Inflammation Research

https://doi.org/10.1007/s00011-018-1161-8 Inflammation Research

ORIGINAL RESEARCH PAPER

Association of hyperhomocysteinemia with genetic variants in key

enzymes of homocysteine metabolism and methotrexate toxicity
in rheumatoid arthritis patients
Souhir Chaabane1 · Meriam Messedi2 · Rim Akrout3 · Mariem Ben Hamad1 · Mouna Turki2 · Sameh Marzouk4 ·
Leila Keskes1 · Zouheir Bahloul4 · Ahmed Rebai5 · Fatma Ayedi2 · Abdellatif Maalej1

Received: 12 May 2017 / Revised: 10 May 2018 / Accepted: 19 May 2018

© Springer International Publishing AG, part of Springer Nature 2018

Abstract
Objectives  The study investigated the association between plasma homocysteine, folate and vitamin B12 with 5,10 meth-
ylenetetrahydrofolate reductase (MTHFR C677T and A1298C), thymidylate synthase (TYMS 2R → 3R) and methionine
synthase (MTR A2756G) polymorphisms and methotrexate (MTX) treatment and toxicity in Tunisian Rheumatoid arthritis
(RA) patients.
Methods  A total of 185 patients with RA were included. Homocysteine (Hcy) was assessed by fluorescence polarization
immunoassay, and folate and vitamin B12 were measured by chemiluminescence immunoassays. The genetic polymorphisms
were analyzed by PCR or PCR-RFLP. Hyperhomocysteinemia (HHC) was considered for Hcy > 15 µmol/L.
Results  MTHFR C677T polymorphism was associated with HHC in RA patients (multi-adjusted OR, 95% CI 2.18, [1.07–
4.57]; p = 0.031). No association was detected with the remaining polymorphisms. Plasma Hcy, folate, and vitamin B12
did not differ according to each polymorphism, or with MTX treatment or toxicity. However, HHC was more prevalent in
patients with than those without MTX toxicity (32.7 vs. 16.7%; p = 0.035).
Conclusions The MTHFR 677TT genotype is an independent risk factor for HHC in Tunisians RA patients. HHC could be
a useful marker of MTX toxicity in RA patients.

Keywords  Rheumatoid arthritis · Homocysteine · Folate · Vitamin B12 · Hyperhomocysteinemia · Genetic polymorphisms

Introduction

Rheumatoid arthritis (RA) is a systemic autoimmune disease

that results in a chronic and systemic inflammatory disorder
that may affect many tissues and organs, especially synovial
Responsible Editor: John Di Battista. joints, leading to functional loss and mortality in the long
term [1]. RA affects approximately 0.5% of the adult popula-
* Souhir Chaabane tion worldwide [2]. Low-dose methotrexate (MTX) therapy
Chaabane.souhir@yahoo.fr
is one of the most commonly prescribed disease-modifying
1
Laboratory of Human Molecular Genetics, Faculty anti-rheumatic drugs (DMARDs) for the treatment of RA
of Medicine, Avenue Majida Boulila, 3029 Sfax, Tunisia [3]. Increased levels of homocysteine (Hcy) have been found
2
UR “Molecular Bases of Human Diseases”, Faculty in patients with RA compared to controls [4, 5]. Further-
of Medicine, Sfax, Tunisia more, hyperhomocysteinemia (HHC) has been proven to be
3
Department of Rheumatology, University Hospital Hedi linked to inflammation in RA [6].
Chaker, Sfax, Tunisia Hcy, is a sulfur-containing amino acid, produced as an
4
Department of Internal Medicine, University Hospital Hedi intermediary of methionine metabolism. Hcy concentrations
Chaker, Sfax, Tunisia are impacted by the dietary intake of folic acid and B vita-
5
Laboratory of Molecular and Cellular Screening Processes, mins, by life style factors such as smoking, and by genetic
Center of Biotechnology, Sfax, Tunisia factors [7–9]. Several common functional polymorphisms of

