- freely available
Int. J. Mol. Sci. 2014, 15(2), 2712-2721; doi:10.3390/ijms15022712
Published: 17 February 2014
Abstract: E26 transformation-specific-1 (ETS1) and WDFY family member 4 (WDFY4) are closely related with systemic lupus erythematosus. We hypothesized that ETS1 and WDFY4 polymorphisms may contribute to rheumatoid arthritis (RA) susceptibility. We studied ETS1 rs1128334 G/A and WDFY4 rs7097397 A/G gene polymorphisms in 329 patients with RA and 697 controls in a Chinese population. Genotyping was done using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. When the WDFY4 rs7097397 AA homozygote genotype was used as the reference group, the AG genotype was associated with a significantly increased risk for RA. In the dominant model, when the WDFY4 rs7097397 AA homozygote genotype was used as the reference group, the AG/GG genotypes were associated with a significant increased susceptibility to RA. In stratification analyses, a significantly increased risk for RA associated with the WDFY4 rs7097397 AG genotype was evident among female patients, younger patients, C-reactive protein (CRP) negative patients and both anti-cyclic citrullinated peptide antibody (ACPA) positive patients and negative patients compared with the WDFY4 rs7097397 AA genotype. These findings suggested that WDFY4 rs7097397 A/G may be associated with the risk of RA, especially among younger, female patients, CRP-negative patients and both ACPA positive and negative patients. However, our results were obtained from a moderate-sized sample, and therefore this is a preliminary conclusion. To confirm these findings, validation by a larger study from a more diverse ethnic population is needed.
Rheumatoid arthritis (RA) is one type of systemic autoimmune diseases due to a failure of immune self-tolerance. RA is characterized by synovial inflammation and hyperplasia, autoantibody production protein antibody, cartilage and bone destruction and systemic features . RA is a complex disease with genetic and environmental predisposing factors. The genetic variants may contribute 50%–60% of the etiology of RA . The highly polymorphic HLA region is a major contributor and accounts for approximately one-third of genetic risk of RA . However, other additional risk alleles of RA remain to be identified .
RA and systemic lupus erythematosus (SLE) are autoimmune rheumatic diseases thought to have a substantial genetic contribution . Recent genome-wide association studies in SLE have identified several novel associated locus including E26 transformation-specific-1 (ETS1) rs1128334 G/A and WDFY family member 4 (WDFY4) rs7097397 A/G polymorphisms, which have not been investigated in RA .
The ETS1 transcription factor is a member of the helix-turn-helix family . ETS1 is required for angiogenesis and cell apoptosis . In the synovial membrane of the joint in active RA patients, ETS1 is produced by endothelial cells and new blood vessels under pathological conditions [9,10]. ETS1 is present in T cells, B cells and natural killer cells [11,12]. A very high level of interleukin-10, an anti-inflammatory cytokine, has been observed in ETS1 deficient type 1 T helper cells . In a recent investigation, ETS1 levels were strongly affected miR-146a promoter activity in vitro; and the knockdown of ETS1 directly impaired the induction of miR-146a . High miR-146a expression levels were correlated with active disease in RA patients [15,16].
WDFY4 is predominantly expressed in the immune tissues. The function of WDFY4 is not well known; rs7097397 in WDFY4 changes an arginine residue to glutamine (R1816Q) .
ETS1 rs1128334 G/A and WDFY4 rs7097397 A/G polymorphisms were distinctly associated with SLE . However, further investigations between ETS1 rs1128334 G/A and WDFY4 rs7097397 A/G polymorphisms and RA risk were not conducted. We therefore undertook genotyping in a hospital-based case–control study in a cohort of 329 patients with RA and 697 controls in a Chinese population.
2.1. Characteristics of the Study Population
Among 329 patients and 697 controls who provided DNA samples, genotyping for the ETS1 rs1128334 G/A polymorphism was successful in 319 (97.0%) patients and 673 (96.6%) controls; genotyping for the WDFY4 rs7097397 A/G polymorphism was successful in 321 (97.6%) patients and 691 (99.1%) controls. The demographic and clinical characteristics of all subjects are summarized in Table 1. Subjects were adequately matched for age and sex (p = 0.829 and 0.190, respectively). The genotype distributions of ETS1 rs1128334 G/A and WDFY4 rs7097397 A/G in all subjects are illustrated in Table 2. The observed genotype frequencies for the polymorphism in controls were in HWE for ETS1 rs1128334 G/A (p = 0.570) and WDFY4 rs7097397 A/G (p = 0.116).
