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Methylene tetrahydrofolate reductase (MTHFR) gene polymorphisms in rheumatoid arthritis patients: Correlation with serum osteopontin levels and disease activity
The Egyptian Rheumatologist (2016) xxx, xxx–xxx
H O S T E D BY
Egyptian Society of Rheumatic Diseases
The Egyptian Rheumatologist www.rheumatology.eg.net www.elsevier.com/locate/ejr
ORIGINAL ARTICLE
Methylene tetrahydrofolate reductase (MTHFR) gene polymorphisms in rheumatoid arthritis patients: Correlation with serum osteopontin levels and disease activity Olfat G. Shaker a, Heba H. El-Demellawy b, Mohamed Nabil Salem b, Nahla Naeem Eesa c,* a
Biochemistry Department, Faculty of Medicine, Cairo University, Egypt Internal Medicine Department, Faculty of Medicine, Beni Sweif University, Egypt c Rheumatology and Rehabilitation Department, Faculty of Medicine, Cairo University, Egypt b
Received 23 February 2016; accepted 7 March 2016
KEYWORDS Rheumatoid arthritis; MTHFR; Osteopontin; Disease activity score
Abstract Background: Rheumatoid arthritis (RA) is a systemic, chronic inflammatory disease with genetic predisposition. Osteopontin (OPN) is overexpressed in RA and plays a key role in the perpetuation of synovitis. Not all RA patients show the same level of response to methotrexate (MTX) suggesting genetic variations in the drug-metabolizing enzymes. Aim of the work: To detect methylene-tetra-hydrofolate reductase (MTHFR) 677C/T and 1298A/C gene polymorphisms in RA patients treated with MTX and to investigate the relationship with serum OPN levels and disease activity. Patients and methods: 62 RA patients and 21 healthy controls were included. Serum OPN was measured using ELISA. Genotyping of MTHFR gene was carried out by polymerase chain reaction-restriction fragment length polymorphism. Disease activity score in 28 joints (DAS28) and the modified health assessment questionnaire (MHAQ) were assessed. Results: The patients’ age was 42.7 ± 12.7 years, F:M (4.6:1) and a disease duration of 5.7 ± 4.6 years. Their DAS28 was 4.1 ± 1.6 and the MHAQ (median 1; range 0–2.3). Serum OPN levels in RA patients (median 8.8; range 4–44.5 ng/ml) were significantly higher than in control (5.6; 2.1–10.9) (p = 0.002). In RA patients, serum OPN significantly correlated with the duration of morning stiffness (p = 0.009), ESR (p < 0.0001) and DAS28 (p < 0.0001). MTHFR (677C>T) polymorphisms significantly correlated with MHAQ (p = 0.012) while (1298A>C) polymorphisms significantly correlated with tender joint count (p = 0.04). OPN levels were higher among patients with MTHFR (1298A/C) AC genotype (8.9; 4.1–33.9 ng/ml), while in those with (677C>T) polymorphisms it was higher among those with CT genotype (8.9; 4.1–44.5).
* Corresponding author at: Rheumatology and Rehabilitation Department, Faculty of Medicine, Cairo University, 11 Ali ben abitaleb st., Haram, Giza, Egypt. Mobile: +20 111 012 1352. E-mail address: [email protected] (N.N. Eesa). Peer review under responsibility of Egyptian Society for Rheumatic Diseases. http://dx.doi.org/10.1016/j.ejr.2016.03.002 1110-1164 Ó 2016 Egyptian Society for Joint Diseases and Arthritis. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Please cite this article in press as: Shaker OG et al. Methylene tetrahydrofolate reductase (MTHFR) gene polymorphisms in rheumatoid arthritis patients: Correlation with serum osteopontin levels and disease activity, The Egyptian Rheumatologist (2016), http://dx.doi.org/10.1016/j.ejr.2016.03.002
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O.G. Shaker et al. Conclusion: Serum OPN level relates with the degree of rheumatoid activity. Ó 2016 Egyptian Society for Joint Diseases and Arthritis. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
1. Introduction
2. Patients and methods
Rheumatoid arthritis (RA) is a chronic progressive inflammatory disease, characterized by synovial inflammation and hyperplasia leading to progressive cartilage and bone destruction [1]. Many factors have been implicated in the pathogenesis of Egyptian RA patients as tumor necrosis factor-a (TNF-a) and IL-6 with their gene polymorphisms [2,3], interleukin-17 (IL-17) and T helper lymphocytes [4], metalloproteinases (MMPs) [5], angiopoitin-2 [6], osteoprotegerin and RANKL [7] as well as CD14++CD16+ monocyte subpopulation [8]. Although the pathogenesis of RA remains unknown and the molecular mechanisms involved are poorly understood, there is evidence suggesting that T cell-mediated inflammation plays an important role [9]. Osteopontin (OPN), also known as early T lymphocyte activation-1, is an extracellular matrix protein with pleiotropic functions, including a proinflammatory function [10]. It has been recognized as a potential biomarker in multiple sclerosis and osteoporosis [11], osteoarthritis [12,13], systemic lupus erythematosus (SLE) [14], psoriasis [15], inflammatory bowel disease (IBD) [16] as well as in RA [17–19]. It is classified as a Th1 cytokine because of its ability to enhance the production of interferon-c (IFN-c) and IL-12 in macrophages [9]. OPN has been involved in joint destruction and arthritis and a direct role in bone re-adsorption beyond its effects on inflammation has been suggested [20]. OPN overexpression in RA synovium was attributable, at least in part, to IL-10 which has been found to correlate with autoantibody production and B cell activation in RA and to contribute to the disease process [21]. OPN also regulates genes related to nuclear factor-jB (NF-jB) transcription pathway and TNF receptor family known to play a critical role in the production of many inflammatory mediators [22,23]. The principal pharmacological effect of methotrexate (MTX) is through folate antagonism. MTX enters the cell through reduced folate carrier-1 and is intracellularly polyglutamated. Polyglutamation enhances the intracellular retention of MTX and promotes inhibition of de novo purine synthesis along with the buildup of adenosine, a potent antiinflammatory agent [24]. MTX inhibits folate pathway enzymes directly as dihydrofolate reductase and indirectly as methylene-tetra-hydrofolate reductase (MTHFR) [25]. Gene polymorphisms related to MTX cell membrane transport and the enzymes involved in their cellular metabolic pathway have been described; with particular note are two MTHFR SNPs 677C/T and 1298A/C [24]. Based on these literature reports, the aim of the present study was to investigate MTHFR gene polymorphisms in RA patients treated with MTX and the possible relationship with plasma OPN levels and disease activity.
