Clin Exp Med

DOI 10.1007/s10238-017-0469-y

ORIGINAL ARTICLE

Nonassociation of homocysteine gene polymorphisms

with treatment outcome in South Indian Tamil Rheumatoid
Arthritis patients
Niveditha Muralidharan1 • Reena Gulati2 • Durga Prasanna Misra1 •

Vir S. Negi1

Received: 12 January 2017 / Accepted: 3 August 2017

Ó Springer International Publishing AG 2017

Abstract The aim of the study was to look for any asso- Abbreviations
ciation of MTR 2756A[G and MTRR 66A[G gene poly- ACPA Anticitrullinated peptide antibody
morphisms with clinical phenotype, methotrexate (MTX) ACR American College of Rheumatology
treatment response, and MTX-induced adverse events in CEU Utah residents with Northern and Western
South Indian Tamil patients with rheumatoid arthritis (RA). European ancestry
A total of 335 patients with RA were investigated. MTR CHD Chinese in Metropolitan Denver
2756A[G gene polymorphism was analyzed by PCR– DAS28 (ESR) Disease Activity Score based on 28 joints
RFLP, and MTRR 66A[G SNP was analyzed by TaqMan score and Erythrocyte Sedimentation Rate
50 nuclease assay. The allele frequencies were compared DMARD Disease-modifying antirheumatic drug
with HapMap groups. MTR 2756G allele was found to be EULAR European League Against Rheumatism
associated with risk of developing RA. The allele fre- GIH Gujarat Indians in Houston
quencies of MTR 2756A[G and MTRR 66A[G SNPs in HWE Hardy Weinberg equilibrium
controls differed significantly when compared with Hap- JPT Japanese in Tokyo Japan
Map groups. Neither of the SNPs influenced the MTX LWK Luhya in Webuye, Kenya
treatment outcome and adverse effects. Neither of the SNPs MKK Maasai in Kinyawa, Kenya
seems to be associated with MTX treatment outcome and MTHFR Methylene tetrahydrofolate reductase
adverse events in South Indian Tamil patients with RA. MTX Methotrexate
PCR Polymerase chain reaction
Keywords Rheumatoid arthritis  MTR  MTRR  RA Rheumatoid arthritis
Methotrexate  Treatment response  Adverse events  RF Rheumatoid factor
South Indian Tamils RFLP Restriction fragment length
polymorphism
MTR 5-Methyltetrahydrofolate-homocysteine
methyltransferase
Electronic supplementary material The online version of this MTRR Methionine synthase reductase
article (doi:10.1007/s10238-017-0469-y) contains supplementary SNP Single nucleotide polymorphism
material, which is available to authorized users.
YRI Yoruba in Ibadan, Nigeria
& Vir S. Negi
vsnegi22@yahoo.co.in
1
Introduction
Department of Clinical Immunology, Jawaharlal Institute of
Postgraduate Medical Education and Research (JIPMER),
Puducherry 605 006, India Rheumatoid arthritis (RA) is the most prevalent chronic
2 systemic inflammatory autoimmune disease [1–3] charac-
Genetic Services Unit, Department of Paediatrics, Jawaharlal
Institute of Postgraduate Medical Education and Research terized by synovial inflammation and joint destruction. If
(JIPMER), Puducherry, India left untreated, the disease leads to severe disability,