2.2. Associations between ETS1 rs1128334 G/A and WDFY4 rs7097397 A/G Polymorphism and the Risk of RA
The genotype frequencies of the ETS1 rs1128334 G/A polymorphism were 42.3% (GG), 44.5% (GA) and 13.2% (AA) in RA patients, and 43.2% (GG), 44.3% (GA) and 12.5% (AA) in controls (p = 0.939) (Table 2). Logistic regression analyses revealed that ETS1 rs1128334 G/A polymorphisms were not associated with the risk of RA (Table 2).
The genotype frequencies of the WDFY4 rs7097397 A/G polymorphism were 38.0% (AA), 49.8% (AG) and 12.1% (GG) in RA patients, and 46.9% (AA), 41.2% (AG) and 11.9% (GG) in controls (p = 0.022) (Table 2).
When the WDFY4 rs7097397 AA homozygote genotype was used as the reference group, the AG genotype was associated with a significantly increased risk for RA (OR = 1.49, 95% CI = 1.12–1.98, p = 0.006). In the dominant model, when the WDFY4 rs7097397 AA homozygote genotype was used as the reference group, the AG/GG genotypes were associated with a significant 1.44-fold increased susceptibility to RA (OR = 1.44, 95% CI = 1.10–1.89, p = 0.008) (Table 2).
2.3. Stratification Analyses of ETS1 rs1128334 G/A and WDFY4 rs7097397 A/G Polymorphisms and the Risk for RA
Stratification analyses were done to evaluate the effects of ETS1 rs1128334 G/A and WDFY4 rs7097397 A/G genotypes on RA risk according to age, sex, C-reactive protein (CRP) status and ACPA status (Table 3). A significantly increased risk for RA associated with the WDFY4 rs7097397 AG genotype was evident among female patients (OR = 1.64, 95% CI = 1.17–2.28, p = 0.004), younger patients (OR = 1.95, 95% CI = 1.29–2.94, p = 0.002), CRP-negative patients (OR = 1.56, 95% CI = 1.07–2.27, p = 0.022) and both ACPA positive patients (OR = 1.49, 95% CI = 1.03–2.16, p = 0.034) and negative patients (OR = 1.49, 95% CI = 1.03–2.16, p = 0.034) compared with the WDFY4 rs7097397 AA genotype. A significantly increased risk for RA associated with the WDFY4 rs7097397 GG genotype was evident among younger patients (OR = 2.45, 95% CI = 1.35–4.43, p = 0.003) and CRP-negative patients (OR = 1.80, 95% CI = 1.07–3.04, p = 0.027) compared with the WDFY4 rs7097397 AA genotype (Table 3).
2.4. Replication and Combination Study of WDFY4 rs7097397 A/G Polymorphism and the Risk of RA
In replication cohort with 100 RA and 100 controls, no positive results were found (data not shown), which might caused by small samples. However, after we had combined discovery cohort and replication cohort, WDFY4 rs7097397 GG homozygote genotype was used as the reference group, the GA/AA genotypes were associated with a significant increased susceptibility to RA (Table S1).
We determined the association between the ETS1 rs1128334 G/A and WDFY4 rs7097397 A/G polymorphisms and the risk of RA in a Chinese population. It was the first positive finding of WDFY4 rs7097397 A/G polymorphism and RA. We found that WDFY4 rs7097397 A/G may be associated with the risk of RA, and that this effect was more evident in female, younger patients, CRP-negative patients and both ACPA positive and negative patients.
The function of WDFY4 is still not well characterized . WDFY4 contains WD40 domains, which covers a wide variety of functions including adaptor/regulatory modules in signal transduction, pre-mRNA processing and cytoskeleton assembly .
WDFY4 rs7097397 causes non-synonymous substitution of Arg1816Gln . We found that the WDFY4 rs7097397 AG allele may increase the risk of RA, particularly in CRP-negative patients and both ACPA positive and negative patients, indicating a gene-environment interaction.