Sixty-two RA patients diagnosed according to the 2010 ACR/ EULAR RA classification criteria [26] were consecutively recruited from the outpatient clinic of Internal medicine department, Beni-Sweif University. They were 51 females, 11 males with a mean age of 42.7 ± 12.7 years and disease duration of 5.7 ± 4.6 years. 21 age (52.7 ± 6.5 years) and gender (15 females, 6 males) matched healthy volunteers served as controls. The study was approved by the Local Research Ethics Committee of Beni-Sweif University and conforms to the provisions of the Declaration of Helsinki in 1995. All patients gave their informed consent prior to their inclusion in the study. All patients had complete medical workup including full history taking and complete physical examination. Assessment included number of tender (TJC) and swollen joint count (SJC), disease activity score in 28 joints (DAS-28) [27] and modified health assessment questionnaire (MHAQ) score [28]. Blood samples were collected for estimation of the erythrocyte sedimentation rate (ESR), C-reactive protein (CRP) and complete blood count (CBC). 2.1. Quantitation of serum osteopontin This was done by ELISA kit provided by quantikine, R&D systems, UK. This assay employs the quantitative sandwich enzyme immunoassay technique. A monoclonal antibody specific for OPN has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and any OPN present is bound by the immobilized antibody. After washing away any unbound substances, an enzyme-linked polyclonal antibody specific for OPN is added to the wells. Following a wash to remove any unbound antibody-enzyme reagent, a substrate solution is added to the wells and color develops in proportion to the amount of OPN bound in the initial step. The color development is stopped and its intensity measured. 2.2. Genotyping of the MTHFR 677C>T and 1298A>C Genomic DNA was extracted from peripheral blood using a QIAamp DNA Blood Mini Kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s protocol. Genotyping was performed by polymerase chain reaction-restriction fragment length polymorphism (PCR–RFLP). Genotyping of MTHFR 677C>T: specific primers ‘‘50 -CAT CCC TAT TGG CAG GTT AC-30 ” and ‘‘50 -GAC GGT GCG GTG AGA GTG-30 ” were used for amplification of a fragment of 265 base pairs. PCR products were digested with HinfI enzyme. PCR profile was: initial denaturation at 94 °C for
Please cite this article in press as: Shaker OG et al. Methylene tetrahydrofolate reductase (MTHFR) gene polymorphisms in rheumatoid arthritis patients: Correlation with serum osteopontin levels and disease activity, The Egyptian Rheumatologist (2016), http://dx.doi.org/10.1016/j.ejr.2016.03.002
Methylene tetrahydrofolate reductase (MTHFR) gene polymorphisms in RA patients 3 min followed by 35 cycles of denaturation at 94 °C for 30 s, annealing at 59 °C for 30 s, extension at 72 °C for 30 s, followed by 10 min at 72 °C. In 677 of MTHFR gene, change of C>T, produces a cut site for HinfI enzyme, which produces two fragments of 171 and 94 bp. Then, CC genotype gives single band of 265 bp (uncut), CT genotype by three bands of 265, 171 and 94 bp, and TT genotypes by two bands of 171 and 94 bp. Genotyping of MTHFR 677C>T: a set of forward ‘‘50 -CTT TGG GGA GCT GAA GGA CTA CTA C-3” and reverse ‘‘50 -CAC TTT GTG ACC ATT CCG GTT TG-3” primers were used for amplification of a fragment of 241 base pairs and then the amplified fragment was digested with MboII enzyme. The PCR profile was: initial denaturation at 95 °C for 5 min, denaturation at 94 °C for 30 s, annealing at 51 °C for 30 s, extension at 72 °C for 30 s for 35 cycles and followed at 72 °C for 10 min. In the position 1298 of MTHFR gene, transversion of ‘‘A” to ‘‘C” produces a cut site for MboII enzyme, which cuts the amplicons into two fragments of 211 and 30 bp. Then, AA genotype results in a single band of 241 bp (uncut), AC genotypes produce three bands of 241, 211 and 30 bp, and CC genotype produces two bands of 211 and 30 bp. Digestion of 10 ll PCR products was carried out with 1.5 U HinfI and MboII restriction enzyme in 37 °C for two hours for MTHFR 677CT and 1298AC genotyping, respectively. Then, products were electrophoresed on 3% agarose gel. Gel was stained with ethidium bromide and presence or absence of products was observed by UV transilluminator. 2.3. Statistical analysis Data were collected, tabulated and statistically analyzed using SPSS (statistical program for social science) version 16 windows. Quantitative variables were described as mean, SD, median and range and qualitative variables as n (number) and %. Chi-square test was used to compare qualitative variables and unpaired t-test was used to compare two independent groups as regards quantitative variables. Correlation between parameters was performed using Pearson correlation coefficient. Strength of association between RA and alleles or genotypes of the MTHFR gene polymorphism was estimated using odds ratios (ORs) and 95% confidence intervals (CIs). Levels of significance were determined using contingency tables by chi-square analysis. Strength of association between osteopontin levels in RA and alleles or genotypes of polymorphisms in the MTHFR gene was estimated using the OR and 95% CI. The data were considered significant if p value was 60.05 and highly significant if p < 0.01. 3. Results Sixty-two RA patients with a mean age of 42.7 ± 12.7 years (F:M 4.6:1) and disease duration of 5.7 ± 4.6 years were included as well as 21 age and gender matched control. Demographic features, plasma osteopontin level and MTHFR gene polymorphisms for the patients and control as well as the patients’ clinical characteristics are shown in Table 1. Demographic, clinical and laboratory characteristics of RA patients according to their MTHFR gene polymorphism are presented in Tables 2 and 3. RA patients with MTHFR 1298AC and 677CT genotypes showed higher serum OPN
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Table 1 Demographic features, serum osteopontin and MTHFR genotype distribution in RA patients and control as well as clinical characteristics of the patients. Variable
RA patients (n = 62) Controls (n = 21)
Age (years) Sex F:M Disease duration (years) Morning stiffness (min) TJC SJC DAS28 MHAQ ESR (mm/h) Osteopontin (ng/ml)
42.7 ± 12.7 4.6:1 5.7 ± 4.6 32.5 ± 39.2 4 (0–20) 1 (0–18) 4.1 ± 1.6 1 (0–2.3) 20 (5–125) 8.8 (4–44.5)
52.7 ± 6.5 2.5:1 – – – – – – – 5.6 (2.1–10.9)
MTHFR polymorphism (677C>T) CC CT TT (1298A>C) AA AC CC
26 (41.9) 30 (48.4) 6 (9.7) 26 (41.9) 22 (35.5) 14 (22.6)
15 (71.4) 5 (23.8) 1 (4.8) 5 (23.8) 13 (61.9) 3 (14.3)
RA: rheumatoid arthritis, TJC: tender joint count, SJC: swollen joint count, DAS28: disease activity score in 28 joints, MHAQ: modified health assessment questionnaire, ESR: erythrocyte sedimentation rate, MTHFR: methylene-tetra-hydrofolate reductase. Values are presented as mean ± SD, median (range) or n (%).
