International Journal of Rheumatic Diseases 2015
ORIGINAL ARTICLE
IL23R gene polymorphism with juvenile idiopathic arthritis and its association with serum IL-17A Samira EMAMI,1 Vahid ZIAEE,2,3 Arezou REZAEI,4 Maryam SADR,5 Marzieh MADDAH,3 Ali Akbar AMIRZARGAR1,5 and Nima REZAEI1,4,5,6 1
Department of Immunology, School of Medicine, 2Pediatric Rheumatology Research Group, Rheumatology Research Center, Pediatrics Center of Excellence, 4Research Center for Immunodeficiencies, Children’s Medical Center, 5Molecular Immunology Research Center, Tehran University of Medical Sciences, and 6Universal Scientific Education and Research Network (USERN), Tehran, Iran 3
Abstract Aim: Interleukin 23 (IL-23) and its receptor (IL-23R) seem to play a major role in differentiation of CD4+ T cells into Th17 cells, induction of IL-17 production, and activation of inflammatory pathways. Recent studies have suggested the association of IL-23R polymorphisms with bone and articular inflammation in diseases such as ankylosing spondylitis and rheumatoid arthritis. The aim of this study was to determine the association between IL-23R polymorphisms and juvenile idiopathic arthritis (JIA). Method: A case–control study on 55 patients with JIA and 78 healthy controls was performed. All samples were genotyped for eight single nucleotide polymorphisms (SNPs) of IL23R (rs1004819, rs2201841, rs10889677, rs1495965, rs7517847, rs10489629, rs11209026 and rs1343151), using real-time polymerase chain reaction Taqman genotyping technique. Forty-two patients and 42 healthy controls were chosen randomly to measure the level of serum IL-17A using enzyme-linked immunosorbent assay. Results: Although the heterozygous genotype of rs1004819 (GA) showed a weak, but statistically significant protective effect on polyarticular subtype (P = 0.03), none of the selected SNPs were associated with JIA overall. Indeed the analysis of haplotypes did not show any significant association with JIA. Serum IL-17A level was not significantly different among patients and healthy controls and between JIA subtypes, as well. Moreover, there was no significant correlation between SNPs and serum IL-17A concentration. Conclusion: This is the first study of the IL-23R gene in Iranian patients with JIA. Our results did not show any strong association between IL-23R polymorphisms and JIA disease or serum IL-17A levels. The only association was seen between rs1004819 and polyarticular JIA. Further larger studies may help clarify the role, if any, of the IL-23/IL-17 pathway in the pathogenesis of JIA. Key words: IL-23R, juvenile idiopathic arthritis, polymorphism.
INTRODUCTION Juvenile idiopathic arthritis (JIA) is a chronic inflammatory articular disease among children under 16 years, with a duration of more than 6 weeks Correspondence: Dr Nima Rezaei, MD, PhD, Children’s Medical Center Hospital, Dr Qarib St, Keshavarz Blvd, Tehran 14194, Iran. Email: [email protected]
which is classified into seven subtypes (systemic JIA [sJIA], oligoarticular JIA [oligo-JIA], rheumatoid factor [RF]-negative polyarticular JIA [poly-JIA], RFpositive polyarticular JIA, juvenile psoriatic arthritis [JPsA], enthesitis-related arthritis [ERA] and undifferentiated arthritis) based on the clinical features in the first 6 months of disease.1 JIA is a complex multifactorial disease in which the interaction of environmental and several unknown genetic factors play
© 2015 Asia Pacific League of Associations for Rheumatology and Wiley Publishing Asia Pty Ltd
S. Emami et al.
critical roles in the pathogenesis of different JIA subtypes.2,3 Interleukin 23 receptor (IL-23R) is a heterodimer protein composed of two subunits: IL-23R and IL-12Rb1. IL-23R subunit is encoded by the IL-23R gene (ch:1p31.2–32.1). IL-23R, expressed on activated T cells, natural killer (NK) cells, macrophages and dendritic cells, binds uniquely to IL-23, produced predominantly by activated macrophages, and dendritic cells from peripheral tissues.4–7 The IL-23/IL-23R signaling pathway induces the differentiation of T CD4+ into Th17 cells and subsequently increases the production of the proinflammatory cytokine, IL-17.6,8 Several studies have reported the role of Th17 cells and their produced cytokines (IL-17A, IL-17F) in the pathogenesis of joint and bone inflammatory diseases such as ankylosing spondylitis,9 rheumatoid arthritis10–13 and JIA.14–16 This finding that the Th17/IL-17 pathway is regulated by IL-23 and its receptor, has prompted studies to investigate the association of IL-23R gene polymorphisms with osteo-articular diseases. Several studies have shown the association of IL-23R polymorphisms with the susceptibility to ankylosing spondilitis17–20 and rheumatoid arthritis.21,22 However, there is only one study considering the association of IL-23R polymorphisms with JpSA, one of the seven subtypes of JIA disease.23 Since there have not been extensive studies detailing the role of IL-23R in JIA, we performed this study with the aim of finding the role of IL-23/IL-17 pathway in JIA disease. We hypothesized that IL-23R polymorphisms may play a critical role in JIA susceptibility and higher IL-17A production in serum of patients.
Single nucleotide polymorphism (SNP) genotyping DNA samples were extracted using the phenol-chloroform method. Genotyping analysis was performed using real-time polymerase chain reaction (PCR) allelic discrimination TaqMan genotyping assays (Applied Biosystems, Foster City, CA, USA). All SNPs were in Hardy–Weinberg equilibrium (HWE) (P > 0.05) in the control group.
Enzyme-linked immunosorbent assay IL-17A was measured in the sera of 42 simple randomly selected patients (oligo-JIA: n = 17, poly JIA: n = 11, sJIA: n = 14) and 42 randomly selected controls, using ELISA (KOMABIOTECH, Seoul, South Korea), based on manufacturer protocols. After sample collection, serum was separated immediately and kept at 20°C. The assay for all the samples was made at the same time between 2–3 months after sampling.
Statistical analysis Chi-square or Fisher’s exact tests were performed in order to determine the association of all SNP alleles and genotypes with disease susceptibility. Two-tailed P-values, odds ratios (OR) and 95% confidence intervals (95% CI) were computed, using OpenEpi online software (http://www.openepi.com). SHEsis online software (http://analysis.bio-x.cn) was used to test for HWE and the analysis of haplotype blocks. The data of serum IL-17A were analyzed using Mann–Witney and Kruskal–Wallis tests.
RESULTS Association analysis of IL-23R variants
PATIENTS AND METHODS Study population Fifty-five Iranian patients with JIA who were referred to the Children’s Medical Center Hospital, Tehran, Iran, were enrolled in this study. The diagnosis of JIA disease was based on International League of Associations for Rheumatology (ILAR) criteria.1 Patients were categorized into three subgroups (oligo-JIA: n = 21, poly-JIA: n = 17, sJIA: n = 17). Seventy-eight healthy controls were recruited in the study. The case and control groups were matched for age, sex and ethnicity; all of them were Iranian. This study was approved by the local Ethics Committee of the University. Written informed consents were taken from all parents before sampling.
2
Analysis of allele and genotype frequency of eight selected SNPs did not show any significant correlation with susceptibility to JIA overall. However, a weak, but statistically significant protective effect of heterozygous variant of rs1004819 (GA) for polyarticular subtype of JIA was revealed in our study (P = 0.03, OR: 0.28). There was no significant association between other studied SNPs and JIA subtypes (Tables 1 and 2).
Haplotype analysis Haplotype block was determined based on D0 value (Fig. 1). Haplotypes with the frequency < 0.03 were ignored in the analysis. Analysis of haplotypes did not show any significant association with JIA susceptibility (Table 3).
