Gene 566 (2015) 18–22
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Research paper
Association between interleukin 17A polymorphisms and susceptibility to rheumatoid arthritis in a Chinese population☆ Li Shen a,1, Hui Zhang b,1, Ting Yan b, Guoxin Zhou b, Ruiping Liu b,c,⁎ a b c
Department of Clinical Laboratory, Changzhou First People's Hospital, Changzhou 213003, China Department of Orthopaedic Trauma, Affiliated Hospital of Nanjing Medical University, Changzhou Second People's Hospital, Changzhou 213003, China Central Laboratory, Changzhou Second People's Hospital, Affiliated Hospital of Nanjing Medical University, Changzhou 213003, China
a r t i c l e
i n f o
Article history: Received 8 October 2014 Received in revised form 2 April 2015 Accepted 8 April 2015 Available online 11 April 2015 Keywords: Interleukin 17A Polymorphisms Rheumatoid arthritis Molecular epidemiology
a b s t r a c t Background: Previous studies have revealed an association between interleukin 17A (IL17A) polymorphisms and the prevalence of rheumatoid arthritis (RA) in Japanese and Caucasian patients. We hypothesized that IL17A polymorphisms might also affect RA susceptibility in the Chinese population. Methods: We studied IL17A rs2275913 G/A, rs3819024 A/G, rs3819025 G/A, rs4711998 A/G, rs8193036 C/T and rs8193037 G/A polymorphisms in 615 RA patients and 839 controls in a Chinese population. Genotyping was performed using a custom-by-design 48-Plex SNP scan™ Kit. Results: Our results indicated that IL17A rs4711998 A/G and IL17A rs8193037 G/A polymorphisms were not associated with RA, and IL17A rs2275913 G/A and IL17A rs3819024 A/G variant alleles decrease the risk of RA, while IL17A rs3819025 G/A and IL17A rs8193036 C/T variant alleles increase the risk of RA. Conclusions: These findings suggest that IL17A polymorphisms may be associated with RA. Future larger studies with other ethnic populations are required to confirm current findings. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Although the etiology of rheumatoid arthritis (RA) is not fully understood, it is believed to result from complex genetic and environmental factors, which trigger and maintain synovial inflammation in affected individuals (Nordang et al., 2009). Genetic factors such as single nucleotide polymorphisms (SNPs) might play important roles in RA pathogenesis (Garcia-Bermudez et al., 2012). Interleukin 17A (IL17A), also known as IL-17, is the signature T helper 17 (Th17) effector cytokine (Korn et al., 2009). IL17A leads to the induction of many factors, including tumor necrosis factor-alpha (TNF-α), IL-6, and IL-1β, which are important in inflammatory processes (Ruddy et al., 2004).
Abbreviations:CI, confidence interval;IL17A, interleukin17A; LD, linkage disequilibrium; OR, odds ratio; SNP, single nucleotide polymorphism. ☆ Condensed abstract: Interleukin 17A polymorphisms are associated with rheumatoid arthritis susceptibility. ⁎ Corresponding author at: Department of Orthopaedic Trauma, Changzhou Second People's Hospital, Changzhou 213003, China. E-mail address: [email protected] (R. Liu). 1 These authors contributed equally to this study.
http://dx.doi.org/10.1016/j.gene.2015.04.028 0378-1119/© 2015 Elsevier B.V. All rights reserved.
IL17A is also produced by many other cell types, including CD8+ T cells and γδ T cells, and is increased locally in the skin and joint diseases in human (Kirkham et al., 2014). High levels of IL17A and its receptor are found in RA synovial fluid and tissue explants, and IL17A promotes joint degradation in ex vivo models (Kotake et al., 1999; Ziolkowska et al., 2000; Cai et al., 2001; Honorati et al., 2001). In RA, leukocyte migration, bone erosions, and angiogenesis are modulated by the IL-23-IL17 cascade (Pope and Shahrara, 2013). Studies of IL-17-deficient mice have suggested that IL17A is crucial in the development of collagen-induced arthritis due to activation of autoantigenspecific cellular and humoral immune responses (Nakae et al., 2003). Accordingly, IL17A and other Th17 related cytokines are considered prime targets of anti-cytokine therapy in autoimmune arthritis (Lubberts, 2008). The IL17A gene is located on Chromosome 6, and the association between its SNPs and certain diseases has been studied recently. Ren et al. previously investigated six functional SNPs of the IL17A gene in gastric cardiac adenocarcinoma (GCA) in a hospital-based case–control study of 243 GCA cases and 476 controls. Their results suggested that an IL17A rs3819024 A/G polymorphism might increase the GCA risk (Ren et al., 2014). Functional variations in the IL17A gene might contribute to the etiology of RA, and may even promote or protect against RA in certain ethnic
L. Shen et al. / Gene 566 (2015) 18–22
groups. To investigate that hypothesis, the current hospital-based case– control genotype study was performed with a cohort of 615 Chinese RA patients and 839 Chinese controls. 2. Patients and methods 2.1. Study subjects A total of 615 RA patients who fulfilled the criteria for RA set by the American College of Rheumatology classification in 1987 (Arnett et al., 1988) were consecutively recruited from the Changzhou Second Hospital — Affiliated Hospital of Nanjing Medical University, the Changzhou First Hospital, and the Changzhou Traditional Chinese Medical Hospital, between September 2010 and October 2013. The controls were patients without RA and other genetic diseases, matched for age (±5 years) and gender, who were recruited from the same institutions during the same time period; most of them had been admitted due to trauma. The study protocol was approved by the Ethics Committee of Nanjing Medical University (Nanjing, China). All patients provided written informed consent prior to participation. Each patient was then interviewed by trained personnel using a pre-tested questionnaire regarding his or her demographics and related risk factors for RA. After the interview, 2 ml of peripheral blood were collected from each subject. Blood samples were collected using vacutainers and transferred to test tubes containing ethylenediaminetetra-acetic acid (EDTA) using the QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany). The SNP genotyping was performed using a custom-by-design 48-Plex SNP scan™ Kit (GeneskyBiotechnologies Inc., Shanghai, China) as described previously (Zheng et al., 2013). 2.2. Statistical analyses Differences in demographics, variables, and genotypes of the IL17A rs2275913 G/A, rs3819024 A/G, rs3819025 G/A, rs4711998 A/G, rs8193036 C/T and rs8193037 G/A polymorphisms were evaluated using chi-squared tests. The associations between the six IL17A genotypes and RA risk were estimated by computing odds ratios (ORs) and 95% confidence intervals (CIs) using logistic regression analyses. The Hardy–Weinberg equilibrium (HWE) was tested using a goodness-offit chi-squared test to compare the observed genotype frequencies to the expected frequencies among controls. All statistical analyses were performed using the SAS software (version 9.1.3; SAS Institute, Cary, NC, USA). 3. Results 3.1. Characteristics of the study population The demographic and clinical characteristics of all subjects are summarized in Table 1. The age ranges from 19 to 90 years (mean 54.51 ± 15.19 years) in case group while 20 to 88 years (mean 55.44 ± 10.80 years) in control group. Subjects were adequately matched for age and sex (P = 0.170 and 0.566, respectively). The genotype distributions of IL17A rs2275913 G/A, rs3819024 A/G, rs3819025 G/A, rs4711998 A/G, rs8193036 C/T and rs8193037 G/A for all subjects are shown in Table 2. The primary information for six genotyped SNPs was in Table 3. For the six SNPs, the genotyping was successful ranging from 95.25% to 98.76% in all 1454 samples. Minor allele frequency (MAF) in our controls was similar to MAF for Chinese in database for all six SNPs (Table 3). The observed genotype frequencies for the polymorphisms in the controls were in HWE for IL17A rs3819024 A/G (P = 0.276), rs3819025 G/A (P = 0.461), rs4711998 A/G (P = 0.129), and rs8193037 G/A (P = 0.630), but not for rs2275913 G/A (P = 0.032) and rs8193036 C/T (P = 0.010) (Table 3).
