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Interactions between IL17A, IL23R, and STAT4 polymorphisms confer susceptibility to intestinal Behcet's disease in Korean population
Life Sciences 90 (2012) 740–746
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Interactions between IL17A, IL23R, and STAT4 polymorphisms confer susceptibility to intestinal Behcet's disease in Korean population Eun Soo Kim a, b, Seung Won Kim a, c,⁎, Chang Mo Moon a, Jae Jun Park a, Tae Il Kim a, Won Ho Kim a, c, Jae Hee Cheon a, c,⁎ a b c
Department of Internal Medicine and Institute of Gastroenterology, Yonsei University College of Medicine, Seoul, Republic of Korea Department of Internal Medicine and Division of Gastroenterology, Keimyung University School of Medicine, Daegu, Republic of Korea Brain Korea 21 Project for Medical Science, Yonsei University, Seoul, Republic of Korea
a r t i c l e
i n f o
Article history: Received 2 December 2011 Accepted 16 March 2012 Keywords: Behcet's disease IL17A IL23R STAT4 Polymorphism
a b s t r a c t Aims: Although polymorphisms in IL23R have recently been proposed to predispose to Behcet's disease (BD), associations between IL23R polymorphisms and intestinal BD have yet to be elucidated. We therefore performed a study to evaluate whether IL17A, IL23R, and STAT4 polymorphisms are associated with susceptibility to intestinal BD in the Korean population. Main methods: Single nucleotide polymorphisms (SNP) in the IL17A, IL23R, and STAT4 genes were analyzed using DNA sequencing, denaturing high performance liquid chromatography, and TaqMan genotyping assays. Key findings: Individual polymorphism analysis revealed that the TT genotype of IL17A rs8193036 (odds ratio (OR) 2.10, 95% confidence interval (CI) (1.12–3.92), p = 0.021), and GG + GT genotype of IL23R rs1884444 (OR 1.92, 95% CI (1.03–3.57), p = 0.034) was associated with the development of intestinal BD. When these two genotypes were combined, the risk of BD increased compared to that of patients with no-risk or one-risk genotype (OR 2.21, 95% CI (1.13–4.34), p = 0.021). Furthermore, statistically significant gene– gene interactions were observed between G149R in IL23R vs. rs11685878 in STAT4, rs2275913 in IL17A vs. rs7574865 in STAT4, and rs11889341 in STAT4 vs. rs2275913 in IL17A. The haplotypes of IL17A had a positive association with intestinal BD risks, whereas those of IL23R were protective for disease development. Significance: Our results indicate that the interaction of specific IL17A, IL23R, and STAT4 SNPs modulate susceptibility to intestinal BD in the Korean population, suggesting that the IL-17/23 axis plays a significant role in disease pathogenesis. © 2012 Elsevier Inc. All rights reserved.
Introduction Behcet's disease (BD) is a multi-systemic, chronic relapsing inflammatory disorder characterized by recurrent ulcerations of the oral cavity and genitalia as well as ocular inflammation. Other organ involvement such as the skin, joints, blood vessels, gastrointestinal tract, and central nervous system may occur concurrently. This disease is predominantly found in East Asia and in the Mediterranean countries and is relatively uncommon in the United States and Northern Europe (Dilsen, 1996; James, 1979). Although epidemiologic studies have shown that the development of the disease might be attributable to both genetic and environmental factors, the exact etiology has yet to be fully identified (Sakane et al., 1999). The disease can be classified
⁎ Corresponding authors at: Department of Internal Medicine, Yonsei University College of Medicine, 134 Shinchon-dong, Seodaemun-gu, Seoul, Republic of Korea. Tel.: +82 2 2228 1990; fax: +82 2 393 6884. E-mail addresses: [email protected] (S.W. Kim), [email protected] (J.H. Cheon). 0024-3205/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2012.03.017
as “intestinal BD” when patients with BD present with gastrointestinal symptoms and show typical intestinal ulcerations (Baba et al., 1976; Kasahara et al., 1981). While the prevalence of intestinal BD varies from 3 to 16% of all patients with BD by area, it appears to be more prevalent in East Asian countries (Kobayashi et al., 2007). The most frequent clinical symptom of intestinal BD is abdominal pain, followed by bleeding and diarrhea (Cheon et al., 2009). The most common site of involvement in the gastrointestinal tract is the ileocecal area, and lesions typically appear as ovoid-shaped deep ulcers with discrete borders (Lee et al., 2001; Sakane et al., 1999). Because of its scarcity and the lack of clinical evidence, it has been a great challenge for clinicians to make an accurate diagnosis of intestinal BD. Given the chronologic delay in systemic manifestations of BD, we have argued that the patients who do not fully satisfy the diagnostic criteria of systemic BD should be included in the category of intestinal BD patients when typical intestinal lesions are identified, and we have reported previously regarding this issue (Cheon et al., 2009; Shin et al., 2010). The cytokines IL-23 and IL-17 are essential inflammatory mediators of various autoimmune pathologies such as multiple sclerosis,