International Journal of Rheumatic Diseases 2015
Control n (%)
n (%)
Pvalue
OR (%95 CI)
n (%)
Pvalue
OR (%95 CI)
International Journal of Rheumatic Diseases 2015 – 0.77 (0.34–1.83) 21 (61.8) – 0.52 (0.23–1.16) 0.53 1.29 (0.54–2.92) 13 (38.2) 0.11 1.91 (0.85–4.19)
– 0.72 (0.41–1.24) 31 (73.8) – 0.90 (0.42–2.04) 24 (70.6) 0.24 1.38 (0.80–2.40) 11 (26.2) 0.79 1.10 (0.48–2.37) 10 (29.4)
– 0.64 (0.12–4.80) 32 (94.1) – 0.64 (0.12–4.80) 0.87 1.55 (0.20–7.74) 2 (5.9) 0.87 1.55 (0.20–7.74) – 1.19 (0.51–3.01) 26 (76.5) – 1.19 (0.51–3.01) 0.70 0.83 (0.33–1.95) 8 (23.5) 0.70 0.83 (0.33–1.95)
– 0.58 (0.18–1.87) 39 (92.9) – 0.52 (0.12–2.65) 32 (94.1) 0.36 1.69 (0.53–5.51) 3 (7.1) 0.38 1.91 (0.37–8.05) 2 (5.9) – 1.31 (0.74–2.36) 34 (81.0) – 1.56 (0.68–3.86) 26 (76.5) 0.34 0.75 (0.42–1.34) 8 (19.0) 0.30 0.64 (0.25–1.46) 8 (23.5)
– 0.96 (0.57–1.64) 29 (69.0) – 1.05 (0.50–2.25) 24 (70.6) – 1.13 (0.50–2.64) 21 (61.8) – 0.76 (0.35–1.68) 0.90 1.03 (0.61–1.73) 13 (31.0) 0.90 0.95 (0.44–1.97) 10 (%29.4) 0.78 0.88 (0.37–1.96) 13 (38.2) 0.49 1.31 (0.59–2.82)
– 0.72 (0.34–1.54) 19 (55.9) – 0.81 (0.38–1.75) 0.39 1.38 (0.64–2.93) 15 (44.1) 0.59 1.22 (0.57–2.61)
1.0 (0.47–2.12) 16 (47.1) – 0.88 (0.41–1.88) 1.0 (0.47–2.12) 18 (52.9) 0.76 1.12 (0.53–2.39)
1.0 (0.47–2.12) 1.0 (0.47–2.12)
– 0.74 (0.45–1.22) 22 (52.4) – 0.70 (0.35–1.41) 18 (52.9) 0.24 1.34 (0.81–2.20) 20 (47.6) 0.32 1.41 (0.70–2.82) 16 (47.1)
1.0 (0.61–1.63) 22 (52.4) – 1.09 (0.55–2.19) 17 (50.0) 1.0 (0.61–1.63) 20 (47.6) 0.78 0.90 (0.45–1.80) 17 (50.0)
1.0 (0.47–2.12) 17 (50.0) – 1.0 (0.47–2.12) 17 (50.0) 1.0
– 1.0
n (%)
– 1.0
OR (%95 CI)
– 1.0
Pvalue
Systemic JIA (n = 17)
– 1.03 (0.63–1.68) 22 (52.4) – 1.09 (0.55–2.19) 17 (50.0) 0.88 0.96 (0.59–1.57) 20 (47.6) 0.78 0.90 (0.45–1.80) 17 (50.0)
n (%)
Poly articular JIA (n = 17)
– 1.15 (0.54–2.45) 21 (61.8) – 1.65 (0.77–3.61) 0.71 0.86 (0.40–1.83) 13 (38.2) 0.19 0.60 (0.27–1.29)
OR (%95 CI)
Oligo articular JIA (n = 21)
– 1.23 (0.75–2.01) 21 (50.0) – 1.02 (0.51–2.04) 18 (52.9) 0.40 0.81 (1.49–1.32) 21 (50.0) 0.94 0.97 (0.48–1.94) 16 (47.1)
Pvalue
JIA (overall) (n = 55)
rs1004819 G 77 (49.4) 60 (54.5) A 79 (50.6) 50 (45.5) rs2201841 A 78 (50.0) 56 (50.9) G 78 (50.0) 54 (49.1) rs10889677 C 78 (50.0) 55 (50.0) A 78 (50.0) 55 (50.0) rs1495965 C 95 (60.9) 59 (53.6) T 61 (39.1) 51 (46.4) rs7517847 T 118 (75.6) 76 (69.1) G 38 (24.4) 34 (30.9) rs10489629 T 106 (67.9) 74 (67.3) C 50 (32.1) 36 (32.7) rs11209026 G 150 (96.2) 103 (93.6) A 6 (3.8) 7 (6.4) rs1343151 G 114 (73.1) 86 (78.2) A 42 (26.9) 24 (21.8)
SNP/ allele
Table 1 Allele frequency of single nucleotide polymorphisms (SNPs) in juvenile idiopathic (JIA) and JIA subtypes versus controls and result of v2 statistics
IL23R polymorphisms and IL17A Level in JIA
3
4
Control n (%)
rs1004819 GG 18 (23.1) GA 41 (52.6) AA 19 (24.3) rs2201841 AA 19 (24.4) AG 40 (51.2) GG 19 (24.4) rs10889677 CC 19 (24.4) CA 40 (51.2) AA 19 (24.4) rs1495965 CC 31 (39.7) CT 33 (42.4) TT 14 (17.9) rs7517847 TT 44 (56.4) TG 30 (38.5) GG 4 (5.1) rs10489629 TT 37 (%47.5) TC 32 (%41.0) CC 9 (%11.5) rs11209026 GG 72 (92.3) GA 6 (7.7) rs1343151 GG 41 (52.6) GA 32 (41.0) AA 5 (6.4)
SNP/ allele Pvalue
OR (%95 CI)
OR (%95 CI)
Pvalue
OR (%95 CI)
0.65 0.77 (0.25–2.27) 0.19 2.03 (0.69–6.23) 0.31 0 (0.0–1.76)
0.87 0.62 (0.11–4.90) 15 (88.2) 0.87 0.62 (0.11–4.90) 0.87 1.59 (0.20–8.36) 2 (11.8) 0.87 1.59 (0.20–8.36)
24 (43.6) 0.67 0.85 (0.42–1.72) 9 (42.9) 0.71 0.83 (0.30–2.22) 7 (41.2) 26 (47.3) 0.48 1.28 (0.63–2.59) 11 (52.4) 0.36 1.57 (0.58–4.25) 10 (58.8) 5 (9.1) 0.67 0.76 (0.22–2.43) 1 (4.8) 0.65 0.38 (0.01–2.56) 0 (0.0)
48 (87.3) 0.35 0.57 (0.17–1.87) 18 (85.7) 0.57 0.50 (0.11–2.67) 15 (88.2) 7 (12.7) 0.35 1.74 (0.53–5.83) 3 (14.3) 0.57 1.98 (0.37–8.79) 2 (11.8)
31 (56.4) 0.67 1.16 (0.57–2.35) 13 (61.9) 0.46 1.46 (0.54–4.09) 24 (43.6) 0.76 1.11 (0.54–2.24) 8 (38.1) 0.82 0.88 (0.31–2.39) 0 (0.0) 0.06 0 (0.0–1.13) 0 (0.0) 0.59 0 (0.0–3.05)
0.49 0.68 (0.23–2.02) 0.52 1.41 (0.47–4.17) 0.99 1.15 (0.04–9.91)
8 (47.1) 8 (47.1) 1 (5.9)
26 (47.3) 0.30 0.69 (0.34–1.39) 11 (52.4) 0.74 0.85 (0.31–2.29) 24 (43.6) 0.55 1.23 (0.60–2.50) 9 (42.9) 0.71 1.19 (0.43–3.22) 5 (9.1) 0.39 1.84 (0.44–8.05) 1 (4.8) 0.99 0.92 (0.03–7.84)
9 (52.9) 0.98 1.01 (0.34–3.01) 8 (47.1) 0.65 1.27 (0.42–3.74) 0 (%0.0) 0.72 0 (0.0–3.81)
0.75 0.82 (0.25–2.47) 0.61 0.74 (0.23–2.22) 0.30 1.89 (0.52–6.22)
9 (52.9) 0.98 1.01 (0.34–3.01) 8 (47.1) 0.65 1.27 (0.42–3.74) 0 (0.0) 0.72 0 (0.0–3.81)
8 (47.1) 0.98 0.98 (0.33–2.88) 5 (29.4) 0.39 0.60 (0.17–1.85) 4 (23.5) 0.35 2.33 (0.55–8.73)
7 (41.2) 0.27 0.54 (0.17–1.59) 7 (41.2) 0.83 1.11 (0.36–3.29) 3 (17.6) 0.21 3.88 (0.66–20.83)
7 (41.2) 0.90 1.06 (0.34–3.12) 5 (29.4) 0.34 0.57 (0.16–1.75) 5 (29.4) 0.30 1.89 (0.52–6.22)
4 (23.5) 0.96 0.95 (0.24–3.20) 8 (47.1) 0.76 0.84 (0.28–2.47) 5 (29.4) 0.66 1.29 (0.36–4.10)
5 (29.4) 0.66 1.29 (0.36–4.10) 7 (41.2) 0.46 0.66 (0.21–1.95) 5 (29.4) 0.66 1.29 (0.36–4.10)
6 (35.3) 6 (35.3) 5 (29.4)
0.66 1.29 (0.36–4.10) 0.46 0.66 (0.21–1.95) 0.66 1.29 (0.36–4.10)