levels. However, the difference was not statistically significant. The significance of difference in osteopontin levels among the studied genotypes is shown in Table 4. The significance of difference in the genotypic frequencies between RA patients and controls is presented in Table 5. Serum OPN levels were significantly higher in RA patients (median: 8.8, range 4–44.5 ng/ml) as compared to healthy controls (median: 5.56, range 2.05–10.85 ng/ml) (p = 0.002). In RA patients, serum osteopontin levels showed a significant correlation with the duration of morning stiffness (r = 0.33, p = 0.009), ESR (r = 0.86, p < 0.0001), DAS 28 (r = 0.55, p < 0.0001) and negative correlation with hemoglobin levels (r = 0.47, p < 0.0001). However serum OPN levels showed a non significant correlation with age, disease duration, TJC, SJC, platelet count and MHAQ. There was a significant correlation between MTHFR (677C>T) gene polymorphisms and MHAQ (p = 0.012) while a non-significant correlation was found with the disease duration (p = 0.22); duration of morning stiffness (p = 0.63); TJC (p = 0.44); SJC (p = 0.48); DAS28 (p = 0.14); ESR (p = 0.22) and serum osteopontin levels (p = 0.42). A significant correlation was found between MTHFR (1298A>C) gene polymorphisms and the TJC (p = 0.04). However a non-significant correlation was found with the disease duration (p = 0.3); duration of morning stiffness (p = 0.79); SJC (p = 0.35); DAS28 (p = 0.42); MHAQ (p = 0.77); ESR (p = 0.42) and serum osteopontin levels (p = 0.95). 4. Discussion The MTHFR enzyme is very important in the regeneration of reduced folate. The MTHFR C677T polymorphism results in a thermolabile variant with decreased enzyme activity [29]. A
Please cite this article in press as: Shaker OG et al. Methylene tetrahydrofolate reductase (MTHFR) gene polymorphisms in rheumatoid arthritis patients: Correlation with serum osteopontin levels and disease activity, The Egyptian Rheumatologist (2016), http://dx.doi.org/10.1016/j.ejr.2016.03.002
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O.G. Shaker et al. Table 2 Demographic, clinical and laboratory characteristics of RA patients according to their MTHFR (1298A>C) gene polymorphism. Mean ± SD or median (range)
MTHFR (1298A>C) in RA patients (n = 62)
Age (years) Disease duration (years) Morning stiffness (min) TJC SJC DAS28 MHAQ ESR (mm/h) Osteopontin (ng/ml)
AA (n = 26)
AC (n = 22)
CC (n = 14)
42.2 ± 12.5 5.04 ± 4.4 39.2 ± 48.1 4 (0–20) 1 (0–15) 4.1 ± 1.9 1 (0–2.3) 14 (5–110) 6.3 (4–44.5)
45.7 ± 12.8 5.7 ± 5.2 34.6 ± 38.1 5 (0–20) 1 (0–18) 4.44 ± 1.6 1 (0–1.8) 20 (7–125) 8.9 (4.1–33.9)
38.7 ± 12.6 7 ± 3.8 16.9 ± 8.3 2 (0–5) 0 (0–3) 3.5 ± 0.1 1 (0.1–1.9) 20 (12–9) 8.8 (4.5–32.4)
MTHFR: methylene-tetra-hydrofolate reductase, RA: rheumatoid arthritis, TJC: tender joint count, SJC: swollen joint count, DAS28: disease activity score in 28 joints, MHAQ: modified health assessment questionnaire, ESR: erythrocyte sedimentation rate.
Table 3 Demographic, clinical and laboratory characteristics of RA patients according to their MTHFR (677C>T) gene polymorphism. Mean ± SD or median (range)
MTHFR (677C>T) in RA patients (n = 62)
Age (years) Disease duration (years) Morning stiffness (min) TJC SJC DAS28 MHAQ ESR (mm/h) Osteopontin (ng/ml)
TT (n = 6)
CT (n = 30)
CC (n = 26)
43.7 ± 18.2 8 ± 3.9 16 ± 12.1 4 (0–5) 0 (0–3) 3.7 ± 0.97 1.1 (0.9–1.6) 20 (19–50) 8.8 (5.4–21)
41.5 ± 12.6 5.9 ± 4.8 36 ± 40.6 4 (0–20) 1 (0–18) 4.4 ± 1.5 0.8 (0–1.8) 20 (7–125) 8.9 (4.1–44.5)
43.8 ± 11.9 5.04 ± 4.3 32.3 ± 41.7 2 (0–13) 1 (0–14) 3.7 ± 1.8 1 (0–2.3) 14 (5–81) 6.3 (4–22.4)
MTHFR: methylene-tetra-hydrofolate reductase, RA: rheumatoid arthritis, TJC: tender joint count, SJC: swollen joint count, DAS28: disease activity score in 28 joints, MHAQ: modified health assessment questionnaire, ESR: erythrocyte sedimentation rate.
Table 4 Significance of difference in osteopontin levels among the studied genotypes.
Table 5 Comparison of the genotypic frequencies between RA patients and controls.
