- Email: [email protected]
C5 and STAT4 genes polymorphisms on susceptibility and severity of rheumatoid arthritis in Egyptian population
Cellular Immunology 273 (2012) 67–72
Contents lists available at SciVerse ScienceDirect
Cellular Immunology journal homepage: www.elsevier.com/locate/ycimm
Influence of TRAF1/C5 and STAT4 genes polymorphisms on susceptibility and severity of rheumatoid arthritis in Egyptian population Rasha H. Mohamed a, Heba F. Pasha b,⇑, Eman E. El-Shahawy c a
Biochemistry Department, Faculty of Pharmacy, Zagazig University, Zagazig, Egypt Medical Biochemistry Department, Faculty of Medicine, Zagazig University, Zagazig, Egypt c Rheumatology and Rehabilitation Department, Faculty of Medicine, Zagazig University, Zagazig, Egypt b
a r t i c l e
i n f o
Article history: Received 20 October 2011 Accepted 23 November 2011 Available online 4 December 2011 Keywords: Rheumatoid arthritis TRAF1/C5 STAT Polymorphism Egyptian population
a b s t r a c t Rheumatoid arthritis (RA) is the most common cause of adult inflammatory arthritis. Recent genomewide association scans have disclosed several single-nucleotide polymorphisms associated with RA susceptibility. The aim of this study was to determine whether the polymorphisms of TRAF1/C5 (tumor necrosis factor (TNF)-receptor associated factor 1)/(complement component 5) and STAT4 (signal transducers and activators of transcription 4) confer susceptibility, activity and severity to RA in Egyptian populations. One hundred and seventy-two RA patients and 160 controls were enrolled in the study. Polymorphisms of TRAF1/C5 and STAT4 genes were determined using restriction fragment length polymorphism–polymerase chain reaction. The TRAF1/C5 A and STAT4 T alleles were significantly associated with RA in Egyptian population. TRAF1/C5 A allele and STAT4 TT genotype were significantly associated with RA severity. In conclusion the mutant alleles or genotypes of both examined polymorphisms are associated with the development of RA in Egyptian population. Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction Rheumatoid arthritis (RA) is one of the most common autoimmune diseases, affecting 1% of the population world wide. Environmental as well as genetic factors are thought to play an important role in both the on set and the progression of the disease [1]. The identification of genes contributing to RA is important for understanding the underlying biologic mechanisms as the genetic contribution to RA has been estimated to be 50–60% [2]. Genetic association of the human leukocyte antigen (HLA) locus and RA has been well established in multiple ethnic groups. However, the HLA region only contributes 30–50% of genetic component for RA [3]. A genome wide association analysis reveals an additional genetic region, TRAF1/C5 containing locus on chromosome 9 [4], and the 2q32 STAT4 gene [5] as a genetic locus associates with RA. The TRAF1/C5 region consists of the TRAF1 (tumor necrosis factor (TNF)-receptor associated factor 1) and C5 (complement component 5), both of which are immune-related genes that may be involved in the onset and/or perpetuation of inflammation. The TRAF1 gene encodes an intracellular protein that mediates signal transduction through TNF receptors1 and 2 and through CD40. TNF is a critical cytokine in the pathogenesis of RA, and TNF antagonists are an effective treatment for RA [6]. ⇑ Corresponding author. Fax: +20 552301523. E-mail address: [email protected] (H.F. Pasha). 0008-8749/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.cellimm.2011.11.005
Many studies in a North American Caucasian and in a Korean population have documented the association of a common STAT4 haplotype with both RA and systemic lupus erythematosus using a combined positional mapping and candidate-gene approach catalyzed by finding a linkage peak on chromosome 2q [7,8]. STAT4 is a transcription factor that is expressed in activated peripheral blood monocytes, dendritic cells and macrophages at sites of inflammation in human beings [9] and lies in the signaling pathway of several important cytokines, including interleukin (IL)12 and type I interferons, as well as IL-23 [10]. STAT4 mediates IL12 signaling that is critical for the development of protective immunity in intracellular infection. The mechanism of STAT4 mediated IL-12 signaling in such protection is dependent on the induction of T helper 1 (Th1) cell differentiation and proliferation [11] and interferon-c production [12]. In addition, it has been reported that STAT4 is necessary for the development of Th17 cells (IL-17producing CD4 (+) T cells) [13]. Since Th1 cells and Th17 cells play an important role in chronic inflammatory disorders and since STAT4 is considered to be a key molecule in both the Th1 and Th17 lineages, STAT4 may play a crucial role in the development of autoimmune diseases such as RA. The allelic frequencies of genes often differ substantially between populations and thus, ethnic-specific association studies are required to confirm genetic associations in different populations. In the present study, we investigated whether the TRAF1/ C5 rs10818488 and STAT4 rs7574865 polymorphisms contribute to RA susceptibility, activity and severity in Egyptian population.
68
R.H. Mohamed et al. / Cellular Immunology 273 (2012) 67–72
2. Subjects and methods
2.3. DNA analysis
2.1. Participants
DNA was isolated and purified from whole blood (EDTA) using QIAamp-spin-columns according to the protocol provided by the manufacturer (QIAamp Blood Kit; Qiagen GmbH, Hilden, Germany).
The study included 172 women with RA. All fulfilled American College of Rheumatology criteria for RA [14]. Their ages ranged from 27 to 65 years of mean ± SD (47.39 ± 9.3), characteristics of the RA patients are presented in Table 1. They were recruited from inpatient and outpatient clinics of Rheumatology & Rehabilitation department of Zagazig University Hospitals. One hundreds of them received low dose of prednisone (15 mg/day); 114 were taking methotrexate, 80 patients were receiving lefluonamide and 40 patients were receiving hydroxychloroquine. Control group was 160 apparently healthy volunteers; their age and ethnic origin were matched with the patients, their ages ranged from 29 to 62 years of mean ± SD (49.35 ± 8.7). An informed written consent was obtained from all participants. All patients were subjected to history taking especially for presenting symptoms, joint affection, together with history of medications. Locomotor examinations were preformed to all patients including tender joint count (TJC) and swollen joint count (SJC). Disease activity was determined on the basis of defined parameters (the number of swollen and tender joints, Disease Activity Score 28-joint assessment (DAS-28) [15], C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR)). X-rays of the hands and feet were obtained in all patients. Disease severity was determined on the basis of defined parameters (Health Assessment Questionnaire (HAQ), seropositivity for rheumatoid factor (RF) and antibodies to cyclic citrullinated peptides (anti-CCP), and X-ray erosion). Patients were deemed to have an erosive arthropathy if one or more definitive erosions were apparent in any of the peripheral joints that have been identified to be predictive of disease progression [16]. Patients were then stratified according to the radiographic findings into 2 groups: those with erosive RA and those with nonerosive RA. 2.2. Biochemical analysis Blood samples were drawn from all subjects after an overnight fast. Sera were separated immediately and stored at 20 °C. CRP was measured using high-sensitivity enzyme-linked immunosorbent assay (ELISA). IgM RF was measured by latex agglutination. RF was considered positive above 20 IU/ml. Anti-CCP was detected in serum samples using enzyme-linked immunosorbent assay test. The assay was performed according to the manufacturer’s instructions. A concentration >25 IU/ml was considered positive.
Table 1 Clinical characteristics of rheumatoid arthritis (RA) patients. RA cases No. of subjects (female) Age (mean ± SD years) Disease duration (mean ± SD years) ESR (mm/h) RF (IU/mL) CRP (lg/ml) Number of swollen joint Number of tender joint RF+ n (%) RF n (%) Anti-CCP+ n (%) Anti-CCP n (%)
172 47.39 ± 9.3 10.65 ± 7.9 47.5 ± 32.9 77.5 ± 79.3 16.17 ± 14.9 5.1 ± 4.3 13.1 ± 7.1 117 (86.0%) 55 (14.0%) 157 (91.3%) 15 (8.7%)
CRP; C-reactive protein, ESR; erythrocyte sedimentation rate, RA; rheumatoid arthritis, RF; rheumatoid factor, anti-CCP; antibodies to cyclic citrullinated peptides.
2.4. Analysis of rs10818488 (TRAF1) polymorphism 226 bp fragment was amplified by polymerase chain reaction (PCR) using upstream primer 50 -GCA GCA GCA GAA CTA CGT GA30 and the downstream primer 50 -GCT TGC TGT TGA AAT CCT GAA GG-30 primers. The PCR reaction mix contained 10 lg genomic DNA, 0.5 lmol/l of each primer (Promega, Madison, WI) and 1X PCR mix (Taq PCR Master Mix Kit, QIAGEN, GmbH, Hilden, Germany) containing (200 mM dNTP, and 1 U Taq polymerase). After the DNA was denatured at 95 °C for 5 min, the reaction mixture was subject to 30 cycles of denaturation at 94 °C for 60 s, annealing at 55 °C for 60 s, and extension at 72 °C for 60 s. This was followed by 7 min of extension at 72 °C. Genotyping for the TRAF1 rs10818488 single nucleotide polymorphism (SNP) was performed by restriction analysis using the SduI restriction enzyme (Ferments). Digestion in the presence of the restriction sites resulted in 2 fragments of 169-bp and 57-bp for (A allele), whereas absence of the restriction site resulted in a fragment of 226 bp (G allele). Both undigested and digested PCR products were visualized in 2.5% agarose gel stained with ethidium bromide [17].
2.5. Analysis of rs7574865 (STAT4) polymorphism The following pair of primers: 50 -AAA GAA GTG GGA TAA AAA GAA GTT TG 30 and 50 -CCA CTG AAA TAA GAT AAC CAC TGT-30 were used to generate a region of 147 bp of the STAT4 gene. The amplification was carried out using 10 lg genomic DNA, 0.5 lmol/l of each primer (Promega, Madison, WI) and 1X PCR mix (Taq PCR Master Mix Kit, QIAGEN, GmbH, Hilden, Germany) containing (200 mM dNTP, and 1 U Taq polymerase). After the DNA was denatured at 95 °C for 5 min, the reaction mixture was subject to 30 cycles of denaturation at 94 °C for 60 s, annealing at 52 °C for 60 s, and extension at 72 °C for 60 s. This was followed by 7 min of extension at 72 °C. Genotyping for the STAT4 rs7574865 PCR product (147-bp) was digested with HpaI (Ferments), the digestion of DNA in the presence of the restriction sites resulted in 2 fragments of 122-bp and 25-bp fragments for (T allele). Both undigested and digested PCR products were visualized in 2.5% agarose gel stained with ethidium bromide [17].