0.66 1.29 (0.36–4.10) 0.46 0.66 (0.21–1.95) 0.66 1.29 (0.36–4.10)
14 (32.7) 0.41 0.73 (0.35–1.52) 5 (23.8) 0.18 0.47 (0.14–1.40) 23 (41.8) 0.95 0.98 (0.48–1.98) 12 (57.1) 0.23 1.80 (0.67–4.95) 18 (25.5) 0.30 1.55 (0.66–3.65) 4 (19.1) 0.99 1.07 (0.27–3.59)
7 (41.2) 0.14 2.31 (0.73–7.06) 7 (41.2) 0.41 0.63 (0.20–1.85) 3 (17.6) 0.81 0.66 (0.14–2.44)
n (%)
5 (29.4) 7 (41.2) 5 (29.4)
0.14 2.31 (0.73–7.06) 0.03 0.28 (0.07–0.90) 0.37 1.68 (0.51–5.19)
Pvalue
14 (25.5) 0.88 1.06 (0.46–2.36) 5 (23.8) 0.98 0.97 (0.28–2.95) 27 (49.0) 0.80 0.91 (0.45–1.83) 12 (57.1) 0.64 1.26 (0.74–3.45) 14 (25.5) 0.88 1.06 (0.46–2.36) 4 (19.1) 0.84 0.73 (0.19–2.36)
n (%)
5 (29.4) 7 (41.2) 5 (29.4)
OR (%95 CI)
Systemic JIA (n = 17)
15 (27.3) 0.70 1.16 (0.52–2.57) 5 (23.8) 0.98 0.97 (0.28–2.95) 26 (47.3) 0.65 0.85 (0.42–1.70) 12 (57.1) 0.64 1.26 (0.47–3.45) 14 (25.4) 0.88 1.06 (0.46–2.36) 4 (19.1) 0.84 0.73 (0.19–2.36)
Pvalue
Poly articular JIA (n = 17)
7 (41.2) 4 (23.5) 6 (35.3)
n (%)
Oligo articular JIA (n = 21)
6 (28.6) 0.60 1.33 (0.41–3.90) 9 (42.9) 0.44 0.67 (0.24–1.81) 6 (28.6) 0.68 1.24 (0.39–3.62)
20 (36.4) 0.10 1.89 (0.88–4.10) 20 (36.4) 0.06 0.51 (0.25–1.05) 15 (27.2) 0.70 1.16 (0.52–2.57)
n (%)
JIA (overall) (n = 55)
Table 2 Genotype frequency of single nucleotide polymorphisms (SNPs) in juvenile idiopathic (JIA) and JIA subtypes versus controls and result of v2 statistics
S. Emami et al.
International Journal of Rheumatic Diseases 2015
IL23R polymorphisms and IL17A Level in JIA
Figure 1 Result of linkage disequilibrium test based on D0 value. Four SNPs (rs10489629, rs2201841, rs11209026, rs1343151, rs10889677) were in high linkage disequilibrium (D0 ≥ 0.85).
Table 3 Haplotype frequencies in cases and controls Haplotype rs10489629
rs2201841
rs112090261
rs134315
rs10889677
A A A A G
A G G G G
A A G G G
C C C C A
C C C T T
Cases (freq)
Controls (freq)
P-value
OR (%95 CI)
0.05 0.16 0.10 0.18 0.49
0.03 0.23 0.05 0.18 0.50
0.52 0.19 0.12 0.93 0.94
1.45 (0.45–4.64) 0.65 (0.35–1.23) 2.07 (0.80–5.34) 1.02 (0.54–1.93) 0.98 (0.60–1.60)
ELISA analysis
DISCUSSION
We did not find significant difference between IL-17A levels in sera of JIA patients and the healthy group (Table 4). The level of IL-17A among JIA subtypes was also not significantly different (Table 5). None of the eight SNPs showed significant association with the level of IL-17A in patients’ sera (Table 6).
Based on the role of IL-23 in joint autoimmune inflammation24 we examined the possible influence of IL-23 receptor gene polymorphisms on JIA predisposition.
Table 4 Interleukin (IL)-17A concentration in patients versus controls
Subgroup
IL-17A (pg/mL
Mean
SD
P-value
JIA patients Healthy controls
753.8 807.73
805.87 739.28
0.335
International Journal of Rheumatic Diseases 2015
Table 5 Interleukin (IL)-17A concentration in juvenile idiopathic arthritis subgroups
Oligo-articular Poly-articular Systemic
IL-17A (pg/mL) n
Mean
SD
P-value
17 11 14
886.76 845.45 520.35
883.36 769.96 736.80
0.61
5
S. Emami et al.
Table 6 Association of single nucleotide polymorphism (SNP) genotypes with the level of interleukin (IL)-17A in sera of juvenile idiopathic arthritis patients SNPs rs1004819
rs2201841
rs10889677
rs1495965
rs7517847
rs10489629
rs11209026 rs1343151
Genotype
n
IL-17A (mean SD) pg/mL
P-value
GG GA AA AA AG GG CC CA AA CC CT TT TT TG GG TT TC CC GG GA GG GA
17 14 11 12 19 11 11 20 11 17 16 9 17 20 5 16 21 5 36 6 21 21
728.82 (809.93) 863.21 (889.44) 653.18 (745.46) 548.33 (737.06) 839.21 (865.21) 830.45 (804.14) 598.18 (751.52) 797.25 (826.79) 830.45 (804.14) 826.47 (805.07) 807.50 (853.64) 521.11 (766.72) 818.23 (838.24) 757.50 (785.81) 520.00 (909.395) 712.18 (797.56) 961.66 (818.83) 14.0 (31.3) 828.05 (823.51) 308.33 (548.10) 697.85 (765.82) 809.76 (859.23)
0.81
0.52
0.72
0.70
0.66
0.05
0.09 0.80
Our results showed a weak, albeit statistically significant protective effect of GA genotype of rs1004819 in polyarticular subtype but not in JIA and other subtypes. Other studied SNPs showed no significant association. There has been only one study investigating the correlation between IL-23R gene polymorphism (rs11209026) and JIA disease. This study revealed the protective role of minor allele of rs11209026 in juvenile psoriatic arthritis subtype but no association with JIA or other subtypes.23 Our findings, in conjunction with this study, suggest that IL-23/IL-17 pathway may play a role in the pathogenesis of some subtypes of JIA disease, not in JIA overall. The possible explanation for finding no association between other studied SNPs and JIA or its subtypes may be the small sample size in our study. The other reason could be that these SNPs do not have any effect on JIA or JIA in our ethnic groups. Since there have not been other numerous studies on JIA disease and our studied SNPs, the comparison of our findings is limited to other osteoarticular diseases such as rheumatoid arthritis and ankylosing spondylitis.