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rheumatoid arthritis (RA), psoriasis, and Crohn's disease (CD). IL-23 is produced mainly by antigen-presenting dendritic cells and macrophages. IL-23 drives and promotes the development of a unique T-helper cell population (Th17 cells) that produces IL-17. These IL-23-driven Th17 cells are highly pathogenic and elicit IL-17-dependent inflammation in autoimmune diseases (Langrish et al., 2005). This effect of IL-23 is mediated through the IL-23 receptor (IL-23R), which is a heterodimer of the IL-12RB1 subunit. IL-17 is a pleiotropic inflammatory cytokine that plays a pivotal role in a variety of pathologic conditions by inducing numerous inflammatory molecules and the recruitment of neutrophils (Kolls and Linden, 2004). IL-17 expression is increased in patients with CD, ulcerative colitis (UC), RA, asthma, and BD compared to healthy individuals (Chabaud et al., 1999; Fujino et al., 2003; Hamzaoui et al., 2002; Linden, 2001; Lubberts et al., 2001; Zhang et al., 2006). In addition, serum IL-17A levels increase in active BD patients, suggesting that Th17 and IL-17 pathway has an important role in acute attacks of the disease (Ekinci et al., 2010). The signal transducer and activator of transcription 4 (STAT4) protein regulates the immune response by transmitting signals activated in response to several cytokines such as IFN, IL-12, and IL-23 (Watford et al., 2004). Furthermore, STAT4 is essential for the expansion of Th17 cells activated by IL-23, which perpetuates chronic inflammation in adaptive and innate immune responses and contributes to the development of many autoimmune diseases (Mathur et al., 2007). Taken together, it can be postulated that interactions among IL17, IL23R, and STAT4 variants may be involved in the pathogenesis of autoimmune diseases such as BD. Recently, genome-wide association (GWA) studies have revealed that variants in IL10 and IL23R-IL12RB2 are associated with BD (Mizuki et al., 2010; Remmers et al., 2010). Other studies have also identified a strong relationship between polymorphisms of IL23R and IL17 and BD (Jang et al., 2008; Jiang et al., 2010). However, to the best of our knowledge, no studies on genetic variants in intestinal BD have been conducted to date. Therefore, our aim in this study was to evaluate the association between polymorphisms of IL17A, IL23R, and STAT4 genes and susceptibility to intestinal BD in the Korean population. Furthermore, we assessed the relationships between these genetic variants and disease phenotypes.
as age, gender, disease location, extraintestinal manifestations (EIM), subtypes of systemic BD, steroid dependency or refractoriness, use of immunosuppressants, and need for surgical intervention were obtained by reviewing the patients’ medical records. Informed consent was obtained from each subject, and this study was approved by the ethics review committee of the Institutional Review Board of Yonsei University College of Medicine. Demographic and clinical characteristics In this study, 141 patients who had ulcerations typical of intestinal BD and 259 normal healthy controls were enrolled. Demographic and clinical characteristics of the subjects are shown in Table 1. The mean age of patients at diagnosis was 38.1 ± 11.5 years and the gender ratio (male:female) was 1:1.07. The most common EIM was oral ulcers (113, 80.1%), followed by skin lesions (60, 42.6%), and the distribution of subtypes of systemic BD according to the Japanese classification system was as follows: 7 complete (5.0%), 60 incomplete (42.6%), and 42 suspected (29.8%) subtypes. Seven patients were assigned to the oral ulcer + gastrointestinal (GI) ulcer group while 25 patients were assigned to the GI ulcer-only group (17.7%). Twenty-one (14.9%) patients were refractory to or dependent on corticosteroids and 44 (31.2%) took immunosuppressants. Surgical management was necessary in 44 (31.2%) patients. Genotyping Considering ethnic differences in the disease, especially between Eastern and Western countries, we decided to scan the entire exons of IL23R and intron/exon junction with the promoter region of IL17A instead of using common tag SNPs. As a result, we found 9 genetic variants in IL17A and 4 in IL23R. For STAT4, tag SNPs that were reported to be strongly related to autoimmune diseases were used (Kobayashi et al., 2008; Korman et al., 2008; Lee et al., 2007). In
Table 1 Demographic and clinical characteristics of patients with intestinal Behcet's disease.