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Table 1 Patient demographics and risk factors in rheumatoid arthritis, all subjects. Variable⁎
Cases (n = 615)
Controls (n = 839)
P
Age (years) Female/male Age at onset, years, mean ± SD Disease duration, years, mean ± SD Treatment duration, years, mean ± SD RF-positive, no. (%) ACPA positive, no. (%) CRP-positive, no. (%) ESR, mm/h DAS28 Functional class, no. (%) I II III IV
54.51 (±15.19) 472/143 46.06 (±13.24) 8.52 (±9.24) 7.30 (±7.91) 486 (79.02%) 321 (52.20%) 165 (26.83%) 35.79 (±28.70) 4.46 (±1.50)
55.44 (±10.80) 633/206 – – – – – – – – – – – – –
0.170 0.566 – – – – – – – – – – – – –
78 (12.68%) 281 (45.69%) 220 (35.77%) 36 (5.85%)
⁎ RF: Rheumatoid factor; ACPA: Anti-cyclic citrullinated peptide antibodies; CRP: C-reactive protein; ESR: Erythrocyte sedimentation rate; DAS28: RA disease activity score.
3.2. Associations between IL17A rs2275913 G/A, rs3819024 A/G, rs3819025 G/A, rs4711998 A/G, rs8193036 C/T and rs8193037 G/A polymorphisms and RA risk Logistic regression analyses revealed that a significantly decreased RA susceptibility was associated with the IL17A rs2275913 AA genotypes (AA vs. GA/GG: OR, 0.77; 95% CI, 0.59–0.99, P = 0.043) and IL17A rs3819024 GG (GG vs. AA/AG: OR, 0.77; 95% CI, 0.60–1.00, P = 0.048), other significantly increased risks for RA associated with the IL17A rs3819025 GA or GA/AA genotypes (GA vs. GG: OR, 1.29; 95% CI, 1.02–1.63, P = 0.036; GA/AA vs. GG: OR, 1.29; 95% CI, 1.02–1.61, P = 0.030), and IL17A rs8193036 CT or CT/TT genotypes (CT vs. CC: OR, 1.36; 95% CI, 1.08–1.71, P = 0.008; CT/TT vs. CC: OR, 1.36; 95% CI, 1.10–1.68, P = 0.005). IL17A rs4711998 A/G and IL17A rs8193037 G/A polymorphisms were not associated with the RA risk (Table 2). 3.3. Stratification analyses of IL17A rs3819025 G/A and IL17A rs8193036 C/T polymorphisms and RA risk Stratification analyses were performed according to age, sex, rheumatoid factor (RF) status, disease activity score in 28 joints (DAS28) status, C-reactive protein (CRP) status, erythrocyte sedimentation rate (ESR) status, functional class, and anti-cyclic citrullinated peptide antibody (ACPA) status (Tables 4–5). The IL17A rs3819025 A allele was associated with a significantly increased RA risk, especially among female patients (GA vs. GG: OR, 1.35; 95% CI, 1.03–1.76, P = 0.027; GA/AA vs. GG: OR, 1.37; 95% CI, 1.06–1.77, P = 0.017), CRP-negative patients, ACPA-positive patients, RF-positive patients, and functional class I/II patients. The IL17A rs8193036 T allele were also correlated to a significantly increased RA risk, especially among younger patients (CT vs. CC: OR, 1.47; 95% CI, 1.05–2.07, P = 0.027; CT/TT vs. CC: OR, 1.42; 95% CI, 1.04–1.96, P = 0.030), CRP-positive patients, ACPA-negative patients, RF-positive patients, those with a DAS28 b3.20, and those with a functional class of I/II. 4. Discussion We investigated the association between the IL17A rs2275913 G/A, rs3819024 A/G, rs3819025 G/A, rs4711998 A/G, rs8193036 C/T and rs8193037 G/A polymorphisms and the RA risk in a Chinese population. We found that IL17A rs2275913 G/A and rs3819024 A/G variant alleles decrease the risk of RA, while IL17A rs3819025 G/A and rs8193036 C/T variant alleles increase the risk of RA. Further studies are warranted to determine which of these functional SNPs really play pivotal roles in RA and to elucidate the underlying mechanisms of action.
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L. Shen et al. / Gene 566 (2015) 18–22
Table 2 Logistic regression analysis of associations between IL17A rs2275913 G/A, rs3819024 A/G, rs3819025 G/A, rs4711998 A/G, rs8193036 C/T, rs8193037 G/A polymorphisms and risk of rheumatoid arthritis. Casesa (n = 615)
Genotype
rs2275913 G/A GA vs. GG AA vs. GG AA vs. GA AA vs. GA vs. GG GA+AA vs. GG AA vs. GG+GA A allele vs. G allele rs3819024 A/G AG vs. AA GG vs. AA GG vs. AG GG vs. AG vs. AA AG+GG vs. AA GG vs. AA+AG G allele vs. A allele rs3819025 G/A GA vs. GG AA vs. GG AA vs. GA AA vs. GA vs. GG GA+AA vs. GG AA vs. GG+GA A allele vs. G allele rs4711998 A/G AG vs. AA GG vs. AA GG vs. AG GG vs. AG vs. AA AG+GG vs. AA GG vs. AA+AG G allele vs. A allele rs8193036 C/T CT vs. CC TT vs. CC TT vs. CT TT vs. CT vs. CC CT+TT vs. CC TT vs. CC+CT T allele vs. C allele rs8193037 G/A GA vs. GG AA vs. GG AA vs. GA AA vs. GA vs. GG GA+AA vs. GG AA vs. GG+GA A allele vs. G allele
Controls (n = 839)
OR (95% CI)
P
46.0/30.7 23.3/30.7 23.3/46.0
0.98 (0.77–1.25) 0.76 (0.56–1.02) 0.77 (0.58–1.02)
577/255 194/638 771/893
69.4/30.7 23.3/76.7 46.3/53.7
0.91 (0.72–1.14) 0.77 (0.59–0.99) 0.88 (0.75–1.02)
0.881 0.066 0.065 0.127 0.391 0.043 0.080
50.6/29.3 20.1/29.3 20.1/50.6
399/227 204/227 204/399
48.1/27.3 24.6/27.3 24.6/48.1
0.98 (0.77–1.26) 0.77 (0.57–1.03) 0.78 (0.59–1.02)
425/176 121/480 546/656
70.7/29.3 20.1/79.9 45.4/54.6
603/227 204/626 807/853
72.7/27.3 24.6/75.4 48.6/51.4
0.91 (0.72–1.15) 0.77 (0.60–1.00) 0.88 (0.75–1.02)
181/404 19/404 19/181
30.0/66.9 3.1/66.9 3.1/30.0
209/600 22/600 22/209
25.2/72.3 2.7/72.3 2.7/25.2
1.29 (1.02–1.63) 1.28 (0.69–2.40) 1.00 (0.52–1.90)
200/404 19/585 219/989
33.1/66.9 3.1/96.9 18.1/81.9
231/600 22/809 253/1409
27.8/72.3 2.7/97.4 15.2/84.8
1.29 (1.02–1.61) 1.19 (0.64–2.23) 1.23 (1.01–1.50)
243/307 44/307 44/243
40.9/18.1 7.4/18.1 7.4/40.9
338/440 49/440 49/338
40.9/53.2 5.9/53.2 5.9/40.9
1.03 (0.83–1.28) 1.29 (0.84–1.98) 1.25 (0.81–1.94)
287/307 44/550 331/857
48.3/18.1 7.4/92.6 27.9/72.1
387/440 49/778 436/1218
46.8/53.2 5.9/94.1 26.4/73.6
1.06 (0.86–1.31) 1.27 (0.83–1.94) 1.08 (0.91–1.28)
234/284 55/284 55/234
40.8/49.6 9.6/49.6 9.6/40.8
281/464 67/464 67/281
34.6/57.1 8.3/57.1 8.3/34.6
1.36 (1.08–1.71) 1.34 (0.91–1.97) 0.99 (0.66–1.47)
289/284 55/518 344/802
50.4/49.6 9.6/90.4 30.0/70.0
348/464 67/745 415/1209
42.9/57.1 8.3/91.7 25.6/74.4
1.36 (1.10–1.68) 1.18 (0.81–1.72) 1.25 (1.06–1.48)
116/475 12/475 12/116
19.2/78.8 2.0/78.8 2.0/19.2
176/642 14/642 14/176
21.2/77.2 1.7/77.2 1.7/21.2
0.89 (0.69–1.16) 1.16 (0.53–2.53) 1.30 (0.58–2.91)
128/475 12/591 140/1066
21.2/78.8 2.0/98.0 11.6/88.4
190/642 14/818 204/1460
22.8/77.2 1.7/98.3 12.3/87.7
0.91 (0.71–1.17) 1.19 (0.55–2.58) 0.94 (0.75–1.18)
n
%
n
%
292/198 114/198 114/292
48.3/32.8 18.9/32.8 18.9/48.3
383/255 194/255 194/383
406/198 114/490 520/688
67.2/32.8 18.9/81.1 43.0/57.0
304/176 121/176 121/304
0.890 0.079 0.069 0.139 0.422 0.048 0.080
0.036 0.436 0.993 0.095 0.030 0.576 0.038
0.790 0.253 0.321 0.519 0.571 0.266 0.373
0.008 0.136 0.944 0.021 0.005 0.384 0.010
0.388 0.712 0.523 0.628 0.469 0.667 0.598
a The genotyping was successful in: 604 cases and 832 controls for rs2275913 G/A; 601 cases and 830 controls for rs3819024 A/G; 604 cases and 831 controls for rs3819025 G/A; 594 cases and 827 controls for rs4711998 A/G; 573 cases and 812 controls for rs8193036 C/T; and 603 cases and 832 controls for rs8193037 G/A.