13
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genes encoding for enzymes acting in the folate/Hcy meta-
bolic pathway are likely to contribute to the variation in Hcy
levels.
Methylenetetrahydrofolate reductase (MTHFR), which
is necessary for the remethylation of Hcy to methionine or
other processes as DNA methylation [10], is a key enzyme
for the generation of reduced tetrahydrofolate. Actually,
the MTHFR catalyzes irreversibly the conversion of 5,10
methylenetetrahydrofolate (5,10-MTHF) to 5-methyltet-
rahydrofolate (5-MTHF), which acts as a methyl donor for
the vitamin B12-dependent remethylation of Hcy to methio-
nine. Hence, an elevated Hcy level is likely to be caused by
genetic defects in this enzyme or by deficiencies in cofac-
tors and substrates involved in this metabolism. Indeed, two
common single-nucleotide polymorphisms (SNPs), C677T
(rs1801133) and A1298C (rs1801131), fundamental to
MTHFR may be accredited to a decline in enzyme activity,
leading to elevated plasma Hcy levels [7, 8]. Thymidylate
synthase (TYMS) competes with MTHFR for one of its sub-
strates 5,10-MTHF, which is used to convert deoxyuridine
monophosphate (dUMP) into deoxythymidine monophos-
phate (dTMP). Nucleotide synthesis is important for DNA
replication and repair. The promoter–enhancer region of
TYMS gene contains a double (2R) or a triple (3R) 28-base-
pair (bp) tandem repeat polymorphism (rs34743033), where
3R genotype is associated with increased TYMS expression
[11, 12]. The methionine synthase (MTR) uses the methyl
group from 5-MTHF to remethylate Hcy. The products of
this reaction are methionine and tetrahydrofolate (THF).
Vitamin B12 is a cofactor for this enzyme. A polymor-
phism in the MTR gene A2756G (rs1805087) results in a
decreased enzyme activity [13]. Hcy levels are also known
to be influenced by anti-RA drugs such as MTX [11]. MTX
is a folic acid antagonist that inhibits key enzymes in several
metabolic pathways, including the Hcy–methionine pathway.
Such mechanisms are responsible for both MTX therapeu-
tic effects and toxicity (Fig. 1). In this regard, increasing
pharmacogenetic studies have examined clinical and genetic
risk factors of MTX outcome in RA patients [14]. Besides
genetic markers, phenotypic markers may determine the
extent of MTX toxicity and could be potential predictors
to identify patients prone to develop MTX toxicity. Some
authors have investigated a possible contribution of phe-
notypic markers such as plasma Hcy, vitamin-B12, folate,
and erythrocyte–folate to the occurrence of MTX toxicity.
Reports are controversial and the relationships between these
parameters are not clearly established [15–18]. The current
study was conducted to evaluate the impact of genetic poly-
morphisms of MTHFR (C677T and A1298C), TYMS (TS
2R → 3R) and MTR (A2756G) on Hcy, folate and B12 levels
and their possible contribution to HHC. We also examined
whether these parameters are associated with MTX treat-
ment and toxicity in a group of Tunisian RA patients.
13
S. Chaabane et al.
Patients and methods
Study population
One hundred and eighty-five Tunisian patients, including
35 men and 136 women affected with RA, were recruited
from March 2012 to February 2016 and followed at the
Department of Internal Medicine and Rheumatology, at Hedi
Chaker University Hospital (Sfax, Tunisia). All patients
were diagnosed with RA according to the 1987 Ameri-
can College of Rheumatology (ACR) criteria for RA and
reclassified in accordance with the 2010 criteria of ACR
and European League Against Rheumatism (EULAR) [19,
20]. Out of all RA patients recruited, 139 patients (75.1%)
were treated with low MTX dose (10–20 mg/week) estab-
lished as a first line DMARD, either orally or subcutane-
ously. Folic acid supplementation with a mean dose of 5 mg/
week was recorded in all patients under MTX therapy. The
remaining 46 patients (24.9%) were treated with other clas-
sic DMARDs. Among MTX-treated patients, 90 patients
(64.7%) were considered MTX tolerant and 49 patients
(35.3%) were considered MTX intolerant, who continued
to receive MTX therapy by subcutaneous administration or
stopped it due to the occurrence of adverse drug reactions
(ADRs). The most prevalent ADR observed was gastrointes-
tinal toxicity (56%). The study was approved by local ethics
committee. All subjects gave informed written consent to
participation in this study.
Laboratory measurements
Blood samples were collected into plain tubes and centri-
fuged at 2000×g at 4 °C within 60 min. Sera were collected
and stored at − 80 °C until analysis. Serum total Hcy was
measured by a fluorescence polarization immunoassay
method on an ­AXSYM® analyser (Abbott, Wiesbaden, Ger-
many) using specific commercial kit. Serum folate and B12
vitamin levels were determined by electrochimilumines-
cence assay using Elecsys 2010 analyser (Roche Diagnos-
tics, Canada). HHC was defined as plasma Hcy ˃ 15 µmol/L
[21].
Genetic evaluation
Total genomic DNA was obtained from peripheral blood leu-
kocyte samples taken from each patient by the standard phe-
nol–chloroform method [22]. The MTHFR C677T, A1298C,
MTR A2756G and TYMS 2R → 3R polymorphisms were
carried out by polymerase chain reaction–restriction frag-
ment length polymorphism (PCR-RFLP) and PCR method
as described previously [23]. It is worthy to note that for
Association of hyperhomocysteinemia with genetic variants in key enzymes of homocysteine…