123

systemic complications, and early death [4–6].
Methotrexate (MTX) is the most commonly used drug for
treatment of RA which is effective in suppressing symp-
toms and reducing disability [7].
A significant inter-patient variability is known to occur
in clinical response to MTX [8] as 30–40% of the patients
on MTX therapy fail to attain remission [9]. The lack of
reliable biomarkers necessitates the identification of
genetic polymorphisms in MTX metabolic pathway to
predict treatment response [10].
Folate metabolism is an important therapeutic target of
MTX, and numerous studies were conducted in diverse
ethnic populations exploring the associations of genetic
variants in folate pathway [11]. Genetic polymorphisms in
MTHFR gene have been thoroughly investigated [12, 13].
The 5-methyltetrahydrofolate-homocysteine methyltrans-
ferase (MTR) and 5-methyltetrahydrofolate-homocysteine
methyltransferase reductase (MTRR) genes play pivotal
roles in homocysteine metabolism. There are fewer studies
conducted in the Caucasian group [14, 15] which investi-
gated the association of MTR and MTRR gene polymor-
phisms with methotrexate treatment outcome; however,
there are no studies in the south Indian cohort of patients
with RA. Hence the aim of this study was to establish allele
frequencies of MTR 2756A[G and MTRR 66A[G SNPs in
348 South Indian Tamil healthy controls and compare the
allele frequencies with HapMap groups and to identify any
associations of these SNPs with clinical phenotype, treat-
ment response, and MTX-induced adverse events in 335
patients with RA. Identification of genetic variants may
help in stratifying patients who are more likely to respond
or develop adverse events to MTX which will in turn aid in
formulating better treatment options and management of
patients with RA.
Patients and methods
Subjects
The study was conducted at the Department of Clinical
Immunology, Jawaharlal Institute of Postgraduate Medical
Education and Research (JIPMER), Puducherry, a major
tertiary referral center for South India. Newly diagnosed,
DMARD naı̈ve patients fulfilling the modified ACR cri-
teria for RA [16] were included in the study. A total of
348 age- and gender-matched healthy individuals of
Tamil ethnicity, without any family history of autoim-
mune disease were enrolled as healthy controls (HC).
Informed consent was taken from all participants, and the
study was approved by the Institute Ethics Committee.
Treatment was initiated with an initial MTX dose of
10 mg/week and escalated by 5 mg every 2 weeks based
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Clin Exp Med
on disease activity and patient tolerance, to a maximum
of 25 mg/week at the end of 3 months. The patients were
followed up every 2 weeks during the course of MTX
dose escalation and once a month after a stable dose was
reached. During each hospital visit, disease activity was
assessed by EULAR DAS28 criteria [17]. Response to
therapy was assessed at 16 weeks by EULAR response
criteria. The response was classified as ‘Good Response,’
‘Partial Response,’ and ‘No Response.’ Nonresponders
were offered combination DMARDs consisting of
methotrexate, sulfasalazine/leflunomide, and hydroxy-
chloroquine. Partial responders were followed up till
24 weeks and offered additional immunomodulator ther-
apy if they failed to improve further. Complete responders
continued with methotrexate.
Patients were monitored for MTX-induced toxicity by
clinical assessment (oral ulcers, nausea, vomiting, diarrhea,
methotrexate flu, methotrexate nodulosis, and methotrex-
ate-induced interstitial lung disease) and laboratory inves-
tigations including complete blood count (CBC) and liver
function tests [to look for cytopenias (anemia, leucopenia,
thrombocytopenia or pancytopenia), rise in aminotrans-
ferases or serum bilirubin, and rise of serum creatinine
greater than 30% from baseline]. Patients were also mon-
itored for development of infections, classifying those as
serious if they required in-hospital treatment.
Data collection
Variables which could possibly influence the treatment
outcome and the development of MTX-induced adverse
events were recorded. These variables included age, gen-
der, disease duration, age at onset (young onset/late onset),
autoantibody status (RF and ACPA), and disease activity.
Genotyping
Five milliliters of venous blood was collected, and DNA
was extracted by the salting-out procedure [18]. MTR
2756A[G genotyping (rs1805087) was performed by PCR
RFLP method as per the published protocol [19]. The PCR
product was digested with Hae III enzyme (New England
BioLabs) and electrophoresed in 3% agarose gel. Wild
types (2756AA) produced a single band at 211 bp.
Heterozygotes (2756AG) produced 211-, 131-, and 80-bp
fragments. Homozygous mutants (2756GG) produced two
fragments of 131 and 80 bp.
MTRR 66A[G (rs1801394) genotyping was performed
by TaqMan 50 nuclease assay consisting of allele-specific
fluorogenic oligonucleotide probes and primers specific for
MTRR 66A[G designed by Applied Biosystems, USA. The
sequences allowed discrimination of genotypes of each
studied pair of alleles.
Clin Exp Med

Laboratory measurement Table 1 Demographic and clinical profile of study subjects

