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Analysis of JAK2 and STAT3 polymorphisms in patients with ankylosing spondylitis in Chinese Han population
Clinical Immunology (2010) 136, 442–446
available at www.sciencedirect.com
Clinical Immunology www.elsevier.com/locate/yclim
Analysis of JAK2 and STAT3 polymorphisms in patients with ankylosing spondylitis in Chinese Han population Chao Chen, Xuesong Zhang, Yan Wang ⁎ Department of Orthopaedics, Chinese PLA General Hospital, No. 28, Fuxing Road, Beijing 100853, PR China Received 13 April 2010; accepted with revision 8 May 2010 Available online 2 June 2010 KEYWORDS Janus kinase 2; Signal transducer and activator of transcription 3; Polymorphism; Ankylosing spondylitis
Abstract JAK2 and STAT3 polymorphisms have been implicated to be associated with inflammatory bowel disease, which might share common immunogenesis and genetic factors with AS. Here, we have investigated the possible relationship of JAK2 and STAT3 polymorphisms with AS in a Chinese Han population. We genotyped 200 AS patients and 200 healthy controls for 4 polymorphisms in JAK2 and 6 in STAT3 using the chip-based matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry. There was no difference in the distribution of allele and genotype in either JAK2 or STAT3 between AS groups and controls. Haplotype analysis revealed an association of haplotype rs1536798/rs10119004/rs7857730-CGT in JAK2 locus with AS. In conclusion, we first demonstrated the association of JAK2 polymorphisms with the susceptibility of AS, which indicated that IL-23 pathway also was an important etiological factor in AS in Chinese population. © 2010 Elsevier Inc. All rights reserved.
Introduction Ankylosing spondylitis (AS) is featured by acute and chronic inflammations of sacroiliac joint and bone insertion sites of tendons or ligaments. The pathogenesis of AS remains poorly understood, but there is little doubt that the disease susceptibility is largely determined by genetic factors. The heritability of AS was evaluated to be beyond 90% [1]. Although human leukocyte antigen-B27 (HLA-B27) has been recognized to be associated with AS for almost 4 decades and its contribution to the genetic of AS is best characterized at present, cumulative evidence proved that a substantial proportion of AS susceptibility is encoded by non-major⁎ Corresponding author. Fax: +86 10 88219862. E-mail address: [email protected] (Y. Wang).
histocompatibility-complex genes in recent years [2]. In particular, interleukin-23 receptor (IL-23R) gene has already been confirmed to be associated with AS in Caucasian population by several studies [3–5] in the last 2 years. Furthermore, the view that clinical and pathological manifestations of AS are closely related with the immunological imbalance is also commonly accepted. As the regulation of both innate and adaptive immune response is altered in AS, inflammatory mediators involved in the immunopathological cascade of this condition might be interesting functional candidate genes [6]. The involvement of IL-23/Th17/IL-17 axis in the pathogenesis of spondyloarthritis has been implicated by multiple studies recently [7]. IL-17, a proinflammatory cytokine produced by osteoclastogenic Th17 cells which was defined as the third subset of helper T cells in 2005, may be directly involved in cartilage destruction and inflammatory subchondral erosions [8]. IL-23
1521-6616/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.clim.2010.05.003
JAK2 and STAT3 polymorphisms in patients with AS in Chinese Han population can promote the expansion and survival of the differentiated Th17 cells [9]. The IL-23/Th17/IL-17 axis has been suggested playing a key role not only in the onset stage but in bone destruction stage of autoimmune arthritis [10]. Moreover, the IL-23 and IL-17 levels are found to be elevated [11], and numbers of Th17 cells are significantly increased [9] in the peripheral blood of AS patients. All of these indicate that IL23/Th17/IL-17 axis may be associated with the pathogenesis of AS. Janus kinase 2 (JAK2) and signal transducer and activator of transcription 3 (STAT3) are both the regulatory factor of IL-23 pathway, and their genes also play a critical role in IL23R signaling. In addition, STAT3 is of great importance for the differentiation and maintenance of Th17 cells [12]. The polymorphisms rs10758669 [12,13] in JAK2 and rs744166 [12,14], rs12948909 [13] in STAT3 were affirmed to be associated with the susceptibility of inflammatory bowel disease (IBD), a condition of which the susceptibility genes are probably overlapped with that of AS [15]. Based on these findings, the question was raised whether the variants of JAK2 and STAT3 may also be associated with the susceptibility of AS. No report was published previously about JAK2 and STAT3 polymorphisms in AS. We hereby analyzed the association of 10 single nucleotide polymorphisms (SNP) of JAK2 and STAT3 gene with AS in Chinese Han population.
Materials and methods Patients and controls A total of 200 patients with AS and 200 unrelated healthy controls were recruited. All patients were of Chinese Han origin with an average age of 29.7 ± 8.7 years (range: 16 years–60 years). The mean duration of the symptoms was 9 years. AS patients were screened to satisfy the modified New York criteria [16] by experienced rheumatologists, and were independent of IBD and/or psoriasis for the purpose of avoiding the probable interferences from overlapping genes. The healthy controls were matched by both nationality and age and sex. All subjects provided their informed consent approved by the local ethic committee of Chinese PLA General Hospital.