MTHFR gene
Genotype
Osteopontin (median)
p
MTHFR alleles
TT/CT TT/CC CT/CC
8.8/8.9 8.8/6.3 8.9/6.3
0.9 0.5 0.2
Frequency (patients:controls)
OR (95% CI)
p
(677C>T)
(677C>T)
18.8/6.3:81.3/93.8
0.24
AA/AC AA/CC AC/CC
6.3/8.9 6.3/8.8 8.9/8.8
0.71 0.95 1
3.4 (0.4– 31.6) 3.4 (1.1– 10.8) 1.11 (0.2– 5.4) 0.36 (0.1– 1.5)
0.89
(1298A>C)
wide range of side effects are associated with this polymorphism as gastrointestinal [30], hepatic toxicity [31], overall adverse events [32] and a less likely response to MTX [24], however, other authors have not found any effects on toxicity and efficacy [33,34]. The A1298C polymorphism also results in decreased MTHFR activity and exhibits other discrepancies in its clinical effects. Some studies of carriers of this allele suggest increased MTX efficacy [31,32], increased susceptibility to RA [33] and increased risk of toxicity [24,35,36] however, another study did not find any effects on efficacy and toxicity [34]. In RA, it is highly appropriate to start directly with adequate therapy to reduce joint damage, radiographic progres-
TT/ CC CT/ CC
(1298A>C) AA/ CC AC/ CC
53.6/25: 46.4/75 65/62.5: 35/37.5 61.1/81.3:38.9/18.8
0.02
0.15
MTHFR: methylene-tetra-hydrofolate reductase. Bold value is significant at p < 0.05.
sion and functional decline. The importance of early and sustained suppression of disease activity in RA has been shown [14,15]. MTX is the most widely used disease-modifying antirheumatic drug (DMARD) for the treatment of RA, and has proven to reduce disease activity and delay or stabilize the development of bone erosions [37]. However, not all patients show the same level of response to MTX. These variations
Please cite this article in press as: Shaker OG et al. Methylene tetrahydrofolate reductase (MTHFR) gene polymorphisms in rheumatoid arthritis patients: Correlation with serum osteopontin levels and disease activity, The Egyptian Rheumatologist (2016), http://dx.doi.org/10.1016/j.ejr.2016.03.002
Methylene tetrahydrofolate reductase (MTHFR) gene polymorphisms in RA patients suggest that RA patients may have genetic variations in drugmetabolizing enzymes. Associations of polymorphisms in the gene coding for MTHFR with MTX efficacy and toxicity have been reported. In this context, pharmacogenetic studies may offer a novel strategy to identify predictors of response to MTX treatment [18,19,38]. Owing to the implication of OPN in amplification and perpetuation of inflammation in the rheumatoid synovium, the aim of the current study was to investigate the possible association between MTHFR gene polymorphisms and serum OPN levels to determine the relationship between polymorphisms in the gene coding for MTHFR with MTX efficacy in RA patients. In the present study, serum OPN levels were significantly higher in RA patients compared to controls (p = 0.002). This was supported by the findings of Bazzichi and colleagues [37] suggesting a peculiar role for this protein in RA pathogenesis. Xu and coworkers [22] revealed that mRNA expression of OPN was highly elevated in CD4+ synovial T cells derived from RA patients which correlated with increased OPN concentrations in synovial fluid. In our study, serum OPN levels showed a significant correlation with the duration of morning stiffness, ESR and DAS 28 in RA patients. This was in agreement with another study [39] which showed a significantly higher OPN levels in RA patients than in healthy controls and were correlated with the CRP levels. Furthermore, Shio and colleagues [40] reported that OPN was significantly higher in RA patients than in healthy controls or OA patients and correlated significantly with CRP and ESR as markers of disease activity. However, Ji and colleagues [41] showed that serum OPN level in RA patients was not significantly correlated with the DAS28 level or responsiveness of RA patients to therapeutic treatments. Serum OPN level in both responders and non-responders after therapy was significantly decreased regardless of responsiveness to therapy. In the current study, serum OPN levels tended to be higher in RA patients with MTHFR 1298AC and 677CT genotypes. This may be explained by the association between MTHFR 1298AC and 677CT genotypes and poor response to MTX. It has been suggested that there are genotype differences in MTX response with increased dosage and longer treatment duration. For instance, patients with MTHFR 1298AA show a higher percentage of good clinical improvement at 6 months when compared with C allele carriers. In contrast, patients with the MTHFR 1298AC genotype do not seem to improve much with increased dosage or with a longer treatment period, whereas for MTHFR 677CT, data suggest that T allele carriers show relatively less clinical improvement upon increased MTX dosage [42]. Dervieux and colleagues [24] reported that the MTHFR C677CT polymorphism results in a decreased enzyme activity and carriers of the MTHFR 677TT genotype are less likely to respond to MTX than those with alternate genotypes. Rego-Perez and coworkers [43] reported that the A1298C polymorphism also results in decreased MTHFR activity and exhibits other discrepancies in its clinical effects. 5. Conclusion The response to therapy is probably partially determined by the genetic makeup of the individual. This study shows that SNPs in MTHFR gene may predict the response to MTX
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treatment. These observations indicate that ascertainment of MTHFR genotype could be useful in establishing the initial MTX dosage to more rapidly reduce disease activity in patients with early RA. Conflict of interest None. References [1] Bazzichi L, Maser J, Piccinni A, Rucci P, Del Debbio A, Vivarelli L, et al. Quality of life in rheumatoid arthritis: impact of disability and lifetime depressive spectrum symptomatology. Clin Exp Rheumatol 2005;23:783–8. [2] Gheita TA, Azkalany GS, Gaber W, Mohey A. Clinical significance of serum TNFa and 308 G/A promoter polymorphism in rheumatoid arthritis. Egypt Rheumatol 2015;37(2):49–54. [3] Gaber W, Azkalany GS, Gheita TA, Mohey A, Sabry R. Clinical significance of serum interleukin-6 and 174 G/C promoter polymorphism in rheumatoid arthritis patients. Egypt Rheumatol 2013;35(2):107–13. [4] Al-Saadany HM, Hussein MS, Gaber RA, Zaytoun HA. Th-17 cells and serum IL-17 in rheumatoid arthritis patients: correlation with disease activity and severity. Egypt Rheumatol 2016;38 (1):1–7. [5] Fadda S, Abolkheir E, Afifi R, Gamal M. Serum matrix metalloproteinase-3 in rheumatoid arthritis patients: correlation with disease activity and joint destruction. Egypt Rheumatol 2016 [in press, Corrected proof, Available online 27 January]. [6] Ghazaly AH, El-Moez KM, El Shorbagy MS, El-Nahrery EM. Angiopoietin-2 as a biomarker for metabolic syndrome and disease activity in rheumatoid arthritis patients. Egypt Rheumatol 2016;38(1):9–13. [7] Ghorbanihaghjo A, Hajialilo M, Shahidi M, Khabazi A, Kolahi S, Nakhjavani MRJ, et al. Osteoprotegerin (OPG) and Matrix Gla protein (MGP) in rheumatoid arthritis patients: relation to disease activity. Egypt Rheumatol 2014;36(3):111–6. [8] Radwan WM, Khalifa KA, Esaily HA, Lashin NA. CD14+ +CD16+ monocyte subset expansion in rheumatoid arthritis patients: relation to disease activity and interleukin-17. Egypt Rheumatol 2016 [in press, Corrected Proof, Available online 12 January]. [9] Ashkar S, Weber GF, Panoutsakopoulou V. Eta-1 (osteopontin): an early component of type-1 (cell-mediated) immunity. Science 2000;287:860–4. [10] O’Regan AW, Nau GJ, Chupp GL, Berman JS, Sanchirico ME, Jansson M, et al. Osteopontin (Eta-1) in cell-mediated immunity: teaching an old dog new tricks. Immunol Today 2000;21:475–8. [11] Altintasß A, Saruhan-Direskeneli G, Benbir G, Demir M, Purisa S. The role of osteopontin: a shared pathway in the pathogenesis of multiple sclerosis and osteoporosis? J Neurol Sci 2009;276:41–4. [12] Mohammed FI, Abd El-Azeem MI, KamalElDin AM. Plasma and synovial fluid osteopontin levels in patients with knee osteoarthritis: relation to radiological grade. Egypt Rheumatol 2012;34(3):131–6. [13] Haider HM, Amin IR, Ahmad KA. Plasma and synovial osteopontin levels, are they associated with disease severity of primary knee osteoarthritis in Egyptian patients? Egypt Rheumatol 2015;37(1):29–34. [14] Xu AP, Bai J, Lu¨ J, Liang YY, Li JG, Lai DY, et al. Osteopontin gene polymorphism in association with systemic lupus erythematosus in Chinese patients. Chin Med J (Engl) 2007;120:2124–8. [15] Buommino E, Tufano MA, Balato N, Canozo N, Donnarumma M, Gallo L, et al. Osteopontin: a new emerging role in psoriasis. Arch Dermatol Res 2009;301:397–404.