MTX

MTXPG

DHF
dUMP

DHFR

TYMS
Homocysteine THF
SHMT
dTMP
De novo
Pyrimidine
B12 synthesis
MTR 5,10-MTHF

MTHFR
Methionine Enzyme Studied enzyme
5-MTHF
Vitamin Substrate
Methionine Folate
pathway Pathway
Drug and acve metabolite
Plasma
Folate Inibion Reacon

Fig. 1  Simplified scheme illustrating folate metabolism and its rela-
tionship to homocysteine methionine metabolism as well as metho-
trexate action mechanism. Methotrexate inhibits multiple enzymes
involved in folate and de novo nucleotides synthesis pathways. This
inhibition is then reflected in other pathways such as homocysteine/
methionine pathway. MTX methotrexate, MTXPG methotrexate poly-
glutamate, DHF dihydrofolate, DHFR dihydrofolate reductase, THF
tetrahydrofolate, SHMT serine hydroxymethyltransferase, 5,10-MTHF
5,10-methenyltetrahydrofolate, 5-MTHF 5-methyltetrahydrofolate,
MTR methionine synthase, TYMS thymidylate synthase, dTMP deox-
ythymidine monophosphate, dUMP deoxyuridine monophosphate

these polymorphisms genotyping, we reused results from our
published data for 96 patients [23]. It is also to be noted that
genotyping was lacking in one patient for the MTR A2756G
polymorphism and in two patients for the MTHFR A1298C
polymorphism.
Statistical analysis
All statistical analyses were conducted with Statistical
Package for Social Services software (SPSS version 20.0
for windows; SPSS Inc. Chicago, IL, USA). Continuous
variables are tested for normality using Shapiro–Wilk test.
Data are expressed as mean (standard deviation, SD) for
normally distributed continuous variables and as median
(Inter quartile range, IQR) for non-Gaussian distributed
variables. Categorical variables are expressed as frequencies
and percentages. Comparison between groups was achieved
using Mann–Whitney U test or Kruskal–Wallis analysis
of variance (ANOVA) as appropriate and Chi-square test.
Two models were used for genotype distribution analysis,