6
In a study conducted by Chang et al.,25 no association was reported between rs11209026 and rheumatoid arthritis. Furthermore, in a study in a Korean population which examined seven SNPs of IL-23R (six of them were common with our study), the lack of association between all SNPs and reumatoid arthritis was reported.26 The results of our study were along the same lines as the results of these two studies. It seems that the location of these polymorphisms, except rs11209026, in introns of IL-23R gene is a factor of their inefficiency on protein expression and as a result, their lack of association with the disease. Contrary to these results, other studies confirmed the association of IL-23R polymorphisms with rheumatoid arthritis21,22 and ankylosing spondylitis.17,27–30 The lack of correlation of our results with the results of these studies may be because of our small sample size. Furthermore, we can conclude that IL-23/Il-17 pathway may play a role in the pathogenesis of other osteoarticular inflammations rather than JIA disease. In the second section of our study we examined the correlation between IL-23R polymorphisms and serum IL-17A level. Although it has been reported that Il-23/ IL-17 play critical roles in joint inflammation,31 our results did not show a higher level of IL-17A in serum of patients in comparison with the control group. Moreover, the level of IL-17A did not differ significantly between JIA subtypes. This finding may be justified by the reason that our patients were under treatment by NSAIDs (non-steroidal anti-inflammatory drugs) and DMARDs (disease-modifying antirheumatic drugs) for more than 6 months. Moreover, we can conclude that IL-17A may play a critical role in the synovia of JIA patients rather than serum. Previously, Hazlett et al.32 showed the associaton between rs11209026 minor allele and lower serum IL-17A expression in patients with rheumatoid arthritis. However, we did not find significant correlation between our studied SNPs and serum levels of IL-17A in JIA patients. Although the level of IL17A was much lower in the rare genotypes of rs10489629 (CC) and rs11209026 (GA), the correlation was not significant (P = 0.05 and 0.09, respectively). The lack of association may result from our small sample size. Furthermore, it can be inferred that intronic SNPs do not have influence on protein expression. Our findings did not support the strong association between IL-23R polymorphisms and JIA disease or IL17A expression. As this gene is not studied widely in JIA patients, we cannot incisively accept or reject the role of
International Journal of Rheumatic Diseases 2015
International Journal of Rheumatic Diseases 2015
ORIGINAL ARTICLE
IL23R gene polymorphism with juvenile idiopathic arthritis and its association with serum IL-17A Samira EMAMI,1 Vahid ZIAEE,2,3 Arezou REZAEI,4 Maryam SADR,5 Marzieh MADDAH,3 Ali Akbar AMIRZARGAR1,5 and Nima REZAEI1,4,5,6 1
Department of Immunology, School of Medicine, 2Pediatric Rheumatology Research Group, Rheumatology Research Center, Pediatrics Center of Excellence, 4Research Center for Immunodeficiencies, Children’s Medical Center, 5Molecular Immunology Research Center, Tehran University of Medical Sciences, and 6Universal Scientific Education and Research Network (USERN), Tehran, Iran 3
Abstract Aim: Interleukin 23 (IL-23) and its receptor (IL-23R) seem to play a major role in differentiation of CD4+ T cells into Th17 cells, induction of IL-17 production, and activation of inflammatory pathways. Recent studies have suggested the association of IL-23R polymorphisms with bone and articular inflammation in diseases such as ankylosing spondylitis and rheumatoid arthritis. The aim of this study was to determine the association between IL-23R polymorphisms and juvenile idiopathic arthritis (JIA). Method: A case–control study on 55 patients with JIA and 78 healthy controls was performed. All samples were genotyped for eight single nucleotide polymorphisms (SNPs) of IL23R (rs1004819, rs2201841, rs10889677, rs1495965, rs7517847, rs10489629, rs11209026 and rs1343151), using real-time polymerase chain reaction Taqman genotyping technique. Forty-two patients and 42 healthy controls were chosen randomly to measure the level of serum IL-17A using enzyme-linked immunosorbent assay. Results: Although the heterozygous genotype of rs1004819 (GA) showed a weak, but statistically significant protective effect on polyarticular subtype (P = 0.03), none of the selected SNPs were associated with JIA overall. Indeed the analysis of haplotypes did not show any significant association with JIA. Serum IL-17A level was not significantly different among patients and healthy controls and between JIA subtypes, as well. Moreover, there was no significant correlation between SNPs and serum IL-17A concentration. Conclusion: This is the first study of the IL-23R gene in Iranian patients with JIA. Our results did not show any strong association between IL-23R polymorphisms and JIA disease or serum IL-17A levels. The only association was seen between rs1004819 and polyarticular JIA. Further larger studies may help clarify the role, if any, of the IL-23/IL-17 pathway in the pathogenesis of JIA. Key words: IL-23R, juvenile idiopathic arthritis, polymorphism.
INTRODUCTION Juvenile idiopathic arthritis (JIA) is a chronic inflammatory articular disease among children under 16 years, with a duration of more than 6 weeks Correspondence: Dr Nima Rezaei, MD, PhD, Children’s Medical Center Hospital, Dr Qarib St, Keshavarz Blvd, Tehran 14194, Iran. Email: [email protected]
which is classified into seven subtypes (systemic JIA [sJIA], oligoarticular JIA [oligo-JIA], rheumatoid factor [RF]-negative polyarticular JIA [poly-JIA], RFpositive polyarticular JIA, juvenile psoriatic arthritis [JPsA], enthesitis-related arthritis [ERA] and undifferentiated arthritis) based on the clinical features in the first 6 months of disease.1 JIA is a complex multifactorial disease in which the interaction of environmental and several unknown genetic factors play
© 2015 Asia Pacific League of Associations for Rheumatology and Wiley Publishing Asia Pty Ltd
S. Emami et al.
29 Rueda B et al. (2008) The IL23R Arg381Gln non-synonymous polymorphism confers susceptibility to ankylosing spondylitis. Ann Rheum Dis 67, 1451–4. 30 Safrany E et al. (2009) Variants of the IL23R gene are associated with ankylosing spondylitis but not with Sj€ ogren syndrome in Hungarian population samples. Scand J Immunol 70 (1), 68–74.
8
31 Cojocaru-Gofita I, Paulina C, Yeremenko N (2012) The cellular source of IL-17A in the pathogenesis of inflammatory joint diseases. Curr Health Sci J 38(2), 53–7. 32 Hazlett J et al. (2011) IL-23R rs11209026 polymorphism modulates IL-17A expression in patients with rheumatoid arthritis. Genes Immun 13, 282–7.