Materials and methods Subject population A total of 400 unrelated Korean subjects (intestinal BD, 141; healthy controls, 259) were enrolled consecutively in this study. All patients were diagnosed at Severance Hospital, Yonsei University, Seoul, Korea. The diagnosis of intestinal BD was based on a combination of clinical criteria for the diagnosis of systemic BD and the identification of intestinal ulcerations by endoscopy or radiographic imaging (Baba et al., 1976; Kobayashi et al., 2007; Lee et al., 2001). To be included in this study, patients had to have intestinal ulcerations typical of intestinal BD: b5 in number, oval in shape, deep with discrete margins, and located in the ileocecal area (Lee et al., 2001). Any patient strongly suggested of having CD, UC, intestinal tuberculosis, or a malignancy through radiographical, endoscopic, or pathological studies was excluded. For CD, in particular, which may be difficult to differentiate from intestinal BD, longitudinal, irregular/ geographic, or cobblestone shaped ulcers and diffuse or segmental distribution of the lesions were considered the typical colonoscopic features of CD (Lee et al., 2009). Patients with concomitant autoimmune diseases including systemic lupus erythematosus, RA, primary Sjogren's syndrome, type 1 diabetes, systemic sclerosis, or asthma were also excluded. The traditional criteria of the BD Research Committee of Japan, revised in 1987, were applied to detect systemic manifestations (Mizushima, 1988). Patients who lacked extraintestinal symptoms that fulfilled the Japanese criteria of BD but had typical intestinal ulcerations were enrolled in this study. Demographic and clinical information such
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Age at diagnosis (years) Sex (M:F) Extraintestinal manifestations (%) Eyes Skin Oral ulcers Genital ulcers Joints Disease location (%) Ileum Ileocecal valve Colon Steroid dependent or refractory Use of immunosuppressants Surgical management Subtypes of systemic BDa Complete Incomplete Suspected Oral ulcer + GI ulcers GI ulcers only Follow-up duration (months)
Intestinal BD (n = 141)
Controls (n = 259)
38.08 ± 11.47 68:73
34.1 ± 10.5 152:107
25 (17.7) 60 (42.6) 113 (80.1) 43 (30.5) 47 (33.3) 71 48 26 21 44 44
(50.4) (34.0) (18.4) (14.9) (31.2) (31.2)
7 (5.0) 60 (42.6) 42 (29.8) 7 (5.0) 25 (17.7) 95.50 ± 60.74
Immunosuppressants: azathioprine, 5-mercaptopurine, cyclosporine, and infliximab. Classification according to the diagnostic criteria of the Research Committee of Japan. BD, Behcet's disease; GI, gastrointestinal. Complete, 4 major symptoms; incomplete, 3 major or 2 major + 2 minor symptoms; suspected, symptoms unsatisfying incomplete criteria. a Complete, incomplete, and suspected subtypes of systemic Behcet's disease were classified according to the diagnostic criteria of the Research Committee of Japan consisting of 4 major (oral ulceration, skin lesion, eye lesion, and genital ulcers) and 5 minor symptoms (arthritis, intestinal ulcers, vascular disease, neuropsychiatric disorders, and epididymitis).
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Table 2 Genotypes of cases and controls and their association with the risk of intestinal BD. Genes
Genotypes
IL17A
rs4711998 AA AG GG rs8193036 CC CT TT TT vs. CC + CT rs9791323 AA AC CC rs3819024 AA AG GG rs2275913 GG GA AA − 168 (novel)⁎ CC GC − 159 (novel)⁎
IL23R
STAT4
TT TA rs8193037 GG GA AA − 112 (novel)⁎ GG GA rs1884444 GG GT TT TT vs. GG + GT V134D (novel)† TT TA G149R (novel)† GG GA IVS4 + 17 (novel)† CC CT rs11889341 CC CT TT rs7574865 GG GT TT rs8179673 TT CT CC rs6752770 AA AG GG rs10168266 GG AG AA rs10181656 CC CG GG rs11685878 CC CT TT
Cases, n (%) Controls
OR
p value 0.130
68 (48.2) 57 (40.4) 16 (11.3)
145 (56.0) 98 (37.8) 16 (6.2)
64 (45.4) 54 (38.3) 23 (16.3)
128 (49.4) 109 (42.1) 22 (8.5)
0.068
2.10 (1.12–3.92) 0.021 0.240 120 (85.1) 20 (14.2) 1 (0.7)
230 (88.8) 29 (11.2) 0 (0)
46 (32.6) 67 (47.5) 28 (19.9)
74 (28.6) 130 (50.2) 55 (21.2)
52 (36.9) 62 (44.0) 27 (19.1)
81 (31.3) 119 (46.0) 59 (22.8)
141 (100) 0 (0)
258 (99.6) 1 (0.4)
140(99.3) 1 (0.7)
257 (99.2) 2 (0.8)
106 (75.7) 33 (23.4) 2 (1.4)
196 (75.7) 59 (22.8) 4 (1.5)
140 (99.3) 1 (0.7)
254 (98.10 5 (1.9)
68 (48.2) 58 (41.1) 15 (10.6)
103 (39.8) 108 (41.7) 48 (18.5)