2.6. Statistical analysis The results for continuous variables were expressed as means ± SD. The means of the three-genotype groups were compared in a one-way analysis of variance (ANOVA). The TRAF1 and STAT4 gene variants under investigation were evaluated for deviation from Hardy–Weinberg equilibrium by comparing observed and expected genotype frequencies by means of v2 test or Fisher’s exact test in the control groups. The statistical difference in genotype distribution and allele frequencies in both control and case subjects was assessed by using Fisher’s exact test. Odds ratios (ORs) and confidence intervals (CIs) were calculated. Because one polymorphism was being investigated for each gene, a P value of 60.05 was defined as significant.
69
R.H. Mohamed et al. / Cellular Immunology 273 (2012) 67–72
3. Results 3.1. Clinical characteristics of rheumatoid arthritis (RA) patients The mean ± SD age of the RA patients was 47.39 ± 9.3 years, and all patients were women. The mean ± SD disease duration was 10.65 ± 7.9 years, the mean ± SD ESR was 47.5 ± 32.9, the mean ± SD rheumatoid factor concentration was 77.5 ± 79.3 IU/ mL, and the mean ± SD CRP concentration was 16.17 ± 14.9 lg/ mL. Eighty-six percent of patients were RF positive, and 91.3% were anti-CCP positive, Table 1. 3.2. Association of TRAF1 (rs10818488) and STAT4 (rs7574865) polymorphisms genotypes with RA susceptibility The genotype frequencies of the TRAF1 and STAT4 were in agreement with Hardy–Weinberg equilibrium in all groups. In RA patients, the frequencies of TRAF1 AA genotypes were significantly increased compared to control group (15.7% versus 10.0%). The frequency of the A allele of TRAF1 was significantly increased in RA compared to control (36.3% versus 27.2%). Carriers of the A allele were significantly more likely to develop RA (OR = 1.53, 95% CI = 1.1–2.13, P = 0.012). In RA group, the frequencies of STAT4 GT genotype were significantly increased compared to control group (42.44% versus 27.5%). Moreover, subjects that carried of the T allele were significantly more likely to develop RA (OR = 1.8, 95% CI = 1.25–2.59, P = 0.001) Table 2. 3.3. Relation between gene polymorphism and clinical and laboratory data of RA patients Patients with TRAF1 AA genotype showed significant increase in the levels of number of swollen joint, ESR and DAS28 as compared
to TRAF1 GG genotype. Meanwhile TRAF1 AA genotype showed a significant decrease in HAQ when compared with GG genotype. Also, STAT4 TT genotype showed significant increase in the levels of number of swollen joint as compared to the STAT4 GG genotype. On the contrary none of the other disease activity parameters showed any correlation with TRAF1 AA or GG and STAT4 GG or TT genotypes, Table 3.
3.4. Association of TRAF1 and STAT4 polymorphisms with autoantibody-positive The magnitude of association for these loci was increased in those patients who were autoantibody positive either RF+ or antiCCP+. TRAF1 A allele and/or STAT4 T allele were significantly associated with RF+ and/or anti-CCP+ RA patients when compared with control. (OR = 1.74 CI: 1.21–2.49, P < 0.003, OR = 1.86 CI: 1.33–2.60, P < 0.001, OR = 2.6 CI: 1.01–9.67, P < 0.04 OR = 3.99 CI: 1.67–9.50, P < 0.001, respectively Tables 4 and 5.
3.5. Association of TRAF1 and STAT4 polymorphisms with severity in RA In patients with erosive RA, the frequencies of TRAF1 GA genotype were significantly increased compared to patients with nonerosive RA (49.2% versus 37%). Subjects with A allele were significantly more likely to have erosive arthropathy (OR = 1.69, 95% CI = 1.07–2.66, P = 0.02). Moreover, the frequencies of STAT4 TT genotype were significantly increased compared to patients with non-erosive RA (14.7% versus 4.5%). The odds ratio of an erosive rheumatoid arthritis for TT genotype carriers was 3.3 (95% CI 1.01–10.7; P = 0.04), Table 6.
Table 2 Distribution of TRAF1 and STAT4 genotypes in RA patients and control. RA subjects(n = 172)
Control subjects(n = 160)
OR (95% CI)
P value
TRAF1 GG, No. (%) GA, No. (%) AA, No. (%) A Allele, No. (%)
74(43.0) 71 (41.3) 27 (15.7) 125 (36.3)
89 55 16 87
0.64 (0.4–1.03) 2.03 (1.02–4.05) 1.53 (1.1–2.13)
0.065 0.04 0.012
STAT4 GG, No. (%) GT, No. (%) TT, No. (%) T Allele, No. (%)
85 (49.42) 73 (42.44) 14 (8.14) 101 (29.4)
108 (67.5) 44 (27.5) 8 (5.0) 60 (18.7)
2.1 (1.32–3.37) 0.45 (0.18–1.12) 1.8 (1.25–2.59)
0.002 0.08 0.001
(55.6) (34.4) (10.0) (27.2)
Table 3 Disease activity and severity parameters in relation to TRAF1 and STAT4 genotypes. TRAF1 (rs10818488)
Age Disease duration Disease activity Number of swollen joint Number of tender joint CRP mg/dl ESR mm/h DAS28 Disease severity HAQ Erosion, No. (%) RF+, No. (%) AntiCCP+, No. (%) P < 0.05. a GG vs. AA. b GG vs. TT.
STAT4 (rs7574865)
GG (n = 74)
GA (n = 71)
GG (n = 85)
GT (n = 73)
TT (n = 14)
45.6 ± 9.8 8.5 ± 8.3
47.8 ± 9.6 10.9 ± 7.8
AA (n = 27) 47.3 ± 10.2 12.9 ± 8.4
46.3 ± 11.2 9.3 ± 7.8
46.3 ± 7.9 10.6 ± 9.2
53.4 ± 7 13.4 ± 3.8
3.5 ± 2.3 13.3 ± 6.9 16.7 ± 18.3 39.5 ± 12.8 4.01 ± 1.6
4.7 ± 4.1 13.6 ± 7.5 17.7 ± 14.4 42.6 ± 23.4 4.8 ± 1.6
6.2 ± 4.4a 15.0 ± 6.2 18.5 ± 18.4 50.1 ± 22.3a 4.9 ± 1.3a
4.8 ± 3.1 12.3 ± 6.4 17.1 ± 16 48 ± 24.8 4.5 ± 1.2
4.8 ± 3.3 13.0 ± 5.4 17.3 ± 18.2 49.2 ± 44.6 4.3 ± 1.5
6.4 ± 5.1 16.3 ± 6.7b 20.4 ± 8.1 53.7 ± 30.5 4.6 ± 1.8
1.71 ± 0.82 19 (31.1) 46 (39.3) 60 (38.22)
1.79 ± 0.73 30 (49.2) 50 (42.7) 65 (41.40)
1.005 ± 0.69a 12 (19.7) 21 (17.9) 32 (20.38)
1.72 ± 0.82 30 (49.2) 56 (47.9) 71 (45.22)
1.6 ± 0.71 22 (36.1) 50 (42.7) 65 (41.40)
1.4 ± 1.3 9 (14.8) 11 (9.4) 21 (13.37)
70
R.H. Mohamed et al. / Cellular Immunology 273 (2012) 67–72
Table 4 Genotype frequencies in rheumatoid factor positive rheumatoid arthritis, relative to controls. Control subjects (n = 160)
RF+ subjects (n = 117)
OR (95% CI)
P value
TRAF1 GG, No. (%) GA, No. (%) AA, No. (%) A allele, No. (%)
89 55 16 87
46 50 21 92
(39.32) (42.74) (17.94) (39.3)
1.76 (1.04–2.97) 2.54 (1.21–5.33) 1.74 (1.21–2.49)
0.03 0.012 0.003
STAT4 GG, No. (%) GT, No. (%) TT, No. (%) T allele, No. (%)
108 (67.5) 44 (27.5) 8 (5.0) 60 (29.1)
56 50 11 72
(47.9) (42.7) (9.4) (30.8)
2.2 (1.31–3.68) 2.6 (1.01–9.67) 1.9 (1.29–2.86)
0.003 0.04 0.001
(55.6) (34.4) (10.0) (27.2)
RF+, Rheumatoid factor-positive.
Table 5 Genotype frequencies in anti-CCP positive rheumatoid arthritis, relative to controls. Control subjects(n = 160)
Anti-CCP+ subjects (n = 157)
OR (95% CI)
P value
TRAF1 GG, No. (%) GA, No. (%) AA, No. (%) A allele, No. (%)
89 55 16 87
60 (38.22) 65 (41.40) 32 (20.38) 129 (41.10)
1.75 (1.07–2.85) 2.96 (1.49–5.87) 1.86 (1.33–2.60)
0.02 0.001 0.0001
STAT4 GG, No. (%) GT, No. (%) TT, No. (%) T allele, No. (%)
108 (67.5) 44 (27.5) 8 (5.0) 60 (29.1)
71 (45.22) 65 (41.40) 21 (13.37) 107 (34.08)
2.24 (1.38–3.65) 3.99 (1.67–9.50) 2.24 (1.55–3.22)
0.001 0.001 0.0001
(55.6) (34.4) (10.0) (27.2)
Anti-CCP+, antibodies to cyclic citrullinated peptide -positive.