International Journal of Rheumatic Diseases 2015
ORIGINAL ARTICLE
IL23R gene polymorphism with juvenile idiopathic arthritis and its association with serum IL-17A Samira EMAMI,1 Vahid ZIAEE,2,3 Arezou REZAEI,4 Maryam SADR,5 Marzieh MADDAH,3 Ali Akbar AMIRZARGAR1,5 and Nima REZAEI1,4,5,6 1
Department of Immunology, School of Medicine, 2Pediatric Rheumatology Research Group, Rheumatology Research Center, Pediatrics Center of Excellence, 4Research Center for Immunodeficiencies, Children’s Medical Center, 5Molecular Immunology Research Center, Tehran University of Medical Sciences, and 6Universal Scientific Education and Research Network (USERN), Tehran, Iran 3
Abstract Aim: Interleukin 23 (IL-23) and its receptor (IL-23R) seem to play a major role in differentiation of CD4+ T cells into Th17 cells, induction of IL-17 production, and activation of inflammatory pathways. Recent studies have suggested the association of IL-23R polymorphisms with bone and articular inflammation in diseases such as ankylosing spondylitis and rheumatoid arthritis. The aim of this study was to determine the association between IL-23R polymorphisms and juvenile idiopathic arthritis (JIA). Method: A case–control study on 55 patients with JIA and 78 healthy controls was performed. All samples were genotyped for eight single nucleotide polymorphisms (SNPs) of IL23R (rs1004819, rs2201841, rs10889677, rs1495965, rs7517847, rs10489629, rs11209026 and rs1343151), using real-time polymerase chain reaction Taqman genotyping technique. Forty-two patients and 42 healthy controls were chosen randomly to measure the level of serum IL-17A using enzyme-linked immunosorbent assay. Results: Although the heterozygous genotype of rs1004819 (GA) showed a weak, but statistically significant protective effect on polyarticular subtype (P = 0.03), none of the selected SNPs were associated with JIA overall. Indeed the analysis of haplotypes did not show any significant association with JIA. Serum IL-17A level was not significantly different among patients and healthy controls and between JIA subtypes, as well. Moreover, there was no significant correlation between SNPs and serum IL-17A concentration. Conclusion: This is the first study of the IL-23R gene in Iranian patients with JIA. Our results did not show any strong association between IL-23R polymorphisms and JIA disease or serum IL-17A levels. The only association was seen between rs1004819 and polyarticular JIA. Further larger studies may help clarify the role, if any, of the IL-23/IL-17 pathway in the pathogenesis of JIA. Key words: IL-23R, juvenile idiopathic arthritis, polymorphism.
INTRODUCTION Juvenile idiopathic arthritis (JIA) is a chronic inflammatory articular disease among children under 16 years, with a duration of more than 6 weeks Correspondence: Dr Nima Rezaei, MD, PhD, Children’s Medical Center Hospital, Dr Qarib St, Keshavarz Blvd, Tehran 14194, Iran. Email: [email protected]
which is classified into seven subtypes (systemic JIA [sJIA], oligoarticular JIA [oligo-JIA], rheumatoid factor [RF]-negative polyarticular JIA [poly-JIA], RFpositive polyarticular JIA, juvenile psoriatic arthritis [JPsA], enthesitis-related arthritis [ERA] and undifferentiated arthritis) based on the clinical features in the first 6 months of disease.1 JIA is a complex multifactorial disease in which the interaction of environmental and several unknown genetic factors play
© 2015 Asia Pacific League of Associations for Rheumatology and Wiley Publishing Asia Pty Ltd
S. Emami et al.
critical roles in the pathogenesis of different JIA subtypes.2,3 Interleukin 23 receptor (IL-23R) is a heterodimer protein composed of two subunits: IL-23R and IL-12Rb1. IL-23R subunit is encoded by the IL-23R gene (ch:1p31.2–32.1). IL-23R, expressed on activated T cells, natural killer (NK) cells, macrophages and dendritic cells, binds uniquely to IL-23, produced predominantly by activated macrophages, and dendritic cells from peripheral tissues.4–7 The IL-23/IL-23R signaling pathway induces the differentiation of T CD4+ into Th17 cells and subsequently increases the production of the proinflammatory cytokine, IL-17.6,8 Several studies have reported the role of Th17 cells and their produced cytokines (IL-17A, IL-17F) in the pathogenesis of joint and bone inflammatory diseases such as ankylosing spondylitis,9 rheumatoid arthritis10–13 and JIA.14–16 This finding that the Th17/IL-17 pathway is regulated by IL-23 and its receptor, has prompted studies to investigate the association of IL-23R gene polymorphisms with osteo-articular diseases. Several studies have shown the association of IL-23R polymorphisms with the susceptibility to ankylosing spondilitis17–20 and rheumatoid arthritis.21,22 However, there is only one study considering the association of IL-23R polymorphisms with JpSA, one of the seven subtypes of JIA disease.23 Since there have not been extensive studies detailing the role of IL-23R in JIA, we performed this study with the aim of finding the role of IL-23/IL-17 pathway in JIA disease. We hypothesized that IL-23R polymorphisms may play a critical role in JIA susceptibility and higher IL-17A production in serum of patients.
Single nucleotide polymorphism (SNP) genotyping DNA samples were extracted using the phenol-chloroform method. Genotyping analysis was performed using real-time polymerase chain reaction (PCR) allelic discrimination TaqMan genotyping assays (Applied Biosystems, Foster City, CA, USA). All SNPs were in Hardy–Weinberg equilibrium (HWE) (P > 0.05) in the control group.
Enzyme-linked immunosorbent assay IL-17A was measured in the sera of 42 simple randomly selected patients (oligo-JIA: n = 17, poly JIA: n = 11, sJIA: n = 14) and 42 randomly selected controls, using ELISA (KOMABIOTECH, Seoul, South Korea), based on manufacturer protocols. After sample collection, serum was separated immediately and kept at 20°C. The assay for all the samples was made at the same time between 2–3 months after sampling.
Statistical analysis Chi-square or Fisher’s exact tests were performed in order to determine the association of all SNP alleles and genotypes with disease susceptibility. Two-tailed P-values, odds ratios (OR) and 95% confidence intervals (95% CI) were computed, using OpenEpi online software (http://www.openepi.com). SHEsis online software (http://analysis.bio-x.cn) was used to test for HWE and the analysis of haplotype blocks. The data of serum IL-17A were analyzed using Mann–Witney and Kruskal–Wallis tests.
RESULTS Association analysis of IL-23R variants
PATIENTS AND METHODS Study population Fifty-five Iranian patients with JIA who were referred to the Children’s Medical Center Hospital, Tehran, Iran, were enrolled in this study. The diagnosis of JIA disease was based on International League of Associations for Rheumatology (ILAR) criteria.1 Patients were categorized into three subgroups (oligo-JIA: n = 21, poly-JIA: n = 17, sJIA: n = 17). Seventy-eight healthy controls were recruited in the study. The case and control groups were matched for age, sex and ethnicity; all of them were Iranian. This study was approved by the local Ethics Committee of the University. Written informed consents were taken from all parents before sampling.
2
Analysis of allele and genotype frequency of eight selected SNPs did not show any significant correlation with susceptibility to JIA overall. However, a weak, but statistically significant protective effect of heterozygous variant of rs1004819 (GA) for polyarticular subtype of JIA was revealed in our study (P = 0.03, OR: 0.28). There was no significant association between other studied SNPs and JIA subtypes (Tables 1 and 2).
Haplotype analysis Haplotype block was determined based on D0 value (Fig. 1). Haplotypes with the frequency < 0.03 were ignored in the analysis. Analysis of haplotypes did not show any significant association with JIA susceptibility (Table 3).