Table 3 Primary information for six genotyped SNPs of IL17A. Genotyped SNPs
Chr
a
Regulome DB score
b
Location
c MAF for Chinese in database
MAF in our controls
P value for dHWE test in our controls
Genotyping value %
rs2275913 GNA rs3819024 ANG rs3819025 GNA rs4711998 ANG rs8193036 CNT rs8193037 GNA
6 6 6 6 6 6
No data No data 5 No data No data No data
Y Y Y Y Y Y
5′-Flanking 5′-Flanking Intron1 5′-Flanking 5′-Flanking 5′-Flanking
0.465 0.477 0.159 0.244 0.265 0.082
0.463 0.486 0.461 0.264 0.256 0.123
0.032 0.276 0.011 0.129 0.010 0.630
98.76 98.42 98.70 97.73 95.25 98.70
a b c d
TFBS
http://www.regulomedb.org/. TFBS: Transcription Factor Binding Site (http://snpinfo.niehs.nih.gov/snpinfo/snpfunc.htm). MAF: minor allele frequency. HWE: Hardy–Weinberg equilibrium.
L. Shen et al. / Gene 566 (2015) 18–22
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Table 4 Stratified analyses between IL-17A rs3819025 G/A polymorphisms and the risk of rheumatoid arthritis. Variable
IL-17A rs3819024 A/G (case/control)
OR (95% CI); P
GG
GA
AA
GA+AA
GA versus GG
AA versus GG
GA+AA versus GG
AA versus GA+GG
Sex Male Female
103/150 301/450
33/45 148/164
4/8 15/14
37/53 163/178
1.07 (0.64–1.79); 0.802 1.35 (1.03–1.76); 0.027
0.73 (0.21–2.48); 0.612 0.60 (0.76–3.37); 0.214
1.02 (0.62–1.66); 0.947 1.37 (1.06–1.77); 0.017
0.72 (0.21–0.43); 0.593 1.47 (0.70–3.07); 0.311
Age (years) b55 ≥55
184/262 220/338
90/95 91/114
7/5 12/17
97/100 103/131
1.35 (0.96–1.90); 0.089 1.23 (0.89–1.70); 0.217
1.99 (0.62–6.38); 0.245 1.08 (0.50–2.32); 0.834
1.38 (0.99–1.94); 0.066 1.21 (0.89–1.65); 0.231
1.82 (0.57–5.81); 0.309 1.03 (0.48–2.18); 0.947
CRP status Positive Negative
237/600 167/600
97/209 84/209
12/22 7/22
109/231 91/231
1.18 (0.89–1.56); 0.266 1.44 (1.06–1.96); 0.019
1.38 (0.67–2.84); 0.379 1.14 (0.48–2.72); 0.763
1.20 (0.91–1.57); 0.202 1.42 (1.05–1.91); 0.022
1.32 (0.65–2.70); 0.445 1.03 (0.43–2.43); 0.954
ACPA status Positive Negative
207/600 197/600
99/209 82/209
10/22 9/22
109/231 91/231
1.37 (1.03–1.83); 0.030 1.20 (0.88–1.68); 0.248
1.32 (0.61–2.83); 0.479 1.25 (0.56–2.75); 0.586
1.37 (1.04–1.81); 0.027 1.20 (0.90–1.61); 0.220
1.20 (0.56–2.57); 0.635 1.19 (0.54–2.61); 0.671
RF status Positive Negative
316/600 88/600
147/209 34/209
16/22 3/22
163/231 37/231
1.34 (1.04–1.72); 0.024 1.11 (0.72–1.70); 0.634
1.38 (0.72–2.67); 0.337 0.93 (0.27–3.17); 0.907
1.34 (1.05–1.71); 0.018 1.9 (0.72–1.65); 0.676
1.27 (0.66–2.44); 0.473 0.90 (0.27–3.07); 0.872
ESR (mm/h) b25.00 ≥25.00
181/600 223/600
82/209 99/209
9/22 10/22
91/231 109/231
1.30 (0.96–1.76); 0.091 1.27 (0.96–1.69); 0.095
1.36 (0.61–3.00); 0.452 1.22 (0.57–2.63); 0.605
1.31 (0.97–1.75); 0.075 1.27 (0.97–1.67); 0.089
1.26 (0.57–2.77); 0.568 1.14 (0.54–2.44); 0.732
DAS28 b3.20 ≥3.20
84/600 320/600
44/209 137/209
3/22 16/22
47/231 153/231
1.50 (1.01–2.24); 0.044 1.23 (0.95–1.59); 0.112
0.97 (0.29–3.33); 0.967 1.36 (0.71–2.63); 0.356
1.45 (0.99–2.14); 0.059 1.24 (0.97–1.59); 0.083
0.86 (0.25–2.92); 0.811 1.29 (0.67–2.48); 0.449
Functional class I+II 229/600 III+IV 175/600
111/209 70/209
14/22 5/22
125/231 75/231
1.39 (1.06–1.83); 0.019 1.15 (0.84–1.58); 0.395
1.67 (0.84–3.32); 0.145 0.78 (0.29–2.09); 0.620
1.42 (1.09–1.85); 0.010 1.11 (0.82–1.52); 0.498
1.51 (0.77–3.00); 0.233 0.75 (0.28–2.00); 0.567
Bold values are statistically significant (P b 0.05).