Please cite this article in press as: Shaker OG et al. Methylene tetrahydrofolate reductase (MTHFR) gene polymorphisms in rheumatoid arthritis patients: Correlation with serum osteopontin levels and disease activity, The Egyptian Rheumatologist (2016), http://dx.doi.org/10.1016/j.ejr.2016.03.002
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Please cite this article in press as: Shaker OG et al. Methylene tetrahydrofolate reductase (MTHFR) gene polymorphisms in rheumatoid arthritis patients: Correlation with serum osteopontin levels and disease activity, The Egyptian Rheumatologist (2016), http://dx.doi.org/10.1016/j.ejr.2016.03.002
H O S T E D BY
Egyptian Society of Rheumatic Diseases
The Egyptian Rheumatologist www.rheumatology.eg.net www.elsevier.com/locate/ejr
ORIGINAL ARTICLE
Methylene tetrahydrofolate reductase (MTHFR) gene polymorphisms in rheumatoid arthritis patients: Correlation with serum osteopontin levels and disease activity Olfat G. Shaker a, Heba H. El-Demellawy b, Mohamed Nabil Salem b, Nahla Naeem Eesa c,* a
Biochemistry Department, Faculty of Medicine, Cairo University, Egypt Internal Medicine Department, Faculty of Medicine, Beni Sweif University, Egypt c Rheumatology and Rehabilitation Department, Faculty of Medicine, Cairo University, Egypt b
Received 23 February 2016; accepted 7 March 2016
KEYWORDS Rheumatoid arthritis; MTHFR; Osteopontin; Disease activity score
Abstract Background: Rheumatoid arthritis (RA) is a systemic, chronic inflammatory disease with genetic predisposition. Osteopontin (OPN) is overexpressed in RA and plays a key role in the perpetuation of synovitis. Not all RA patients show the same level of response to methotrexate (MTX) suggesting genetic variations in the drug-metabolizing enzymes. Aim of the work: To detect methylene-tetra-hydrofolate reductase (MTHFR) 677C/T and 1298A/C gene polymorphisms in RA patients treated with MTX and to investigate the relationship with serum OPN levels and disease activity. Patients and methods: 62 RA patients and 21 healthy controls were included. Serum OPN was measured using ELISA. Genotyping of MTHFR gene was carried out by polymerase chain reaction-restriction fragment length polymorphism. Disease activity score in 28 joints (DAS28) and the modified health assessment questionnaire (MHAQ) were assessed. Results: The patients’ age was 42.7 ± 12.7 years, F:M (4.6:1) and a disease duration of 5.7 ± 4.6 years. Their DAS28 was 4.1 ± 1.6 and the MHAQ (median 1; range 0–2.3). Serum OPN levels in RA patients (median 8.8; range 4–44.5 ng/ml) were significantly higher than in control (5.6; 2.1–10.9) (p = 0.002). In RA patients, serum OPN significantly correlated with the duration of morning stiffness (p = 0.009), ESR (p < 0.0001) and DAS28 (p < 0.0001). MTHFR (677C>T) polymorphisms significantly correlated with MHAQ (p = 0.012) while (1298A>C) polymorphisms significantly correlated with tender joint count (p = 0.04). OPN levels were higher among patients with MTHFR (1298A/C) AC genotype (8.9; 4.1–33.9 ng/ml), while in those with (677C>T) polymorphisms it was higher among those with CT genotype (8.9; 4.1–44.5).