International Journal of Rheumatic Diseases 2015
Control n (%)
n (%)
Pvalue
OR (%95 CI)
n (%)
Pvalue
OR (%95 CI)
International Journal of Rheumatic Diseases 2015 – 0.77 (0.34–1.83) 21 (61.8) – 0.52 (0.23–1.16) 0.53 1.29 (0.54–2.92) 13 (38.2) 0.11 1.91 (0.85–4.19)
– 0.72 (0.41–1.24) 31 (73.8) – 0.90 (0.42–2.04) 24 (70.6) 0.24 1.38 (0.80–2.40) 11 (26.2) 0.79 1.10 (0.48–2.37) 10 (29.4)
– 0.64 (0.12–4.80) 32 (94.1) – 0.64 (0.12–4.80) 0.87 1.55 (0.20–7.74) 2 (5.9) 0.87 1.55 (0.20–7.74) – 1.19 (0.51–3.01) 26 (76.5) – 1.19 (0.51–3.01) 0.70 0.83 (0.33–1.95) 8 (23.5) 0.70 0.83 (0.33–1.95)
– 0.58 (0.18–1.87) 39 (92.9) – 0.52 (0.12–2.65) 32 (94.1) 0.36 1.69 (0.53–5.51) 3 (7.1) 0.38 1.91 (0.37–8.05) 2 (5.9) – 1.31 (0.74–2.36) 34 (81.0) – 1.56 (0.68–3.86) 26 (76.5) 0.34 0.75 (0.42–1.34) 8 (19.0) 0.30 0.64 (0.25–1.46) 8 (23.5)
– 0.96 (0.57–1.64) 29 (69.0) – 1.05 (0.50–2.25) 24 (70.6) – 1.13 (0.50–2.64) 21 (61.8) – 0.76 (0.35–1.68) 0.90 1.03 (0.61–1.73) 13 (31.0) 0.90 0.95 (0.44–1.97) 10 (%29.4) 0.78 0.88 (0.37–1.96) 13 (38.2) 0.49 1.31 (0.59–2.82)
– 0.72 (0.34–1.54) 19 (55.9) – 0.81 (0.38–1.75) 0.39 1.38 (0.64–2.93) 15 (44.1) 0.59 1.22 (0.57–2.61)
1.0 (0.47–2.12) 16 (47.1) – 0.88 (0.41–1.88) 1.0 (0.47–2.12) 18 (52.9) 0.76 1.12 (0.53–2.39)
1.0 (0.47–2.12) 1.0 (0.47–2.12)
– 0.74 (0.45–1.22) 22 (52.4) – 0.70 (0.35–1.41) 18 (52.9) 0.24 1.34 (0.81–2.20) 20 (47.6) 0.32 1.41 (0.70–2.82) 16 (47.1)
1.0 (0.61–1.63) 22 (52.4) – 1.09 (0.55–2.19) 17 (50.0) 1.0 (0.61–1.63) 20 (47.6) 0.78 0.90 (0.45–1.80) 17 (50.0)
1.0 (0.47–2.12) 17 (50.0) – 1.0 (0.47–2.12) 17 (50.0) 1.0
– 1.0
n (%)
– 1.0
OR (%95 CI)
– 1.0
Pvalue
Systemic JIA (n = 17)
– 1.03 (0.63–1.68) 22 (52.4) – 1.09 (0.55–2.19) 17 (50.0) 0.88 0.96 (0.59–1.57) 20 (47.6) 0.78 0.90 (0.45–1.80) 17 (50.0)
n (%)
Poly articular JIA (n = 17)
– 1.15 (0.54–2.45) 21 (61.8) – 1.65 (0.77–3.61) 0.71 0.86 (0.40–1.83) 13 (38.2) 0.19 0.60 (0.27–1.29)
OR (%95 CI)
Oligo articular JIA (n = 21)
– 1.23 (0.75–2.01) 21 (50.0) – 1.02 (0.51–2.04) 18 (52.9) 0.40 0.81 (1.49–1.32) 21 (50.0) 0.94 0.97 (0.48–1.94) 16 (47.1)
Pvalue
JIA (overall) (n = 55)
rs1004819 G 77 (49.4) 60 (54.5) A 79 (50.6) 50 (45.5) rs2201841 A 78 (50.0) 56 (50.9) G 78 (50.0) 54 (49.1) rs10889677 C 78 (50.0) 55 (50.0) A 78 (50.0) 55 (50.0) rs1495965 C 95 (60.9) 59 (53.6) T 61 (39.1) 51 (46.4) rs7517847 T 118 (75.6) 76 (69.1) G 38 (24.4) 34 (30.9) rs10489629 T 106 (67.9) 74 (67.3) C 50 (32.1) 36 (32.7) rs11209026 G 150 (96.2) 103 (93.6) A 6 (3.8) 7 (6.4) rs1343151 G 114 (73.1) 86 (78.2) A 42 (26.9) 24 (21.8)
SNP/ allele
Table 1 Allele frequency of single nucleotide polymorphisms (SNPs) in juvenile idiopathic (JIA) and JIA subtypes versus controls and result of v2 statistics
IL23R polymorphisms and IL17A Level in JIA
3
4
Control n (%)
rs1004819 GG 18 (23.1) GA 41 (52.6) AA 19 (24.3) rs2201841 AA 19 (24.4) AG 40 (51.2) GG 19 (24.4) rs10889677 CC 19 (24.4) CA 40 (51.2) AA 19 (24.4) rs1495965 CC 31 (39.7) CT 33 (42.4) TT 14 (17.9) rs7517847 TT 44 (56.4) TG 30 (38.5) GG 4 (5.1) rs10489629 TT 37 (%47.5) TC 32 (%41.0) CC 9 (%11.5) rs11209026 GG 72 (92.3) GA 6 (7.7) rs1343151 GG 41 (52.6) GA 32 (41.0) AA 5 (6.4)
SNP/ allele Pvalue
OR (%95 CI)
OR (%95 CI)
Pvalue
OR (%95 CI)
0.65 0.77 (0.25–2.27) 0.19 2.03 (0.69–6.23) 0.31 0 (0.0–1.76)
0.87 0.62 (0.11–4.90) 15 (88.2) 0.87 0.62 (0.11–4.90) 0.87 1.59 (0.20–8.36) 2 (11.8) 0.87 1.59 (0.20–8.36)
24 (43.6) 0.67 0.85 (0.42–1.72) 9 (42.9) 0.71 0.83 (0.30–2.22) 7 (41.2) 26 (47.3) 0.48 1.28 (0.63–2.59) 11 (52.4) 0.36 1.57 (0.58–4.25) 10 (58.8) 5 (9.1) 0.67 0.76 (0.22–2.43) 1 (4.8) 0.65 0.38 (0.01–2.56) 0 (0.0)
48 (87.3) 0.35 0.57 (0.17–1.87) 18 (85.7) 0.57 0.50 (0.11–2.67) 15 (88.2) 7 (12.7) 0.35 1.74 (0.53–5.83) 3 (14.3) 0.57 1.98 (0.37–8.79) 2 (11.8)
31 (56.4) 0.67 1.16 (0.57–2.35) 13 (61.9) 0.46 1.46 (0.54–4.09) 24 (43.6) 0.76 1.11 (0.54–2.24) 8 (38.1) 0.82 0.88 (0.31–2.39) 0 (0.0) 0.06 0 (0.0–1.13) 0 (0.0) 0.59 0 (0.0–3.05)
0.49 0.68 (0.23–2.02) 0.52 1.41 (0.47–4.17) 0.99 1.15 (0.04–9.91)
8 (47.1) 8 (47.1) 1 (5.9)
26 (47.3) 0.30 0.69 (0.34–1.39) 11 (52.4) 0.74 0.85 (0.31–2.29) 24 (43.6) 0.55 1.23 (0.60–2.50) 9 (42.9) 0.71 1.19 (0.43–3.22) 5 (9.1) 0.39 1.84 (0.44–8.05) 1 (4.8) 0.99 0.92 (0.03–7.84)
9 (52.9) 0.98 1.01 (0.34–3.01) 8 (47.1) 0.65 1.27 (0.42–3.74) 0 (%0.0) 0.72 0 (0.0–3.81)
0.75 0.82 (0.25–2.47) 0.61 0.74 (0.23–2.22) 0.30 1.89 (0.52–6.22)
9 (52.9) 0.98 1.01 (0.34–3.01) 8 (47.1) 0.65 1.27 (0.42–3.74) 0 (0.0) 0.72 0 (0.0–3.81)
8 (47.1) 0.98 0.98 (0.33–2.88) 5 (29.4) 0.39 0.60 (0.17–1.85) 4 (23.5) 0.35 2.33 (0.55–8.73)
7 (41.2) 0.27 0.54 (0.17–1.59) 7 (41.2) 0.83 1.11 (0.36–3.29) 3 (17.6) 0.21 3.88 (0.66–20.83)
7 (41.2) 0.90 1.06 (0.34–3.12) 5 (29.4) 0.34 0.57 (0.16–1.75) 5 (29.4) 0.30 1.89 (0.52–6.22)
4 (23.5) 0.96 0.95 (0.24–3.20) 8 (47.1) 0.76 0.84 (0.28–2.47) 5 (29.4) 0.66 1.29 (0.36–4.10)
5 (29.4) 0.66 1.29 (0.36–4.10) 7 (41.2) 0.46 0.66 (0.21–1.95) 5 (29.4) 0.66 1.29 (0.36–4.10)
6 (35.3) 6 (35.3) 5 (29.4)
0.66 1.29 (0.36–4.10) 0.46 0.66 (0.21–1.95) 0.66 1.29 (0.36–4.10)