SNP selection The SNPs detected in this study included the 3 polymorphisms in recent IBD findings and 7 tag SNPs. Among these sites, 3 polymorphisms were found to be associated with IBD in the recent genome-wide scan by WTCCC [12] and association study by Anderson et al. [13]. Tag SNPs in the 2 genes were then screened by SNP Browser version 3.5 with the limitation parameters set as follows: population = CHB (Han-Chinese, Beijing), minimal allele frequency (MAF) N 0.1 and pairwise r2 b 0.85. The polymorphisms with higher MAF (N 0.3) were randomly selected for current study.
Genotyping DNA was isolated from whole blood using the AxyPrep Blood Genomic DNA Miniprep kit (Axygen Biosciences, Union City,
443
CA, USA). Detection of SNPs was performed by MassARRAY system (Sequenom, San Diego, CA, USA) using the chip-based matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry technology. Primers were obtained from Sangon Biotech (Shanghai, China). Briefly, multiplex reaction was designed using Assay Designer software version 3.0 (Sequenom) and was processed following standard protocols for iPLEX chemistry. The reaction products were then cleaned and dispensed onto a SpectroCHIP bioarray. The chip was scanned using MassARRAY workstation version 3.3 and the resulting spectra were analyzed using the Sequenom TYPER software.
Statistical analysis Hardy–Weinberg equilibrium (HWE) testing was carried out for all 10 SNPs. Single marker differences were accessed using χ2 tests, as well as the 3 genotypes in cases and controls. Data of odds ratio (OD) and 95% confidence intervals (CI) were calculated. Haploview software version 4.1 was used to analyze the association between haplotypes and the disease.
Results A total of 400 subjects (200 patients with AS, 200 controls) were successfully genotyped for polymorphisms in JAK2 and STAT3. 89.5% (179/200) of AS patients were male. Data for HLA-B27 status were available in 187 patients, of which 175 patients presented B27 positive (93.6%) and 12 were tested negative (6.4%). All participants were genotyped for a total of 10 SNPs, 4 in JAK2 and 6 in STAT3 gene. 3 of the 10 SNPs were the sites reported to be associated with IBD, including rs10758669 in JAK2 and rs744166, rs12948909 in STAT3. Another 7 polymorphisms were tag SNPs with MAF N 0.3. These SNPs were rs1536798, rs10119004, rs7857730 in JAK2 and rs9912773, rs12601982, rs8078731, rs1887427 in STAT3. The studied population was in HWE with the p values of 0.19 to 0.81 in case group and 0.27 to 0.85 in control group. No association with AS was observed for allele and genotype frequency in either JAK2 or STAT3 (Tables 1 and 2). Haploview identified 3 blocks (Fig. 1), in which the frequency of the haplotype rs1536798/rs10119004/rs7857730-CGT of JAK2 locus was significantly lower in cases vs. controls (0.055 vs. 0.095, p = 0.0323).
Discussion We undertook the present study to investigate the association between AS and the polymorphisms in JAK2 and STAT3 in a Chinese Han population. Our findings show that although no single marker of JAK2 and STAT3 contributed to the susceptibility risk of AS, a haplotype within JAK2 locus was related to the disease, indicating that JAK2 locus is associated with the susceptibility of AS. This result added to our knowledge of Th17 pathway in the immunogenesis of AS. A potential reason why the haplotypic finding is stronger than individual SNP associations could be because the markers chosen by this study may not be causally associated with disease or may encode only part of the susceptibility
444
C. Chen et al.
Table 1 Odds ratios (OR) and 95% confidence intervals (95% CI) for disease association of 10 single-nucleotide polymorphisms (SNP) in the JAK2 and STAT3 gene in a Chinese AS case–control cohort. Gene
SNP rs no.
Allele
Frequency Case
Control
JAK2
rs10758669
JAK2
rs1536798
JAK2
rs10119004
JAK2
rs7857730
STAT3
rs744166
STAT3
rs12948909
STAT3
rs9912773
STAT3
rs12601982
STAT3
rs8078731
STAT3
rs1887427
A C A C A G T G T C A C C G A G A T A G
0.695 0.305 0.347 0.652 0.399 0.601 0.460 0.540 0.637 0.362 0.907 0.092 0.652 0.347 0.685 0.315 0.693 0.308 0.808 0.193
0.670 0.330 0.328 0.672 0.395 0.605 0.485 0.515 0.603 0.398 0.900 0.100 0.625 0.375 0.680 0.320 0.675 0.325 0.838 0.163
effect [17]. Unfortunately, there was almost no data on the biological implication of polymorphisms in JAK2 and STAT3 in international literature. We believe that how these SNPs affect the function of the 2 genes is very significant for thorough understanding the associations between them and AS. Therefore, functional study of the SNPs should really be considered in future studies. JAKs/STATs signaling coordinate multiple signaling events in T cells leading to their differentiation into distinct subpopulations as well as regulation of pro- and antiapoptotic cascades [18]. During the differentiating process of Th17, innate cytokines signal through STAT3 to establish lineage specific transcriptional programs. STAT3 functions as a critical transcription factor for