Table 6 Association of TRAF1 and STAT4 genotypes with RA severity. Non erosive subjects (n = 111)
Erosive subjects (n = 61)
OR (95% CI)
P value
TRAF1 GG, No. (%) GA, No. (%) AA, No. (%) A allele, No. (%)
55 (49.5) 41 (37) 15 (13.5) 71(32)
19 30 12 54
(31.1) (49.2) (19.7) (44.3)
2.12 (1.05–4.28) 0.58 (0.22–1.53) 1.69 (1.07–2.66)
0.03 0.2 0.02
STAT4 GG, No. (%) GT, No. (%) TT, No. (%) T allele, No. (%)
55 (49.5) 51 (46) 5 (4.5) 61 (27.5)
30 (49.2) 22 (36.1) 9 (14.7) 40 (32.8)
0.67 (0.35–1.30) 3.3 (1.01–10.7) 0.77 (0.48–1.25)
0.2 0.04 0.3
4. Discussion
4.1. TRAF1/C5 Region (rs10818488) and RA
Recent large-scale whole-genome and candidate gene association studies have identified a number of SNP markers that reproducibly associate with RA susceptibility, substantially improving our understanding of the genetic component of disease susceptibility. While this is important, it is unlikely to have an immediate clinical impact unless these markers also associate with prognosis or treatment response [18]. Several risk alleles for rheumatoid arthritis have been identified in gene regions containing TRAF1 (C5 locus), STAT4, and OLIG3AIP3 genes [7,19]. These new findings, and data from complementary candidate gene studies [4,20], indicate how series of variations together make up the genetic risk for rheumatoid arthritis, and they show how different patterns of genetic risk factors have emerged for subsets of disease positive and negative for anti-CCP or RF [21]. Interestingly, the frequency of the A allele of the TRAF1/C5 SNP was lower in the Egyptian healthy controls (27.2%) as compared with either the Dutch (40%) [4] or Spanish controls (36%) [22]. We also observed a much lower frequency of the T allele of the STAT4 SNP in the Egyptian healthy individuals (18.7%) as compared with the North American NARAC cohort (22%) [7]. Taken together, these findings indicate that there are distinct population specific differences in the prevalence of these alleles.
The TRAF1 rs10818488 SNP is located within a region encoding a putative binding site for the transcription factor P300. P300 frequently plays important roles in a broad spectrum of biologic processes, including cell proliferation and differentiation [23]. Our results showed significant increase in TRAF1 A allele in RA patients suggesting an association between the presence of the polymorphism and the disease. This confirmed the results of Kurreeman et al. (2007) and Zervou et al. (2008) [4,17] who reported this region as RA associated region in a large-scale genetic association study. It is, therefore, in doing so provide strong evidence for the TRAF1/C5 region as a true RA-associated genetic variant. On the contrary a genome-wide study performed by the Wellcome Trust failed to identify this region as a candidate for RA [24]. RA is a heterogeneous disease with a considerable variation in phenotype as evidenced by the fact that some patients are autoantibody-positive whereas others are not. Anti-CCP has gained much interest as current data suggests that anti-CCP positive and negative RA may have different genetic risk factors [25]. To investigate whether the TRAF1/C5 region is associated with a specific phenotype of RA, we next stratified patients for autoantibody status. Of particular interest is the observation that for TRAF1 loci the associ-
R.H. Mohamed et al. / Cellular Immunology 273 (2012) 67–72
ation was most marked in the autoantibody-positive subgroup carrying of the A allele than normal subject who carried the same allele. Moreover although we also observed an increase in the frequency of the A allele in autoantibody-positive as compared to autoantibody-negative disease, this difference did not reach formal statistical significance. This agree with the results obtained by Kurreeman et al. (2007), who found association of rs10818488 with autoantibody-positive subgroup carrying of the A allele than normal subject who carried the same allele and no significant difference in the frequency of the A allele in autoantibody-positive as compared to autoantibodynegative RA patients. Moreover another study stated that the significant association between TRAF1-C1 and RA was independent of the concentrations of anti-CCP and RF [26]. Because the clinical course of RA can vary considerably ranging from nonerosive disease to rapidly progressive joint damage, we also analyzed whether the SNPs in the TRAF1/C5 region were involved with RA progression. Annual X-rays of the hands and feet of patients were assigned Sharp–van der Heijde units, a combined score for bone erosions and joint space narrowing. Rheumatic patient with A allele had an almost two-fold higher severe disease as compared to the non erosive patients carried the same allele. Moreover, carriers of the minor AA genotype of rs10818488 at this locus showed a significant decrease in joint function (HAQ) than carriers of GG genotype. More interestingly, these data on joint destruction not only indicated that the TRAF1/C5 region predisposes to RA, but also suggested that within the RA population, patients harboring the A allele predisposes not only to RA susceptibility, but also to severity. Carriers of the minor A allele of rs10818488 at this locus were previously shown to have increased radiographic progression, whereas previous study stated that individuals homozygosity for the G allele may be associated with mortality [27].
71
the same allele. This agree with Orozco et al. (2008) and Barton et al. (2008) who found significant association of anti CCP-positive RA patients carrying the T allele than normal subject which carried the same allele [29,34] and disagree with Lee et al. (2007) who found no association [8]. Stratification by the presence of antibody revealed that the association between the STAT4 rs7574865 polymorphism and RA is not dependant of the presence of this antibody (data not shown). This confirmed pervious studies [34–36] that showed no evidence for association between the minor T allele of STAT4 7574865 SNP and antibody positively. These findings supported the mounting evidence that different genetic loci are associated with autoantibody-positive and autoantibody-negative RA. Many of the genes identified to date may therefore predispose to autoimmunity in general, or more specifically the immunological processes involved in the breakdown of self-tolerance and autoantibody production [37]. Several studies have focused on the relationship between the severity of RA and STAT4 polymorphisms. Results obtained in this study suggest that rheumatic patients with TT genotype were significantly more likely to have erosive arthropathy than non erosive patients who carried the same allele. In conclusion TRAF1 and STAT4 are emerging as a novel common risk factor for diverse complex diseases. The current study provided evidence showing the important role of TRAF1 and STAT4 genes polymorphisms in the development of RA. Knowing genetic factors determining disease susceptibility and severity may facilitate personalized medicine. It also remains important to identify the immunological and biochemical pathways that determine outcome, which can then be targeted therapeutically. The TRAF1/C5 and STAT4 locus warrant further investigation as a potential disease susceptibility and severity locus in RA.
4.2. STAT4 (rs7574865) and RA References The rs7574865 SNP examined is located in the third intron of the STAT4 gene, and its actual functional consequence remains to be identified. The polymorphic site was not found to disrupt any transcription factor binding site [17]. Our results showed significant increase in the frequency in STAT4 T allele in RA patients suggesting an association between the presence of the polymorphism and the disease. These results are consistent with the previous studies which observed a significant difference in allele frequency and genotype distribution of the STAT4 polymorphism between RA patients and controls in Japanese and European populations [28,29]. Another study from South American showed that this population has 31% minor T allele frequency of STAT4 and this STAT4 SNP give a similar degree of risk to this population compared with Asian population and population of European origin [30]. The transcription factor encoded by STAT4 is downstream of several cytokines that play a crucial role in the development of Th1 and Th17 responses, such as IL-12, IL-15 and IL-23 [13,31] and the type I interferons [13,32]. Whilst this gene undoubtedly contributes to susceptibility to some autoantibody-associated diseases (RA, systemic lupus erythematosus and type I diabetes), there are recent reports that it may be associated with both clinical forms of inflammatory bowel diseases [33] suggesting that rather than contributing to autoantibody production, it may be a common risk factor for inflammatory disease per se. This is consistent with the apparent association of STAT4 with both autoantibody-positive and autoantibody-negative RA in our study. Of particular interest is the observation that for STAT4 loci the association was most marked in the autoantibody-positive subgroup carrying of the T allele than normal subject which carried
[1] G.S. Firestein, Evolving concepts of rheumatoid arthritis [review], Nature 423 (2003) 356–361. [2] A.H. Van, J.Z. Wesoly, T.W. Huizinga, Understanding the genetic contribution to rheumatoid arthritis, Curr. Opin. Rheumatol. 17 (2005) 299–304. [3] J. Bowes, A. Barton, Recent advances in the genetics of RA susceptibility, Rheumatology (Oxford) 47 (2008) 399–402. [4] F.A. Kurreeman, L. Padyukov, R.B. Marques, S.J. Schrodi, M. Seddighzadeh, G. Stoeken-Rijsbergen, A.H. Helm-van, C.F. Allaart, W. Verduyn, J. HouwingDuistermaat, L. Alfredsson, A.B. Begovich, L. Klareskog, T.W. Huizinga, R.E. Toes, A candidate gene approach identifies the TRAF1/C5 region as a risk factor for rheumatoid arthritis, PLoS Med. 4 (2007) 1515–1524. [5] H.S. Lee, E.F. Remmers, J.M. Le, D.L. Kastner, S.C. Bae, P.K. Gregersen, Association of STAT4 with rheumatoid arthritis in the Korean population, Arthritis Rheum. 58 (2008) 2598–2602. [6] M.E. Weinblatt, J.M. Kremer, A.D. Bankhurst, K.J. Bulpitt, R.M. Fleischmann, R.I. Fox, C.G. Jackson, M. Lange, D.J. Burge, A trial of Etanercept, a recombinant tumor necrosis factor receptor: Fc fusion protein, in patients with rheumatoid arthritis receiving methotrexate, N. Engl. J. Med. 340 (1999) 253–259. [7] E.F. Remmers, R.M. Plenge, A.T. Lee, R.R. Graham, G. Hom, T.W. Behrens, STAT4 and the risk of rheumatoid arthritis and systemic lupus erythematosus, N. Engl. J. Med. 357 (2007) 977–986. [8] H.S. Lee, E.F. Remmers, J.M. Le, D.L. Kastner, S.C. Bae, P.K. Gregersen, Association of STAT4 with rheumatoid arthritis in the Korean population, Mol. Med. 13 (2007) 455–460. [9] D.M. Frucht, M. Aringer, J. Galon, C. Danning, M. Akrad, M. Brown, M. Centola, C.Y. Wu, N. Yamada, T. Cheng, S. Fan, G. Steiner, H. El-Gabalawy, J.J. O’Shea, STAT4 is expressed in activated peripheral blood monocytes, dendritic cells, and macrophages at sites of Th1-mediated inflammation, J. Immunol. 164 (2000) 4659–4664. [10] K.M. Murphy, S.L. Reiner, The lineage decisions of helper T cells, Nat. Rev. Immunol. 2 (2002) 933–944. [11] R. Nishikomori, T. Usui, C.Y. Wu, A. Morinobu, J.J. O’Shea, W. Strober, Activated STAT4 has an essential role in Th1 differentiation and proliferation that is independent of its role in the maintenance of IL-12R beta 2 chain expression and signaling, J. Immunol. 169 (2002) 4388–4398. [12] G. Cai, T. Radzanowski, E.N. Villegas, R. Kastelein, C.A. Hunter, Identification of STAT4-dependent and independent mechanisms of resistance to Toxoplasma gondii, J. Immunol. 165 (2000) 2619–2627.