* Corresponding author at: Rheumatology and Rehabilitation Department, Faculty of Medicine, Cairo University, 11 Ali ben abitaleb st., Haram, Giza, Egypt. Mobile: +20 111 012 1352. E-mail address: [email protected] (N.N. Eesa). Peer review under responsibility of Egyptian Society for Rheumatic Diseases. http://dx.doi.org/10.1016/j.ejr.2016.03.002 1110-1164 Ó 2016 Egyptian Society for Joint Diseases and Arthritis. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Please cite this article in press as: Shaker OG et al. Methylene tetrahydrofolate reductase (MTHFR) gene polymorphisms in rheumatoid arthritis patients: Correlation with serum osteopontin levels and disease activity, The Egyptian Rheumatologist (2016), http://dx.doi.org/10.1016/j.ejr.2016.03.002
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O.G. Shaker et al. Conclusion: Serum OPN level relates with the degree of rheumatoid activity. Ó 2016 Egyptian Society for Joint Diseases and Arthritis. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
1. Introduction
2. Patients and methods
Rheumatoid arthritis (RA) is a chronic progressive inflammatory disease, characterized by synovial inflammation and hyperplasia leading to progressive cartilage and bone destruction [1]. Many factors have been implicated in the pathogenesis of Egyptian RA patients as tumor necrosis factor-a (TNF-a) and IL-6 with their gene polymorphisms [2,3], interleukin-17 (IL-17) and T helper lymphocytes [4], metalloproteinases (MMPs) [5], angiopoitin-2 [6], osteoprotegerin and RANKL [7] as well as CD14++CD16+ monocyte subpopulation [8]. Although the pathogenesis of RA remains unknown and the molecular mechanisms involved are poorly understood, there is evidence suggesting that T cell-mediated inflammation plays an important role [9]. Osteopontin (OPN), also known as early T lymphocyte activation-1, is an extracellular matrix protein with pleiotropic functions, including a proinflammatory function [10]. It has been recognized as a potential biomarker in multiple sclerosis and osteoporosis [11], osteoarthritis [12,13], systemic lupus erythematosus (SLE) [14], psoriasis [15], inflammatory bowel disease (IBD) [16] as well as in RA [17–19]. It is classified as a Th1 cytokine because of its ability to enhance the production of interferon-c (IFN-c) and IL-12 in macrophages [9]. OPN has been involved in joint destruction and arthritis and a direct role in bone re-adsorption beyond its effects on inflammation has been suggested [20]. OPN overexpression in RA synovium was attributable, at least in part, to IL-10 which has been found to correlate with autoantibody production and B cell activation in RA and to contribute to the disease process [21]. OPN also regulates genes related to nuclear factor-jB (NF-jB) transcription pathway and TNF receptor family known to play a critical role in the production of many inflammatory mediators [22,23]. The principal pharmacological effect of methotrexate (MTX) is through folate antagonism. MTX enters the cell through reduced folate carrier-1 and is intracellularly polyglutamated. Polyglutamation enhances the intracellular retention of MTX and promotes inhibition of de novo purine synthesis along with the buildup of adenosine, a potent antiinflammatory agent [24]. MTX inhibits folate pathway enzymes directly as dihydrofolate reductase and indirectly as methylene-tetra-hydrofolate reductase (MTHFR) [25]. Gene polymorphisms related to MTX cell membrane transport and the enzymes involved in their cellular metabolic pathway have been described; with particular note are two MTHFR SNPs 677C/T and 1298A/C [24]. Based on these literature reports, the aim of the present study was to investigate MTHFR gene polymorphisms in RA patients treated with MTX and the possible relationship with plasma OPN levels and disease activity.
Sixty-two RA patients diagnosed according to the 2010 ACR/ EULAR RA classification criteria [26] were consecutively recruited from the outpatient clinic of Internal medicine department, Beni-Sweif University. They were 51 females, 11 males with a mean age of 42.7 ± 12.7 years and disease duration of 5.7 ± 4.6 years. 21 age (52.7 ± 6.5 years) and gender (15 females, 6 males) matched healthy volunteers served as controls. The study was approved by the Local Research Ethics Committee of Beni-Sweif University and conforms to the provisions of the Declaration of Helsinki in 1995. All patients gave their informed consent prior to their inclusion in the study. All patients had complete medical workup including full history taking and complete physical examination. Assessment included number of tender (TJC) and swollen joint count (SJC), disease activity score in 28 joints (DAS-28) [27] and modified health assessment questionnaire (MHAQ) score [28]. Blood samples were collected for estimation of the erythrocyte sedimentation rate (ESR), C-reactive protein (CRP) and complete blood count (CBC). 2.1. Quantitation of serum osteopontin This was done by ELISA kit provided by quantikine, R&D systems, UK. This assay employs the quantitative sandwich enzyme immunoassay technique. A monoclonal antibody specific for OPN has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and any OPN present is bound by the immobilized antibody. After washing away any unbound substances, an enzyme-linked polyclonal antibody specific for OPN is added to the wells. Following a wash to remove any unbound antibody-enzyme reagent, a substrate solution is added to the wells and color develops in proportion to the amount of OPN bound in the initial step. The color development is stopped and its intensity measured. 2.2. Genotyping of the MTHFR 677C>T and 1298A>C Genomic DNA was extracted from peripheral blood using a QIAamp DNA Blood Mini Kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s protocol. Genotyping was performed by polymerase chain reaction-restriction fragment length polymorphism (PCR–RFLP). Genotyping of MTHFR 677C>T: specific primers ‘‘50 -CAT CCC TAT TGG CAG GTT AC-30 ” and ‘‘50 -GAC GGT GCG GTG AGA GTG-30 ” were used for amplification of a fragment of 265 base pairs. PCR products were digested with HinfI enzyme. PCR profile was: initial denaturation at 94 °C for
Please cite this article in press as: Shaker OG et al. Methylene tetrahydrofolate reductase (MTHFR) gene polymorphisms in rheumatoid arthritis patients: Correlation with serum osteopontin levels and disease activity, The Egyptian Rheumatologist (2016), http://dx.doi.org/10.1016/j.ejr.2016.03.002