Patients/controls (no.) 335/348
IgM rheumatoid factor (RF) in sera was measured at Sex (female/male) (%) 93/7
baseline by nephelometry (BN Prospec, Dade Behring,
Mean age (years) ± SEM 42.72 ± 0.55 years
Germany). Serum anticyclic citrullinated peptide antibody
Mean disease duration (years) ± SEM 3.75 ± 0.23 years
(ACPA) levels were examined by second-generation
Young onset disease (YORA) 314 (93.73%)
commercial ELISA kits (Biosystems, Barcelona, Spain).
Late onset disease (LORA) 21 (6.26%)
Antibody levels [10 and [20 IU/ml were regarded as RF
Erosive, deforming disease 237 (70.74%)
positive and ACPA positive, respectively.
Extra-articular manifestations 87 (25.97%)
Levels of homocysteine in plasma were measured at
Autoantibody phenotype
baseline by nephelometry (BN Prospec, Dade Behring,
RF positive 262 (78.20%)
Germany). Homocysteine levels of [15 lmol/L were
ACPA positive 250 (74.62%)
considered to be elevated.
Disease activity

Statistical analysis High disease activity 248 (74.02%)

Statistical analysis was carried out using GraphPad Instat
version 3.0 and SPSS version 16.0. Differences in genotype
and allele frequencies of MTR 2756A[G and MTRR
66A[G between responders and nonresponders were ana-
lyzed by Chi-square test with Yate’s continuity correction.
Odds ratio and 95% confidence interval were calculated for
each allele and genotype. p \ 0.05 was considered to be
statistically significant. Sample size and power of the study
(80%) were calculated using CaTS software (Power cal-
culator for genetic studies, compiled and published by
Centre for Statistical Genetics, Michigan University,
USA).
Univariate logistic regression analysis for the associa-
tion between methotrexate nonresponse and development
of MTX-induced adverse events with respective SNPs,
gender, and other clinical characteristics were tested, and
those factors that were significant in the univariate analysis
were included in a second multivariate logistic analysis.
The binary outcome variables were ‘responders’ (good and
partial responders) and ‘nonresponders’ for treatment out-
come and ‘with adverse events’ and ‘without adverse
events’ for development of adverse events.
Results
Characteristics of study participants
Moderate disease activity 81 (24.17%)
Low disease activity 6 (1.79%)
Baseline DAS28 (mean ± SEM) 5.77 ± 0.05
Treatment response
Mean dose of MTX (mg/week) 16.79 ± 0.21
Good responders 121 (36.11%)
Partial responders 107 (31.94%)
Nonresponders 107 (31.94%)
YORA—young onset RA (onset \55 years); LORA—late onset RA
(onset 55 years or above); high disease activity (DAS28 score [5.1),
moderate disease activity (DAS28 3.2 B 5.1); good response—(an
improvement of[1.2 in DAS28 score and a DAS of B2.6 (remission)
on follow-up); no response—(B0.6 change in DAS score or a change
between 0.6 and 1.2 with DAS score [3.7 on follow-up); moderate
response—(an improvement 0.6–1.2 in DAS score with overall DAS
between 2.6 and 3.7)
gastrointestinal (GI) side effects like oral ulcers, nausea,
vomiting, or diarrhea. Thirteen (19.11%) had hematologi-
cal manifestations in the form of anemia, leucopenia, or
pancytopenia. Five (7.35%) developed hepatotoxicity as
evidenced by an increase in amino transferase levels more
than two times the upper limit of normal range which
improved on discontinuation of MTX. Another nine
(13.23%) developed infections such as herpes zoster, oral
candidiasis, cellulitis, or urinary tract infection. Three
patients (4.41%) had pulmonary toxicity, and one (1.47%)
developed MTX-induced nodulosis.