72
R.H. Mohamed et al. / Cellular Immunology 273 (2012) 67–72
[13] A.N. Mathur, H.C. Chang, D.G. Zisoulis, G.L. Stritesky, Q. Yu, J.T. O’Malley, R. Kapur, D.E. Levy, G.S. Kansas, M.H. Kaplan, Stat3 and Stat4 direct development of IL-17-secreting Th cells, J. Immunol. 178 (2007) 4901–4907. [14] F.C. Arnett, S.M. Edworthy, D.A. Bloch, D.J. McShane, J.F. Fries, N.S. Cooper, L.A. Healey, S.R. Kaplan, M.H. Liang, H.S. Luthra, The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis, Arthritis Rheum. 31 (1988) 315–324. [15] M.L. Prevoo, M.A. van ‘t Hof, H.H. Kuper, M.A. van Leeuwen, L.B. vande Putte, P.L. van Riel, Modified disease activity scores that include twentyeight-joint counts: development and validationin a prospective longitudinal study of patients with rheumatoid arthritis, Arthritis Rheum. 38 (1995) 44– 48. [16] J.T. Sharp, An overview of radiographic analysis of joint damage in rheumatoid arthritis and its use in misanalysis, J. Rheumatol. 27 (2000) 254–260. [17] M.I. Zervou, P. Sidiropoulos, E. Petraki, V. Vazgiourakis, E. Krasoudaki, A. Raptopoulou, H. Kritikos, E. Choustoulaki, D.T. Boumpas, G.N. Goulielmos, Association of a TRAF1 and a STAT4 gene polymorphism with increased risk for rheumatoid arthritis in a genetically homogeneous population, Hum. Immunol. 69 (2008) 567–571. [18] D. Plant, W. Thomson, M. Lunt, E. Flynn, P. Martin, S. Eyre, T. Farragher, D. Bunn, J. Worthington, D. Symmons, A. Barton, The role of rheumatoid arthritis genetic susceptibility markers in the prediction of erosive disease in patients with early inflammatory polyarthritis: results from the Norfolk Arthritis Register, Rheumatology 50 (2011) 78–84. [19] R.M. Plenge, C. Cotsapas, L. Davies, A.L. Price, P.I. de Bakker, J. Maller, I. Pe’er, N.P. Burtt, B. Blumenstiel, M. DeFelice, M. Parkin, R. Barry, W. Winslow, C. Healy, R.R. Graham, B.M. Neale, E. Izmailova, R. Roubenoff, A.N. Parker, R. Glass, E.W. Karlson, N. Maher, D.A. Hafler, D.M. Lee, M.F. Seldin, E.F. Remmers, A.T. Lee, L. Padyukov, L. Alfredsson, J. Coblyn, M.E. Weinblatt, S.B. Gabriel, S. Purcell, L. Klareskog, P.K. Gregersen, N.A. Shadick, M.J. Daly, D. Altshuler, Two independent alleles at 6q23 associated with risk of rheumatoid arthritis, Nat. Genet. 39 (2007) 1477–1482. [20] S. Sigurdsson, L. Padyukov, F.A. Kurreeman, U. Liljedahl, A.C. Wiman, L. Alfredsson, R. Toes, J. Rönnelid, L. Klareskog, T.W. Huizinga, G. Alm, A.C. Syvänen, L. Rönnblom, Association of a haplotype in the promoter region of the interferon regulatory factor 5 gene with rheumatoid arthritis, Arthritis Rheum. 56 (2007) 2202–2210. [21] A.H. Van der Helm-van Mil, T.W. Huizinga, Advances in the genetics of rheumatoid arthritis point to sub classification into distinct disease subsets, Arthritis Res. Ther. 10 (2008) 205. [22] F.A.S. Kurreeman, D. Rocha, J. Houwing-Duistermaat, S. Vrijmoet, V.H. Teixeira, P. Migliorini, A. Balsa, R. Westhovens, P. Barrera, H. Alves, C. Vaz, M. Fernandes, D. Pascual-Salcedo, L. Michou, S. Bombardieri, T. Radstake, P. Van Riel, L. van de Putte, A. Lopes-Vaz, B. Prum, T. Bardin, I. Gut, F. Cornelis, T.W.J. Huizinga, E. Petit-Teixeira, R.E.M. Toes, Replication of the tumor necrosis factor receptor_associated factor 1/complement component 5 region as a susceptibility locus for rheumatoid arthritis in a European family-based study, Arthritis Rheum. 58 (2008) 2670–2674. [23] D. Chakravarti, V.J. La-Morte, M.C. Nelson, T. Nakajima, I.G. Schulman, H. Juguilon, M. Montminy, R.M. Evans, Role of CBP/P300 in nuclear receptor signaling, Nature 383 (1996) 99–108. [24] The Wellcome Trust Case Control Consortium, Genome-wide association study of 14,000 cases of seven common diseases and 3000 shared controls, Nature 447 (2007) 661–678.
[25] F. Van Gaalen, A. Ioan-Facsinay, T.W. Huizinga, R.E. Toes, The devil in the details: the emerging role of anticitrulline autoimmunity in rheumatoid arthritis, J. Immunol. 175 (2005) 5575–5580. [26] T.U. Han, S.Y. Bang, C. Kang, S.C. Bae, TRAF1 polymorphisms associated with rheumatoid arthritis susceptibility in Asians and in Caucasians, Arthritis Rheum. 175 (2009) 2577–2584. [27] V.F. Panoulas, J.P. Smith, P. Nightingale, G.D. Kitas, Association of the TRAF1/C5 locus with increased mortality, particularly from malignancy or sepsis, in patients with rheumatoid arthritis, Arthritis Rheum. 60 (2009) 39–46. [28] S. Kobayashi, K. Ikari, H. Kaneko, Y. Kochi, K. Yamamoto, K. Shimane, Y. Nakamura, Y. Toyama, T. Mochizuki, S. Tsukahara, Y. Kawaguchi, C. Terai, M. Hara, T. Tomatsu, H. Yamanaka, T. Horiuchi, K. Tao, K. Yasutomo, D. Hamada, N. Yasui, H. Inoue, M. Itakura, H. Okamoto, N. Kamatani, S. Momohara, Association of STAT4 with susceptibility to rheumatoid arthritis and systemic lupus erythematosus in the Japanese population, Arthritis Rheum. 58 (2008) 1940–1946. [29] G. Orozco, B.Z. Alizadeh, A.M. Delgado-Vega, M.A. González-Gay, A. Balsa, D. Pascual-Salcedo, B. Fernández-Gutierrez, M.F. González-Escribano, I.F. Petersson, P.L. van Riel, P. Barrera, M.J. Coenen, T.R. Radstake, M.A. van Leeuwen, C. Wijmenga, B.P. Koeleman, M. Alarcón-Riquelme, J. Martín, Association of STAT4 with rheumatoid arthritis: a replication study in three European populations, Arthritis Rheum. 58 (2008) 1974–1980. [30] R.J. Palomino-Morales, A. Rojas-Villarraga, C.I. Gonzalez, G. Ramirez, J.M. Anaya, J. Martin, STAT4 but not TRAF1/C5 variants influence the risk of developing rheumatoid arthritis and systemic lupus erythematosus in Colombians, Genes Immun. 9 (2008) 379–382. [31] M. Kaplan, STAT4: a critical regulator of inflammation in vivo, Immunol. Res. 31 (2005) 231–242. [32] S.N. Kariuki, K.A. Kirou, E.J. MacDermott, L. Barillas-Arias, M.K. Crow, T.B. Niewold, Cutting edge: autoimmune disease risk variant of STAT4 confers increased sensitivity to IFN-a in lupus patients in vivo, J. Immunol. 182 (2009) 34–38. [33] A. Martnez, J. Varadé, A. Márquez, M.C. Cénit, L. Espino, N. Perdigones, J.L. Santiago, M. Ferndndez-Arquero, H. de la Calle, R. Arroyo, J.L. Mendoza, B. Fernández-Gutiérrez, E.G. de la Concha, Association of the STAT4 gene with increased susceptibility for some immune-mediated diseases, Arthritis Rheum. 58 (2008) 2598–2602. [34] A. Barton, W. Thomson, X. Ke, S. Eyre, A. Hinks, J. Bowes, L.J. Gibbons, D. Plant, Wellcome Trust Case Control Consortium, A. Wilson, I. Marinou, A. Morgan, P. Emery, S. Steer, L. Hocking, D. Reid, P. Wordsworth, P. Harrison, J. Worthington, Re-evaluation of putative rheumatoid arthritis susceptibility genes in the postgenome wide association study era and hypothesis of a key pathway underlying susceptibility, Hum. Mol. Genet. 17 (2008) 2274–2279. [35] J.D. Ji, W.J. Lee, K.A. Kong, J.H. Woo, S.J. Choi, Y.H. Lee, G.G. Song, Association of STAT4 polymorphism with rheumatoid arthritis and systemic lupus erythematosus: a meta-analysis, Mol. Biol. Rep. 37 (2010) 141–147. [36] Y.H. Lee, J.H. Woo, S.J. Choi, J.D. Ji, G. Gyu, Association between the rs7574865 polymorphism of STAT4 and rheumatoid arthritis: a meta-analysis, Rheumatol. Int. 30 (2010) 661–666. [37] A.W. Morgan, J.I. Robinson, P.G. Conaghan, S.G. Martin, E.A. Hensor, M.D. Morgan, L. Steiner, H.A. Erlich, H.C. Gooi, A. Barton, J. Worthington, P. Emery, Evaluation of the rheumatoid arthritis susceptibility loci HLA-DRB1, PTPN22, OLIG3/TNFAIP3, STAT4 and TRAF1/C5 in an inception cohort, Arthritis Res. Ther. 12 (2010) 57–62.