Ethnic differences may play a part in the conflicting results seen in associated studies. Our results, using the same genetic markers with subjects of the same ethnic backgrounds as those in the original studies, suggest that WDFY4 rs7097397 A/G confers susceptibility for RA in the Chinese population.
Genetic polymorphisms often vary between ethnic groups. In the present study with 697 controls, we reported that the minor allele frequency of ETS1 rs1128334 G/A was similar to that reported in Hong Kong and Shanghai of Chinese populations, but a little higher than in Hefei of Chinese population and Bangkok . The minor allele frequency of WDFY4 rs7097397 A/G was similar to that reported in Shanghai and Hefei of Chinese populations and Bangkok, but a little higher than in Hong Kong of Chinese population .
Considering mutant alleles in the control group, OR, case samples and control samples, the power of our study (α = 0.05) was 0.071 and 0.835 for ETS1 rs1128334 G/A and WDFY4 rs7097397 A/G respectively.
Several limitations of the present study need to be addressed. First, this was a hospital-based case–control study, so selection bias was unavoidable and the subjects were not fully representative of the general population. Second, the polymorphisms we investigated, based on their functional considerations, may not offer a comprehensive view of the genetic variability of ETS1 and WDFY4, further fine mapping studies are recommend. Third, a single case–control study is not sufficient to fully interpret the relationship between the ETS1 rs1128334 G/A and WDFY4 rs7097397 A/G polymorphisms and susceptibility to RA because of the relatively moderate number of patients evaluated. Larger numbers of subjects are necessary to confirm our findings, especially for the results of ETS1 rs1128334 G/A and WDFY4 rs7097397 A/G polymorphisms and RA. Finally, we did not obtain detailed information about RA severity and the outcomes of treatment, which restricted our analyses.
4. Experimental Section
4.1. Study Populations
We obtained approval of the study protocol from the Ethics Committee of Nanjing Medical University (Nanjing, China). All patients provided written informed consent to be included in the study.
Three hundred and twenty-nine RA patients who fulfilled the criteria for RA set by the American College of Rheumatology classification in 1987  were consecutively recruited from the Changzhou Second Hospital-Affiliated Hospital of Nanjing Medical University (Changzhou, China), the Changzhou First Hospital (Changzhou, China), and the Changzhou Traditional Chinese Medical Hospital (Changzhou, China), between September 2010 and October 2011. The controls were patients without RA, matched for age (±5 years) and sex, and recruited from the same institutions during the same time period; most of the controls were admitted to the hospitals for the treatment of trauma. All cases and controls were Chinese Han population. We also recruited another 100 RA cases and 100 controls without RA, matched for age (±5 years) and sex, between June 2013 and December 2013 for replication study purpose.
Each patient was interviewed by trained personnel using a pre-tested questionnaire to obtain information on demographic data and related risk factors for RA. After the interview, 2 mL of peripheral blood was collected from each subject.
Isolation of DNA and genotyping by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS)
Blood samples were collected using vacutainers and transferred to test tubes containing ethylenediamine tetra-acetic acid (EDTA). Genomic DNA was isolated from whole blood using the QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany). Genotyping was done by MALDI-TOF MS using the MassARRAY system (Sequenom, San Diego, CA, USA) as previously described (Figure S1) . For quality control, repeated analyses were undertaken on 10% of randomly selected samples.
4.2. Statistical Analyses
Differences in demographics, variables, and genotypes of the ETS1 rs1128334 G/A and WDFY4 rs7097397 A/G polymorphism variants were evaluated using a chi-squared test. The associations between ETS1 rs1128334 G/A and WDFY4 rs7097397 A/G genotypes and risk of RA were estimated by computing odds ratios (ORs) and 95% confidence intervals (CIs) using logistic regression analyses, and by using crude ORs. The Hardy–Weinberg equilibrium (HWE) was tested by a goodness-of-fit chi-squared test. All statistical analyses were done with SAS software (version 9.1.3; SAS Institute, Cary, NC, USA).