The study included 335 patients with rheumatoid arthritis
treated with methotrexate, of which 93% were females.
The baseline demographic details of the patients are sum-
marized in Table 1. At the time of enrollment all patients
were DMARD naı̈ve with active disease.
All patients received 5 mg two times per week folic acid
supplementation. Sixty-eight patients (20.29%) developed
adverse events (AEs) related to MTX administration during
the course of treatment. Thirty-seven of them (54.41%) had
Clinical variables influencing the treatment outcome
and development of adverse events
Univariate analysis showed that patients who were positive
for RF [p = 0.01, OR 2.37, 95% CI (1.23–4.57)] and
ACPA [p = 0.03, OR 1.86, 95% CI (1.04–3.31)] showed
poor response to treatment. However, the statistical sig-
nificance was retained only for RF [p = 0.04, OR 2.03,
95% CI (1.01–4.09)] after multivariate analysis

123

(Supplementary Table 1). None of the variables influenced
the development of MTX-induced adverse events.
Relationship between homocysteine levels and gene
polymorphisms
The mean homocysteine levels were 10.29 ± 0.35 lmol/L
(n = 108). Eleven patients (10.18%) had hyperhomocys-
teinemia. No association was found between levels of
homocysteine and any of the gene polymorphisms studied.
MTR 2756A>G and MTRR 66A>G gene
polymorphisms
The MTR 2756A[G genotype distribution in cases were
found to be AA 166 (50.30%), AG 154 (46.66%) and GG
10 (3.03%), and in controls it was found to be AA 227
(65.22%), AG 106 (30.45%) and GG 15 (4.31%). The
ancestral A allele frequency was 73.63% in cases and
80.45% in controls, and mutant G allele frequency was
26.36% in cases and 19.54% in controls. The genotype
frequencies of MTR 2756A[G SNP among controls were
in HWE, and in cases there was a minor deviation.
The genotype and allele frequency of MTR 2756A[G
SNP in South Indians were compared with HapMap pop-
ulations, and it was found that the allele frequency were
significantly different from YRI, GIH, LWK, and MKK
ethnic groups (Supplementary Table 2).The mutant MTR
2756 G allele was associated with risk of developing RA
[p = 0.003, OR 1.47, 95% CI (1.14–1.90)] (Table 2).
The MTRR 66A[G genotype distribution in cases were
found to be AA 87 (25.97%), AG 178 (53.13%), and GG
70 (20.89%), and in controls it was found to be AA 82
(25.94%), AG 149 (47.15%), and GG 85 (26.89%). The
ancestral A allele frequency was 52.53% in cases and
49.52% in controls, and mutant G allele frequency was
47.46% in cases and 50.47% in controls. The cases and
controls of MTRR 66A[G SNP were in HWE. The geno-
type and allele frequency of MTRR 66A[G SNP in South
Indians were compared with HapMap populations, and it
was found that the MTRR 66A[G allele frequencies were
Table 2 Genotype and allele frequencies of MTR 2756A[G in cases and controls
MTR 2756A[G genotype Cases (n = 330)
AA 166 (50.30%)
AG 154 (46.66%)
GG 10 (3.03%)
Allele
A 486 (73.63%)
G 174 (26.36%)
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Clin Exp Med
concordant with CEU and statistically different from JPT,
YRI, and CHD ethnic groups (Supplementary Table 3).
The major and minor allele frequencies of MTR
2756A[G and MTRR 66A[G SNPs remained same in
good and nonresponders groups, and hence neither of the
SNPs was found be associated with MTX treatment
response (Tables 3, 4). Similarly, no association was seen
for both the SNPs with development of MTX adverse
events (Supplementary Tables 4, 5).
Discussion
The present study on South Indian Tamil patients with RA
found no significant association of MTR 2756A[G and
MTRR 66A[G SNPs with treatment outcome and adverse
events. Also neither of the SNPs influenced the plasma
homocysteine levels. However, MTR 2756G allele was
found to be associated with susceptibility to RA.
The folate metabolism is critical in regulating the
intracellular folate pool needed for synthesis and methy-
lation of DNA [20]. MTR located at 1q 43 is essential for
maintaining adequate intracellular methionine, intracellular
folate, and normal homocysteine concentrations [21–23]. A
common polymorphism in MTR gene involves a base
transition near the crucial vitamin B12-binding site
resulting in a change of aspartic acid to glycine (2756A[G,
D919G) [21, 24, 25]. This SNP influence the enzyme’s
secondary structure [24, 25]. The enzyme is maintained in
an active state by MTRR [26]. MTRR located on chromo-
some 5q15.3–p15.2 is a dual flavo protein that catalyzes the
reductive activation of cobalamin(II) to cobalamin(I), thus
making it available for remethylation of homocysteine to
methionine [26–29]. The common polymorphism in MTRR
gene is A[G substitution, leading to a change of isoleucine
to methionine at amino acid 22 [30]. This substitution is
thought to disrupt the binding of MTRR to the MTR-cob (I)-
alanine complex which could decrease the rate of homo-
cysteine remethylation [27].
MTR 2756A[G SNP result in increased homocysteine
levels, decreased folate levels, and decreased cobalamin
Controls (n = 348) PC (Yates corrected) OR (95% CI)
227 (65.22%) 0.0001 0.53 (0.39–0.73)
106 (30.45%) \0.0001 1.99 (1.45–2.73)
15 (4.31%) 0.49 0.69 (0.30–1.56)
560 (80.45%) 0.003 0.67 (0.52–0.87)
136 (19.54%) 0.003 1.47 (1.14–1.90)
Clin Exp Med