addition, we have previously reported the association of these STAT4 variants with IBD (Moon et al., 2010). Genomic DNA was isolated from whole blood samples using commercially available kits (Qiagen, Hilden, Germany). DNA sequence analysis of the promoter region of IL17A, including exon 1 (~1.2 kb), was carried out, and identification of variants in other exons of IL17A and IL23R, including splice junctions, was performed by denaturing high performance liquid chromatography (DHPLC). Primer sequences are provided in Supplementary Table 1. All variants detected by DHPLC analysis were confirmed by subsequent full DNA sequencing. Genotyping for the STAT4 variants (rs11889341, rs7574865, rs8179673, rs6752770, rs10168266, rs10181656, and rs11685878) was performed using TaqMan technology (Applied Biosystems, Foster City, California, USA) as described previously (Moon et al., 2010).
0.700
Statistical analysis 0.470
0.350
0.940
0.990
0.310
We tested all single nucleotide polymorphisms (SNPs) to determine if they were in Hardy–Weinberg equilibrium (HWE) and assessed whether differences in the genotype frequencies between cases and controls were significant using the Chi-square test or Fisher's exact test. Gene–gene interaction was analyzed using an interaction test in logistic regression models (Thornton-Wells et al., 2004). Logistic regression models were used to assess relationships between SNPs and phenotypes after adjustment for possible confounding factors (gender, age at diagnosis, disease location, total follow-up period, immunosuppressant use, and requirement for an operation). For multiple comparisons, the Bonferroni correction was applied. P values of less than 0.05 were considered significant. Statistical analysis was performed using SPSS version 14.0 (SPSS, Chicago, IL, USA). Haplotypes were analyzed using Haploview 4.1 software.
0.069
Results 0.52 (0.28–0.97) 0.034 0.680
138 (97.9) 3 (2.1)
255 (98.5) 4 (1.5)
125 (88.7) 16 (11.3)
226 (87.3) 33 (12.7)
134 (95) 7 (5)
252 (97.3) 7 (2.7)
0.630
The distributions of the genotypes of SNPs in IL17A, IL23R, and STAT4 among patients and controls are presented in Table 2. The genotype distributions of all SNPs among the controls were in HWE. IL17A rs8193036 was associated with a significantly increased risk of intestinal BD under a recessive model for the variant T allele (OR 2.10, 95% CI 1.12–3.92, p = 0.021). Among the SNPs in IL23R, rs1884444 was associated with a significantly decreased risk of intestinal BD under a recessive model for the variant T allele (OR 0.52, 95% CI 0.28–0.97, p =0.034). When this was shown as a risk genotype (OR> 1.00) for better comparison in the combined genotype analysis, the OR of the GG+GT genotype compared with the TT genotype was 1.92 (95% CI 1.03–3.57, p= 0.034). There was no significant difference in the genotype frequencies of SNPs in STAT4 between the patients and controls.
0.5
Combined effect of risk genotypes, gene–gene interaction, and haplotypes for risk of intestinal BD
0.680
0.250
0.620 58 (45.7) 57 (44.9) 12 (9.4)
117 (51.1) 93 (40.6) 19 (8.3)
55 (43.3) 55 (43.3) 17 (13.4)
106 (46.3) 100 (43.7) 23 (10.0)
55 (43.3) 55 (43.3) 17 (13.4)
105 (45.9) 101 (44.1) 23 (10.0)
0.620
76 (59.8) 42 (33.1) 9 (7.1)
135 (59.0) 84 (36.7) 10 (4.4) 0.590
57 (44.9) 57 (44.9) 13 (10.2)
114 (49.8) 97 (42.4) 18 (7.9)
55 (43.3) 55 (43.3) 17 (13.4)
105 (45.9) 101 (44.1) 23 (10.0)
41 (32.2) 58 (45.7) 28 (22.1)
82 (35.8) 114 (49.8) 33 (14.4)
Association of IL17A, IL23R, and STAT4 polymorphisms with intestinal BD susceptibility
0.630
We investigated the combined effects of genetic variants of IL17A and IL23R that were found to be associated with susceptibility to intestinal BD in individual polymorphism analyses. The IL17A rs8193036 TT and IL23R rs1884444 GG + GT genotypes were considered to be risk genotypes for intestinal BD. When subjects were divided into three groups based on the number of risk genotypes they possessed, the risk Table 2 (continued)
0.190
Genes Genotypes Cases, n (%) Controls OR p value Notes to Table 2: “Novel” indicates SNPs that have not been reported previously. The number shows the nucleotide positions of the SNP calculated from ⁎the translation start site or †amino acid position. Significant associations with p b 0.05 are shown in bold.