0.66 1.29 (0.36–4.10) 0.46 0.66 (0.21–1.95) 0.66 1.29 (0.36–4.10)
14 (32.7) 0.41 0.73 (0.35–1.52) 5 (23.8) 0.18 0.47 (0.14–1.40) 23 (41.8) 0.95 0.98 (0.48–1.98) 12 (57.1) 0.23 1.80 (0.67–4.95) 18 (25.5) 0.30 1.55 (0.66–3.65) 4 (19.1) 0.99 1.07 (0.27–3.59)
7 (41.2) 0.14 2.31 (0.73–7.06) 7 (41.2) 0.41 0.63 (0.20–1.85) 3 (17.6) 0.81 0.66 (0.14–2.44)
n (%)
5 (29.4) 7 (41.2) 5 (29.4)
0.14 2.31 (0.73–7.06) 0.03 0.28 (0.07–0.90) 0.37 1.68 (0.51–5.19)
Pvalue
14 (25.5) 0.88 1.06 (0.46–2.36) 5 (23.8) 0.98 0.97 (0.28–2.95) 27 (49.0) 0.80 0.91 (0.45–1.83) 12 (57.1) 0.64 1.26 (0.74–3.45) 14 (25.5) 0.88 1.06 (0.46–2.36) 4 (19.1) 0.84 0.73 (0.19–2.36)
n (%)
5 (29.4) 7 (41.2) 5 (29.4)
OR (%95 CI)
Systemic JIA (n = 17)
15 (27.3) 0.70 1.16 (0.52–2.57) 5 (23.8) 0.98 0.97 (0.28–2.95) 26 (47.3) 0.65 0.85 (0.42–1.70) 12 (57.1) 0.64 1.26 (0.47–3.45) 14 (25.4) 0.88 1.06 (0.46–2.36) 4 (19.1) 0.84 0.73 (0.19–2.36)
Pvalue
Poly articular JIA (n = 17)
7 (41.2) 4 (23.5) 6 (35.3)
n (%)
Oligo articular JIA (n = 21)
6 (28.6) 0.60 1.33 (0.41–3.90) 9 (42.9) 0.44 0.67 (0.24–1.81) 6 (28.6) 0.68 1.24 (0.39–3.62)
20 (36.4) 0.10 1.89 (0.88–4.10) 20 (36.4) 0.06 0.51 (0.25–1.05) 15 (27.2) 0.70 1.16 (0.52–2.57)
n (%)
JIA (overall) (n = 55)
Table 2 Genotype frequency of single nucleotide polymorphisms (SNPs) in juvenile idiopathic (JIA) and JIA subtypes versus controls and result of v2 statistics
S. Emami et al.
International Journal of Rheumatic Diseases 2015
IL23R polymorphisms and IL17A Level in JIA
Figure 1 Result of linkage disequilibrium test based on D0 value. Four SNPs (rs10489629, rs2201841, rs11209026, rs1343151, rs10889677) were in high linkage disequilibrium (D0 ≥ 0.85).
Table 3 Haplotype frequencies in cases and controls Haplotype rs10489629
rs2201841
rs112090261
rs134315
rs10889677
A A A A G
A G G G G
A A G G G
C C C C A
C C C T T
Cases (freq)
Controls (freq)
P-value
OR (%95 CI)
0.05 0.16 0.10 0.18 0.49
0.03 0.23 0.05 0.18 0.50
0.52 0.19 0.12 0.93 0.94
1.45 (0.45–4.64) 0.65 (0.35–1.23) 2.07 (0.80–5.34) 1.02 (0.54–1.93) 0.98 (0.60–1.60)
ELISA analysis
DISCUSSION
We did not find significant difference between IL-17A levels in sera of JIA patients and the healthy group (Table 4). The level of IL-17A among JIA subtypes was also not significantly different (Table 5). None of the eight SNPs showed significant association with the level of IL-17A in patients’ sera (Table 6).
Based on the role of IL-23 in joint autoimmune inflammation24 we examined the possible influence of IL-23 receptor gene polymorphisms on JIA predisposition.
Table 4 Interleukin (IL)-17A concentration in patients versus controls
Subgroup
IL-17A (pg/mL
Mean
SD
P-value
JIA patients Healthy controls
753.8 807.73
805.87 739.28
0.335
International Journal of Rheumatic Diseases 2015
Table 5 Interleukin (IL)-17A concentration in juvenile idiopathic arthritis subgroups
Oligo-articular Poly-articular Systemic
IL-17A (pg/mL) n
Mean
SD
P-value
17 11 14
886.76 845.45 520.35
883.36 769.96 736.80
0.61
5
S. Emami et al.
Table 6 Association of single nucleotide polymorphism (SNP) genotypes with the level of interleukin (IL)-17A in sera of juvenile idiopathic arthritis patients SNPs rs1004819
rs2201841
rs10889677
rs1495965
rs7517847
rs10489629
rs11209026 rs1343151
Genotype
n
IL-17A (mean SD) pg/mL
P-value
GG GA AA AA AG GG CC CA AA CC CT TT TT TG GG TT TC CC GG GA GG GA
17 14 11 12 19 11 11 20 11 17 16 9 17 20 5 16 21 5 36 6 21 21
728.82 (809.93) 863.21 (889.44) 653.18 (745.46) 548.33 (737.06) 839.21 (865.21) 830.45 (804.14) 598.18 (751.52) 797.25 (826.79) 830.45 (804.14) 826.47 (805.07) 807.50 (853.64) 521.11 (766.72) 818.23 (838.24) 757.50 (785.81) 520.00 (909.395) 712.18 (797.56) 961.66 (818.83) 14.0 (31.3) 828.05 (823.51) 308.33 (548.10) 697.85 (765.82) 809.76 (859.23)
0.81
0.52
0.72
0.70
0.66
0.05
0.09 0.80
Our results showed a weak, albeit statistically significant protective effect of GA genotype of rs1004819 in polyarticular subtype but not in JIA and other subtypes. Other studied SNPs showed no significant association. There has been only one study investigating the correlation between IL-23R gene polymorphism (rs11209026) and JIA disease. This study revealed the protective role of minor allele of rs11209026 in juvenile psoriatic arthritis subtype but no association with JIA or other subtypes.23 Our findings, in conjunction with this study, suggest that IL-23/IL-17 pathway may play a role in the pathogenesis of some subtypes of JIA disease, not in JIA overall. The possible explanation for finding no association between other studied SNPs and JIA or its subtypes may be the small sample size in our study. The other reason could be that these SNPs do not have any effect on JIA or JIA in our ethnic groups. Since there have not been other numerous studies on JIA disease and our studied SNPs, the comparison of our findings is limited to other osteoarticular diseases such as rheumatoid arthritis and ankylosing spondylitis.