In conclusion, the present study provided strong evidence that WDFY4 rs7097397 A/G functional polymorphisms may contribute to the risk of RA. However, our results were obtained from a moderate-sized sample, and therefore this is a preliminary conclusion. Validation by a larger study from a more diverse ethnic population is needed to confirm these findings.
This study was supported in part by National Natural Science Foundation of China (81371927) and Nanjing Medical University Foundation for Development of Science and Technology (2012NJMU128).
Conflicts of Interest
The authors declare no conflict of interest.
WDFY family member 4
single nucleotide polymorphism
- McInnes, I.B.; Schett, G. The pathogenesis of rheumatoid arthritis. N. Engl. J. Med 2011, 365, 2205–2219. [Google Scholar]
- MacGregor, A.J.; Snieder, H.; Rigby, A.S.; Koskenvuo, M.; Kaprio, J.; Aho, K.; Silman, A.J. Characterizing the quantitative genetic contribution to rheumatoid arthritis using data from twins. Arthritis Rheum 2000, 43, 30–37. [Google Scholar]
- Hasstedt, S.J.; Clegg, D.O.; Ingles, L.; Ward, R.H. HLA-linked rheumatoid arthritis. Am. J. Hum. Genet 1994, 55, 738–746. [Google Scholar]
- Stahl, E.A.; Raychaudhuri, S.; Remmers, E.F.; Xie, G.; Eyre, S.; Thomson, B.P.; Li, Y.; Kurreeman, F.A.; Zhernakova, A.; Hinks, A.; et al. Genome-wide association study meta-analysis identifies seven new rheumatoid arthritis risk loci. Nat. Genet 2010, 42, 508–514. [Google Scholar]
- Orozco, G.; Eyre, S.; Hinks, A.; Bowes, J.; Morgan, A.W.; Wilson, A.G.; Wordsworth, P.; Steer, S.; Hocking, L.; Thomson, W.; et al. Study of the common genetic background for rheumatoid arthritis and systemic lupus erythematosus. Ann. Rheum. Dis 2011, 70, 463–468. [Google Scholar]
- Yang, W.; Shen, N.; Ye, D.Q.; Liu, Q.; Zhang, Y.; Qian, X.X.; Hirankarn, N.; Ying, D.; Pan, H.F.; Mok, C.C.; et al. Genome-wide association study in Asian populations identifies variants in ETS1 and WDFY4 associated with systemic lupus erythematosus. PLoS Genet 2010, 6, e1000841. [Google Scholar]
- Leprince, D.; Gegonne, A.; Coll, J.; de Taisne, C.; Schneeberger, A.; Lagrou, C.; Stehelin, D. A putative second cell-derived oncogene of the avian leukaemia retrovirus E26. Nature 1983, 306, 395–397. [Google Scholar]
- Dittmer, J. The biology of the Ets1 proto-oncogene. Mol. Cancer 2003, 2, 29. [Google Scholar]
- Redlich, K.; Kiener, H.P.; Schett, G.; Tohidast-Akrad, M.; Selzer, E.; Radda, I.; Stummvoll, G.H.; Steiner, C.W.; Groger, M.; Bitzan, P.; et al. Overexpression of transcription factor Ets-1 in rheumatoid arthritis synovial membrane: Regulation of expression and activation by interleukin-1 and tumor necrosis factor alpha. Arthritis Rheum 2001, 44, 266–274. [Google Scholar]
- Wernert, N.; Justen, H.P.; Rothe, M.; Behrens, P.; Dreschers, S.; Neuhaus, T.; Florin, A.; Sachinidis, A.; Vetter, H.; Ko, Y. The Ets 1 transcription factor is upregulated during inflammatory angiogenesis in rheumatoid arthritis. J. Mol. Med. (Berl.) 2002, 80, 258–266. [Google Scholar]