E.S. Kim et al. / Life Sciences 90 (2012) 740–746
increased in a dose-dependent manner as the number of risk genotypes increased. Compared with subjects with no-risk genotype, the OR was 2.029 (95% CI 1.03–4.00, p = 0.09) for those with 1 risk genotype and 4.074 (95% CI 1.65–10.04, p = 0.004) for those with 2 risk genotypes (Fig. 1A). When participants were classified into two groups (zero to one vs. two risk genotypes), the risk of the participants with two risk genotypes was significantly greater than those with zero or one-risk genotype (OR 2.21, 95% CI 1.24–4.34, p = 0.021) (Fig. 1B). We also investigated potential gene–gene interactions between SNPs in IL23R, IL17A, and STAT4. There was a significant interaction between G149R in IL23R and rs11685878 in STAT4 (p = 0.004 in codominant model; p = 0.002 in recessive model; Table 3), rs2275913 in IL17A and rs7574865 in STAT4 (p= 0.003 in codominant model; p = 0.006 in recessive model; Table 3), and rs1188941 in STAT4 and rs2275913 in IL17A (p= 0.008 in codominant model; p = 0.021 in dominant model; Table 3). Haplotype analysis revealed that the frequencies of the GCAAGCTGG and ACCGACTGG haplotypes of IL17A were significantly higher in intestinal BD patients than in controls (OR 2.05, 95% CI 1.01–4.16, p = 0.047 and OR 12.12, 95% CI 1.39–105.77, p = 0.025, respectively) (Table 4). Meanwhile, the haplotype TTGC of IL23R was inversely related with disease susceptibility (OR 0.71, 95% CI 0.52–0.98, p = 0.038) (Table 5). Associations of IL17A, IL23R, and STAT4 genetic polymorphisms and the phenotype of intestinal BD To analyze associations between genotypes and disease phenotypes, multivariate logistic regression was performed after adjusting
Fig. 1. Combined effect of polymorphisms in the IL17A and IL23R genes on the risk of intestinal Behcet's disease. The odds ratio of each group according to the number of risk genotypes (A). The odds ratio of patients with 2 risk genotypes compared with those with 0–1 risk genotype (B). Risk genotypes: IL17A rs8193036 TT and IL23R rs1884444 GG + GT.
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for other covariates such as gender, age at diagnosis, EIMs, disease locations, total follow-up period, use of immunosuppressants, and need for an operation. In terms of the location of GI tract involvement, STAT4 rs10168266 was associated with the involvement of the ileocecal valve area under a dominant model (AA + AG vs. GG, OR 2.309, 95% CI 1.028–5.181, p = 0.042). In addition, a dominant model of IL17A rs9791323 was inversely associated with ileocecal valve involvement (AC + CC vs. AA, OR 0.25, 95% CI 0.07–0.94, p = 0.040). Subjects with the GT + TT genotype of IL23R rs1884444 were less likely to exhibit systemic BD manifestations than those with a GG genotype (dominant model; OR 0.36, 95% CI 0.15–0.90, p = 0.028). Steroid dependency or refractoriness was more often observed in patients with the TT+ CT genotype of IL17A rs8193036 than those with the CC genotype (dominant model; OR 3.98, 95% CI 1.23–12.84, p = 0.021). However, all significance of above associations between SNPs and phenotype did not remain after correction for multiple testing (Supplementary Table 2). Discussion IL17-producing Th17 cells, a novel T cell population, play a major role in autoimmunity. Th17 cell differentiation from naïve CD4+ T cells is facilitated by various cytokines, including IL–1ß, IL-6, IL-21, and IL-23 (Aggarwal et al., 2003; Bettelli et al., 2006). A critical feature of Th17 cells is the expression of IL-17A and IL-17F. The association between IL-17F genetic polymorphisms and autoimmune diseases has been extensively evaluated in various ethnic populations (Chen et al., 2009; Paradowska-Gorycka et al., 2010; Saitoh et al., 2011). IL-17A is a powerful proinflammatory