Contents lists available at SciVerse ScienceDirect
Cellular Immunology journal homepage: www.elsevier.com/locate/ycimm
Influence of TRAF1/C5 and STAT4 genes polymorphisms on susceptibility and severity of rheumatoid arthritis in Egyptian population Rasha H. Mohamed a, Heba F. Pasha b,⇑, Eman E. El-Shahawy c a
Biochemistry Department, Faculty of Pharmacy, Zagazig University, Zagazig, Egypt Medical Biochemistry Department, Faculty of Medicine, Zagazig University, Zagazig, Egypt c Rheumatology and Rehabilitation Department, Faculty of Medicine, Zagazig University, Zagazig, Egypt b
a r t i c l e
i n f o
Article history: Received 20 October 2011 Accepted 23 November 2011 Available online 4 December 2011 Keywords: Rheumatoid arthritis TRAF1/C5 STAT Polymorphism Egyptian population
a b s t r a c t Rheumatoid arthritis (RA) is the most common cause of adult inflammatory arthritis. Recent genomewide association scans have disclosed several single-nucleotide polymorphisms associated with RA susceptibility. The aim of this study was to determine whether the polymorphisms of TRAF1/C5 (tumor necrosis factor (TNF)-receptor associated factor 1)/(complement component 5) and STAT4 (signal transducers and activators of transcription 4) confer susceptibility, activity and severity to RA in Egyptian populations. One hundred and seventy-two RA patients and 160 controls were enrolled in the study. Polymorphisms of TRAF1/C5 and STAT4 genes were determined using restriction fragment length polymorphism–polymerase chain reaction. The TRAF1/C5 A and STAT4 T alleles were significantly associated with RA in Egyptian population. TRAF1/C5 A allele and STAT4 TT genotype were significantly associated with RA severity. In conclusion the mutant alleles or genotypes of both examined polymorphisms are associated with the development of RA in Egyptian population. Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction Rheumatoid arthritis (RA) is one of the most common autoimmune diseases, affecting 1% of the population world wide. Environmental as well as genetic factors are thought to play an important role in both the on set and the progression of the disease [1]. The identification of genes contributing to RA is important for understanding the underlying biologic mechanisms as the genetic contribution to RA has been estimated to be 50–60% [2]. Genetic association of the human leukocyte antigen (HLA) locus and RA has been well established in multiple ethnic groups. However, the HLA region only contributes 30–50% of genetic component for RA [3]. A genome wide association analysis reveals an additional genetic region, TRAF1/C5 containing locus on chromosome 9 [4], and the 2q32 STAT4 gene [5] as a genetic locus associates with RA. The TRAF1/C5 region consists of the TRAF1 (tumor necrosis factor (TNF)-receptor associated factor 1) and C5 (complement component 5), both of which are immune-related genes that may be involved in the onset and/or perpetuation of inflammation. The TRAF1 gene encodes an intracellular protein that mediates signal transduction through TNF receptors1 and 2 and through CD40. TNF is a critical cytokine in the pathogenesis of RA, and TNF antagonists are an effective treatment for RA [6]. ⇑ Corresponding author. Fax: +20 552301523. E-mail address: [email protected] (H.F. Pasha). 0008-8749/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.cellimm.2011.11.005
Many studies in a North American Caucasian and in a Korean population have documented the association of a common STAT4 haplotype with both RA and systemic lupus erythematosus using a combined positional mapping and candidate-gene approach catalyzed by finding a linkage peak on chromosome 2q [7,8]. STAT4 is a transcription factor that is expressed in activated peripheral blood monocytes, dendritic cells and macrophages at sites of inflammation in human beings [9] and lies in the signaling pathway of several important cytokines, including interleukin (IL)12 and type I interferons, as well as IL-23 [10]. STAT4 mediates IL12 signaling that is critical for the development of protective immunity in intracellular infection. The mechanism of STAT4 mediated IL-12 signaling in such protection is dependent on the induction of T helper 1 (Th1) cell differentiation and proliferation [11] and interferon-c production [12]. In addition, it has been reported that STAT4 is necessary for the development of Th17 cells (IL-17producing CD4 (+) T cells) [13]. Since Th1 cells and Th17 cells play an important role in chronic inflammatory disorders and since STAT4 is considered to be a key molecule in both the Th1 and Th17 lineages, STAT4 may play a crucial role in the development of autoimmune diseases such as RA. The allelic frequencies of genes often differ substantially between populations and thus, ethnic-specific association studies are required to confirm genetic associations in different populations. In the present study, we investigated whether the TRAF1/ C5 rs10818488 and STAT4 rs7574865 polymorphisms contribute to RA susceptibility, activity and severity in Egyptian population.
68
R.H. Mohamed et al. / Cellular Immunology 273 (2012) 67–72
2. Subjects and methods
2.3. DNA analysis
2.1. Participants
DNA was isolated and purified from whole blood (EDTA) using QIAamp-spin-columns according to the protocol provided by the manufacturer (QIAamp Blood Kit; Qiagen GmbH, Hilden, Germany).
The study included 172 women with RA. All fulfilled American College of Rheumatology criteria for RA [14]. Their ages ranged from 27 to 65 years of mean ± SD (47.39 ± 9.3), characteristics of the RA patients are presented in Table 1. They were recruited from inpatient and outpatient clinics of Rheumatology & Rehabilitation department of Zagazig University Hospitals. One hundreds of them received low dose of prednisone (15 mg/day); 114 were taking methotrexate, 80 patients were receiving lefluonamide and 40 patients were receiving hydroxychloroquine. Control group was 160 apparently healthy volunteers; their age and ethnic origin were matched with the patients, their ages ranged from 29 to 62 years of mean ± SD (49.35 ± 8.7). An informed written consent was obtained from all participants. All patients were subjected to history taking especially for presenting symptoms, joint affection, together with history of medications. Locomotor examinations were preformed to all patients including tender joint count (TJC) and swollen joint count (SJC). Disease activity was determined on the basis of defined parameters (the number of swollen and tender joints, Disease Activity Score 28-joint assessment (DAS-28) [15], C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR)). X-rays of the hands and feet were obtained in all patients. Disease severity was determined on the basis of defined parameters (Health Assessment Questionnaire (HAQ), seropositivity for rheumatoid factor (RF) and antibodies to cyclic citrullinated peptides (anti-CCP), and X-ray erosion). Patients were deemed to have an erosive arthropathy if one or more definitive erosions were apparent in any of the peripheral joints that have been identified to be predictive of disease progression [16]. Patients were then stratified according to the radiographic findings into 2 groups: those with erosive RA and those with nonerosive RA. 2.2. Biochemical analysis Blood samples were drawn from all subjects after an overnight fast. Sera were separated immediately and stored at 20 °C. CRP was measured using high-sensitivity enzyme-linked immunosorbent assay (ELISA). IgM RF was measured by latex agglutination. RF was considered positive above 20 IU/ml. Anti-CCP was detected in serum samples using enzyme-linked immunosorbent assay test. The assay was performed according to the manufacturer’s instructions. A concentration >25 IU/ml was considered positive.
Table 1 Clinical characteristics of rheumatoid arthritis (RA) patients. RA cases No. of subjects (female) Age (mean ± SD years) Disease duration (mean ± SD years) ESR (mm/h) RF (IU/mL) CRP (lg/ml) Number of swollen joint Number of tender joint RF+ n (%) RF n (%) Anti-CCP+ n (%) Anti-CCP n (%)
172 47.39 ± 9.3 10.65 ± 7.9 47.5 ± 32.9 77.5 ± 79.3 16.17 ± 14.9 5.1 ± 4.3 13.1 ± 7.1 117 (86.0%) 55 (14.0%) 157 (91.3%) 15 (8.7%)
CRP; C-reactive protein, ESR; erythrocyte sedimentation rate, RA; rheumatoid arthritis, RF; rheumatoid factor, anti-CCP; antibodies to cyclic citrullinated peptides.
2.4. Analysis of rs10818488 (TRAF1) polymorphism 226 bp fragment was amplified by polymerase chain reaction (PCR) using upstream primer 50 -GCA GCA GCA GAA CTA CGT GA30 and the downstream primer 50 -GCT TGC TGT TGA AAT CCT GAA GG-30 primers. The PCR reaction mix contained 10 lg genomic DNA, 0.5 lmol/l of each primer (Promega, Madison, WI) and 1X PCR mix (Taq PCR Master Mix Kit, QIAGEN, GmbH, Hilden, Germany) containing (200 mM dNTP, and 1 U Taq polymerase). After the DNA was denatured at 95 °C for 5 min, the reaction mixture was subject to 30 cycles of denaturation at 94 °C for 60 s, annealing at 55 °C for 60 s, and extension at 72 °C for 60 s. This was followed by 7 min of extension at 72 °C. Genotyping for the TRAF1 rs10818488 single nucleotide polymorphism (SNP) was performed by restriction analysis using the SduI restriction enzyme (Ferments). Digestion in the presence of the restriction sites resulted in 2 fragments of 169-bp and 57-bp for (A allele), whereas absence of the restriction site resulted in a fragment of 226 bp (G allele). Both undigested and digested PCR products were visualized in 2.5% agarose gel stained with ethidium bromide [17].
2.5. Analysis of rs7574865 (STAT4) polymorphism The following pair of primers: 50 -AAA GAA GTG GGA TAA AAA GAA GTT TG 30 and 50 -CCA CTG AAA TAA GAT AAC CAC TGT-30 were used to generate a region of 147 bp of the STAT4 gene. The amplification was carried out using 10 lg genomic DNA, 0.5 lmol/l of each primer (Promega, Madison, WI) and 1X PCR mix (Taq PCR Master Mix Kit, QIAGEN, GmbH, Hilden, Germany) containing (200 mM dNTP, and 1 U Taq polymerase). After the DNA was denatured at 95 °C for 5 min, the reaction mixture was subject to 30 cycles of denaturation at 94 °C for 60 s, annealing at 52 °C for 60 s, and extension at 72 °C for 60 s. This was followed by 7 min of extension at 72 °C. Genotyping for the STAT4 rs7574865 PCR product (147-bp) was digested with HpaI (Ferments), the digestion of DNA in the presence of the restriction sites resulted in 2 fragments of 122-bp and 25-bp fragments for (T allele). Both undigested and digested PCR products were visualized in 2.5% agarose gel stained with ethidium bromide [17].