- Anderson, M.K.; Hernandez-Hoyos, G.; Diamond, R.A.; Rothenberg, E.V. Precise developmental regulation of Ets family transcription factors during specification and commitment to the T cell lineage. Development 1999, 126, 3131–3148. [Google Scholar]
- Barton, K.; Muthusamy, N.; Fischer, C.; Ting, C.N.; Walunas, T.L.; Lanier, L.L.; Leiden, J.M. The Ets-1 transcription factor is required for the development of natural killer cells in mice. Immunity 1998, 9, 555–563. [Google Scholar]
- Grenningloh, R.; Kang, B.Y.; Ho, I.C. Ets-1, a functional cofactor of T-bet, is essential for Th1 inflammatory responses. J. Exp. Med 2005, 201, 615–626. [Google Scholar]
- Luo, X.; Yang, W.; Ye, D.Q.; Cui, H.; Zhang, Y.; Hirankarn, N.; Qian, X.; Tang, Y.; Lau, Y.L.; de Vries, N.; et al. A functional variant in microRNA-146a promoter modulates its expression and confers disease risk for systemic lupus erythematosus. PLoS Genet 2011, 7, e1002128. [Google Scholar]
- Murata, K.; Yoshitomi, H.; Tanida, S.; Ishikawa, M.; Nishitani, K.; Ito, H.; Nakamura, T. Plasma and synovial fluid microRNAs as potential biomarkers of rheumatoid arthritis and osteoarthritis. Arthritis Res. Ther 2010, 12, R86. [Google Scholar]
- Pauley, K.M.; Satoh, M.; Chan, A.L.; Bubb, M.R.; Reeves, W.H.; Chan, E.K. Upregulated miR-146a expression in peripheral blood mononuclear cells from rheumatoid arthritis patients. Arthritis Res. Ther 2008, 10, R101. [Google Scholar]
- Yuan, Y.J.; Luo, X.B.; Shen, N. Current advances in lupus genetic and genomic studies in Asia. Lupus 2010, 19, 1374–1383. [Google Scholar]
- Arnett, F.C.; Edworthy, S.M.; Bloch, D.A.; McShane, D.J.; Fries, J.F.; Cooper, N.S.; Healey, L.A.; Kaplan, S.R.; Liang, M.H.; Luthra, H.S.; et al. The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis. Arthritis Rheum 1988, 31, 315–324. [Google Scholar]
- Liu, R.; Xu, N.; Wang, X.; Shen, L.; Zhao, G.; Zhang, H.; Fan, W. Influence of MIF, CD40, and CD226 polymorphisms on risk of rheumatoid arthritis. Mol. Biol. Rep 2012, 39, 6915–6922. [Google Scholar]
|Table 1. Patient demographics and risk factors in rheumatoid arthritis, all subjects.|
|Variable||Cases (n = 329)||Controls (n = 697)||p|
|Age (years)||53.64 (±15.52)||53.45 (±11.35)||0.829|
|Age at onset, years, mean ± SD||44.93 (±12.55)||–||–|
|Disease duration, years, mean ± SD||8.76 (±9.31)||–||–|
|Treatment duration, years, mean ± SD||7.07 (±7.38)||–||–|
|RF-positive, no. (%)||266 (80.9%)||–||–|
|ACPA-positive, no. (%)||163 (49.5%)||–||–|
|CRP-positive, no. (%)||165 (50.2%)||–||–|
|ESR, mm/h||34.00 (±23.96)||–||–|
|Functional class, no. (%)||–||–|
RF: Rheumatoid factor; ACPA: Anti-cyclic citrullinated peptide; CRP: C-reactive protein; ESR: Erythrocyte sedimentation rate; DAS28: RA disease activity score.