Methylene tetrahydrofolate reductase (MTHFR) gene polymorphisms in RA patients 3 min followed by 35 cycles of denaturation at 94 °C for 30 s, annealing at 59 °C for 30 s, extension at 72 °C for 30 s, followed by 10 min at 72 °C. In 677 of MTHFR gene, change of C>T, produces a cut site for HinfI enzyme, which produces two fragments of 171 and 94 bp. Then, CC genotype gives single band of 265 bp (uncut), CT genotype by three bands of 265, 171 and 94 bp, and TT genotypes by two bands of 171 and 94 bp. Genotyping of MTHFR 677C>T: a set of forward ‘‘50 -CTT TGG GGA GCT GAA GGA CTA CTA C-3” and reverse ‘‘50 -CAC TTT GTG ACC ATT CCG GTT TG-3” primers were used for amplification of a fragment of 241 base pairs and then the amplified fragment was digested with MboII enzyme. The PCR profile was: initial denaturation at 95 °C for 5 min, denaturation at 94 °C for 30 s, annealing at 51 °C for 30 s, extension at 72 °C for 30 s for 35 cycles and followed at 72 °C for 10 min. In the position 1298 of MTHFR gene, transversion of ‘‘A” to ‘‘C” produces a cut site for MboII enzyme, which cuts the amplicons into two fragments of 211 and 30 bp. Then, AA genotype results in a single band of 241 bp (uncut), AC genotypes produce three bands of 241, 211 and 30 bp, and CC genotype produces two bands of 211 and 30 bp. Digestion of 10 ll PCR products was carried out with 1.5 U HinfI and MboII restriction enzyme in 37 °C for two hours for MTHFR 677CT and 1298AC genotyping, respectively. Then, products were electrophoresed on 3% agarose gel. Gel was stained with ethidium bromide and presence or absence of products was observed by UV transilluminator. 2.3. Statistical analysis Data were collected, tabulated and statistically analyzed using SPSS (statistical program for social science) version 16 windows. Quantitative variables were described as mean, SD, median and range and qualitative variables as n (number) and %. Chi-square test was used to compare qualitative variables and unpaired t-test was used to compare two independent groups as regards quantitative variables. Correlation between parameters was performed using Pearson correlation coefficient. Strength of association between RA and alleles or genotypes of the MTHFR gene polymorphism was estimated using odds ratios (ORs) and 95% confidence intervals (CIs). Levels of significance were determined using contingency tables by chi-square analysis. Strength of association between osteopontin levels in RA and alleles or genotypes of polymorphisms in the MTHFR gene was estimated using the OR and 95% CI. The data were considered significant if p value was 60.05 and highly significant if p < 0.01. 3. Results Sixty-two RA patients with a mean age of 42.7 ± 12.7 years (F:M 4.6:1) and disease duration of 5.7 ± 4.6 years were included as well as 21 age and gender matched control. Demographic features, plasma osteopontin level and MTHFR gene polymorphisms for the patients and control as well as the patients’ clinical characteristics are shown in Table 1. Demographic, clinical and laboratory characteristics of RA patients according to their MTHFR gene polymorphism are presented in Tables 2 and 3. RA patients with MTHFR 1298AC and 677CT genotypes showed higher serum OPN
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Table 1 Demographic features, serum osteopontin and MTHFR genotype distribution in RA patients and control as well as clinical characteristics of the patients. Variable
RA patients (n = 62) Controls (n = 21)
Age (years) Sex F:M Disease duration (years) Morning stiffness (min) TJC SJC DAS28 MHAQ ESR (mm/h) Osteopontin (ng/ml)
42.7 ± 12.7 4.6:1 5.7 ± 4.6 32.5 ± 39.2 4 (0–20) 1 (0–18) 4.1 ± 1.6 1 (0–2.3) 20 (5–125) 8.8 (4–44.5)
52.7 ± 6.5 2.5:1 – – – – – – – 5.6 (2.1–10.9)
MTHFR polymorphism (677C>T) CC CT TT (1298A>C) AA AC CC
26 (41.9) 30 (48.4) 6 (9.7) 26 (41.9) 22 (35.5) 14 (22.6)
15 (71.4) 5 (23.8) 1 (4.8) 5 (23.8) 13 (61.9) 3 (14.3)
RA: rheumatoid arthritis, TJC: tender joint count, SJC: swollen joint count, DAS28: disease activity score in 28 joints, MHAQ: modified health assessment questionnaire, ESR: erythrocyte sedimentation rate, MTHFR: methylene-tetra-hydrofolate reductase. Values are presented as mean ± SD, median (range) or n (%).
levels. However, the difference was not statistically significant. The significance of difference in osteopontin levels among the studied genotypes is shown in Table 4. The significance of difference in the genotypic frequencies between RA patients and controls is presented in Table 5. Serum OPN levels were significantly higher in RA patients (median: 8.8, range 4–44.5 ng/ml) as compared to healthy controls (median: 5.56, range 2.05–10.85 ng/ml) (p = 0.002). In RA patients, serum osteopontin levels showed a significant correlation with the duration of morning stiffness (r = 0.33, p = 0.009), ESR (r = 0.86, p < 0.0001), DAS 28 (r = 0.55, p < 0.0001) and negative correlation with hemoglobin levels (r = 0.47, p < 0.0001). However serum OPN levels showed a non significant correlation with age, disease duration, TJC, SJC, platelet count and MHAQ. There was a significant correlation between MTHFR (677C>T) gene polymorphisms and MHAQ (p = 0.012) while a non-significant correlation was found with the disease duration (p = 0.22); duration of morning stiffness (p = 0.63); TJC (p = 0.44); SJC (p = 0.48); DAS28 (p = 0.14); ESR (p = 0.22) and serum osteopontin levels (p = 0.42). A significant correlation was found between MTHFR (1298A>C) gene polymorphisms and the TJC (p = 0.04). However a non-significant correlation was found with the disease duration (p = 0.3); duration of morning stiffness (p = 0.79); SJC (p = 0.35); DAS28 (p = 0.42); MHAQ (p = 0.77); ESR (p = 0.42) and serum osteopontin levels (p = 0.95). 4. Discussion The MTHFR enzyme is very important in the regeneration of reduced folate. The MTHFR C677T polymorphism results in a thermolabile variant with decreased enzyme activity [29]. A
Please cite this article in press as: Shaker OG et al. Methylene tetrahydrofolate reductase (MTHFR) gene polymorphisms in rheumatoid arthritis patients: Correlation with serum osteopontin levels and disease activity, The Egyptian Rheumatologist (2016), http://dx.doi.org/10.1016/j.ejr.2016.03.002
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O.G. Shaker et al. Table 2 Demographic, clinical and laboratory characteristics of RA patients according to their MTHFR (1298A>C) gene polymorphism. Mean ± SD or median (range)
MTHFR (1298A>C) in RA patients (n = 62)
Age (years) Disease duration (years) Morning stiffness (min) TJC SJC DAS28 MHAQ ESR (mm/h) Osteopontin (ng/ml)
AA (n = 26)
AC (n = 22)
CC (n = 14)
42.2 ± 12.5 5.04 ± 4.4 39.2 ± 48.1 4 (0–20) 1 (0–15) 4.1 ± 1.9 1 (0–2.3) 14 (5–110) 6.3 (4–44.5)
45.7 ± 12.8 5.7 ± 5.2 34.6 ± 38.1 5 (0–20) 1 (0–18) 4.44 ± 1.6 1 (0–1.8) 20 (7–125) 8.9 (4.1–33.9)
38.7 ± 12.6 7 ± 3.8 16.9 ± 8.3 2 (0–5) 0 (0–3) 3.5 ± 0.1 1 (0.1–1.9) 20 (12–9) 8.8 (4.5–32.4)
MTHFR: methylene-tetra-hydrofolate reductase, RA: rheumatoid arthritis, TJC: tender joint count, SJC: swollen joint count, DAS28: disease activity score in 28 joints, MHAQ: modified health assessment questionnaire, ESR: erythrocyte sedimentation rate.