IL17A, the key cytokine of Th17 cells, can induce pro-inflammatory cytokines and promote rheumatoid arthritis (Gaffen, 2009). IL17A is produced by many cell types, including Th17 cells, CD8+ T cells, and
γδ T cells, and is elevated along with numbers of mast cells and neutrophils at sites of skin and joint diseases in humans (Kirkham et al., 2014). IL17A, along with TNF-α, was found to be a predictor of a poor outcome
Table 5 Stratified analyses between IL-17A rs8193036 C/T polymorphisms and the risk of rheumatoid arthritis. Variable
IL-17A rs8193036/(case/control)
OR (95% CI); P
CC
CT
TT
CT+TT
CT versus CC
TT versus CC
CT+TT versus CC
TT versus CT+CC
Sex Male Female
63/108 221/356
60/64 174/217
7/21 48/46
67/85 222/263
1.61 (1.01–2.57); 0.048 1.29 (1.00–1.68); 0.054
0.57 (0.23–1.42); 0.228 1.68 (1.09–2.60); 0.020
1.35 (0.87–2.11); 0.186 1.36 (1.06–1.74); 0.014
0.67 (0.19–1.13); 0.091 1.51 (0.99–2.31); 0.056
Age (years) b55 ≥55
132/203 152/261
110/115 124/166
28/34 27/33
138/149 151/199
1.47 (1.05–2.07); 0.027 1.28 (0.94–1.74); 0.112
1.27 (0.73–2.19); 0.397 1.41 (0.81–2.43); 0.223
1.42 (1.04–1.96); 0.030 1.30 (0.97–1.74); 0.075
1.08 (0.64–1.83); 0.768 1.27 (0.75–2.15); 0.384
CRP status Positive Negative
155/464 129/464
138/281 96/281
31/67 24/67
169/348 120/348
1.47 (1.12–1.93); 0.006 1.23 (0.91–1.66); 0.182
1.39 (0.87–2.20); 0.168 1.29 (0.78–2.14); 0.326
1.45 (1.12–1.88); 0.005 1.24 (0.93–1.64); 0.138
1.18 (0.73–1.84); 0.476 1.19 (0.73–1.94); 0.494
ACPA status Positive Negative
149/464 118/464
183/281 96/281
5/67 24/67
188/348 120/348
1.30 (0.98–1.72); 0.073 1.43 (1.07–1.91); 0.015
1.30 (0.81–2.10); 0.280 1.39 (0.85–2.25); 0.189
1.30 (0.99–1.70); 0.056 1.42 (1.08–1.87); 0.011
1.17 (0.74–1.86); 0.505 1.19 (0.75–1.90); 0.464
RF status Positive Negative
222/464 62/464
184/281 50/281
48/67 7/67
232/348 57/348
1.37 (1.07–1.75); 0.012 1.33 (0.89–1.99); 0.161
1.50 (1.00–2.24); 0.050 0.78 (0.34–1.78); 0.559
1.39 (1.11–1.76); 0.005 1.23 (0.83–1.80); 0.301
1.32 (0.89–1.94); 0.669 0.70 (0.31–1.55); 0.376
ESR (mm/h) b25.00 136/464 ≥25.00 148/464
109/281 125/281
18/67 37/67
127/348 162/348
1.32 (0.99–1.77); 0.060 1.40 (1.05–1.85); 0.020
0.92 (0.53–1.60); 0.758 1.73 (1.11–2.69); 0.015
1.25 (0.94–1.65); 0.124 1.46 (1.12–1.90); 0.005
0.82 (0.48–1.40); 0.463 1.51 (0.99–2.31); 0.058
DAS28 b3.20 ≥3.20
57/464 227/464
60/281 174/281
11/67 44/67
71/348 218/348
1.74 (1.18–2.57); 0.006 1.27 (0.99–1.62); 0.061
1.34 (0.67–2.68); 0.413 1.34 (0.89–2.03); 0.161
1.66 (1.14–2.42); 0.008 1.28 (1.02–1.62); 0.037
1.05 (0.54–2.04); 0.895 1.22 (0.82–1.82); 0.329
Functional class I+II 162/464 III+IV 122/464
148/281 86/281
31/67 24/67
179/348 110/348
1.51 (1.15–1.97); 0.003 1.16 (0.85–1.59); 0.342
1.33 (0.84–2.10); 0.232 1.36 (0.82–2.26); 0.232
1.47 (1.14–1.90); 0.003 1.20 (0.90–1.61); 0.218
1.11 (0.71–1.74); 0.641 1.28 (0.79–2.10); 0.320
Bold values are statistically significant (P b 0.05).
22
L. Shen et al. / Gene 566 (2015) 18–22
in RA cases (Kirkham et al., 2006). Fortunately, therapies targeting IL-17 in autoimmune diseases ameliorated the inadequate response to antiTNF-α therapy (Yan et al., 2014). Studies have shown that IL17A polymorphisms are associated with an increased risk of gastric cancer, breast cancer and cervical cancer (Shibata et al., 2009; Quan et al., 2012; Wang et al., 2012; Han et al., 2014). In a recent study, Han et al. showed that polymorphisms of IL17A G-197A may be closely associated with susceptibility to osteoarthritis in the Korean population (Han et al., 2014). A recent Japanese study reported a weak association between an IL17A intronic SNP, rs3804513, and radiographic progression of RA at 2 years (P = 0.049), which indicated that IL-17 might be associated with joint destruction in Japanese patients with early RA (Furuya et al., 2007). IL17A rs3804513 MAF was 0.131 in this Japanese study and no linkage disequilibrium (LD) relation was found between IL17A rs3804513 and other SNPs (Furuya et al., 2007). However, in our present study, we found that IL17A rs3819025 and rs8193036 polymorphisms increased the RA risk among functional class I/II patients but not functional class III + IV patients. Nordang et al. reported an association between IL17A SNPs and RA in Caucasian patients from Norway and New Zealand. They found a modest association between RA prevalence and rs2275913 in the IL17A gene among Norwegian RA patients; however, their findings were not replicated in an independent New Zealand RA cohort (Nordang et al., 2009). MAF of IL17A rs2275913 was 0.463 in our study and 0.385 in New Zealand and Norwegian studies (Nordang et al., 2009). In our study, we discovered a significantly decreased risk of RA associated with the IL17A rs2275913 AA genotypes (AA vs. GA/GG: OR, 0.77; 95% CI, 0.59–0.99, P = 0.043). Our study also revealed that the rs3819024 and rs8193036 polymorphisms in the IL17A gene are related to RA; in contrast, Nordang et al. stated that rs3819024 and rs8193036 polymorphisms in the IL17A gene were not associated with RA in Norwegian patients. Several limitations of our study should be addressed. First, as a hospital-based case–control study, the participants were not fully representative of the general population. Second, the polymorphisms investigated, based on their functional considerations, might not offer a comprehensive view of IL17A genetic variability, necessitating further fine-mapping studies. Third, the sample size of this study was not sufficiently large to evaluate the low penetrance effect of the SNPs. Fourth, the observed genotype frequencies for IL17A rs2275913 G/A and IL17A rs8193036 C/T polymorphisms in the controls did not conform to HWE. In conclusion, the present study demonstrated that the IL17A rs2275913 G/A variant allele and IL17A rs3819024 A/G decrease the RA risk, whereas the IL17A rs3819025 G/A and IL17A rs8193036 C/T variant alleles increase the RA risk. However, our results were obtained from only a moderate-sized sample. Larger replication studies from more diverse ethnic populations are needed to confirm these findings. Declaration of interest The authors have no declarations of interest. Acknowledgments This study was supported in part by National Natural Science Foundation of China (81371927) and Changzhou Foundation for Applied Basic Research (CJ20130027).