mediator that presents a number of proinflammatory effects on intestinal epithelial cells, endothelial cells, macrophages, fibroblasts and colonic myofibroblasts with increased release of proinflammatory chemokines, cytokines, and other mediators such as prostaglandins, matrix metalloproteinases, nitric oxide synthase-2, and granulocyte colony stimulating factor (Brand, 2009). In addition, IL-17A exerts a pathologic role in intestinal inflammation through activation of nuclear factor kappa B (NF-kB) and mitogen-activated protein kinase (MAPK) pathways (Awane et al., 1999; Shalom-Barak et al., 1998). Recent GWA studies, demonstrating IL23R genes as IBD susceptibility genes, validate the important role of Th17 cells that express the IL-23R on their surface in the pathogenesis of CD (Barrett et al., 2008; Duerr et al., 2006). STAT4 is a transcription factor that transduces IL-12, IL-23, and type 1 interferonmediated signals into Th1 and Th17 differentiation, monocyte activation, and interferon-gamma production. STAT4-dependent cytokine regulation plays a critical role in the pathogenesis of autoimmune diseases. Thus, it is possible that genetic variants in genes of these mediators could lead to dysregulation of the IL-23/IL-17 pathway, thereby increasing the risk for autoimmunity. Here, we investigated the relationships of the variants of IL17A, IL23R, and STAT4 with susceptibility to intestinal BD and BD phenotypes. This is the first genetic variant study conducted on intestinal BD. We identified two individual polymorphisms (IL17A rs8193036C> T and IL23R rs1884444G > T) associated with the risk of intestinal BD. Although it is challenging to know the exact mechanisms of these polymorphisms involving intestinal BD, functional variant of rs1884444 of IL23R changing glutamine into histidine may affect the structure and the biological function or stability of IL-23R. Polymorphism rs8193036 of IL17A may give a significant influence on binding affinity of transcription factors. We previously reported that rs8193036 of IL17A was significantly related to a higher level of IL-17A mRNA expression in patients with inflammatory bowel disease and showed a substantial activity to transcription factor complexes as a kind of regulatory mechanism (Kim et al., 2011). Notably, the joint effects of these two SNPs (IL17A and IL23R) raised the risk of intestinal BD as the number of risk genotypes increased. Furthermore, significant synergistic interactions between genes were observed (G149R in IL23R and rs11685878 in STAT4; rs2275913 in IL17A and
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Table 3 Synergistic interactions between IL23R, IL17A, and STAT4 genotypes in intestinal Behcet's disease. G149R Codominant
Dominant
rs11685878 GG
GA
CC CT TT Interactive P value
0.79 (0.28–2.21) 1.75 (0.62–4.95)
1.00 0.90 (0.53–1.54) 2.12 (1.08–4.17)
AA
0.004
Recessive
GG
GA
1.00
0.79 (0.28–2.21)
AA
GG
GA
1.00
1.22 (0.60–2.49)
1.14 (0.69–1.89) 0.99 (0.38–2.53)
2.26 (1.25–4.10)
0.00
0.900
0.002
AA
rs2275913 Codominant rs7574865
GG
GG GT TT Interactive P value
Dominant GA
AA
Recessive
GG
GA
AA
GG
1.00 1.47 (0.71–3.05) 0.77 (0.31–1.96) 1.41 (0.64–3.09) 0.80 (0.38–1.67) 1.64 (0.67–4.03) 7.52 (1.43–39.52) 1.93 (0.68–5.50) 0.20 (0.02–1.62)
1.00
1.47 (0.71–3.05) 0.77 (0.31–1.96)
GA
1.00
0.92 (0.55–1.56) 0.97 (0.51–1.85)
AA
1.80 (0.86–3.79) 0.97 (0.49–1.94) 1.07 (0.47–2.47)
6.46 (1.28–32.62) 1.66 (0.62–4.43) 0.17 (0.02–1.35)
0.003
0.12
0.006
rs11889341 Codominant
Dominant TT
CC
Recessive
rs2275913
CC
CT
GG GA AA Interactive P value
1.00 1.40 (0.68–2.87) 1.34 (0.57–3.17)
2.19 (1.01–4.74) 9.89 (1.04–94.37) 1.00 2.19 (1.01–4.74) 9.89 (1.04–94.37) 1.00 1.18 (0.57–2.47) 1.73 (0.57–5.22) 1.18 (0.47–2.96) 0.00 1.38 (0.71–2.67) 1.18 (0.60–2.33) 0.96 (0.35–2.68) 1.12 (0.52–2.40)