Th17 differentiation. Four mechanisms were suggested in the literature describing in detail as follows. Firstly, the IL-17A/F locus has putative STAT-binding sites. By using chromatin immunoprecipitation assays, Chen et al. [19] found that STAT3 directly binds to this promoter. Thus, STAT3 appears to be a direct regulator of IL-17. Secondly, IL-6, IL-21 and IL-23 all activate Janus family kinases and preferentially activate STAT3. Since the production of IL-21 by IL-6 is also STAT3-dependent, STAT3 appears to be a direct regulator of IL-21 production. Thirdly, STAT3 plays an important role in the expression of retinoic acid receptor-related orphan receptor (ROR) γt which is a key transcription factor for Th17 differentiation. Heterozygous STAT3 gene mutations reduce the availability of functional STAT3 dimers to 25% and concordantly reduce RORγt expression by fourfold [20]. Lastly, IL-6, IL-21 and IL23 can up-regulate expression of the IL-23R which is also likely to be STAT3-dependent. However, STAT3 functioning can only be initiated via phosphorylating by JAK2 after the binding of IL-23 to its receptor. JAK2 is a gene encoding an IL-23R signaling
p value
OR
95% CI
0.4476
1.122
0.833–1.512
0.5497
1.094
0.816–1.466
0.8967
1.019
0.767–1.353
0.4788
0.905
0.685–1.194
0.3079
1.16
0.872–1.544
0.7191
1.09
0.681–1.745
0.4182
1.127
0.844–1.504
0.8793
1.023
0.760–1.378
0.5946
1.084
0.805–1.461
0.2668
0.814
0.566–1.171
component up-stream of STAT3. It is therefore closely related to STAT3 in IL-17 signaling. IL-23 binding to IL-23R results in the activation of JAK2, which can phosphorylate IL-23R at discrete locations and thus form docking sites for the STAT3. STAT3 are then phosphorylated by the JAK2 and capable of dimerising and translocating to the nucleus where they activate the transcription of key proinflammatory genes IL-17 [21]. Inhibition of JAK2 will dramatically reduce STAT3 phosphorylation [22]. JAK2, tyrosine kinase 2 (TYK2), interleukin 12 receptor (IL-12RB1) and interleukin 12 precursor (IL-12B) all belong to a gene network which is typically referred to as the “IL-23 pathway” [23], and IL23R is also constitutively associated with JAK2 and STAT3 [24]. Several investigations demonstrated the association between the variants of JAK2, STAT3, IL-23R and IBD in Caucasian populations [12,13,25]. Since IBD is closely related to AS clinically and there might be a strong genetic overlap involved in the two conditions, JAK2 and STAT3 may likewise have possible impact on the susceptibility of AS. This inference was partially supported by the present study, which showed that JAK2 was a potential gene associated with AS in Chinese population. On the other hand, our findings also further affirmed the assumption that AS shares common genetic background with IBD. In addition, despite the confirmation of relationship between IL-23R and AS in Caucasian patients [26], this gene is not associated with AS patients in Chinese [27]. Davidson et al. [27] presumed that this difference between ethnic groups could be possibly explained by different disease mechanisms independent of IL-23R in Chinese, or due to an association with a different gene also involved in the IL-23 signaling pathway. Inasmuch as JAK2 belongs to IL-23 pathway, the current study provided some evidence that supported the latter supposition while
JAK2 and STAT3 polymorphisms in patients with AS in Chinese Han population Table 2 Genotype frequency of the 10 SNPs in case group and control group. Gene
rs
Genotype
Frequency Case
Control
JAK2
rs10758669
JAK2
rs1536798
JAK2
rs10119004
STAT3
rs7857730
STAT3
rs744166
STAT3
rs12948909
AC CC AA AC CC AA AG AA GG TT GG GT CC TT CT AC CC AA CC GG CG AA AG GG AA TT AT AG AA GG
0.400 0.105 0.495 0.435 0.435 0.130 0.437 0.181 0.382 0.235 0.315 0.450 0.125 0.400 0.475 0.165 0.010 0.825 0.415 0.110 0.475 0.460 0.450 0.090 0.465 0.080 0.455 0.285 0.665 0.050
0.430 0.115 0.455 0.475 0.435 0.090 0.490 0.150 0.360 0.230 0.260 0.510 0.155 0.360 0.485 0.190 0.005 0.805 0.380 0.130 0.490 0.470 0.420 0.110 0.465 0.115 0.420 0.255 0.710 0.035
STAT3
rs9912773
STAT3
rs12601982
STAT3
rs8078731
STAT3
rs1887427
p value
0.7245
0.4053
0.5207
0.4039
To conclude, we are the first to investigate the association of JAK2 and STAT3 and AS. We demonstrated that JAK2 played an indirect role in the susceptibility of AS in Chinese Han population. Resequencing of the gene and genome wide scan studies in large samples in multiple races are expected to refine this relationship. Particularly, functional exploring of the polymorphisms should urgently be processed for deeper understanding of the mechanisms of AS.
Acknowledgment We are grateful to Dr. Yanyan Wang and Lixia Feng for their kind help in patient screening. We also thank Shanghai Benegene Biotechnology Co, Ltd for their excellent technical support.
0.5814
References 0.6926
0.7089
0.7304
0.4638
0.5607
denied the former. We accordingly may still consider IL-23 pathway to be an important etiological factor for AS in Chinese population.
