therapy.
CONCLUSION
In summary, the overall results of our meta-analysis
showed no association between MTHFR A1298C poly-
morphism and outcome in RA patients receiving MTX.
However, multiple factors might have confounded the
results and limited the power of the current meta-analy-
sis. Thus, larger, well-designed, prospective studies
which take into account the described genetic and non-
genetic factors, will be important to improve our under-
standing of the association between MTHFR A1298C
SNP and MTX outcome in RA patients.
ETHICS APPROVAL
This article does not contain any studies with animals
or human participants performed directly by any of its
authors. Authors of the included studies have declared
in their published articles that institutional review
boards or ethics committee at each participating site
approved their protocols.
INFORMED CONSENT
The authors of this article did not directly involve any
human subjects; however, the individuals have declared
obtaining informed consent from the patients.
CONFLICT OF INTEREST
The authors declare that they have no conflict of inter-
est.
REFERENCES
1 Smolen JS, Aletaha D, McInnes IB (2016) Rheumatoid
arthritis. Lancet 388, 2023–38.
2 Cross M, Smith E, Hoy D et al. (2014) The global burden
of rheumatoid arthritis: estimates from the global burden
of disease 2010 study. Ann Rheum Dis 73, 1316–22.
538
3 Smolen JS, Aletaha D (2015) Rheumatoid arthritis therapy
reappraisal: strategies, opportunities and challenges. Nat
Rev Rheumatol 11, 276–89.
4 Kremer JM (2004) Toward a better understanding of
methotrexate. Arthritis Rheum 50, 1370–82.
5 Smolen JS, Landewe R, Breedveld FC et al. (2014) EULAR
recommendations for the management of rheumatoid
arthritis with synthetic and biological disease-modifying
antirheumatic drugs: 2013 update. Ann Rheum Dis 73,
492–509.
6 Kooloos WM, Huizinga TW, Guchelaar HJ et al. (2010)
Pharmacogenetics in treatment of rheumatoid arthritis.
Curr Pharm Des 16, 164–75.
7 Cronstein BN (2005) Low-dose methotrexate: a mainstay
in the treatment of rheumatoid arthritis. Pharmacol Rev 57,
163–72.
8 Zhu H, Deng FY, Mo XB et al. (2014) Pharmacogenetics
and pharmacogenomics for rheumatoid arthritis respon-
siveness to methotrexate treatment: the 2013 update. Phar-
macogenomics 15, 551–66.
9 Zhang LL, Yang S, Wei W et al. (2014) Genetic polymor-
phisms affect efficacy and adverse drug reactions of
DMARDs in rheumatoid arthritis. Pharmacogenet Geomics
24, 531–8.
10 Owen SA, Hider SL, Martin P et al. (2013) Genetic poly-
morphisms in key methotrexate pathway genes are associ-
ated with response to treatment in rheumatoid arthritis
patients. Pharmacogenomics J 13, 227–34.
11 Lima A, Bernardes M, Azevedo R et al. (2016) Moving
toward personalized medicine in rheumatoid arthritis:
SNPs in methotrexate intracellular pathways are associated
with methotrexate therapeutic outcome. Pharmacogenomics
17, 1649–74.
12 Blount BC, Mack MM, Wehr CM et al. (1997) Folate
deficiency causes uracil misincorporation into human
DNA and chromosome breakage: implications for cancer
and neuronal damage. Proc Natl Acad Sci USA 94,
3290–5.
13 van der Put NM, Gabreels F, Stevens EM et al. (1998) A
second common mutation in the methylenetetrahydrofo-
late reductase gene: an additional risk factor for neural-
tube defects? Am J Hum Genet 62, 1044–51.
14 Fisher MC, Cronstein BN (2009) Metaanalysis of
methylenetetrahydrofolate reductase (MTHFR) polymor-
phisms affecting methotrexate toxicity. J Rheumatol 36,
539–45.
15 Owen SA, Lunt M, Bowes J et al. (2013) MTHFR gene
polymorphisms and outcome of methotrexate treatment
in patients with rheumatoid arthritis: analysis of key poly-
morphisms and meta-analysis of C677T and A1298C
polymorphisms. Pharmacogenomics J 13, 137–47.
16 Song GG, Bae SC, Lee YH (2014) Association of the
MTHFR C677T and A1298C polymorphisms with
methotrexate toxicity in rheumatoid arthritis: a meta-ana-
lysis. Clin Rheumatol 33, 1715–24.
International Journal of Rheumatic Diseases 2017; 20: 526–540

17 Spyridopoulou KP, Dimou NL, Hamodrakas SJ et al.
(2012) Methylene tetrahydrofolate reductase gene
polymorphisms and their association with methotrexate
toxicity: a meta-analysis. Pharmacogenet Geomics 22,
117–33.