13
 S. Chaabane et al.

including dominant model (for isolated SNPs: homozygote Table 1  Demographic and clinical characteristics of patients with
rare + heterozygote vs. homozygote common) and recessive rheumatoid arthritis (RA) according to methotrexate (MTX) treat-
ment
model (for isolated SNPs: homozygote rare vs. heterozy-
gote + homozygote common). The Hardy–Weinberg equi- RA patients with RA patients
librium (HWE) was tested using Chi-square method. Binary MTX (n = 139) without MTX
(n = 46)
logistic regression models were applied to test how the asso-
ciation of MTHFR C677T polymorphism was independent Age, ­yearsa 53.6 (43.5–62) 63 (49–69)
of potential confounding factors. Adjustment was performed Male/female, n 26/113 10/36
for folate, vitamin B12 levels and MTX treatment. Unad- Disease duration, y­ earsa 9 (4–16) 3.5 (2–7)
justed and multi-adjusted odds ratio (OR) with 95% con- C-reactive protein, mg/La 24.4 (7.6–57.3) 25.5 (9.5–71.2)
fidence interval (95% CI) were calculated to estimate the Erythrocytes sedimentation rate, 45.5 ± 26.3 42.7 ± 26.9
association between HHC and different variables. The level ­mmb
of significance was set to 0.05. Rheumatoid factor p­ ositivec 105 (75.4) 31 (68.4)
Anti-CCP ­positivec 94 (67.6) 24 (52.6)
Power calculation Disease activity score ­28b 5.10 ± 1.72 4.4 ± 1.53
Bone ­erosionsc 97 (70) 26 (56.2)
We calculated the empirical power of the study by bootstrap- Carrying other autoimmune 51 (36.5) 12 (26.6)
­diseasec
ping from the data set of patients with and without HHC
(random sampling with replacement). For each bootstrap, a
 Data are expressed as median (IQR)
a genotype relative risk (GRR) test was calculated; 10,000 b
 Mean ± SD
bootstraps are used and the empirical power is estimated as C
 Number (%)
the number of times the GRR test exceeds the Chi-square Anti-CCP anti-cyclic citrullinated peptide
threshold (when a true association is declared) at 5 × 10−5
and 5% significance levels are divided by the total number
of bootstraps. Calculations were performed using a program The MTHFR C677T polymorphism remains associated
written in R language (http://www.r-proje​ct.org) [24]. with HHC after adjusting for potential confounders (multi-
adjusted OR, 2.18 [1.07–4.57], p = 0.031) (Table 4). No
significant differences in genotype distributions and allele
Results frequencies were observed for the remaining polymorphisms
(MTHFR A1298C, MTR A2756G and TYMS 2R → 3R) with
The study included 185 patients with RA, of which 46 HHC (data not shown).
patients were treated with MTX. Demographic and clinical
characteristics of patients according to MTX treatment are
summarized in Table 1. Plasma folate concentrations were Discussion
significantly higher in patients who were treated with MTX
compared to patient who were not (p = 0.039) (Table 2). This study showed a significant association between MTHFR
However, Hcy and vitamin B12 concentrations did not dif- 677T carriers and HHC in RA patients. The HHC was more
fer between the two groups. All these parameters did not frequent in RA carrying the T allele of MTHFR C677T poly-
significantly differ according to MTX toxicity. HHC was morphism than those carrying the C allele. The association
significantly more frequent in patients with MTX toxic- of 677TT genotype remained significant after adjusting for
ity (p = 0.035) (Table 2). Distribution of C677T, A1298C potential confounding factors. To assess the validity of our
and MTR A2756G genotypes among patients did not devi- sample, empirical power, which is the probability of detect-
ate from those predicted by the HWE (p > 0.05). Yet, the ing an association, was computed. Power calculations indi-
TYMS 2R → 3R polymorphism was not in HWE (p < 0.05) cated that our sample size provided 63% for the genotypic
for the studied group of RA patients. No significant differ- association of MTHFR C677T polymorphism.
ences were observed for plasma Hcy, folate and vitamin These observations are compatible with the results of
B12 concentrations between MTHFR (C677T and A1298C), the previous studies conducted in RA patients, in which
TYMS (2R → 3R) and MTR (A2756G) genotypes (data not HHC were associated with MTHFR 677TT genotype [6,
shown). However, the MTHFR 677T allele was associated 25, 26]. The association of the TT and CT genotypes with
with increased risk for HHC when compared to the C allele HHC was also reported in healthy adults [27] as well as
(T vs. C; OR = 2.06, CI [1.24–3.40], p = 0.004). Besides, the in other chronic disorders such as Behçet disease [28] and
association was also detected in the recessive model (TT vs. coronary artery disease [29]. This study found no signifi-
TC + CC; OR = 3.09, CI [1.07–8.89], p = 0.029) (Table 3). cant association between plasma HHC and MTHFR A1298C

13
Association of hyperhomocysteinemia with genetic variants in key enzymes of homocysteine…

Table 2  Plasma Homocysteine, folate and vitamin B12 in Rheumatoid Arthritis patients according to Methotrexate treatment and toxicity
Methotrexate treatment Methotrexate toxicity
Yes (n = 139) No (n = 46) p value Yes (n = 49) No (n = 90) p value