Figure 1
445
[1] J.D. Reveille, E.J. Ball, M.A. Khan, HLA-B27 and genetic predisposing factors in spondyloarthropathies, Curr. Opin. Rheumatol. 13 (2001) 265–272. [2] J.D. Reveille, Recent studies on the genetic basis of ankylosing spondylitis, Curr. Rheumatol. Rep. 11 (2009) 340–348. [3] P.R. Burton, D.G. Clayton, L.R. Cardon, et al., Association scan of 14, 500 nonsynonymous SNPs in four diseases identifies autoimmunity variants, Nat. Genet. 39 (2007) 1329–1337. [4] T. Karaderi, D. Harvey, C. Farrar, et al., Association between the interleukin 23 receptor and ankylosing spondylitis is confirmed by a new UK case–control study and meta-analysis of published series, Rheumatology (Oxford) 48 (2009) 386–389. [5] P. Rahman, R.D. Inman, D.D. Gladman, J.P. Reeve, L. Peddle, W.P. Maksymowych, Association of interleukin-23 receptor variants with ankylosing spondylitis, Arthritis Rheum. 58 (2008) 1020–1025. [6] B. Rueda, G. Orozco, E. Raya, et al., The IL23R Arg381Gln non-synonymous polymorphism confers susceptibility to ankylosing spondylitis, Ann. Rheum. Dis. 67 (2008) 1451–1454. [7] G. Layh-Schmitt, R.A. Colbert, The interleukin-23/interleukin-17 axis in spondyloarthritis, Curr. Opin. Rheumatol. 20 (2008) 392–397.
Haplotype structure of all markers. Darker color indicates higher linkage disequilibrium (LD), lighter color indicates less LD.
446 [8] S.L. Gaffen, The role of interleukin-17 in the pathogenesis of rheumatoid arthritis, Curr. Rheumatol. Rep. 11 (2009) 365–370. [9] H. Shen, J.C. Goodall, J.S.H. Gaston, Frequency and phenotype of peripheral blood Th17 cells in ankylosing spondylitis and rheumatoid arthritis, Arthritis Rheum. 60 (2009) 1647–1656. [10] K. Sato, A. Suematsu, K. Okamoto, et al., Th17 functions as an osteoclastogenic helper T cell subset that links T cell activation and bone destruction, J. Exp. Med. 203 (2006) 2673–2682. [11] X. Wang, Z. Lin, Q. Wei, Y. Jiang, J. Gu, Expression of IL-23 and IL-17 and effect of IL-23 on IL-17 production in ankylosing spondylitis, Rheumatol. Int. 29 (2009) 1343–1347. [12] J.C. Barrett, S. Hansoul, D.L. Nicolae, et al., Genome-wide association defines more than 30 distinct susceptibility loci for Crohn's disease, Nat. Genet. 40 (2008) 955–962. [13] C.A. Anderson, D.C. Massey, J.C. Barrett, et al., Investigation of Crohn's disease risk loci in ulcerative colitis further defines their molecular relationship, Gastroenterology 136 (2009) 523–529.e3. [14] A. Franke, T. Balschun, T.H. Karlsen, et al., Replication of signals from recent studies of Crohn's disease identifies previously unknown disease loci for ulcerative colitis, Nat. Genet. 40 (2008) 713–715. [15] E. Colombo, A. Latiano, O. Palmieri, F. Bossa, A. Andriulli, V. Annese, Enteropathic spondyloarthropathy: a common genetic background with inflammatory bowel disease? World J. Gastroenterol. 15 (2009) 2456–2462. [16] S. van der Linden, H.A. Valkenburg, A. Cats, Evaluation of diagnostic criteria for ankylosing spondylitis. A proposal for modification of the New York criteria, Arthritis Rheum. 27 (1984) 361–368. [17] T.J. Kim, T.H. Kim, H.J. Lee, et al., Interleukin 1 polymorphisms in patients with ankylosing spondylitis in Korea, J. Rheumatol. 35 (2008) 1603–1608.
C. Chen et al. [18] S.M. Stepkowski, W. Chen, J.A. Ross, Z.S. Nagy, R.A. Kirken, STAT3: an important regulator of multiple cytokine functions, Transplantation 85 (2008) 1372–1377. [19] Z. Chen, A. Laurence, J.J. O'Shea, Signal transduction pathways and transcriptional regulation in the control of Th17 differentiation, Semin. Immunol. 19 (2007) 400–408. [20] A. Fischer, Human immunodeficiency: connecting STAT3, Th17 and human mucosal immunity, Immunol. Cell Biol. 86 (2008) 549–551. [21] E. Safrany, B. Pazar, V. Csongei, et al., Variants of the IL23R gene are associated with ankylosing spondylitis but not with Sjogren syndrome in Hungarian population samples, Scand. J. Immunol. 70 (2009) 68–74. [22] A. Saleh, L. Shan, A.J. Halayko, S. Kung, A.S. Gounni, Critical role for STAT3 in IL-17A-mediated CCL11 expression in human airway smooth muscle cells, J. Immunol. 182 (2009) 3357–3365. [23] C. Abraham, J.H. Cho, IL-23 and autoimmunity: new insights into the pathogenesis of inflammatory bowel disease, Annu. Rev. Med. 60 (2009) 97–110. [24] C. Parham, M. Chirica, J. Timans, et al., A receptor for the heterodimeric cytokine IL-23 is composed of IL-12Rbeta1 and a novel cytokine receptor subunit, IL-23R, J. Immunol. 168 (2002) 5699–5708. [25] C.W. Lees, J. Satsangi, Genetics of inflammatory bowel disease: implications for disease pathogenesis and natural history, Expert Rev. Gastroenterol. Hepatol. 3 (2009) 513–534. [26] G.P. Thomas, M.A. Brown, Genetics and genomics of ankylosing spondylitis, Immunol. Rev. 233 (2010) 162–180. [27] S.I. Davidson, X. Wu, Y. Liu, et al., Association of ERAP1, but not IL23R, with ankylosing spondylitis in a Han Chinese population, Arthritis Rheum. 60 (2009) 3263–3268.