0.008
CT
TT
CC
0.021
CT
TT
1.29 (0.77–2.16) 2.07 (0.83–5.13) 0.98 (0.43–2.26) 0.00
0.015
Significant associations with p b 0.05 are shown in bold. BD, Behcet's disease
rs7574865 in STAT4; rs11889341 in STAT4 and rs2275913 in IL17A). Altogether, these results highlight the importance of combined analysis of polymorphisms for risk prediction rather than analysis of each SNP individually and also suggest that interactions of variants of different genes in the same pathway may modulate susceptibility to intestinal BD. Increasing evidence has suggested that IL23R polymorphisms are associated with a number of inflammatory and immune-linked diseases including inflammatory bowel disease, psoriasis, ankylosing spondylitis, and BD (Cargill et al., 2007; Duerr et al., 2006; Remmers et al., 2010; Rueda et al., 2008). Two recent genome-wide association studies of BD identified a significant association between two IL23R polymorphisms (rs924080 and rs1495965) and disease susceptibility (Mizuki et al., 2010; Remmers et al., 2010). In the present study, the locus rs1884444G > T of the IL23R gene protected the development of intestinal BD. These discrepancies in the results concerning the role of genetic variants in IL23R for disease risk are interesting. For instance, some SNPs of IL23R are associated with a greater risk for
CD (Newman et al., 2009; Okazaki et al., 2008), whereas others appear to protect against the development of CD, which are similar to our results (Dubinsky et al., 2007; Glas et al., 2007; Taylor et al., 2008). Schmechel et al. (2008) reported that the level of IL-22 that correlates with the disease activity was higher in carriers of CD riskincreasing IL23R variants than in those of CD risk-decreasing IL23R variants, suggesting that IL23R genotypes influence IL-22 serum expression. Regarding the IL17 gene, while a recent Chinese study failed to find an association between IL17A SNP rs2275913 and BD susceptibility (Shu et al., 2010), we found a significant relationship between an IL17A variant and the intestinal BD risk and synergistic interaction between rs2275913 of IL17A and rs7574865 of STAT4. Interestingly, the IL17A gene and the HLA B51, TNFα, and SUMO4 genes that are known to increase the BD risk are located on the same chromosome (chromosome 6), suggesting an association between IL17A variants and disease susceptibility (Hou et al., 2008; Remmers et al., 2010; Verity et al., 1999). As the evidence of IL17A genetic variants having a potential risk of the autoimmune disease development, our very
Table 4 Association of IL17A haplotypes with intestinal Behcet's disease. rs4711998
rs8193036
rs9791323
rs3819024
rs2275913
− 168
− 159
rs8193037
− 112
Freq
OR (95% CI)
p value
A G A A A G A A G A A G A A
C T C T C C C T C C T C C T
A A A A A A A C A A A A C A
G A A A A A G A G A A A G G
A G G G G G G G A A G G A A
C C C C C C C C C C C C C C
T T T T T T T T T T T T T T
G G A G G G G G G G A A G G
G G G G G G G G G G G G G G
0.3307 0.1328 0.0807 0.0705 0.0704 0.0555 0.045 0.0366 0.0232 0.0209 0.019 0.0168 0.0114 0.0102
1 1.45 (0.88–2.39) 0.92 (0.50–1.69) 1.53 (0.83–2.84) 0.46 (0.21–1.02) 2.05 (1.01–4.16) 0.59 (0.24–1.42) 0.93 (0.39–2.22) 0.54 (0.12–2.44) 0.14 (0.02–1.12) 1.61 (0.46–5.69) 1.12 (0.28–4.55) 12.12 (1.39–105.77) 0.65 (0.11–3.83)
0.14 0.79 0.17 0.056 0.047 0.24 0.86 0.42 0.065 0.46 0.87 0.025 0.64
Significant associations with p b 0.05 are shown in bold.
E.S. Kim et al. / Life Sciences 90 (2012) 740–746
745
Table 5 Association of IL23R haplotypes with intestinal Behcet's disease. rs1884444
E4.V134D
E4.G149R
E4.IVS4 + 17
Freq
OR (95% CI)
p value
G T G T
T T T T
G G A A
C C C C
0.5883 0.3277 0.0343 0.0247
1 0.71 (0.52–0.98) 0.78 (0.30–2.00) 0.76 (0.24–2.39)
0.038 0.6 0.64
Significant associations with p b 0.05 are shown in bold.