Homocysteine, µmol/L 12.3 (8.61–15.1) 12.1 (9.52–14.5) 0.351 12.4 (9.32–17.1) 11.5(8–15) 0.386
Folate, ng/L 6.29 (3.37–11.2) 4.16 (2.24–8.56) 0.039 9.58 (3.56–19.5) 5.50 (3.11–10.3) 0.233
Vitamin B12, pg/L 288 (227–382) 293 (225–398) 0.893 299 (234–419) 284 (228–395) 0.461
HHC, % 22.3% 21.7% 1.000 32.7% 16.7% 0.035

Results are expressed as median (IQR)

HHC hyperhomocysteinemia

Table 3  Genotype distribution and allele frequency of MTHFR an early study that found no association with HHC in RA
C677T polymorphism in Rheumatoid arthritis patients according to patients [32]. However, a recent study performed on Chinese
hyperhomocysteinemia
hypertensive patients suggested that MTR A2756G polymor-
Hyperhomocysteinemia phism was independently associated with HHC [31].
Yes No OR [CI 95%] p value Moreover, we observed no association of the TYMS
n (freq) n (freq) 2R/3R polymorphism with plasma Hcy levels nor HHC in
RA patients, which is in accordance with the findings of
MTHFR C677T
Borman et al. [33] carried out in Turkish RA patients. On the
 CC 10 (0.24) 68 (0.47) –
contrary, Trinh et al. [9] reported that the TYMS 3R/3R gen-
 CT 24 (0.59) 67 (0.47) 2.43 [1.08–5.48] 0.028
otype was associated with non-statistically significant ele-
 TT 7 (0.17) 9 (0.06) 5.28 [1.60–17.38] 0.003
vated Hcy levels among individuals with low dietary folate
 C 44 (0.54) 203 (0.70) –
intake. These divergent research findings may be related to
 T 38 (0.46) 85 (0.30) 2.06 [1.24–3.40] 0.004
the ethnic background, the detection methods, the sample
Dominant model
sizes, and/or characteristics of the different study cohorts.
 CC 10 (0.24) 68 (0.47) –
Interactions between folate and vitamin B12 levels and
 CT + TT 31 (0.76) 76 (0.53) 2.77 [1.26–6.07] 0.009
Hcy metabolism-related genes polymorphisms were also
Recessive model
evaluated in this study. Although the concentrations of these
 CT + CC 34 (0.83) 135 (0.94) –
two cofactors were not different among the genotypes of
 TT 7 (0.17) 9 (0.06) 3.09 [1.07–8.89] 0.029
each polymorphism, a sub-optimal vitamin B12 level was
Hyperhomocysteinemia (plasma homocysteine > 15 µmol/L) observed in patients with MTHFR 677T homozygotes along
n number, freq frequency, OR odds ratio, CI confidence interval with a non-significant higher Hcy level.
Earlier studies have already examined genetic risk factors
of the MTX toxicity in RA patients [14]. Our previous report
Table 4  Multivariate logistic modeling using hyperhomocysteinemia suggest that the MTHFR 677TT genotype could be an effec-
as dependent variable (n = 185) tive biomarker for the occurrence of MTX-related toxicity
Multi-adjusted OR (95% p value [23]. This led us to the hypothesis that low MTHFR activity
CI)a due to the carrying of TT MTHFR genotype might potential-
ize the effects of MTX, leading to toxicity. Therefore, it is
MTHFR C677T 2.18 (1.07–4.57) 0.031
justifiable to suspect that Hcy plasma could be a potential
Plasma folate 0.94 (0.88–1.01) 0.15
marker of MTX toxicity [26, 34].
Plasma vitamin B12 1.05 (0.98–1.11) 0.17
Conflicting data have been reported on the link between
Methotrexate treatment 1.05 (0.36–3.06) 0.92
HHC and MTX treatment in RA patients. Several authors
OR odds ratio, CI confidence interval reported that MTX-treated RA patients had significantly
a
 Adjusted for plasma folate, plasma vitamin B12 and methotrexate higher plasma Hcy levels which may be counteracted by
treatment folic acid administration [16, 35, 36]. Similar to our findings,
Hernanz et al. [4] found no difference in Hcy concentra-
polymorphism in RA patients, which is in agreement with a tions between RA patients with and without MTX treatment.
previous report [30]. Controversially, there are some other These conflicting data may be explained by the timing of
studies that have shown significant association of 1298AA MTX treatment and blood samples collection [37].
genotype with a higher risk for HHC [26, 31]. With respect The present study also showed a significant association
to MTR A2756G polymorphism, our findings are in line with between HHC and MTX toxicity in RA patients. Literature