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Garcia-Bermudez, M., Lopez-Mejias, R., Gonzalez-Juanatey, C., Castaneda, S., MirandaFilloy, J.A., Blanco, R., Fernandez-Gutierrez, B., Balsa, A., Gonzalez-Alvaro, I., GomezVaquero, C., Llorca, J., Martin, J., Gonzalez-Gay, M.A., 2012. Association of the methionine sulfoxide reductase A rs10903323 gene polymorphism with cardiovascular disease in patients with rheumatoid arthritis. Scand. J. Rheumatol. 41, 350–353. Han, L., Lee, H.S., Yoon, J.H., Choi, W.S., Park, Y.G., Nam, S.W., Lee, J.Y., Park, W.S., 2014. Association of IL-17A and IL-17 F single nucleotide polymorphisms with susceptibility to osteoarthritis in a Korean population. Gene 533, 119–122. Honorati, M.C., Meliconi, R., Pulsatelli, L., Cane, S., Frizziero, L., Facchini, A., 2001. High in vivo expression of interleukin-17 receptor in synovial endothelial cells and chondrocytes from arthritis patients. Rheumatology (Oxford) 40, 522–527. Kirkham, B.W., Lassere, M.N., Edmonds, J.P., Juhasz, K.M., Bird, P.A., Lee, C.S., Shnier, R., Portek, I.J., 2006. Synovial membrane cytokine expression is predictive of joint damage progression in rheumatoid arthritis: a two-year prospective study (the DAMAGE study cohort). Arthritis Rheum. 54, 1122–1131. Kirkham, B.W., Kavanaugh, A., Reich, K., 2014. Interleukin-17A: a unique pathway in immune-mediated diseases: psoriasis, psoriatic arthritis and rheumatoid arthritis. Immunology 141, 133–142. Korn, T., Bettelli, E., Oukka, M., Kuchroo, V.K., 2009. IL-17 and Th17 cells. Annu. Rev. Immunol. 27, 485–517. Kotake, S., Udagawa, N., Takahashi, N., Matsuzaki, K., Itoh, K., Ishiyama, S., Saito, S., Inoue, K., Kamatani, N., Gillespie, M.T., Martin, T.J., Suda, T., 1999. IL-17 in synovial fluids from patients with rheumatoid arthritis is a potent stimulator of osteoclastogenesis. J. Clin. Invest. 103, 1345–1352. Lubberts, E., 2008. IL-17/Th17 targeting: on the road to prevent chronic destructive arthritis? Cytokine 41, 84–91. Nakae, S., Nambu, A., Sudo, K., Iwakura, Y., 2003. Suppression of immune induction of collagen-induced arthritis in IL-17-deficient mice. J. Immunol. 171, 6173–6177. Nordang, G.B., Viken, M.K., Hollis-Moffatt, J.E., Merriman, T.R., Forre, O.T., Helgetveit, K., Kvien, T.K., Lie, B.A., 2009. Association analysis of the interleukin 17A gene in Caucasian rheumatoid arthritis patients from Norway and New Zealand. Rheumatology (Oxford) 48, 367–370. Pope, R.M., Shahrara, S., 2013. Possible roles of IL-12-family cytokines in rheumatoid arthritis. Nat. Rev. Rheumatol. 9, 252–256. Quan, Y., Zhou, B., Wang, Y., Duan, R., Wang, K., Gao, Q., Shi, S., Song, Y., Zhang, L., Xi, M., 2012. Association between IL17 polymorphisms and risk of cervical cancer in Chinese women. Clin. Dev. Immunol. 2012, 258293. Ren, Z., Li, M., Liu, R., Wang, Y., Gu, H., 2014. Interleukin 17A rs3819024 A N G polymorphism is associated with an increased risk of gastric cardia adenocarcinoma in a Chinese population. Biomarkers 1–6. Ruddy, M.J., Wong, G.C., Liu, X.K., Yamamoto, H., Kasayama, S., Kirkwood, K.L., Gaffen, S.L., 2004. Functional cooperation between interleukin-17 and tumor necrosis factoralpha is mediated by CCAAT/enhancer-binding protein family members. J. Biol. Chem. 279, 2559–2567. Shibata, T., Tahara, T., Hirata, I., Arisawa, T., 2009. Genetic polymorphism of interleukin17A and -17 F genes in gastric carcinogenesis. Hum. Immunol. 70, 547–551. Wang, L., Jiang, Y., Zhang, Y., Wang, Y., Huang, S., Wang, Z., Tian, B., Yang, Y., Jiang, W., Pang, D., 2012. Association analysis of IL-17A and IL-17 F polymorphisms in Chinese Han women with breast cancer. PLoS One 7, e34400. Yan, J.W., Wang, Y.J., Peng, W.J., Tao, J.H., Wan, Y.N., Li, B.Z., Mei, B., Chen, B., Yao, H., Yang, G.J., Li, X.P., Ye, D.Q., Wang, J., 2014. Therapeutic potential of interleukin-17 in inflammation and autoimmune diseases. Expert Opin. Ther. Targets 18, 29–41. Zheng, L., Yin, J., Wang, L., Wang, X., Shi, Y., Shao, A., Tang, W., Ding, G., Liu, C., Chen, S., Gu, H., 2013. Interleukin 1B rs16944 GNA polymorphism was associated with a decreased risk of esophageal cancer in a Chinese population. Clin. Biochem. 46, 1469–1473. Ziolkowska, M., Koc, A., Luszczykiewicz, G., Ksiezopolska-Pietrzak, K., Klimczak, E., Chwalinska-Sadowska, H., Maslinski, W., 2000. High levels of IL-17 in rheumatoid arthritis patients: IL-15 triggers in vitro IL-17 production via cyclosporin A-sensitive mechanism. J. Immunol. 164, 2832–2838.
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Research paper
Association between interleukin 17A polymorphisms and susceptibility to rheumatoid arthritis in a Chinese population☆ Li Shen a,1, Hui Zhang b,1, Ting Yan b, Guoxin Zhou b, Ruiping Liu b,c,⁎ a b c
Department of Clinical Laboratory, Changzhou First People's Hospital, Changzhou 213003, China Department of Orthopaedic Trauma, Affiliated Hospital of Nanjing Medical University, Changzhou Second People's Hospital, Changzhou 213003, China Central Laboratory, Changzhou Second People's Hospital, Affiliated Hospital of Nanjing Medical University, Changzhou 213003, China
a r t i c l e
i n f o
Article history: Received 8 October 2014 Received in revised form 2 April 2015 Accepted 8 April 2015 Available online 11 April 2015 Keywords: Interleukin 17A Polymorphisms Rheumatoid arthritis Molecular epidemiology
a b s t r a c t Background: Previous studies have revealed an association between interleukin 17A (IL17A) polymorphisms and the prevalence of rheumatoid arthritis (RA) in Japanese and Caucasian patients. We hypothesized that IL17A polymorphisms might also affect RA susceptibility in the Chinese population. Methods: We studied IL17A rs2275913 G/A, rs3819024 A/G, rs3819025 G/A, rs4711998 A/G, rs8193036 C/T and rs8193037 G/A polymorphisms in 615 RA patients and 839 controls in a Chinese population. Genotyping was performed using a custom-by-design 48-Plex SNP scan™ Kit. Results: Our results indicated that IL17A rs4711998 A/G and IL17A rs8193037 G/A polymorphisms were not associated with RA, and IL17A rs2275913 G/A and IL17A rs3819024 A/G variant alleles decrease the risk of RA, while IL17A rs3819025 G/A and IL17A rs8193036 C/T variant alleles increase the risk of RA. Conclusions: These findings suggest that IL17A polymorphisms may be associated with RA. Future larger studies with other ethnic populations are required to confirm current findings. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Although the etiology of rheumatoid arthritis (RA) is not fully understood, it is believed to result from complex genetic and environmental factors, which trigger and maintain synovial inflammation in affected individuals (Nordang et al., 2009). Genetic factors such as single nucleotide polymorphisms (SNPs) might play important roles in RA pathogenesis (Garcia-Bermudez et al., 2012). Interleukin 17A (IL17A), also known as IL-17, is the signature T helper 17 (Th17) effector cytokine (Korn et al., 2009). IL17A leads to the induction of many factors, including tumor necrosis factor-alpha (TNF-α), IL-6, and IL-1β, which are important in inflammatory processes (Ruddy et al., 2004).
Abbreviations:CI, confidence interval;IL17A, interleukin17A; LD, linkage disequilibrium; OR, odds ratio; SNP, single nucleotide polymorphism. ☆ Condensed abstract: Interleukin 17A polymorphisms are associated with rheumatoid arthritis susceptibility. ⁎ Corresponding author at: Department of Orthopaedic Trauma, Changzhou Second People's Hospital, Changzhou 213003, China. E-mail address: [email protected] (R. Liu). 1 These authors contributed equally to this study.
http://dx.doi.org/10.1016/j.gene.2015.04.028 0378-1119/© 2015 Elsevier B.V. All rights reserved.