2.6. Statistical analysis The results for continuous variables were expressed as means ± SD. The means of the three-genotype groups were compared in a one-way analysis of variance (ANOVA). The TRAF1 and STAT4 gene variants under investigation were evaluated for deviation from Hardy–Weinberg equilibrium by comparing observed and expected genotype frequencies by means of v2 test or Fisher’s exact test in the control groups. The statistical difference in genotype distribution and allele frequencies in both control and case subjects was assessed by using Fisher’s exact test. Odds ratios (ORs) and confidence intervals (CIs) were calculated. Because one polymorphism was being investigated for each gene, a P value of 60.05 was defined as significant.
69
R.H. Mohamed et al. / Cellular Immunology 273 (2012) 67–72
3. Results 3.1. Clinical characteristics of rheumatoid arthritis (RA) patients The mean ± SD age of the RA patients was 47.39 ± 9.3 years, and all patients were women. The mean ± SD disease duration was 10.65 ± 7.9 years, the mean ± SD ESR was 47.5 ± 32.9, the mean ± SD rheumatoid factor concentration was 77.5 ± 79.3 IU/ mL, and the mean ± SD CRP concentration was 16.17 ± 14.9 lg/ mL. Eighty-six percent of patients were RF positive, and 91.3% were anti-CCP positive, Table 1. 3.2. Association of TRAF1 (rs10818488) and STAT4 (rs7574865) polymorphisms genotypes with RA susceptibility The genotype frequencies of the TRAF1 and STAT4 were in agreement with Hardy–Weinberg equilibrium in all groups. In RA patients, the frequencies of TRAF1 AA genotypes were significantly increased compared to control group (15.7% versus 10.0%). The frequency of the A allele of TRAF1 was significantly increased in RA compared to control (36.3% versus 27.2%). Carriers of the A allele were significantly more likely to develop RA (OR = 1.53, 95% CI = 1.1–2.13, P = 0.012). In RA group, the frequencies of STAT4 GT genotype were significantly increased compared to control group (42.44% versus 27.5%). Moreover, subjects that carried of the T allele were significantly more likely to develop RA (OR = 1.8, 95% CI = 1.25–2.59, P = 0.001) Table 2. 3.3. Relation between gene polymorphism and clinical and laboratory data of RA patients Patients with TRAF1 AA genotype showed significant increase in the levels of number of swollen joint, ESR and DAS28 as compared
to TRAF1 GG genotype. Meanwhile TRAF1 AA genotype showed a significant decrease in HAQ when compared with GG genotype. Also, STAT4 TT genotype showed significant increase in the levels of number of swollen joint as compared to the STAT4 GG genotype. On the contrary none of the other disease activity parameters showed any correlation with TRAF1 AA or GG and STAT4 GG or TT genotypes, Table 3.
3.4. Association of TRAF1 and STAT4 polymorphisms with autoantibody-positive The magnitude of association for these loci was increased in those patients who were autoantibody positive either RF+ or antiCCP+. TRAF1 A allele and/or STAT4 T allele were significantly associated with RF+ and/or anti-CCP+ RA patients when compared with control. (OR = 1.74 CI: 1.21–2.49, P < 0.003, OR = 1.86 CI: 1.33–2.60, P < 0.001, OR = 2.6 CI: 1.01–9.67, P < 0.04 OR = 3.99 CI: 1.67–9.50, P < 0.001, respectively Tables 4 and 5.
3.5. Association of TRAF1 and STAT4 polymorphisms with severity in RA In patients with erosive RA, the frequencies of TRAF1 GA genotype were significantly increased compared to patients with nonerosive RA (49.2% versus 37%). Subjects with A allele were significantly more likely to have erosive arthropathy (OR = 1.69, 95% CI = 1.07–2.66, P = 0.02). Moreover, the frequencies of STAT4 TT genotype were significantly increased compared to patients with non-erosive RA (14.7% versus 4.5%). The odds ratio of an erosive rheumatoid arthritis for TT genotype carriers was 3.3 (95% CI 1.01–10.7; P = 0.04), Table 6.
Table 2 Distribution of TRAF1 and STAT4 genotypes in RA patients and control. RA subjects(n = 172)
Control subjects(n = 160)
OR (95% CI)
P value
TRAF1 GG, No. (%) GA, No. (%) AA, No. (%) A Allele, No. (%)
74(43.0) 71 (41.3) 27 (15.7) 125 (36.3)
89 55 16 87
0.64 (0.4–1.03) 2.03 (1.02–4.05) 1.53 (1.1–2.13)
0.065 0.04 0.012
STAT4 GG, No. (%) GT, No. (%) TT, No. (%) T Allele, No. (%)
85 (49.42) 73 (42.44) 14 (8.14) 101 (29.4)
108 (67.5) 44 (27.5) 8 (5.0) 60 (18.7)
2.1 (1.32–3.37) 0.45 (0.18–1.12) 1.8 (1.25–2.59)
0.002 0.08 0.001
(55.6) (34.4) (10.0) (27.2)
Table 3 Disease activity and severity parameters in relation to TRAF1 and STAT4 genotypes. TRAF1 (rs10818488)
Age Disease duration Disease activity Number of swollen joint Number of tender joint CRP mg/dl ESR mm/h DAS28 Disease severity HAQ Erosion, No. (%) RF+, No. (%) AntiCCP+, No. (%) P < 0.05. a GG vs. AA. b GG vs. TT.
STAT4 (rs7574865)
GG (n = 74)
GA (n = 71)
GG (n = 85)
GT (n = 73)
TT (n = 14)
45.6 ± 9.8 8.5 ± 8.3
47.8 ± 9.6 10.9 ± 7.8
AA (n = 27) 47.3 ± 10.2 12.9 ± 8.4
46.3 ± 11.2 9.3 ± 7.8
46.3 ± 7.9 10.6 ± 9.2
53.4 ± 7 13.4 ± 3.8
3.5 ± 2.3 13.3 ± 6.9 16.7 ± 18.3 39.5 ± 12.8 4.01 ± 1.6
4.7 ± 4.1 13.6 ± 7.5 17.7 ± 14.4 42.6 ± 23.4 4.8 ± 1.6
6.2 ± 4.4a 15.0 ± 6.2 18.5 ± 18.4 50.1 ± 22.3a 4.9 ± 1.3a
4.8 ± 3.1 12.3 ± 6.4 17.1 ± 16 48 ± 24.8 4.5 ± 1.2
4.8 ± 3.3 13.0 ± 5.4 17.3 ± 18.2 49.2 ± 44.6 4.3 ± 1.5
6.4 ± 5.1 16.3 ± 6.7b 20.4 ± 8.1 53.7 ± 30.5 4.6 ± 1.8
1.71 ± 0.82 19 (31.1) 46 (39.3) 60 (38.22)
1.79 ± 0.73 30 (49.2) 50 (42.7) 65 (41.40)
1.005 ± 0.69a 12 (19.7) 21 (17.9) 32 (20.38)
1.72 ± 0.82 30 (49.2) 56 (47.9) 71 (45.22)
1.6 ± 0.71 22 (36.1) 50 (42.7) 65 (41.40)
1.4 ± 1.3 9 (14.8) 11 (9.4) 21 (13.37)
70
R.H. Mohamed et al. / Cellular Immunology 273 (2012) 67–72
Table 4 Genotype frequencies in rheumatoid factor positive rheumatoid arthritis, relative to controls. Control subjects (n = 160)
RF+ subjects (n = 117)
OR (95% CI)
P value
TRAF1 GG, No. (%) GA, No. (%) AA, No. (%) A allele, No. (%)
89 55 16 87
46 50 21 92
(39.32) (42.74) (17.94) (39.3)
1.76 (1.04–2.97) 2.54 (1.21–5.33) 1.74 (1.21–2.49)
0.03 0.012 0.003
STAT4 GG, No. (%) GT, No. (%) TT, No. (%) T allele, No. (%)
108 (67.5) 44 (27.5) 8 (5.0) 60 (29.1)
56 50 11 72
(47.9) (42.7) (9.4) (30.8)
2.2 (1.31–3.68) 2.6 (1.01–9.67) 1.9 (1.29–2.86)
0.003 0.04 0.001
(55.6) (34.4) (10.0) (27.2)
RF+, Rheumatoid factor-positive.
Table 5 Genotype frequencies in anti-CCP positive rheumatoid arthritis, relative to controls. Control subjects(n = 160)
Anti-CCP+ subjects (n = 157)
OR (95% CI)
P value
TRAF1 GG, No. (%) GA, No. (%) AA, No. (%) A allele, No. (%)
89 55 16 87
60 (38.22) 65 (41.40) 32 (20.38) 129 (41.10)
1.75 (1.07–2.85) 2.96 (1.49–5.87) 1.86 (1.33–2.60)
0.02 0.001 0.0001
STAT4 GG, No. (%) GT, No. (%) TT, No. (%) T allele, No. (%)
108 (67.5) 44 (27.5) 8 (5.0) 60 (29.1)
71 (45.22) 65 (41.40) 21 (13.37) 107 (34.08)
2.24 (1.38–3.65) 3.99 (1.67–9.50) 2.24 (1.55–3.22)
0.001 0.001 0.0001
(55.6) (34.4) (10.0) (27.2)
Anti-CCP+, antibodies to cyclic citrullinated peptide -positive.