|Table 2. Logistic regression analysis of associations between E26 transformation-specific-1 (ETS1) rs1128334 G/A and WDFY4 rs7097397 A/G polymorphisms and risk of rheumatoid arthritis.|
|Genotype||Cases (n = 329)||Controls (n = 697)||Crude OR (95% CI)||p||Adjusted OR (95% CI)||p|
|ETS1 rs1128334 G/A|
|A allele||226||35.4||466||34.6||1.04 (0.85–1.26)||0.726|
|GA||142||44.5||298||44.3||1.03 (0.77–1.37)||0.854||1.02 (0.76–1.35)||0.921|
|AA||42||13.2||84||12.5||1.08 (0.71–1.65)||0.729||1.06 (0.69–1.62)||0.792|
|AA vs. GA vs. GG||0.939|
|GA + AA||184||57.7||382||56.8||1.04 (0.79–1.36)||0.785||1.02 (0.78–1.34)||0.862|
|GG + GA||277||86.8||589||87.5||1.00||–||1.00||–|
|AA||42||13.2||84||12.5||1.06 (0.72–1.58)||0.762||1.05 (0.71–1.57)||0.807|
|WDFY4 rs7097397 A/G|
|G allele||238||37.1||449||32.5||1.22 (1.01–1.49)||0.043|
|AG||160||49.8||285||41.2||1.49 (1.12–1.98)||0.006||1.50 (1.13–1.99)||0.005|
|GG||39||12.1||82||11.9||1.26 (0.82–1.95)||0.292||1.26 (0.82–1.95)||0.294|
|GG vs. AG vs. AA||0.022|
|AG + GG||199||62.0||367||53.1||1.44 (1.10–1.89)||0.008||1.45 (1.10–1.90)||0.008|
|AA + AG||282||87.9||609||88.1||1.00||–||1.00||–|
|GG||39||12.1||82||11.9||1.03 (0.68–1.54)||0.897||1.02 (0.68–1.54)||0.911|
The genotyping was successful in: 319 cases and 673 controls for ETS1 rs1128334 G/A; 321 cases and 691 controls for WDFY4 rs7097397 A/G. Adjusted for age and sex.
|Table 3. Stratified analyses between ETS1 rs1128334 G/A and WDFY4 rs7097397 A/G polymorphisms and risk of rheumatoid arthritis.|
|Variable||ETS1 rs1128334 G/A (Case/Control)||OR (95% CI)||WDFY4 rs7097397 A/G (Case/Control)||OR (95% CI)|
|Male||33/99||37/75||8/20||1.00||1.48 (0.85–2.58); p:0.168||1.20 (0.48–2.98); p:0.695||33/87||39/88||9/22||1.00||1.17 (0.67–2.03); p:0.579||1.08 (0.45–2.58); p:0.865|
|Female||102/192||105/223||34/64||1.00||0.89 (0.64–1.24); p:0.479||1.00 (0.62–1.62); p:1.000||89/237||121/197||30/60||1.00||1.64 (1.17–2.28); p:0.0037||1.33 (0.81–2.20); p:0.263|
|<55||69/143||68/156||23/34||1.00||0.90 (0.60–1.35); p:0.623||1.40 (0.77–2.56); p:0.271||55/176||79/130||26/34||1.00||1.95 (1.29–2.94); p:0.0016, pcorrect:0.0032||2.45 (1.35–4.43); p:0.0031, pcorrect:0.0062|
|≥55||66/148||74/142||19/50||1.00||1.17 (0.78–1.75); p:0.450||0.85 (0.47–1.56); p:0.603||67/148||81/155||13/48||1.00||1.15 (0.78–1.71); p:0.476||0.60 (0.30–1.18); p:0.137|
|Positive||67/291||72/298||20/84||1.00||1.05 (0.73–1.52); p:0.799||1.03 (0.59–1.80); p:0.906||65/324||82/285||13/82||1.00||1.43 (1.00–2.06); p:0.051||0.79 (0.42–1.50); p:0.473|
|Negative||68/291||70/298||22/84||1.00||1.01 (0.69–1.46); p:0.978||1.12 (0.65–1.92); p:0.678||57/324||78/285||26/82||1.00||1.56(1.07–2.27); p:0.0216|
|1.80 (1.07–3.04); p:0.0273|
|Positive||66/291||76/298||17/84||1.00||1.12 (0.78–1.62); p:0.531||0.89 (0.50–1.60); p:0.703||61/324||80/285||19/82||1.00||1.49 (1.03–2.16); p:0.0340|
|1.23 (0.70–2.17); p:0.475|
|Negative||69/291||66/298||25/84||1.00||0.93 (0.64–1.36); p:0.721||1.26 (0.75–2.11); p:0.390||61/324||80/285||20/82||1.00||1.49 (1.03–2.16); p:0.0340|
|1.30 (0.74–2.27); p:0.365|
The genotyping was successful in: 319 cases and 673 controls for ETS1 rs1128334 G/A; 321 cases and 691 controls for WDFY4 rs7097397 A/G; Bonferroni correction was performed to correct the p value (pcorrect).
© 2014 by the authors; licensee MDPI, Basel, Switzerland This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).