13

yielded confusing data on the subject. Indeed, our findings
are consistent with a previous study performed by Bor-
man et al. [33] showing that the higher levels of Hcy were
observed in patients with ADRs due to MTX, while other
studies found no association [16, 30].
When Hcy was considered as a categorical variable, HHC
was significantly more prevalent in patients with MTX-
related toxicity carrying the T allele of MTHFR C677T poly-
morphism. However, the lack of significant association with
plasma Hcy is likely due to unmarked difference between
the groups and the large dispersion of plasma levels in each
group. This might also be a consequence of treatment itself.
MTX adverse effects are thought to be mediated through
folate antagonism. By inhibiting dihydrofolate reductase
enzyme, MTX could consume reduced folic acid and pro-
duce functional folate deficiency. Decreased levels of folic
acid may, in turn, reduce remethylation of Hcy to methionine
and, therefore, induce HHC [38].
Furthermore, folate deficiency is recognized as a risk fac-
tor for MTX toxicity in RA, mainly gastrointestinal toxic-
ity which was also previously correlated with HHC in RA
patients [39]. It is noteworthy to note that gastrointestinal
toxicity is the most common ADR related to MTX in this
study (56%) and in another reported RA study [14]. This
gastrointestinal disorder, reported in RA patients, is asso-
ciated with mucosal inflammation and damage in barrier
dysfunction [40]. This may cause malabsorption of several
nutrients. In such conditions, folate function may be com-
promised with important consequences on Hcy metabolism
[41]. Besides, the most plausible causes of high Hcy lev-
els are increased Hcy production or/and decreased its renal
excretion. Indeed, an important amount of Hcy is metabo-
lized in the kidney and plasma Hcy levels are negatively
correlated with the glomerular filtration rate [42]. There are
several side effects accompanying gastrointestinal disorders,
one of which is increasing oxidative stress and inflamma-
tion, resulting in damage renal function [43]. Decreased
renal blood flow, glomerular filtration rates in kidney, could
lead to elevated levels of Hcy in patients with MTX gastro-
intestinal toxicity.
So far, the relationship between these polymorphisms,
HHC as well as the risk to developing an MTX- related
toxicity in RA patients under MTX therapy has remained
inconclusive. Therefore, further investigation is needed to
clarify these interactions.
This study has several limitations, the major one is the
relatively small cohort of patients. Moreover, the sectional
design of our study might allow association between the
studied parameters to be assessed but does not assert cau-
sality. It is well known that inflammation linked to RA and
dietary habits of patients may influence Hcy and B-vitamin
status. Therefore, in our study, only genetic polymorphisms
were found to be non-modifiable parameters among the
13
S. Chaabane et al.
studied parameters. Despite these limitations, the observa-
tion appears to be solid as we found significant differences
in several parameters.
In conclusion, MTHFR 677TT genotype is associated
with HHC in Tunisian RA patients. HHC may reflect bio-
logical effects of MTX and may be considered as a marker
of toxicity of MTX therapy in RA patients. Further studies
are required to investigate other polymorphisms in enzymes
related to HHC and to clarify the mechanism underlying
HHC and MTX toxicity in patients with RA treated with
MTX.
Acknowledgements  We thank the patients for their cooperation. This
work was supported by the Ministry of Higher Education and Scientific
Research and the Ministry of Health, Tunisia. This paper is dedicated to
the memory of Pr Abdellatif Maalej who passed away on august 2017.
Compliance with ethical standards 
Conflict of interest  The authors declare that they have no conflicts of
interest.
Ethical approval  All procedures performed in studies involving human
participants were in accordance with the ethical standards of the insti-
tutional and/or national research committee and with the 1964 Helsinki
declaration and its later amendments or comparable ethical standards.
For this type of study, formal consent is not required.
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