Table 3 Genotype and allele frequencies of MTR 2756A[G in good, partial and nonresponders
MTR 2756A[G genotype Good responders (n = 121) Partial responders (n = 103) Nonresponders (n = 106)
n (%) n (%) PC OR (95% CI) n (%) PC OR (95% CI)

AA 55 (45.45%) 58 (56.31%) 0.13 0.64 (0.38–1.09) 53 (50%) 0.58 0.83 (0.49–1.40)

AG 64 (52.89%) 42 (40.77%) 0.09 1.63 (0.95–2.77) 48 (45.28%) 0.31 1.35 (0.80–2.29)
GG 2 (1.65%) 3 (2.91%) 0.85 0.56 (0.09–3.42) 5 (4.71%) 0.34 0.33 (0.06–1.78)
Allele
A 174 (71.90%) 158 (76.69%) 0.29 0.77 (0.50—1.19) 154 (72.64%) 0.94 0.96 (0.63–1.45)
G 68 (28.09%) 48 (23.30%) 0.29 1.28 (0.83—1.97) 58 (27.35%) 0.94 1.03 (0.68–1.56)

Table 4 Genotype and allele frequencies of MTRR 66A[G in good, partial and nonresponders
MTRR 66A[G genotype Good responders (n = 121) Partial responders (n = 107) Nonresponders (n = 107)
n (%) n (%) PC OR (95% CI) n (%) PC OR (95% CI)

AA 27 (22.31%) 27 (25.23%) 0.71 0.85 (0.46–1.56) 33 (30.84%) 0.19 0.64 (0.35–1.16)

AG 69 (57.02%) 59 (55.14%) 0.87 1.08 (0.63–1.82) 50 (46.72%) 0.15 1.51 (0.89–2.55)
GG 25 (20.66%) 21 (19.62%) 0.97 1.06 (0.55–2.04) 24 (22.42%) 0.87 0.90 (0.47–1.69)
Allele
A 123 (50.82%) 113 (52.80%) 0.74 0.92 (0.63–1.33) 116 (54.20%) 0.53 0.87 (0.60–1.26)
G 119 (49.17%) 101 (47.19%) 0.74 1.08 (0.74–1.56) 98 (45.79%) 0.53 1.14 (0.79–1.65)