available at www.sciencedirect.com
Clinical Immunology www.elsevier.com/locate/yclim
Analysis of JAK2 and STAT3 polymorphisms in patients with ankylosing spondylitis in Chinese Han population Chao Chen, Xuesong Zhang, Yan Wang ⁎ Department of Orthopaedics, Chinese PLA General Hospital, No. 28, Fuxing Road, Beijing 100853, PR China Received 13 April 2010; accepted with revision 8 May 2010 Available online 2 June 2010 KEYWORDS Janus kinase 2; Signal transducer and activator of transcription 3; Polymorphism; Ankylosing spondylitis
Abstract JAK2 and STAT3 polymorphisms have been implicated to be associated with inflammatory bowel disease, which might share common immunogenesis and genetic factors with AS. Here, we have investigated the possible relationship of JAK2 and STAT3 polymorphisms with AS in a Chinese Han population. We genotyped 200 AS patients and 200 healthy controls for 4 polymorphisms in JAK2 and 6 in STAT3 using the chip-based matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry. There was no difference in the distribution of allele and genotype in either JAK2 or STAT3 between AS groups and controls. Haplotype analysis revealed an association of haplotype rs1536798/rs10119004/rs7857730-CGT in JAK2 locus with AS. In conclusion, we first demonstrated the association of JAK2 polymorphisms with the susceptibility of AS, which indicated that IL-23 pathway also was an important etiological factor in AS in Chinese population. © 2010 Elsevier Inc. All rights reserved.
Introduction Ankylosing spondylitis (AS) is featured by acute and chronic inflammations of sacroiliac joint and bone insertion sites of tendons or ligaments. The pathogenesis of AS remains poorly understood, but there is little doubt that the disease susceptibility is largely determined by genetic factors. The heritability of AS was evaluated to be beyond 90% [1]. Although human leukocyte antigen-B27 (HLA-B27) has been recognized to be associated with AS for almost 4 decades and its contribution to the genetic of AS is best characterized at present, cumulative evidence proved that a substantial proportion of AS susceptibility is encoded by non-major⁎ Corresponding author. Fax: +86 10 88219862. E-mail address: [email protected] (Y. Wang).
histocompatibility-complex genes in recent years [2]. In particular, interleukin-23 receptor (IL-23R) gene has already been confirmed to be associated with AS in Caucasian population by several studies [3–5] in the last 2 years. Furthermore, the view that clinical and pathological manifestations of AS are closely related with the immunological imbalance is also commonly accepted. As the regulation of both innate and adaptive immune response is altered in AS, inflammatory mediators involved in the immunopathological cascade of this condition might be interesting functional candidate genes [6]. The involvement of IL-23/Th17/IL-17 axis in the pathogenesis of spondyloarthritis has been implicated by multiple studies recently [7]. IL-17, a proinflammatory cytokine produced by osteoclastogenic Th17 cells which was defined as the third subset of helper T cells in 2005, may be directly involved in cartilage destruction and inflammatory subchondral erosions [8]. IL-23
1521-6616/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.clim.2010.05.003
JAK2 and STAT3 polymorphisms in patients with AS in Chinese Han population can promote the expansion and survival of the differentiated Th17 cells [9]. The IL-23/Th17/IL-17 axis has been suggested playing a key role not only in the onset stage but in bone destruction stage of autoimmune arthritis [10]. Moreover, the IL-23 and IL-17 levels are found to be elevated [11], and numbers of Th17 cells are significantly increased [9] in the peripheral blood of AS patients. All of these indicate that IL23/Th17/IL-17 axis may be associated with the pathogenesis of AS. Janus kinase 2 (JAK2) and signal transducer and activator of transcription 3 (STAT3) are both the regulatory factor of IL-23 pathway, and their genes also play a critical role in IL23R signaling. In addition, STAT3 is of great importance for the differentiation and maintenance of Th17 cells [12]. The polymorphisms rs10758669 [12,13] in JAK2 and rs744166 [12,14], rs12948909 [13] in STAT3 were affirmed to be associated with the susceptibility of inflammatory bowel disease (IBD), a condition of which the susceptibility genes are probably overlapped with that of AS [15]. Based on these findings, the question was raised whether the variants of JAK2 and STAT3 may also be associated with the susceptibility of AS. No report was published previously about JAK2 and STAT3 polymorphisms in AS. We hereby analyzed the association of 10 single nucleotide polymorphisms (SNP) of JAK2 and STAT3 gene with AS in Chinese Han population.
Materials and methods Patients and controls A total of 200 patients with AS and 200 unrelated healthy controls were recruited. All patients were of Chinese Han origin with an average age of 29.7 ± 8.7 years (range: 16 years–60 years). The mean duration of the symptoms was 9 years. AS patients were screened to satisfy the modified New York criteria [16] by experienced rheumatologists, and were independent of IBD and/or psoriasis for the purpose of avoiding the probable interferences from overlapping genes. The healthy controls were matched by both nationality and age and sex. All subjects provided their informed consent approved by the local ethic committee of Chinese PLA General Hospital.
SNP selection The SNPs detected in this study included the 3 polymorphisms in recent IBD findings and 7 tag SNPs. Among these sites, 3 polymorphisms were found to be associated with IBD in the recent genome-wide scan by WTCCC [12] and association study by Anderson et al. [13]. Tag SNPs in the 2 genes were then screened by SNP Browser version 3.5 with the limitation parameters set as follows: population = CHB (Han-Chinese, Beijing), minimal allele frequency (MAF) N 0.1 and pairwise r2 b 0.85. The polymorphisms with higher MAF (N 0.3) were randomly selected for current study.