Table 6 Association of TRAF1 and STAT4 genotypes with RA severity. Non erosive subjects (n = 111)
Erosive subjects (n = 61)
OR (95% CI)
P value
TRAF1 GG, No. (%) GA, No. (%) AA, No. (%) A allele, No. (%)
55 (49.5) 41 (37) 15 (13.5) 71(32)
19 30 12 54
(31.1) (49.2) (19.7) (44.3)
2.12 (1.05–4.28) 0.58 (0.22–1.53) 1.69 (1.07–2.66)
0.03 0.2 0.02
STAT4 GG, No. (%) GT, No. (%) TT, No. (%) T allele, No. (%)
55 (49.5) 51 (46) 5 (4.5) 61 (27.5)
30 (49.2) 22 (36.1) 9 (14.7) 40 (32.8)
0.67 (0.35–1.30) 3.3 (1.01–10.7) 0.77 (0.48–1.25)
0.2 0.04 0.3
4. Discussion
4.1. TRAF1/C5 Region (rs10818488) and RA
Recent large-scale whole-genome and candidate gene association studies have identified a number of SNP markers that reproducibly associate with RA susceptibility, substantially improving our understanding of the genetic component of disease susceptibility. While this is important, it is unlikely to have an immediate clinical impact unless these markers also associate with prognosis or treatment response [18]. Several risk alleles for rheumatoid arthritis have been identified in gene regions containing TRAF1 (C5 locus), STAT4, and OLIG3AIP3 genes [7,19]. These new findings, and data from complementary candidate gene studies [4,20], indicate how series of variations together make up the genetic risk for rheumatoid arthritis, and they show how different patterns of genetic risk factors have emerged for subsets of disease positive and negative for anti-CCP or RF [21]. Interestingly, the frequency of the A allele of the TRAF1/C5 SNP was lower in the Egyptian healthy controls (27.2%) as compared with either the Dutch (40%) [4] or Spanish controls (36%) [22]. We also observed a much lower frequency of the T allele of the STAT4 SNP in the Egyptian healthy individuals (18.7%) as compared with the North American NARAC cohort (22%) [7]. Taken together, these findings indicate that there are distinct population specific differences in the prevalence of these alleles.
The TRAF1 rs10818488 SNP is located within a region encoding a putative binding site for the transcription factor P300. P300 frequently plays important roles in a broad spectrum of biologic processes, including cell proliferation and differentiation [23]. Our results showed significant increase in TRAF1 A allele in RA patients suggesting an association between the presence of the polymorphism and the disease. This confirmed the results of Kurreeman et al. (2007) and Zervou et al. (2008) [4,17] who reported this region as RA associated region in a large-scale genetic association study. It is, therefore, in doing so provide strong evidence for the TRAF1/C5 region as a true RA-associated genetic variant. On the contrary a genome-wide study performed by the Wellcome Trust failed to identify this region as a candidate for RA [24]. RA is a heterogeneous disease with a considerable variation in phenotype as evidenced by the fact that some patients are autoantibody-positive whereas others are not. Anti-CCP has gained much interest as current data suggests that anti-CCP positive and negative RA may have different genetic risk factors [25]. To investigate whether the TRAF1/C5 region is associated with a specific phenotype of RA, we next stratified patients for autoantibody status. Of particular interest is the observation that for TRAF1 loci the associ-
R.H. Mohamed et al. / Cellular Immunology 273 (2012) 67–72
ation was most marked in the autoantibody-positive subgroup carrying of the A allele than normal subject who carried the same allele. Moreover although we also observed an increase in the frequency of the A allele in autoantibody-positive as compared to autoantibody-negative disease, this difference did not reach formal statistical significance. This agree with the results obtained by Kurreeman et al. (2007), who found association of rs10818488 with autoantibody-positive subgroup carrying of the A allele than normal subject who carried the same allele and no significant difference in the frequency of the A allele in autoantibody-positive as compared to autoantibodynegative RA patients. Moreover another study stated that the significant association between TRAF1-C1 and RA was independent of the concentrations of anti-CCP and RF [26]. Because the clinical course of RA can vary considerably ranging from nonerosive disease to rapidly progressive joint damage, we also analyzed whether the SNPs in the TRAF1/C5 region were involved with RA progression. Annual X-rays of the hands and feet of patients were assigned Sharp–van der Heijde units, a combined score for bone erosions and joint space narrowing. Rheumatic patient with A allele had an almost two-fold higher severe disease as compared to the non erosive patients carried the same allele. Moreover, carriers of the minor AA genotype of rs10818488 at this locus showed a significant decrease in joint function (HAQ) than carriers of GG genotype. More interestingly, these data on joint destruction not only indicated that the TRAF1/C5 region predisposes to RA, but also suggested that within the RA population, patients harboring the A allele predisposes not only to RA susceptibility, but also to severity. Carriers of the minor A allele of rs10818488 at this locus were previously shown to have increased radiographic progression, whereas previous study stated that individuals homozygosity for the G allele may be associated with mortality [27].
71
the same allele. This agree with Orozco et al. (2008) and Barton et al. (2008) who found significant association of anti CCP-positive RA patients carrying the T allele than normal subject which carried the same allele [29,34] and disagree with Lee et al. (2007) who found no association [8]. Stratification by the presence of antibody revealed that the association between the STAT4 rs7574865 polymorphism and RA is not dependant of the presence of this antibody (data not shown). This confirmed pervious studies [34–36] that showed no evidence for association between the minor T allele of STAT4 7574865 SNP and antibody positively. These findings supported the mounting evidence that different genetic loci are associated with autoantibody-positive and autoantibody-negative RA. Many of the genes identified to date may therefore predispose to autoimmunity in general, or more specifically the immunological processes involved in the breakdown of self-tolerance and autoantibody production [37]. Several studies have focused on the relationship between the severity of RA and STAT4 polymorphisms. Results obtained in this study suggest that rheumatic patients with TT genotype were significantly more likely to have erosive arthropathy than non erosive patients who carried the same allele. In conclusion TRAF1 and STAT4 are emerging as a novel common risk factor for diverse complex diseases. The current study provided evidence showing the important role of TRAF1 and STAT4 genes polymorphisms in the development of RA. Knowing genetic factors determining disease susceptibility and severity may facilitate personalized medicine. It also remains important to identify the immunological and biochemical pathways that determine outcome, which can then be targeted therapeutically. The TRAF1/C5 and STAT4 locus warrant further investigation as a potential disease susceptibility and severity locus in RA.
4.2. STAT4 (rs7574865) and RA References The rs7574865 SNP examined is located in the third intron of the STAT4 gene, and its actual functional consequence remains to be identified. The polymorphic site was not found to disrupt any transcription factor binding site [17]. Our results showed significant increase in the frequency in STAT4 T allele in RA patients suggesting an association between the presence of the polymorphism and the disease. These results are consistent with the previous studies which observed a significant difference in allele frequency and genotype distribution of the STAT4 polymorphism between RA patients and controls in Japanese and European populations [28,29]. Another study from South American showed that this population has 31% minor T allele frequency of STAT4 and this STAT4 SNP give a similar degree of risk to this population compared with Asian population and population of European origin [30]. The transcription factor encoded by STAT4 is downstream of several cytokines that play a crucial role in the development of Th1 and Th17 responses, such as IL-12, IL-15 and IL-23 [13,31] and the type I interferons [13,32]. Whilst this gene undoubtedly contributes to susceptibility to some autoantibody-associated diseases (RA, systemic lupus erythematosus and type I diabetes), there are recent reports that it may be associated with both clinical forms of inflammatory bowel diseases [33] suggesting that rather than contributing to autoantibody production, it may be a common risk factor for inflammatory disease per se. This is consistent with the apparent association of STAT4 with both autoantibody-positive and autoantibody-negative RA in our study. Of particular interest is the observation that for STAT4 loci the association was most marked in the autoantibody-positive subgroup carrying of the T allele than normal subject which carried
[1] G.S. Firestein, Evolving concepts of rheumatoid arthritis [review], Nature 423 (2003) 356–361. [2] A.H. Van, J.Z. Wesoly, T.W. Huizinga, Understanding the genetic contribution to rheumatoid arthritis, Curr. Opin. Rheumatol. 17 (2005) 299–304. [3] J. Bowes, A. Barton, Recent advances in the genetics of RA susceptibility, Rheumatology (Oxford) 47 (2008) 399–402. [4] F.A. Kurreeman, L. Padyukov, R.B. Marques, S.J. Schrodi, M. Seddighzadeh, G. Stoeken-Rijsbergen, A.H. Helm-van, C.F. Allaart, W. Verduyn, J. HouwingDuistermaat, L. Alfredsson, A.B. Begovich, L. Klareskog, T.W. Huizinga, R.E. Toes, A candidate gene approach identifies the TRAF1/C5 region as a risk factor for rheumatoid arthritis, PLoS Med. 4 (2007) 1515–1524. [5] H.S. Lee, E.F. Remmers, J.M. Le, D.L. Kastner, S.C. Bae, P.K. Gregersen, Association of STAT4 with rheumatoid arthritis in the Korean population, Arthritis Rheum. 58 (2008) 2598–2602. [6] M.E. Weinblatt, J.M. Kremer, A.D. Bankhurst, K.J. Bulpitt, R.M. Fleischmann, R.I. Fox, C.G. Jackson, M. Lange, D.J. Burge, A trial of Etanercept, a recombinant tumor necrosis factor receptor: Fc fusion protein, in patients with rheumatoid arthritis receiving methotrexate, N. Engl. J. Med. 340 (1999) 253–259. [7] E.F. Remmers, R.M. Plenge, A.T. Lee, R.R. Graham, G. Hom, T.W. Behrens, STAT4 and the risk of rheumatoid arthritis and systemic lupus erythematosus, N. Engl. J. Med. 357 (2007) 977–986. [8] H.S. Lee, E.F. Remmers, J.M. Le, D.L. Kastner, S.C. Bae, P.K. Gregersen, Association of STAT4 with rheumatoid arthritis in the Korean population, Mol. Med. 13 (2007) 455–460. [9] D.M. Frucht, M. Aringer, J. Galon, C. Danning, M. Akrad, M. Brown, M. Centola, C.Y. Wu, N. Yamada, T. Cheng, S. Fan, G. Steiner, H. El-Gabalawy, J.J. O’Shea, STAT4 is expressed in activated peripheral blood monocytes, dendritic cells, and macrophages at sites of Th1-mediated inflammation, J. Immunol. 164 (2000) 4659–4664. [10] K.M. Murphy, S.L. Reiner, The lineage decisions of helper T cells, Nat. Rev. Immunol. 2 (2002) 933–944. [11] R. Nishikomori, T. Usui, C.Y. Wu, A. Morinobu, J.J. O’Shea, W. Strober, Activated STAT4 has an essential role in Th1 differentiation and proliferation that is independent of its role in the maintenance of IL-12R beta 2 chain expression and signaling, J. Immunol. 169 (2002) 4388–4398. [12] G. Cai, T. Radzanowski, E.N. Villegas, R. Kastelein, C.A. Hunter, Identification of STAT4-dependent and independent mechanisms of resistance to Toxoplasma gondii, J. Immunol. 165 (2000) 2619–2627.