Table 3 Demographic, clinical and laboratory characteristics of RA patients according to their MTHFR (677C>T) gene polymorphism. Mean ± SD or median (range)
MTHFR (677C>T) in RA patients (n = 62)
Age (years) Disease duration (years) Morning stiffness (min) TJC SJC DAS28 MHAQ ESR (mm/h) Osteopontin (ng/ml)
TT (n = 6)
CT (n = 30)
CC (n = 26)
43.7 ± 18.2 8 ± 3.9 16 ± 12.1 4 (0–5) 0 (0–3) 3.7 ± 0.97 1.1 (0.9–1.6) 20 (19–50) 8.8 (5.4–21)
41.5 ± 12.6 5.9 ± 4.8 36 ± 40.6 4 (0–20) 1 (0–18) 4.4 ± 1.5 0.8 (0–1.8) 20 (7–125) 8.9 (4.1–44.5)
43.8 ± 11.9 5.04 ± 4.3 32.3 ± 41.7 2 (0–13) 1 (0–14) 3.7 ± 1.8 1 (0–2.3) 14 (5–81) 6.3 (4–22.4)
MTHFR: methylene-tetra-hydrofolate reductase, RA: rheumatoid arthritis, TJC: tender joint count, SJC: swollen joint count, DAS28: disease activity score in 28 joints, MHAQ: modified health assessment questionnaire, ESR: erythrocyte sedimentation rate.
Table 4 Significance of difference in osteopontin levels among the studied genotypes.
Table 5 Comparison of the genotypic frequencies between RA patients and controls.
MTHFR gene
Genotype
Osteopontin (median)
p
MTHFR alleles
TT/CT TT/CC CT/CC
8.8/8.9 8.8/6.3 8.9/6.3
0.9 0.5 0.2
Frequency (patients:controls)
OR (95% CI)
p
(677C>T)
(677C>T)
18.8/6.3:81.3/93.8
0.24
AA/AC AA/CC AC/CC
6.3/8.9 6.3/8.8 8.9/8.8
0.71 0.95 1
3.4 (0.4– 31.6) 3.4 (1.1– 10.8) 1.11 (0.2– 5.4) 0.36 (0.1– 1.5)
0.89
(1298A>C)
wide range of side effects are associated with this polymorphism as gastrointestinal [30], hepatic toxicity [31], overall adverse events [32] and a less likely response to MTX [24], however, other authors have not found any effects on toxicity and efficacy [33,34]. The A1298C polymorphism also results in decreased MTHFR activity and exhibits other discrepancies in its clinical effects. Some studies of carriers of this allele suggest increased MTX efficacy [31,32], increased susceptibility to RA [33] and increased risk of toxicity [24,35,36] however, another study did not find any effects on efficacy and toxicity [34]. In RA, it is highly appropriate to start directly with adequate therapy to reduce joint damage, radiographic progres-
TT/ CC CT/ CC
(1298A>C) AA/ CC AC/ CC
53.6/25: 46.4/75 65/62.5: 35/37.5 61.1/81.3:38.9/18.8
0.02
0.15
MTHFR: methylene-tetra-hydrofolate reductase. Bold value is significant at p < 0.05.
sion and functional decline. The importance of early and sustained suppression of disease activity in RA has been shown [14,15]. MTX is the most widely used disease-modifying antirheumatic drug (DMARD) for the treatment of RA, and has proven to reduce disease activity and delay or stabilize the development of bone erosions [37]. However, not all patients show the same level of response to MTX. These variations
Please cite this article in press as: Shaker OG et al. Methylene tetrahydrofolate reductase (MTHFR) gene polymorphisms in rheumatoid arthritis patients: Correlation with serum osteopontin levels and disease activity, The Egyptian Rheumatologist (2016), http://dx.doi.org/10.1016/j.ejr.2016.03.002
Methylene tetrahydrofolate reductase (MTHFR) gene polymorphisms in RA patients suggest that RA patients may have genetic variations in drugmetabolizing enzymes. Associations of polymorphisms in the gene coding for MTHFR with MTX efficacy and toxicity have been reported. In this context, pharmacogenetic studies may offer a novel strategy to identify predictors of response to MTX treatment [18,19,38]. Owing to the implication of OPN in amplification and perpetuation of inflammation in the rheumatoid synovium, the aim of the current study was to investigate the possible association between MTHFR gene polymorphisms and serum OPN levels to determine the relationship between polymorphisms in the gene coding for MTHFR with MTX efficacy in RA patients. In the present study, serum OPN levels were significantly higher in RA patients compared to controls (p = 0.002). This was supported by the findings of Bazzichi and colleagues [37] suggesting a peculiar role for this protein in RA pathogenesis. Xu and coworkers [22] revealed that mRNA expression of OPN was highly elevated in CD4+ synovial T cells derived from RA patients which correlated with increased OPN concentrations in synovial fluid. In our study, serum OPN levels showed a significant correlation with the duration of morning stiffness, ESR and DAS 28 in RA patients. This was in agreement with another study [39] which showed a significantly higher OPN levels in RA patients than in healthy controls and were correlated with the CRP levels. Furthermore, Shio and colleagues [40] reported that OPN was significantly higher in RA patients than in healthy controls or OA patients and correlated significantly with CRP and ESR as markers of disease activity. However, Ji and colleagues [41] showed that serum OPN level in RA patients was not significantly correlated with the DAS28 level or responsiveness of RA patients to therapeutic treatments. Serum OPN level in both responders and non-responders after therapy was significantly decreased regardless of responsiveness to therapy. In the current study, serum OPN levels tended to be higher in RA patients with MTHFR 1298AC and 677CT genotypes. This may be explained by the association between MTHFR 1298AC and 677CT genotypes and poor response to MTX. It has been suggested that there are genotype differences in MTX response with increased dosage and longer treatment duration. For instance, patients with MTHFR 1298AA show a higher percentage of good clinical improvement at 6 months when compared with C allele carriers. In contrast, patients with the MTHFR 1298AC genotype do not seem to improve much with increased dosage or with a longer treatment period, whereas for MTHFR 677CT, data suggest that T allele carriers show relatively less clinical improvement upon increased MTX dosage [42]. Dervieux and colleagues [24] reported that the MTHFR C677CT polymorphism results in a decreased enzyme activity and carriers of the MTHFR 677TT genotype are less likely to respond to MTX than those with alternate genotypes. Rego-Perez and coworkers [43] reported that the A1298C polymorphism also results in decreased MTHFR activity and exhibits other discrepancies in its clinical effects. 5. Conclusion The response to therapy is probably partially determined by the genetic makeup of the individual. This study shows that SNPs in MTHFR gene may predict the response to MTX
5
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Please cite this article in press as: Shaker OG et al. Methylene tetrahydrofolate reductase (MTHFR) gene polymorphisms in rheumatoid arthritis patients: Correlation with serum osteopontin levels and disease activity, The Egyptian Rheumatologist (2016), http://dx.doi.org/10.1016/j.ejr.2016.03.002