Genotyping DNA was isolated from whole blood using the AxyPrep Blood Genomic DNA Miniprep kit (Axygen Biosciences, Union City,
443
CA, USA). Detection of SNPs was performed by MassARRAY system (Sequenom, San Diego, CA, USA) using the chip-based matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry technology. Primers were obtained from Sangon Biotech (Shanghai, China). Briefly, multiplex reaction was designed using Assay Designer software version 3.0 (Sequenom) and was processed following standard protocols for iPLEX chemistry. The reaction products were then cleaned and dispensed onto a SpectroCHIP bioarray. The chip was scanned using MassARRAY workstation version 3.3 and the resulting spectra were analyzed using the Sequenom TYPER software.
Statistical analysis Hardy–Weinberg equilibrium (HWE) testing was carried out for all 10 SNPs. Single marker differences were accessed using χ2 tests, as well as the 3 genotypes in cases and controls. Data of odds ratio (OD) and 95% confidence intervals (CI) were calculated. Haploview software version 4.1 was used to analyze the association between haplotypes and the disease.
Results A total of 400 subjects (200 patients with AS, 200 controls) were successfully genotyped for polymorphisms in JAK2 and STAT3. 89.5% (179/200) of AS patients were male. Data for HLA-B27 status were available in 187 patients, of which 175 patients presented B27 positive (93.6%) and 12 were tested negative (6.4%). All participants were genotyped for a total of 10 SNPs, 4 in JAK2 and 6 in STAT3 gene. 3 of the 10 SNPs were the sites reported to be associated with IBD, including rs10758669 in JAK2 and rs744166, rs12948909 in STAT3. Another 7 polymorphisms were tag SNPs with MAF N 0.3. These SNPs were rs1536798, rs10119004, rs7857730 in JAK2 and rs9912773, rs12601982, rs8078731, rs1887427 in STAT3. The studied population was in HWE with the p values of 0.19 to 0.81 in case group and 0.27 to 0.85 in control group. No association with AS was observed for allele and genotype frequency in either JAK2 or STAT3 (Tables 1 and 2). Haploview identified 3 blocks (Fig. 1), in which the frequency of the haplotype rs1536798/rs10119004/rs7857730-CGT of JAK2 locus was significantly lower in cases vs. controls (0.055 vs. 0.095, p = 0.0323).
Discussion We undertook the present study to investigate the association between AS and the polymorphisms in JAK2 and STAT3 in a Chinese Han population. Our findings show that although no single marker of JAK2 and STAT3 contributed to the susceptibility risk of AS, a haplotype within JAK2 locus was related to the disease, indicating that JAK2 locus is associated with the susceptibility of AS. This result added to our knowledge of Th17 pathway in the immunogenesis of AS. A potential reason why the haplotypic finding is stronger than individual SNP associations could be because the markers chosen by this study may not be causally associated with disease or may encode only part of the susceptibility
444
C. Chen et al.
Table 1 Odds ratios (OR) and 95% confidence intervals (95% CI) for disease association of 10 single-nucleotide polymorphisms (SNP) in the JAK2 and STAT3 gene in a Chinese AS case–control cohort. Gene
SNP rs no.
Allele
Frequency Case
Control
JAK2
rs10758669
JAK2
rs1536798
JAK2
rs10119004
JAK2
rs7857730
STAT3
rs744166
STAT3
rs12948909
STAT3
rs9912773
STAT3
rs12601982
STAT3
rs8078731
STAT3
rs1887427
A C A C A G T G T C A C C G A G A T A G
0.695 0.305 0.347 0.652 0.399 0.601 0.460 0.540 0.637 0.362 0.907 0.092 0.652 0.347 0.685 0.315 0.693 0.308 0.808 0.193
0.670 0.330 0.328 0.672 0.395 0.605 0.485 0.515 0.603 0.398 0.900 0.100 0.625 0.375 0.680 0.320 0.675 0.325 0.838 0.163
effect [17]. Unfortunately, there was almost no data on the biological implication of polymorphisms in JAK2 and STAT3 in international literature. We believe that how these SNPs affect the function of the 2 genes is very significant for thorough understanding the associations between them and AS. Therefore, functional study of the SNPs should really be considered in future studies. JAKs/STATs signaling coordinate multiple signaling events in T cells leading to their differentiation into distinct subpopulations as well as regulation of pro- and antiapoptotic cascades [18]. During the differentiating process of Th17, innate cytokines signal through STAT3 to establish lineage specific transcriptional programs. STAT3 functions as a critical transcription factor for Th17 differentiation. Four mechanisms were suggested in the literature describing in detail as follows. Firstly, the IL-17A/F locus has putative STAT-binding sites. By using chromatin immunoprecipitation assays, Chen et al. [19] found that STAT3 directly binds to this promoter. Thus, STAT3 