18 Lee YH, Song GG (2010) Associations between the C677T
and A1298C polymorphisms of MTHFR and the efficacy
and toxicity of methotrexate in rheumatoid arthritis: a
meta-analysis. Clin Drug Investig 30, 101–8.
19 Morgan MD, Al-Shaarawy N, Martin S et al. (2014)
MTHFR functional genetic variation and methotrexate
treatment response in rheumatoid arthritis: a meta-analy-
sis. Pharmacogenomics 15, 467–75.
20 Chaabane S, Marzouk S, Akrout R et al. (2015) Genetic
determinants of methotrexate toxicity in tunisian patients
with rheumatoid arthritis: a study of polymorphisms
involved in the MTX metabolic pathway. Eur J Drug Metab
Pharmacokinet 41, 385–93.
21 Davis LA, Polk B, Mann A et al. (2014) Folic acid pathway
single nucleotide polymorphisms associated with
methotrexate significant adverse events in United States
veterans with rheumatoid arthritis. Clin Exp Rheumatol 32,
324–32.
22 Ghodke-Puranik Y, Puranik AS, Shintre P et al. (2015)
Folate metabolic pathway single nucleotide polymor-
phisms: a predictive pharmacogenetic marker of
methotrexate response in Indian (Asian) patients with
rheumatoid arthritis. Pharmacogenomics 16, 2019–34.
23 Iqbal MPAA, Mehboobali N, Iqbal K (2015) Lack of asso-
ciation between MTHFR gene polymorphisms and
response to methotrexate treatment in Pakistani patients
with rheumatoid arthritis. Pak J Pharm Sci 28, 4.
24 Salazar J, Moya P, Altes A et al. (2014) Polymorphisms in
genes involved in the mechanism of action of methotrex-
ate: are they associated with outcome in rheumatoid
arthritis patients? Pharmacogenomics 15, 1079–90.
25 Soukup T, Dosedel M, Pavek P et al. (2015) The impact of
C677T and A1298C MTHFR polymorphisms on
methotrexate therapeutic response in East Bohemian
region rheumatoid arthritis patients. Rheumatol Int 35,
1149–61.
26 Swierkot J, Slezak R, Karpinski P et al. (2015) Associations
between single-nucleotide polymorphisms of RFC-1,
GGH, MTHFR, TYMS, and TCII genes and the efficacy and
toxicity of methotrexate treatment in patients with
rheumatoid arthritis. Pol Arch Med Wewn 125, 152–61.
27 Wessels JA, de Vries-Bouwstra JK, Heijmans BT et al.
(2006) Efficacy and toxicity of methotrexate in early
rheumatoid arthritis are associated with single-nucleotide
polymorphisms in genes coding for folate pathway
enzymes. Arthritis Rheum 54, 1087–95.
28 Xiao H, Xu J, Zhou X et al. (2010) Associations between
the genetic polymorphisms of MTHFR and outcomes of
methotrexate treatment in rheumatoid arthritis. Clin Exp
Rheumatol 28, 728–33.
International Journal of Rheumatic Diseases 2017; 20: 526–540
MTHFR polymorphism and RA
29 Urano W, Taniguchi A, Yamanaka H et al. (2002) Poly-
morphisms in the methylenetetrahydrofolate reductase
gene were associated with both the efficacy and the toxic-
ity of methotrexate used for the treatment of rheumatoid
arthritis, as evidenced by single locus and haplotype analy-
ses. Pharmacogenetics 12, 183–90.
30 Tasbas O, Borman P, Gurhan Karabulut H et al. (2011)
The frequency of A1298C and C677T polymorphisms of
the methylentetrahydrofolate gene in Turkish patients
with rheumatoid arthritis: relationship with methotrexate
toxicity. Open Rheumatol J 5, 30–5.
31 Taraborelli M, Andreoli L, Archetti S et al. (2009)
Methylenetetrahydrofolate reductase polymorphisms and
methotrexate: no association with response to therapy nor
with drug-related adverse events in an Italian population
of rheumatic patients. Clin Exp Rheumatol 27, 499–502.
32 Taniguchi A, Urano W, Tanaka E et al. (2007) Validation
of the associations between single nucleotide polymor-
phisms or haplotypes and responses to disease-modifying
antirheumatic drugs in patients with rheumatoid arthritis:
a proposal for prospective pharmacogenomic study in
clinical practice. Pharmacogenet Geomics 17, 383–90.
33 Stamp LK, Chapman PT, O’Donnell JL et al. (2010) Poly-
morphisms within the folate pathway predict folate con-
centrations but are not associated with disease activity in
rheumatoid arthritis patients on methotrexate. Pharmaco-
genet Geomics 20, 367–76.
34 Plaza-Plaza JC, Aguilera M, Canadas-Garre M et al. (2012)
Pharmacogenetic polymorphisms contributing to toxicity
induced by methotrexate in the southern Spanish popula-
tion with rheumatoid arthritis. OMICS 16, 589–95.