IL17A is also produced by many other cell types, including CD8+ T cells and γδ T cells, and is increased locally in the skin and joint diseases in human (Kirkham et al., 2014). High levels of IL17A and its receptor are found in RA synovial fluid and tissue explants, and IL17A promotes joint degradation in ex vivo models (Kotake et al., 1999; Ziolkowska et al., 2000; Cai et al., 2001; Honorati et al., 2001). In RA, leukocyte migration, bone erosions, and angiogenesis are modulated by the IL-23-IL17 cascade (Pope and Shahrara, 2013). Studies of IL-17-deficient mice have suggested that IL17A is crucial in the development of collagen-induced arthritis due to activation of autoantigenspecific cellular and humoral immune responses (Nakae et al., 2003). Accordingly, IL17A and other Th17 related cytokines are considered prime targets of anti-cytokine therapy in autoimmune arthritis (Lubberts, 2008). The IL17A gene is located on Chromosome 6, and the association between its SNPs and certain diseases has been studied recently. Ren et al. previously investigated six functional SNPs of the IL17A gene in gastric cardiac adenocarcinoma (GCA) in a hospital-based case–control study of 243 GCA cases and 476 controls. Their results suggested that an IL17A rs3819024 A/G polymorphism might increase the GCA risk (Ren et al., 2014). Functional variations in the IL17A gene might contribute to the etiology of RA, and may even promote or protect against RA in certain ethnic
L. Shen et al. / Gene 566 (2015) 18–22
groups. To investigate that hypothesis, the current hospital-based case– control genotype study was performed with a cohort of 615 Chinese RA patients and 839 Chinese controls. 2. Patients and methods 2.1. Study subjects A total of 615 RA patients who fulfilled the criteria for RA set by the American College of Rheumatology classification in 1987 (Arnett et al., 1988) were consecutively recruited from the Changzhou Second Hospital — Affiliated Hospital of Nanjing Medical University, the Changzhou First Hospital, and the Changzhou Traditional Chinese Medical Hospital, between September 2010 and October 2013. The controls were patients without RA and other genetic diseases, matched for age (±5 years) and gender, who were recruited from the same institutions during the same time period; most of them had been admitted due to trauma. The study protocol was approved by the Ethics Committee of Nanjing Medical University (Nanjing, China). All patients provided written informed consent prior to participation. Each patient was then interviewed by trained personnel using a pre-tested questionnaire regarding his or her demographics and related risk factors for RA. After the interview, 2 ml of peripheral blood were collected from each subject. Blood samples were collected using vacutainers and transferred to test tubes containing ethylenediaminetetra-acetic acid (EDTA) using the QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany). The SNP genotyping was performed using a custom-by-design 48-Plex SNP scan™ Kit (GeneskyBiotechnologies Inc., Shanghai, China) as described previously (Zheng et al., 2013). 2.2. Statistical analyses Differences in demographics, variables, and genotypes of the IL17A rs2275913 G/A, rs3819024 A/G, rs3819025 G/A, rs4711998 A/G, rs8193036 C/T and rs8193037 G/A polymorphisms were evaluated using chi-squared tests. The associations between the six IL17A genotypes and RA risk were estimated by computing odds ratios (ORs) and 95% confidence intervals (CIs) using logistic regression analyses. The Hardy–Weinberg equilibrium (HWE) was tested using a goodness-offit chi-squared test to compare the observed genotype frequencies to the expected frequencies among controls. All statistical analyses were performed using the SAS software (version 9.1.3; SAS Institute, Cary, NC, USA). 3. Results 3.1. Characteristics of the study population The demographic and clinical characteristics of all subjects are summarized in Table 1. The age ranges from 19 to 90 years (mean 54.51 ± 15.19 years) in case group while 20 to 88 years (mean 55.44 ± 10.80 years) in control group. Subjects were adequately matched for age and sex (P = 0.170 and 0.566, respectively). The genotype distributions of IL17A rs2275913 G/A, rs3819024 A/G, rs3819025 G/A, rs4711998 A/G, rs8193036 C/T and rs8193037 G/A for all subjects are shown in Table 2. The primary information for six genotyped SNPs was in Table 3. For the six SNPs, the genotyping was successful ranging from 95.25% to 98.76% in all 1454 samples. Minor allele frequency (MAF) in our controls was similar to MAF for Chinese in database for all six SNPs (Table 3). The observed genotype frequencies for the polymorphisms in the controls were in HWE for IL17A rs3819024 A/G (P = 0.276), rs3819025 G/A (P = 0.461), rs4711998 A/G (P = 0.129), and rs8193037 G/A (P = 0.630), but not for rs2275913 G/A (P = 0.032) and rs8193036 C/T (P = 0.010) (Table 3).
19
Table 1 Patient demographics and risk factors in rheumatoid arthritis, all subjects. Variable⁎
Cases (n = 615)
Controls (n = 839)
P
Age (years) Female/male Age at onset, years, mean ± SD Disease duration, years, mean ± SD Treatment duration, years, mean ± SD RF-positive, no. (%) ACPA positive, no. (%) CRP-positive, no. (%) ESR, mm/h DAS28 Functional class, no. (%) I II III IV
54.51 (±15.19) 472/143 46.06 (±13.24) 8.52 (±9.24) 7.30 (±7.91) 486 (79.02%) 321 (52.20%) 165 (26.83%) 35.79 (±28.70) 4.46 (±1.50)
55.44 (±10.80) 633/206 – – – – – – – – – – – – –
0.170 0.566 – – – – – – – – – – – – –
78 (12.68%) 281 (45.69%) 220 (35.77%) 36 (5.85%)
⁎ RF: Rheumatoid factor; ACPA: Anti-cyclic citrullinated peptide antibodies; CRP: C-reactive protein; ESR: Erythrocyte sedimentation rate; DAS28: RA disease activity score.
3.2. Associations between IL17A rs2275913 G/A, rs3819024 A/G, rs3819025 G/A, rs4711998 A/G, rs8193036 C/T and rs8193037 G/A polymorphisms and RA risk Logistic regression analyses revealed that a significantly decreased RA susceptibility was associated with the IL17A rs2275913 AA genotypes (AA vs. GA/GG: OR, 0.77; 95% CI, 0.59–0.99, P = 0.043) and IL17A rs3819024 GG (GG vs. AA/AG: OR, 0.77; 95% CI, 0.60–1.00, P = 0.048), other significantly increased risks for RA associated with the IL17A rs3819025 GA or GA/AA genotypes (GA vs. GG: OR, 1.29; 95% CI, 1.02–1.63, P = 0.036; GA/AA vs. GG: OR, 1.29; 95% CI, 1.02–1.61, P = 0.030), and IL17A rs8193036 CT or CT/TT genotypes (CT vs. CC: OR, 1.36; 95% CI, 1.08–1.71, P = 0.008; CT/TT vs. CC: OR, 1.36; 95% CI, 1.10–1.68, P = 0.005). IL17A rs4711998 A/G and IL17A rs8193037 G/A polymorphisms were not associated with the RA risk (Table 2). 3.3. Stratification analyses of IL17A rs3819025 G/A and IL17A rs8193036 C/T polymorphisms and RA risk Stratification analyses were performed according to age, sex, rheumatoid factor (RF) status, disease activity score in 28 joints (DAS28) status, C-reactive protein (CRP) status, erythrocyte sedimentation rate (ESR) status, functional class, and anti-cyclic citrullinated peptide antibody (ACPA) status (Tables 4–5). The IL17A rs3819025 A allele was associated with a significantly increased RA risk, especially among female patients (GA vs. GG: OR, 1.35; 95% CI, 1.03–1.76, P = 0.027; GA/AA vs. GG: OR, 1.37; 95% CI, 1.06–1.77, P = 0.017), CRP-negative patients, ACPA-positive patients, RF-positive patients, and functional class I/II patients. The IL17A rs8193036 T allele were also correlated to a significantly increased RA risk, especially among younger patients (CT vs. CC: OR, 1.47; 95% CI, 1.05–2.07, P = 0.027; CT/TT vs. CC: OR, 1.42; 95% CI, 1.04–1.96, P = 0.030), CRP-positive patients, ACPA-negative patients, RF-positive patients, those with a DAS28 b3.20, and those with a functional class of I/II. 4. Discussion We investigated the association between the IL17A rs2275913 G/A, rs3819024 A/G, rs3819025 G/A, rs4711998 A/G, rs8193036 C/T and rs8193037 G/A polymorphisms and the RA risk in a Chinese population. We found that IL17A rs2275913 G/A and rs3819024 A/G variant alleles decrease the risk of RA, while IL17A rs3819025 G/A and rs8193036 C/T variant alleles increase the risk of RA. Further studies are warranted to determine which of these functional SNPs really play pivotal roles in RA and to elucidate the underlying mechanisms of action.