6
In a study conducted by Chang et al.,25 no association was reported between rs11209026 and rheumatoid arthritis. Furthermore, in a study in a Korean population which examined seven SNPs of IL-23R (six of them were common with our study), the lack of association between all SNPs and reumatoid arthritis was reported.26 The results of our study were along the same lines as the results of these two studies. It seems that the location of these polymorphisms, except rs11209026, in introns of IL-23R gene is a factor of their inefficiency on protein expression and as a result, their lack of association with the disease. Contrary to these results, other studies confirmed the association of IL-23R polymorphisms with rheumatoid arthritis21,22 and ankylosing spondylitis.17,27–30 The lack of correlation of our results with the results of these studies may be because of our small sample size. Furthermore, we can conclude that IL-23/Il-17 pathway may play a role in the pathogenesis of other osteoarticular inflammations rather than JIA disease. In the second section of our study we examined the correlation between IL-23R polymorphisms and serum IL-17A level. Although it has been reported that Il-23/ IL-17 play critical roles in joint inflammation,31 our results did not show a higher level of IL-17A in serum of patients in comparison with the control group. Moreover, the level of IL-17A did not differ significantly between JIA subtypes. This finding may be justified by the reason that our patients were under treatment by NSAIDs (non-steroidal anti-inflammatory drugs) and DMARDs (disease-modifying antirheumatic drugs) for more than 6 months. Moreover, we can conclude that IL-17A may play a critical role in the synovia of JIA patients rather than serum. Previously, Hazlett et al.32 showed the associaton between rs11209026 minor allele and lower serum IL-17A expression in patients with rheumatoid arthritis. However, we did not find significant correlation between our studied SNPs and serum levels of IL-17A in JIA patients. Although the level of IL17A was much lower in the rare genotypes of rs10489629 (CC) and rs11209026 (GA), the correlation was not significant (P = 0.05 and 0.09, respectively). The lack of association may result from our small sample size. Furthermore, it can be inferred that intronic SNPs do not have influence on protein expression. Our findings did not support the strong association between IL-23R polymorphisms and JIA disease or IL17A expression. As this gene is not studied widely in JIA patients, we cannot incisively accept or reject the role of
International Journal of Rheumatic Diseases 2015
International Journal of Rheumatic Diseases 2015
ORIGINAL ARTICLE
IL23R gene polymorphism with juvenile idiopathic arthritis and its association with serum IL-17A Samira EMAMI,1 Vahid ZIAEE,2,3 Arezou REZAEI,4 Maryam SADR,5 Marzieh MADDAH,3 Ali Akbar AMIRZARGAR1,5 and Nima REZAEI1,4,5,6 1
Department of Immunology, School of Medicine, 2Pediatric Rheumatology Research Group, Rheumatology Research Center, Pediatrics Center of Excellence, 4Research Center for Immunodeficiencies, Children’s Medical Center, 5Molecular Immunology Research Center, Tehran University of Medical Sciences, and 6Universal Scientific Education and Research Network (USERN), Tehran, Iran 3
Abstract Aim: Interleukin 23 (IL-23) and its receptor (IL-23R) seem to play a major role in differentiation of CD4+ T cells into Th17 cells, induction of IL-17 production, and activation of inflammatory pathways. Recent studies have suggested the association of IL-23R polymorphisms with bone and articular inflammation in diseases such as ankylosing spondylitis and rheumatoid arthritis. The aim of this study was to determine the association between IL-23R polymorphisms and juvenile idiopathic arthritis (JIA). Method: A case–control study on 55 patients with JIA and 78 healthy controls was performed. All samples were genotyped for eight single nucleotide polymorphisms (SNPs) of IL23R (rs1004819, rs2201841, rs10889677, rs1495965, rs7517847, rs10489629, rs11209026 and rs1343151), using real-time polymerase chain reaction Taqman genotyping technique. Forty-two patients and 42 healthy controls were chosen randomly to measure the level of serum IL-17A using enzyme-linked immunosorbent assay. Results: Although the heterozygous genotype of rs1004819 (GA) showed a weak, but statistically significant protective effect on polyarticular subtype (P = 0.03), none of the selected SNPs were associated with JIA overall. Indeed the analysis of haplotypes did not show any significant association with JIA. Serum IL-17A level was not significantly different among patients and healthy controls and between JIA subtypes, as well. Moreover, there was no significant correlation between SNPs and serum IL-17A concentration. Conclusion: This is the first study of the IL-23R gene in Iranian patients with JIA. Our results did not show any strong association between IL-23R polymorphisms and JIA disease or serum IL-17A levels. The only association was seen between rs1004819 and polyarticular JIA. Further larger studies may help clarify the role, if any, of the IL-23/IL-17 pathway in the pathogenesis of JIA. Key words: IL-23R, juvenile idiopathic arthritis, polymorphism.
INTRODUCTION Juvenile idiopathic arthritis (JIA) is a chronic inflammatory articular disease among children under 16 years, with a duration of more than 6 weeks Correspondence: Dr Nima Rezaei, MD, PhD, Children’s Medical Center Hospital, Dr Qarib St, Keshavarz Blvd, Tehran 14194, Iran. Email: [email protected]
which is classified into seven subtypes (systemic JIA [sJIA], oligoarticular JIA [oligo-JIA], rheumatoid factor [RF]-negative polyarticular JIA [poly-JIA], RFpositive polyarticular JIA, juvenile psoriatic arthritis [JPsA], enthesitis-related arthritis [ERA] and undifferentiated arthritis) based on the clinical features in the first 6 months of disease.1 JIA is a complex multifactorial disease in which the interaction of environmental and several unknown genetic factors play
© 2015 Asia Pacific League of Associations for Rheumatology and Wiley Publishing Asia Pty Ltd
S. Emami et al.
29 Rueda B et al. (2008) The IL23R Arg381Gln non-synonymous polymorphism confers susceptibility to ankylosing spondylitis. Ann Rheum Dis 67, 1451–4. 30 Safrany E et al. (2009) Variants of the IL23R gene are associated with ankylosing spondylitis but not with Sj€ ogren syndrome in Hungarian population samples. Scand J Immunol 70 (1), 68–74.
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31 Cojocaru-Gofita I, Paulina C, Yeremenko N (2012) The cellular source of IL-17A in the pathogenesis of inflammatory joint diseases. Curr Health Sci J 38(2), 53–7. 32 Hazlett J et al. (2011) IL-23R rs11209026 polymorphism modulates IL-17A expression in patients with rheumatoid arthritis. Genes Immun 13, 282–7.
International Journal of Rheumatic Diseases 2015