72
R.H. Mohamed et al. / Cellular Immunology 273 (2012) 67–72
[13] A.N. Mathur, H.C. Chang, D.G. Zisoulis, G.L. Stritesky, Q. Yu, J.T. O’Malley, R. Kapur, D.E. Levy, G.S. Kansas, M.H. Kaplan, Stat3 and Stat4 direct development of IL-17-secreting Th cells, J. Immunol. 178 (2007) 4901–4907. [14] F.C. Arnett, S.M. Edworthy, D.A. Bloch, D.J. McShane, J.F. Fries, N.S. Cooper, L.A. Healey, S.R. Kaplan, M.H. Liang, H.S. Luthra, The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis, Arthritis Rheum. 31 (1988) 315–324. [15] M.L. Prevoo, M.A. van ‘t Hof, H.H. Kuper, M.A. van Leeuwen, L.B. vande Putte, P.L. van Riel, Modified disease activity scores that include twentyeight-joint counts: development and validationin a prospective longitudinal study of patients with rheumatoid arthritis, Arthritis Rheum. 38 (1995) 44– 48. [16] J.T. Sharp, An overview of radiographic analysis of joint damage in rheumatoid arthritis and its use in misanalysis, J. Rheumatol. 27 (2000) 254–260. [17] M.I. Zervou, P. Sidiropoulos, E. Petraki, V. Vazgiourakis, E. Krasoudaki, A. Raptopoulou, H. Kritikos, E. Choustoulaki, D.T. Boumpas, G.N. Goulielmos, Association of a TRAF1 and a STAT4 gene polymorphism with increased risk for rheumatoid arthritis in a genetically homogeneous population, Hum. Immunol. 69 (2008) 567–571. [18] D. Plant, W. Thomson, M. Lunt, E. Flynn, P. Martin, S. Eyre, T. Farragher, D. Bunn, J. Worthington, D. Symmons, A. Barton, The role of rheumatoid arthritis genetic susceptibility markers in the prediction of erosive disease in patients with early inflammatory polyarthritis: results from the Norfolk Arthritis Register, Rheumatology 50 (2011) 78–84. [19] R.M. Plenge, C. Cotsapas, L. Davies, A.L. Price, P.I. de Bakker, J. Maller, I. Pe’er, N.P. Burtt, B. Blumenstiel, M. DeFelice, M. Parkin, R. Barry, W. Winslow, C. Healy, R.R. Graham, B.M. Neale, E. Izmailova, R. Roubenoff, A.N. Parker, R. Glass, E.W. Karlson, N. Maher, D.A. Hafler, D.M. Lee, M.F. Seldin, E.F. Remmers, A.T. Lee, L. Padyukov, L. Alfredsson, J. Coblyn, M.E. Weinblatt, S.B. Gabriel, S. Purcell, L. Klareskog, P.K. Gregersen, N.A. Shadick, M.J. Daly, D. Altshuler, Two independent alleles at 6q23 associated with risk of rheumatoid arthritis, Nat. Genet. 39 (2007) 1477–1482. [20] S. Sigurdsson, L. Padyukov, F.A. Kurreeman, U. Liljedahl, A.C. Wiman, L. Alfredsson, R. Toes, J. Rönnelid, L. Klareskog, T.W. Huizinga, G. Alm, A.C. Syvänen, L. Rönnblom, Association of a haplotype in the promoter region of the interferon regulatory factor 5 gene with rheumatoid arthritis, Arthritis Rheum. 56 (2007) 2202–2210. [21] A.H. Van der Helm-van Mil, T.W. Huizinga, Advances in the genetics of rheumatoid arthritis point to sub classification into distinct disease subsets, Arthritis Res. Ther. 10 (2008) 205. [22] F.A.S. Kurreeman, D. Rocha, J. Houwing-Duistermaat, S. Vrijmoet, V.H. Teixeira, P. Migliorini, A. Balsa, R. Westhovens, P. Barrera, H. Alves, C. Vaz, M. Fernandes, D. Pascual-Salcedo, L. Michou, S. Bombardieri, T. Radstake, P. Van Riel, L. van de Putte, A. Lopes-Vaz, B. Prum, T. Bardin, I. Gut, F. Cornelis, T.W.J. Huizinga, E. Petit-Teixeira, R.E.M. Toes, Replication of the tumor necrosis factor receptor_associated factor 1/complement component 5 region as a susceptibility locus for rheumatoid arthritis in a European family-based study, Arthritis Rheum. 58 (2008) 2670–2674. [23] D. Chakravarti, V.J. La-Morte, M.C. Nelson, T. Nakajima, I.G. Schulman, H. Juguilon, M. Montminy, R.M. Evans, Role of CBP/P300 in nuclear receptor signaling, Nature 383 (1996) 99–108. [24] The Wellcome Trust Case Control Consortium, Genome-wide association study of 14,000 cases of seven common diseases and 3000 shared controls, Nature 447 (2007) 661–678.
[25] F. Van Gaalen, A. Ioan-Facsinay, T.W. Huizinga, R.E. Toes, The devil in the details: the emerging role of anticitrulline autoimmunity in rheumatoid arthritis, J. Immunol. 175 (2005) 5575–5580. [26] T.U. Han, S.Y. Bang, C. Kang, S.C. Bae, TRAF1 polymorphisms associated with rheumatoid arthritis susceptibility in Asians and in Caucasians, Arthritis Rheum. 175 (2009) 2577–2584. [27] V.F. Panoulas, J.P. Smith, P. Nightingale, G.D. Kitas, Association of the TRAF1/C5 locus with increased mortality, particularly from malignancy or sepsis, in patients with rheumatoid arthritis, Arthritis Rheum. 60 (2009) 39–46. [28] S. Kobayashi, K. Ikari, H. Kaneko, Y. Kochi, K. Yamamoto, K. Shimane, Y. Nakamura, Y. Toyama, T. Mochizuki, S. Tsukahara, Y. Kawaguchi, C. Terai, M. Hara, T. Tomatsu, H. Yamanaka, T. Horiuchi, K. Tao, K. Yasutomo, D. Hamada, N. Yasui, H. Inoue, M. Itakura, H. Okamoto, N. Kamatani, S. Momohara, Association of STAT4 with susceptibility to rheumatoid arthritis and systemic lupus erythematosus in the Japanese population, Arthritis Rheum. 58 (2008) 1940–1946. [29] G. Orozco, B.Z. Alizadeh, A.M. Delgado-Vega, M.A. González-Gay, A. Balsa, D. Pascual-Salcedo, B. Fernández-Gutierrez, M.F. González-Escribano, I.F. Petersson, P.L. van Riel, P. Barrera, M.J. Coenen, T.R. Radstake, M.A. van Leeuwen, C. Wijmenga, B.P. Koeleman, M. Alarcón-Riquelme, J. Martín, Association of STAT4 with rheumatoid arthritis: a replication study in three European populations, Arthritis Rheum. 58 (2008) 1974–1980. [30] R.J. Palomino-Morales, A. Rojas-Villarraga, C.I. Gonzalez, G. Ramirez, J.M. Anaya, J. Martin, STAT4 but not TRAF1/C5 variants influence the risk of developing rheumatoid arthritis and systemic lupus erythematosus in Colombians, Genes Immun. 9 (2008) 379–382. [31] M. Kaplan, STAT4: a critical regulator of inflammation in vivo, Immunol. Res. 31 (2005) 231–242. [32] S.N. Kariuki, K.A. Kirou, E.J. MacDermott, L. Barillas-Arias, M.K. Crow, T.B. Niewold, Cutting edge: autoimmune disease risk variant of STAT4 confers increased sensitivity to IFN-a in lupus patients in vivo, J. Immunol. 182 (2009) 34–38. [33] A. Martnez, J. Varadé, A. Márquez, M.C. Cénit, L. Espino, N. Perdigones, J.L. Santiago, M. Ferndndez-Arquero, H. de la Calle, R. Arroyo, J.L. Mendoza, B. Fernández-Gutiérrez, E.G. de la Concha, Association of the STAT4 gene with increased susceptibility for some immune-mediated diseases, Arthritis Rheum. 58 (2008) 2598–2602. [34] A. Barton, W. Thomson, X. Ke, S. Eyre, A. Hinks, J. Bowes, L.J. Gibbons, D. Plant, Wellcome Trust Case Control Consortium, A. Wilson, I. Marinou, A. Morgan, P. Emery, S. Steer, L. Hocking, D. Reid, P. Wordsworth, P. Harrison, J. Worthington, Re-evaluation of putative rheumatoid arthritis susceptibility genes in the postgenome wide association study era and hypothesis of a key pathway underlying susceptibility, Hum. Mol. Genet. 17 (2008) 2274–2279. [35] J.D. Ji, W.J. Lee, K.A. Kong, J.H. Woo, S.J. Choi, Y.H. Lee, G.G. Song, Association of STAT4 polymorphism with rheumatoid arthritis and systemic lupus erythematosus: a meta-analysis, Mol. Biol. Rep. 37 (2010) 141–147. [36] Y.H. Lee, J.H. Woo, S.J. Choi, J.D. Ji, G. Gyu, Association between the rs7574865 polymorphism of STAT4 and rheumatoid arthritis: a meta-analysis, Rheumatol. Int. 30 (2010) 661–666. [37] A.W. Morgan, J.I. Robinson, P.G. Conaghan, S.G. Martin, E.A. Hensor, M.D. Morgan, L. Steiner, H.A. Erlich, H.C. Gooi, A. Barton, J. Worthington, P. Emery, Evaluation of the rheumatoid arthritis susceptibility loci HLA-DRB1, PTPN22, OLIG3/TNFAIP3, STAT4 and TRAF1/C5 in an inception cohort, Arthritis Res. Ther. 12 (2010) 57–62.