20
L. Shen et al. / Gene 566 (2015) 18–22
Table 2 Logistic regression analysis of associations between IL17A rs2275913 G/A, rs3819024 A/G, rs3819025 G/A, rs4711998 A/G, rs8193036 C/T, rs8193037 G/A polymorphisms and risk of rheumatoid arthritis. Casesa (n = 615)
Genotype
rs2275913 G/A GA vs. GG AA vs. GG AA vs. GA AA vs. GA vs. GG GA+AA vs. GG AA vs. GG+GA A allele vs. G allele rs3819024 A/G AG vs. AA GG vs. AA GG vs. AG GG vs. AG vs. AA AG+GG vs. AA GG vs. AA+AG G allele vs. A allele rs3819025 G/A GA vs. GG AA vs. GG AA vs. GA AA vs. GA vs. GG GA+AA vs. GG AA vs. GG+GA A allele vs. G allele rs4711998 A/G AG vs. AA GG vs. AA GG vs. AG GG vs. AG vs. AA AG+GG vs. AA GG vs. AA+AG G allele vs. A allele rs8193036 C/T CT vs. CC TT vs. CC TT vs. CT TT vs. CT vs. CC CT+TT vs. CC TT vs. CC+CT T allele vs. C allele rs8193037 G/A GA vs. GG AA vs. GG AA vs. GA AA vs. GA vs. GG GA+AA vs. GG AA vs. GG+GA A allele vs. G allele
Controls (n = 839)
OR (95% CI)
P
46.0/30.7 23.3/30.7 23.3/46.0
0.98 (0.77–1.25) 0.76 (0.56–1.02) 0.77 (0.58–1.02)
577/255 194/638 771/893
69.4/30.7 23.3/76.7 46.3/53.7
0.91 (0.72–1.14) 0.77 (0.59–0.99) 0.88 (0.75–1.02)
0.881 0.066 0.065 0.127 0.391 0.043 0.080
50.6/29.3 20.1/29.3 20.1/50.6
399/227 204/227 204/399
48.1/27.3 24.6/27.3 24.6/48.1
0.98 (0.77–1.26) 0.77 (0.57–1.03) 0.78 (0.59–1.02)
425/176 121/480 546/656
70.7/29.3 20.1/79.9 45.4/54.6
603/227 204/626 807/853
72.7/27.3 24.6/75.4 48.6/51.4
0.91 (0.72–1.15) 0.77 (0.60–1.00) 0.88 (0.75–1.02)
181/404 19/404 19/181
30.0/66.9 3.1/66.9 3.1/30.0
209/600 22/600 22/209
25.2/72.3 2.7/72.3 2.7/25.2
1.29 (1.02–1.63) 1.28 (0.69–2.40) 1.00 (0.52–1.90)
200/404 19/585 219/989
33.1/66.9 3.1/96.9 18.1/81.9
231/600 22/809 253/1409
27.8/72.3 2.7/97.4 15.2/84.8
1.29 (1.02–1.61) 1.19 (0.64–2.23) 1.23 (1.01–1.50)
243/307 44/307 44/243
40.9/18.1 7.4/18.1 7.4/40.9
338/440 49/440 49/338
40.9/53.2 5.9/53.2 5.9/40.9
1.03 (0.83–1.28) 1.29 (0.84–1.98) 1.25 (0.81–1.94)
287/307 44/550 331/857
48.3/18.1 7.4/92.6 27.9/72.1
387/440 49/778 436/1218
46.8/53.2 5.9/94.1 26.4/73.6
1.06 (0.86–1.31) 1.27 (0.83–1.94) 1.08 (0.91–1.28)
234/284 55/284 55/234
40.8/49.6 9.6/49.6 9.6/40.8
281/464 67/464 67/281
34.6/57.1 8.3/57.1 8.3/34.6
1.36 (1.08–1.71) 1.34 (0.91–1.97) 0.99 (0.66–1.47)
289/284 55/518 344/802
50.4/49.6 9.6/90.4 30.0/70.0
348/464 67/745 415/1209
42.9/57.1 8.3/91.7 25.6/74.4
1.36 (1.10–1.68) 1.18 (0.81–1.72) 1.25 (1.06–1.48)
116/475 12/475 12/116
19.2/78.8 2.0/78.8 2.0/19.2
176/642 14/642 14/176
21.2/77.2 1.7/77.2 1.7/21.2
0.89 (0.69–1.16) 1.16 (0.53–2.53) 1.30 (0.58–2.91)
128/475 12/591 140/1066
21.2/78.8 2.0/98.0 11.6/88.4
190/642 14/818 204/1460
22.8/77.2 1.7/98.3 12.3/87.7
0.91 (0.71–1.17) 1.19 (0.55–2.58) 0.94 (0.75–1.18)
n
%
n
%
292/198 114/198 114/292
48.3/32.8 18.9/32.8 18.9/48.3
383/255 194/255 194/383
406/198 114/490 520/688
67.2/32.8 18.9/81.1 43.0/57.0
304/176 121/176 121/304
0.890 0.079 0.069 0.139 0.422 0.048 0.080
0.036 0.436 0.993 0.095 0.030 0.576 0.038
0.790 0.253 0.321 0.519 0.571 0.266 0.373
0.008 0.136 0.944 0.021 0.005 0.384 0.010
0.388 0.712 0.523 0.628 0.469 0.667 0.598
a The genotyping was successful in: 604 cases and 832 controls for rs2275913 G/A; 601 cases and 830 controls for rs3819024 A/G; 604 cases and 831 controls for rs3819025 G/A; 594 cases and 827 controls for rs4711998 A/G; 573 cases and 812 controls for rs8193036 C/T; and 603 cases and 832 controls for rs8193037 G/A.
Table 3 Primary information for six genotyped SNPs of IL17A. Genotyped SNPs
Chr
a
Regulome DB score
b
Location
c MAF for Chinese in database
MAF in our controls
P value for dHWE test in our controls
Genotyping value %
rs2275913 GNA rs3819024 ANG rs3819025 GNA rs4711998 ANG rs8193036 CNT rs8193037 GNA
6 6 6 6 6 6
No data No data 5 No data No data No data
Y Y Y Y Y Y
5′-Flanking 5′-Flanking Intron1 5′-Flanking 5′-Flanking 5′-Flanking
0.465 0.477 0.159 0.244 0.265 0.082
0.463 0.486 0.461 0.264 0.256 0.123
0.032 0.276 0.011 0.129 0.010 0.630
98.76 98.42 98.70 97.73 95.25 98.70
a b c d
TFBS
http://www.regulomedb.org/. TFBS: Transcription Factor Binding Site (http://snpinfo.niehs.nih.gov/snpinfo/snpfunc.htm). MAF: minor allele frequency. HWE: Hardy–Weinberg equilibrium.
L. Shen et al. / Gene 566 (2015) 18–22
21
Table 4 Stratified analyses between IL-17A rs3819025 G/A polymorphisms and the risk of rheumatoid arthritis. Variable
IL-17A rs3819024 A/G (case/control)
OR (95% CI); P
GG
GA
AA
GA+AA
GA versus GG
AA versus GG
GA+AA versus GG
AA versus GA+GG
Sex Male Female
103/150 301/450
33/45 148/164
4/8 15/14
37/53 163/178
1.07 (0.64–1.79); 0.802 1.35 (1.03–1.76); 0.027
0.73 (0.21–2.48); 0.612 0.60 (0.76–3.37); 0.214
1.02 (0.62–1.66); 0.947 1.37 (1.06–1.77); 0.017
0.72 (0.21–0.43); 0.593 1.47 (0.70–3.07); 0.311
Age (years) b55 ≥55
184/262 220/338
90/95 91/114
7/5 12/17
97/100 103/131
1.35 (0.96–1.90); 0.089 1.23 (0.89–1.70); 0.217
1.99 (0.62–6.38); 0.245 1.08 (0.50–2.32); 0.834
1.38 (0.99–1.94); 0.066 1.21 (0.89–1.65); 0.231
1.82 (0.57–5.81); 0.309 1.03 (0.48–2.18); 0.947
CRP status Positive Negative
237/600 167/600
97/209 84/209
12/22 7/22
109/231 91/231
1.18 (0.89–1.56); 0.266 1.44 (1.06–1.96); 0.019
1.38 (0.67–2.84); 0.379 1.14 (0.48–2.72); 0.763
1.20 (0.91–1.57); 0.202 1.42 (1.05–1.91); 0.022
1.32 (0.65–2.70); 0.445 1.03 (0.43–2.43); 0.954
ACPA status Positive Negative
207/600 197/600
99/209 82/209
10/22 9/22
109/231 91/231
1.37 (1.03–1.83); 0.030 1.20 (0.88–1.68); 0.248
1.32 (0.61–2.83); 0.479 1.25 (0.56–2.75); 0.586
1.37 (1.04–1.81); 0.027 1.20 (0.90–1.61); 0.220
1.20 (0.56–2.57); 0.635 1.19 (0.54–2.61); 0.671
RF status Positive Negative
316/600 88/600
147/209 34/209
16/22 3/22
163/231 37/231
1.34 (1.04–1.72); 0.024 1.11 (0.72–1.70); 0.634
1.38 (0.72–2.67); 0.337 0.93 (0.27–3.17); 0.907
1.34 (1.05–1.71); 0.018 1.9 (0.72–1.65); 0.676
1.27 (0.66–2.44); 0.473 0.90 (0.27–3.07); 0.872
ESR (mm/h) b25.00 ≥25.00
181/600 223/600
82/209 99/209
9/22 10/22
91/231 109/231
1.30 (0.96–1.76); 0.091 1.27 (0.96–1.69); 0.095
1.36 (0.61–3.00); 0.452 1.22 (0.57–2.63); 0.605
1.31 (0.97–1.75); 0.075 1.27 (0.97–1.67); 0.089
1.26 (0.57–2.77); 0.568 1.14 (0.54–2.44); 0.732
DAS28 b3.20 ≥3.20
84/600 320/600
44/209 137/209
3/22 16/22
47/231 153/231
1.50 (1.01–2.24); 0.044 1.23 (0.95–1.59); 0.112
0.97 (0.29–3.33); 0.967 1.36 (0.71–2.63); 0.356
1.45 (0.99–2.14); 0.059 1.24 (0.97–1.59); 0.083
0.86 (0.25–2.92); 0.811 1.29 (0.67–2.48); 0.449
Functional class I+II 229/600 III+IV 175/600
111/209 70/209
14/22 5/22
125/231 75/231
1.39 (1.06–1.83); 0.019 1.15 (0.84–1.58); 0.395
1.67 (0.84–3.32); 0.145 0.78 (0.29–2.09); 0.620
1.42 (1.09–1.85); 0.010 1.11 (0.82–1.52); 0.498
1.51 (0.77–3.00); 0.233 0.75 (0.28–2.00); 0.567
Bold values are statistically significant (P b 0.05).