appears to be a direct regulator of IL-17. Secondly, IL-6, IL-21 and IL-23 all activate Janus family kinases and preferentially activate STAT3. Since the production of IL-21 by IL-6 is also STAT3-dependent, STAT3 appears to be a direct regulator of IL-21 production. Thirdly, STAT3 plays an important role in the expression of retinoic acid receptor-related orphan receptor (ROR) γt which is a key transcription factor for Th17 differentiation. Heterozygous STAT3 gene mutations reduce the availability of functional STAT3 dimers to 25% and concordantly reduce RORγt expression by fourfold [20]. Lastly, IL-6, IL-21 and IL23 can up-regulate expression of the IL-23R which is also likely to be STAT3-dependent. However, STAT3 functioning can only be initiated via phosphorylating by JAK2 after the binding of IL-23 to its receptor. JAK2 is a gene encoding an IL-23R signaling
p value
OR
95% CI
0.4476
1.122
0.833–1.512
0.5497
1.094
0.816–1.466
0.8967
1.019
0.767–1.353
0.4788
0.905
0.685–1.194
0.3079
1.16
0.872–1.544
0.7191
1.09
0.681–1.745
0.4182
1.127
0.844–1.504
0.8793
1.023
0.760–1.378
0.5946
1.084
0.805–1.461
0.2668
0.814
0.566–1.171
component up-stream of STAT3. It is therefore closely related to STAT3 in IL-17 signaling. IL-23 binding to IL-23R results in the activation of JAK2, which can phosphorylate IL-23R at discrete locations and thus form docking sites for the STAT3. STAT3 are then phosphorylated by the JAK2 and capable of dimerising and translocating to the nucleus where they activate the transcription of key proinflammatory genes IL-17 [21]. Inhibition of JAK2 will dramatically reduce STAT3 phosphorylation [22]. JAK2, tyrosine kinase 2 (TYK2), interleukin 12 receptor (IL-12RB1) and interleukin 12 precursor (IL-12B) all belong to a gene network which is typically referred to as the “IL-23 pathway” [23], and IL23R is also constitutively associated with JAK2 and STAT3 [24]. Several investigations demonstrated the association between the variants of JAK2, STAT3, IL-23R and IBD in Caucasian populations [12,13,25]. Since IBD is closely related to AS clinically and there might be a strong genetic overlap involved in the two conditions, JAK2 and STAT3 may likewise have possible impact on the susceptibility of AS. This inference was partially supported by the present study, which showed that JAK2 was a potential gene associated with AS in Chinese population. On the other hand, our findings also further affirmed the assumption that AS shares common genetic background with IBD. In addition, despite the confirmation of relationship between IL-23R and AS in Caucasian patients [26], this gene is not associated with AS patients in Chinese [27]. Davidson et al. [27] presumed that this difference between ethnic groups could be possibly explained by different disease mechanisms independent of IL-23R in Chinese, or due to an association with a different gene also involved in the IL-23 signaling pathway. Inasmuch as JAK2 belongs to IL-23 pathway, the current study provided some evidence that supported the latter supposition while
JAK2 and STAT3 polymorphisms in patients with AS in Chinese Han population Table 2 Genotype frequency of the 10 SNPs in case group and control group. Gene
rs
Genotype
Frequency Case
Control
JAK2
rs10758669
JAK2
rs1536798
JAK2
rs10119004
STAT3
rs7857730
STAT3
rs744166
STAT3
rs12948909
AC CC AA AC CC AA AG AA GG TT GG GT CC TT CT AC CC AA CC GG CG AA AG GG AA TT AT AG AA GG
0.400 0.105 0.495 0.435 0.435 0.130 0.437 0.181 0.382 0.235 0.315 0.450 0.125 0.400 0.475 0.165 0.010 0.825 0.415 0.110 0.475 0.460 0.450 0.090 0.465 0.080 0.455 0.285 0.665 0.050
0.430 0.115 0.455 0.475 0.435 0.090 0.490 0.150 0.360 0.230 0.260 0.510 0.155 0.360 0.485 0.190 0.005 0.805 0.380 0.130 0.490 0.470 0.420 0.110 0.465 0.115 0.420 0.255 0.710 0.035
STAT3
rs9912773
STAT3
rs12601982
STAT3
rs8078731
STAT3
rs1887427
p value
0.7245
0.4053
0.5207
0.4039
To conclude, we are the first to investigate the association of JAK2 and STAT3 and AS. We demonstrated that JAK2 played an indirect role in the susceptibility of AS in Chinese Han population. Resequencing of the gene and genome wide scan studies in large samples in multiple races are expected to refine this relationship. Particularly, functional exploring of the polymorphisms should urgently be processed for deeper understanding of the mechanisms of AS.
Acknowledgment We are grateful to Dr. Yanyan Wang and Lixia Feng for their kind help in patient screening. We also thank Shanghai Benegene Biotechnology Co, Ltd for their excellent technical support.
0.5814
References 0.6926
0.7089
0.7304
0.4638
0.5607
denied the former. We accordingly may still consider IL-23 pathway to be an important etiological factor for AS in Chinese population.