levels in patient carriers of the 2756A variant versus those
with the 2756G variant. On the other hand, an 66A[G
polymorphism in MTRR was associated with decreased
homocysteine levels, increased folate levels, and increased
cobalamin levels in those with the 66A variant versus those
with the 66G variant [31, 32]. Thus, both variants seem to
have opposite contributions to homocysteine remethylation
activity [33].
MTR 2756A allele frequency was 80.45%, and mutant G
allele frequency was 19.54% in controls. In 324 healthy
subjects from the Jewish cohort [34], the MTR 2756A and
G allele frequency was 86 and 14%, respectively. In 144
Western Indian healthy subjects [35], MTR 2756 G allele
frequency was 34%. MTR 2756 allele frequencies were
significantly different from YRI, GIH, LWK, and MKK
ethnic groups.
MTRR 66G allele frequency was 50% in this study and
58% in volunteers of Uttar Pradesh [27]. In 144 Western
Indian healthy subjects [35], MTRR G allele frequency was
50%. In 294 South Indian healthy volunteers [36], MTRR
66 A and G allele frequency was 33 and 67%, respectively.
MTRR 66A[G allele frequencies were concordant with
CEU and statistically different from JPT, YRI, and CHD
ethnic groups.
The mutant MTR 2756 G allele was associated with risk
of developing RA [p = 0.003, OR 1.47, 95% CI
(1.14–1.90)]. Similar to our results, MTR 2756 GG geno-
type and MTR 2756 G allele were associated with sus-
ceptibility to RA [34]. Also in 106 Polish group of patients
with SLE, MTR 2756G allele was associated with SLE
susceptibility [37]. The present finding of how MTR
2756A[G gene polymorphism confers susceptibility to RA
could be interpreted by the shared chromosomal location of
MTR with PTPN22, IL23R, and TGFb genes at chromo-
some 1. Any of these immune-related genes could be in
linkage disequilibrium with MTR 2756A[G SNP which in
turn could be responsible for the observed susceptibility in
RA [38–40].
In the present study on South Indian Tamils, MTR
2756A[G and MTRR 66A[G gene polymorphisms did not
influence the MTX treatment response and adverse events.
Our results are concordant to reports from Chaabane et al.
[41] and Wessels et al. [15]. Chaabane et al. [41] in a group
of 141 Tunisian patients with RA reported no significant
association of these SNPs with development of toxicities.
Wessels et al. in 205 patients with active RA [15] found
that neither of the SNPs had any association with clinical
response or with the development of toxicities. Grabar
et al. [14] in 213 Caucasians reported that MTR 2756A[G
and MTRR 66A[G SNPs had no role in predicting the
treatment outcome, MTX adverse events, and total homo-
cysteine concentrations.

123

On the contrary, James et al. [33] observed that
patients with MTR 2756 A allele were more likely to
respond to methotrexate [OR 2.6, 95% CI (1.1–6.1)].
Similarly, in a cross-sectional study involving 86 patients
treated with MTX [34], it was found that MTR 2756GG
genotype was associated with MIARN (p = 0.013). In
213 patients with RA [14], MTRR 66A[G gene poly-
morphism was associated with lower risk of developing
dermatologic complaints (p = 0.007, OR 0.191, 95% CI
0.057–637). Stamp et al. [42] found that MTRR 66A[G
gene polymorphism (p \ 0.0001) influenced RBC folate
concentrations.
Differences in the heterogeneity of results could be due
to differences in demographic features, variations in the
measures of treatment outcome, interethnic variations, and
lack of adequate sample size. Establishing pharmacoge-
netic models [43] could provide better insights into the role
of folate metabolizing gene polymorphisms with MTX
treatment outcome in RA. Measuring the intracellular
folate levels and analyzing other functional genetic variants
in homocysteine metabolism may provide better insights
into the role of genetic variants with MTX treatment
outcome.
The strength of the present study is that this is a
prospective genetic study conducted in South Indian
Tamils with adequate sample size (statistical power of
[80%). However, the limitation of the study is that not all
functionally relevant genetic variants in MTR and MTRR
genes were analyzed.
Conclusion
MTR 2756G allele confers susceptibility to RA in South
Indians. MTR 2756A[G and MTRR 66A[G gene poly-
morphisms had no role with MTX treatment outcome and
adverse events.
Funding The study was supported by an intramural research grant by
JIPMER, Puducherry, India. In addition, the infrastructure and other
support provided by DST-FIST and ICMR-INSERM programs are
also acknowledged.
Author contributions VSN and RG conceived and planned the
study. VSN recruited the patients and collected clinical data. NM
performed laboratory evaluation and molecular analysis on patient
samples. NM and DPM carried out the statistical analysis and wrote
the manuscript. RG critically evaluated and gave useful suggestions to
improve the manuscript. VSN is the lead author and takes primary
responsibility for the data and the study.
Compliance with ethical standards
Conflict of interest All authors declare that there are no conflicts of
interest.
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Clin Exp Med
Ethical approval The study was carried out in compliance with
international, national and institutional regulations. Institute Ethics
committee approved the study.
Informed consent All persons gave informed consent prior to the
inclusion in the study.
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