IL17A, the key cytokine of Th17 cells, can induce pro-inflammatory cytokines and promote rheumatoid arthritis (Gaffen, 2009). IL17A is produced by many cell types, including Th17 cells, CD8+ T cells, and
γδ T cells, and is elevated along with numbers of mast cells and neutrophils at sites of skin and joint diseases in humans (Kirkham et al., 2014). IL17A, along with TNF-α, was found to be a predictor of a poor outcome
Table 5 Stratified analyses between IL-17A rs8193036 C/T polymorphisms and the risk of rheumatoid arthritis. Variable
IL-17A rs8193036/(case/control)
OR (95% CI); P
CC
CT
TT
CT+TT
CT versus CC
TT versus CC
CT+TT versus CC
TT versus CT+CC
Sex Male Female
63/108 221/356
60/64 174/217
7/21 48/46
67/85 222/263
1.61 (1.01–2.57); 0.048 1.29 (1.00–1.68); 0.054
0.57 (0.23–1.42); 0.228 1.68 (1.09–2.60); 0.020
1.35 (0.87–2.11); 0.186 1.36 (1.06–1.74); 0.014
0.67 (0.19–1.13); 0.091 1.51 (0.99–2.31); 0.056
Age (years) b55 ≥55
132/203 152/261
110/115 124/166
28/34 27/33
138/149 151/199
1.47 (1.05–2.07); 0.027 1.28 (0.94–1.74); 0.112
1.27 (0.73–2.19); 0.397 1.41 (0.81–2.43); 0.223
1.42 (1.04–1.96); 0.030 1.30 (0.97–1.74); 0.075
1.08 (0.64–1.83); 0.768 1.27 (0.75–2.15); 0.384
CRP status Positive Negative
155/464 129/464
138/281 96/281
31/67 24/67
169/348 120/348
1.47 (1.12–1.93); 0.006 1.23 (0.91–1.66); 0.182
1.39 (0.87–2.20); 0.168 1.29 (0.78–2.14); 0.326
1.45 (1.12–1.88); 0.005 1.24 (0.93–1.64); 0.138
1.18 (0.73–1.84); 0.476 1.19 (0.73–1.94); 0.494
ACPA status Positive Negative
149/464 118/464
183/281 96/281
5/67 24/67
188/348 120/348
1.30 (0.98–1.72); 0.073 1.43 (1.07–1.91); 0.015
1.30 (0.81–2.10); 0.280 1.39 (0.85–2.25); 0.189
1.30 (0.99–1.70); 0.056 1.42 (1.08–1.87); 0.011
1.17 (0.74–1.86); 0.505 1.19 (0.75–1.90); 0.464
RF status Positive Negative
222/464 62/464
184/281 50/281
48/67 7/67
232/348 57/348
1.37 (1.07–1.75); 0.012 1.33 (0.89–1.99); 0.161
1.50 (1.00–2.24); 0.050 0.78 (0.34–1.78); 0.559
1.39 (1.11–1.76); 0.005 1.23 (0.83–1.80); 0.301
1.32 (0.89–1.94); 0.669 0.70 (0.31–1.55); 0.376
ESR (mm/h) b25.00 136/464 ≥25.00 148/464
109/281 125/281
18/67 37/67
127/348 162/348
1.32 (0.99–1.77); 0.060 1.40 (1.05–1.85); 0.020
0.92 (0.53–1.60); 0.758 1.73 (1.11–2.69); 0.015
1.25 (0.94–1.65); 0.124 1.46 (1.12–1.90); 0.005
0.82 (0.48–1.40); 0.463 1.51 (0.99–2.31); 0.058
DAS28 b3.20 ≥3.20
57/464 227/464
60/281 174/281
11/67 44/67
71/348 218/348
1.74 (1.18–2.57); 0.006 1.27 (0.99–1.62); 0.061
1.34 (0.67–2.68); 0.413 1.34 (0.89–2.03); 0.161
1.66 (1.14–2.42); 0.008 1.28 (1.02–1.62); 0.037
1.05 (0.54–2.04); 0.895 1.22 (0.82–1.82); 0.329
Functional class I+II 162/464 III+IV 122/464
148/281 86/281
31/67 24/67
179/348 110/348
1.51 (1.15–1.97); 0.003 1.16 (0.85–1.59); 0.342
1.33 (0.84–2.10); 0.232 1.36 (0.82–2.26); 0.232
1.47 (1.14–1.90); 0.003 1.20 (0.90–1.61); 0.218
1.11 (0.71–1.74); 0.641 1.28 (0.79–2.10); 0.320
Bold values are statistically significant (P b 0.05).
22
L. Shen et al. / Gene 566 (2015) 18–22
in RA cases (Kirkham et al., 2006). Fortunately, therapies targeting IL-17 in autoimmune diseases ameliorated the inadequate response to antiTNF-α therapy (Yan et al., 2014). Studies have shown that IL17A polymorphisms are associated with an increased risk of gastric cancer, breast cancer and cervical cancer (Shibata et al., 2009; Quan et al., 2012; Wang et al., 2012; Han et al., 2014). In a recent study, Han et al. showed that polymorphisms of IL17A G-197A may be closely associated with susceptibility to osteoarthritis in the Korean population (Han et al., 2014). A recent Japanese study reported a weak association between an IL17A intronic SNP, rs3804513, and radiographic progression of RA at 2 years (P = 0.049), which indicated that IL-17 might be associated with joint destruction in Japanese patients with early RA (Furuya et al., 2007). IL17A rs3804513 MAF was 0.131 in this Japanese study and no linkage disequilibrium (LD) relation was found between IL17A rs3804513 and other SNPs (Furuya et al., 2007). However, in our present study, we found that IL17A rs3819025 and rs8193036 polymorphisms increased the RA risk among functional class I/II patients but not functional class III + IV patients. Nordang et al. reported an association between IL17A SNPs and RA in Caucasian patients from Norway and New Zealand. They found a modest association between RA prevalence and rs2275913 in the IL17A gene among Norwegian RA patients; however, their findings were not replicated in an independent New Zealand RA cohort (Nordang et al., 2009). MAF of IL17A rs2275913 was 0.463 in our study and 0.385 in New Zealand and Norwegian studies (Nordang et al., 2009). In our study, we discovered a significantly decreased risk of RA associated with the IL17A rs2275913 AA genotypes (AA vs. GA/GG: OR, 0.77; 95% CI, 0.59–0.99, P = 0.043). Our study also revealed that the rs3819024 and rs8193036 polymorphisms in the IL17A gene are related to RA; in contrast, Nordang et al. stated that rs3819024 and rs8193036 polymorphisms in the IL17A gene were not associated with RA in Norwegian patients. Several limitations of our study should be addressed. First, as a hospital-based case–control study, the participants were not fully representative of the general population. Second, the polymorphisms investigated, based on their functional considerations, might not offer a comprehensive view of IL17A genetic variability, necessitating further fine-mapping studies. Third, the sample size of this study was not sufficiently large to evaluate the low penetrance effect of the SNPs. Fourth, the observed genotype frequencies for IL17A rs2275913 G/A and IL17A rs8193036 C/T polymorphisms in the controls did not conform to HWE. In conclusion, the present study demonstrated that the IL17A rs2275913 G/A variant allele and IL17A rs3819024 A/G decrease the RA risk, whereas the IL17A rs3819025 G/A and IL17A rs8193036 C/T variant alleles increase the RA risk. However, our results were obtained from only a moderate-sized sample. Larger replication studies from more diverse ethnic populations are needed to confirm these findings. Declaration of interest The authors have no declarations of interest. Acknowledgments This study was supported in part by National Natural Science Foundation of China (81371927) and Changzhou Foundation for Applied Basic Research (CJ20130027).
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