Figure 1
445
[1] J.D. Reveille, E.J. Ball, M.A. Khan, HLA-B27 and genetic predisposing factors in spondyloarthropathies, Curr. Opin. Rheumatol. 13 (2001) 265–272. [2] J.D. Reveille, Recent studies on the genetic basis of ankylosing spondylitis, Curr. Rheumatol. Rep. 11 (2009) 340–348. [3] P.R. Burton, D.G. Clayton, L.R. Cardon, et al., Association scan of 14, 500 nonsynonymous SNPs in four diseases identifies autoimmunity variants, Nat. Genet. 39 (2007) 1329–1337. [4] T. Karaderi, D. Harvey, C. Farrar, et al., Association between the interleukin 23 receptor and ankylosing spondylitis is confirmed by a new UK case–control study and meta-analysis of published series, Rheumatology (Oxford) 48 (2009) 386–389. [5] P. Rahman, R.D. Inman, D.D. Gladman, J.P. Reeve, L. Peddle, W.P. Maksymowych, Association of interleukin-23 receptor variants with ankylosing spondylitis, Arthritis Rheum. 58 (2008) 1020–1025. [6] B. Rueda, G. Orozco, E. Raya, et al., The IL23R Arg381Gln non-synonymous polymorphism confers susceptibility to ankylosing spondylitis, Ann. Rheum. Dis. 67 (2008) 1451–1454. [7] G. Layh-Schmitt, R.A. Colbert, The interleukin-23/interleukin-17 axis in spondyloarthritis, Curr. Opin. Rheumatol. 20 (2008) 392–397.
Haplotype structure of all markers. Darker color indicates higher linkage disequilibrium (LD), lighter color indicates less LD.
446 [8] S.L. Gaffen, The role of interleukin-17 in the pathogenesis of rheumatoid arthritis, Curr. Rheumatol. Rep. 11 (2009) 365–370. [9] H. Shen, J.C. Goodall, J.S.H. Gaston, Frequency and phenotype of peripheral blood Th17 cells in ankylosing spondylitis and rheumatoid arthritis, Arthritis Rheum. 60 (2009) 1647–1656. [10] K. Sato, A. Suematsu, K. Okamoto, et al., Th17 functions as an osteoclastogenic helper T cell subset that links T cell activation and bone destruction, J. Exp. Med. 203 (2006) 2673–2682. [11] X. Wang, Z. Lin, Q. Wei, Y. Jiang, J. Gu, Expression of IL-23 and IL-17 and effect of IL-23 on IL-17 production in ankylosing spondylitis, Rheumatol. Int. 29 (2009) 1343–1347. [12] J.C. Barrett, S. Hansoul, D.L. Nicolae, et al., Genome-wide association defines more than 30 distinct susceptibility loci for Crohn's disease, Nat. Genet. 40 (2008) 955–962. [13] C.A. Anderson, D.C. Massey, J.C. Barrett, et al., Investigation of Crohn's disease risk loci in ulcerative colitis further defines their molecular relationship, Gastroenterology 136 (2009) 523–529.e3. [14] A. Franke, T. Balschun, T.H. Karlsen, et al., Replication of signals from recent studies of Crohn's disease identifies previously unknown disease loci for ulcerative colitis, Nat. Genet. 40 (2008) 713–715. [15] E. Colombo, A. Latiano, O. Palmieri, F. Bossa, A. Andriulli, V. Annese, Enteropathic spondyloarthropathy: a common genetic background with inflammatory bowel disease? World J. Gastroenterol. 15 (2009) 2456–2462. [16] S. van der Linden, H.A. Valkenburg, A. Cats, Evaluation of diagnostic criteria for ankylosing spondylitis. A proposal for modification of the New York criteria, Arthritis Rheum. 27 (1984) 361–368. [17] T.J. Kim, T.H. Kim, H.J. Lee, et al., Interleukin 1 polymorphisms in patients with ankylosing spondylitis in Korea, J. Rheumatol. 35 (2008) 1603–1608.
C. Chen et al. [18] S.M. Stepkowski, W. Chen, J.A. Ross, Z.S. Nagy, R.A. Kirken, STAT3: an important regulator of multiple cytokine functions, Transplantation 85 (2008) 1372–1377. [19] Z. Chen, A. Laurence, J.J. O'Shea, Signal transduction pathways and transcriptional regulation in the control of Th17 differentiation, Semin. Immunol. 19 (2007) 400–408. [20] A. Fischer, Human immunodeficiency: connecting STAT3, Th17 and human mucosal immunity, Immunol. Cell Biol. 86 (2008) 549–551. [21] E. Safrany, B. Pazar, V. Csongei, et al., Variants of the IL23R gene are associated with ankylosing spondylitis but not with Sjogren syndrome in Hungarian population samples, Scand. J. Immunol. 70 (2009) 68–74. [22] A. Saleh, L. Shan, A.J. Halayko, S. Kung, A.S. Gounni, Critical role for STAT3 in IL-17A-mediated CCL11 expression in human airway smooth muscle cells, J. Immunol. 182 (2009) 3357–3365. [23] C. Abraham, J.H. Cho, IL-23 and autoimmunity: new insights into the pathogenesis of inflammatory bowel disease, Annu. Rev. Med. 60 (2009) 97–110. [24] C. Parham, M. Chirica, J. Timans, et al., A receptor for the heterodimeric cytokine IL-23 is composed of IL-12Rbeta1 and a novel cytokine receptor subunit, IL-23R, J. Immunol. 168 (2002) 5699–5708. [25] C.W. Lees, J. Satsangi, Genetics of inflammatory bowel disease: implications for disease pathogenesis and natural history, Expert Rev. Gastroenterol. Hepatol. 3 (2009) 513–534. [26] G.P. Thomas, M.A. Brown, Genetics and genomics of ankylosing spondylitis, Immunol. Rev. 233 (2010) 162–180. [27] S.I. Davidson, X. Wu, Y. Liu, et al., Association of ERAP1, but not IL23R, with ankylosing spondylitis in a Han Chinese population, Arthritis Rheum. 60 (2009) 3263–3268.