35 Mena JP, Salazar-Paramo M, Gonzalez-Lopez L et al.
(2011) Polymorphisms C677T and A1298C in the
MTHFR gene in Mexican patients with rheumatoid arthri-
tis treated with methotrexate: implication with elevation
of transaminases. Pharmacogenomics J 11, 287–91.
36 Kurzawski M, Pawlik A, Safranow K et al. (2007) 677C>T
and 1298A>C MTHFR polymorphisms affect methotrexate
treatment outcome in rheumatoid arthritis. Pharmacoge-
nomics 8, 1551–9.
37 Kumagai K, Hiyama K, Oyama T et al. (2003) Polymor-
phisms in the thymidylate synthase and methylenetetrahy-
drofolate reductase genes and sensitivity to the low-dose
methotrexate therapy in patients with rheumatoid arthri-
tis. Int J Mol Med 11, 593–600.
38 Choe JY, Lee H, Jung HY et al. (2012) Methylenetetrahy-
drofolate reductase polymorphisms, C677T and A1298C,
are associated with methotrexate-related toxicities in Kor-
ean patients with rheumatoid arthritis. Rheumatol Int 32,
1837–42.
39 Berkun Y, Levartovsky D, Rubinow A et al. (2004)
Methotrexate related adverse effects in patients with
rheumatoid arthritis are associated with the A1298C poly-
morphism of the MTHFR gene. Ann Rheum Dis 63, 1227–
31.
539
H. Fan et al.
40 Caliz R, del Amo J, Balsa A et al. (2012) The C677T poly-
morphism in the MTHFR gene is associated with the toxic-
ity of methotrexate in a Spanish rheumatoid arthritis
population. Scand J Rheumatol 41, 10–4.
41 Eissa DS, Ahmed TM (2013) C677T and A1298C poly-
morphisms of the methylenetetrahydrofolate reductase
gene: effect on methotrexate-related toxicity in adult acute
lymphoblastic leukaemia. Blood Coagul Fibrinolysis 24,
181–8.
42 Yang L, Hu X, Xu L (2012) Impact of methylenetetrahy-
drofolate reductase (MTHFR) polymorphisms on
methotrexate-induced toxicities in acute lymphoblastic
leukemia: a meta-analysis. Tumour Biol 33, 1445–54.
43 Hayashi H, Tazoe Y, Tsuboi S et al. (2013) A single
nucleotide polymorphism of reduced folate carrier 1 pre-
dicts methotrexate efficacy in Japanese patients with
rheumatoid arthritis. Drug Metab Pharmacokinet 28, 164–8.
44 Szekanecz Z, Mesko B, Poliska S et al. (2013) Pharmacoge-
netics and pharmacogenomics in rheumatology. Immunol
Res 56, 325–33.
45 Ongaro A, De Mattei M, Della Porta MG et al. (2009)
Gene polymorphisms in folate metabolizing enzymes in
adult acute lymphoblastic leukemia: effects on methotrex-
ate-related toxicity and survival. Haematologica 94, 1391–
8.
46 Anderson JJ, Wells G, Verhoeven AC et al. (2000) Factors
predicting response to treatment in rheumatoid arthritis:
540
the importance of disease duration. Arthritis Rheum 43,
22–9.
47 Hoekstra M, van Ede AE, Haagsma CJ et al. (2003) Factors
associated with toxicity, final dose, and efficacy of
methotrexate in patients with rheumatoid arthritis. Ann
Rheum Dis 62, 423–6.
48 Hider SL, Silman AJ, Thomson W et al. (2009) Can clini-
cal factors at presentation be used to predict outcome of
treatment with methotrexate in patients with early inflam-
matory polyarthritis? Ann Rheum Dis 68, 57–62.
49 van Ede AE, Laan RF, Rood MJ et al. (2001) Effect of folic
or folinic acid supplementation on the toxicity and effi-
cacy of methotrexate in rheumatoid arthritis: a forty-eight
week, multicenter, randomized, double-blind, placebo-
controlled study. Arthritis Rheum 44, 1515–24.
50 Rosenberg N, Murata M, Ikeda Y et al. (2002) The fre-
quent 5,10-methylenetetrahydrofolate reductase C677T
polymorphism is associated with a common haplotype in
whites, Japanese, and Africans. Am J Hum Genet 70, 758–
62.
51 Harten P (2005) Reducing toxicity of methotrexate with
folic acid. Z Rheumatol 64, 353–8.
52 Donin AS, Nightingale CM, Owen CG et al. (2010) Nutri-
tional composition of the diets of South Asian, black Afri-
can-Caribbean and white European children in the United
Kingdom: the Child Heart and Health Study in England
(CHASE). Br J Nutr 104, 276–85.
International Journal of Rheumatic Diseases 2017; 20: 526–540