recent study investigating the association of genetic variants of IL23R and IL17A with inflammatory bowel disease identified the relationship of rs2275913 of IL17A with the risk of UC (Kim et al., 2011). Although we were not able to find any association of SNPs of STAT4 with the risk of intestinal BD in the individual polymorphism analysis, they were found to exert a significant synergistic effect both with IL17A and IL23R in gene–gene interaction analysis. There are several studies suggesting the association of STAT4 SNP rs7574865, which showed synergistic interaction with rs2275913 of IL17A in our study, with autoimmune diseases. The Chinese study identified a significant association between STAT4 SNP rs7574865 and the BD risk (Hu et al., 2010), and another study suggested that this variant was associated with colonic CD (Glas et al., 2010). Inconsistent results among different genetic studies on BD susceptibility may be due to differences in patient characteristics because of the different clinical settings of each study. Because of the wide range of BD systemic manifestations, patients with BD may visit various clinics such as dermatology, rheumatology, ophthalmology, or gastroenterology clinics based on their main symptoms. Therefore, subjects may be recruited from different clinical settings, leading to heterogeneity in the severity of symptoms in the different studies. We previously reported that a significant number of patients with typical intestinal lesions who did not fully satisfy the diagnostic criteria of systemic BD at the time of initial presentation presented with systemic manifestations at the end of the follow-up period (Cheon et al., 2009; Shin et al., 2010). Based on these studies, we included patients with typical intestinal lesions irrespective of systemic manifestations in our study, in contrast to prior BD studies, which could perhaps explain the differences in our findings compared to the studies mentioned above. An alternative explanation could be ethnic differences between the diverse populations examined in the above studies. Intriguingly, functional variants in the IL23R gene, which were identified as susceptibility loci in Caucasian IBD patients, showed no association with IBD in Asian populationbased studies (Bin et al., 2009; Yamazaki et al., 2007). Likewise, although GWA studies identified an association between SNPs in IL23R and the risk of BD in Turkish and Japanese cohorts, no significant association was found in a Korean cohort (Mizuki et al., 2010; Remmers et al., 2010). Importantly, combined effect analysis of multiple genetic variants of IL23R and IL17A resulted in better detection of high-risk individuals susceptible to intestinal BD than analysis of a single polymorphism. Compared to patients with no-risk genotypes, patients with one or two risk genotypes had an intestinal BD risk 2.03-fold or 4.07-fold greater, respectively. Furthermore, gene–gene interaction analysis revealed evidence for epistatic interactions among IL17A, IL23R, and STAT4 genes, indicating that the IL-17/23 axis plays a significant role in the pathogenesis of intestinal BD. Although significant associations appeared to be observed between SNPs in the IL17A, IL23R, and STAT4 genes and disease phenotypes, statistical significance failed to remain after correction for multiple testing. Further studies with a larger sample population than that examined in our study are warranted to clarify this issue. We acknowledge the small sample size as a significant limitation of the current study. In comparison to results of genome-wide association studies, the p values obtained were not very impressive, which is mainly attributed to the limited sample size of the present study. However, given that Behcet's disease is rarer than CD at least in
Western European populations, we assumed that the number of cases of this study might be acceptable. Regarding the diagnostic criteria of systemic BD, one might think that the International Study Group of BD (ISGBD) criteria should be used because they have been widely accepted and used among clinicians worldwide since its description. However, we decided to use the Japanese criteria instead of the ISGBD criteria for the following several reasons. First, the ISGBD criteria failed to identify patients if they did not show preceding oral ulcerations. It has been reported that 3% of 914 patients with definite BD who lacked oral ulcerations were excluded in the original survey for the ISGBD criteria (1990). Second, in Korea, the rate of positive pathergy test, which is one of the major factors included in ISGBD criteria, has been reported to be lower than 40% (Chang and Cheon, 2002). Third, Korea and Japan have numerous similarities in terms of ethnic and geographical backgrounds. Fourth, the Japanese criteria have a gradual diagnostic system that can reflect a temporal change better than the ISGBD criteria during the follow-up period. Finally, the gastrointestinal manifestation is included in the Japanese criteria but not in the ISGBD criteria. Conclusions Our results indicate that the joint effect of SNPs of IL17A, IL23R, and STAT4 may modulate susceptibility to intestinal BD in the Korean population, suggesting a potential significance of the IL-17/23 axis in the pathogenesis of intestinal BD. Conflict of interest statement The authors declare no conflict of interest.
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Interactions between IL17A, IL23R, and STAT4 polymorphisms confer susceptibility to intestinal Behcet's disease in Korean population Eun Soo Kim a, b, Seung Won Kim a, c,⁎, Chang Mo Moon a, Jae Jun Park a, Tae Il Kim a, Won Ho Kim a, c, Jae Hee Cheon a, c,⁎ a b c
Department of Internal Medicine and Institute of Gastroenterology, Yonsei University College of Medicine, Seoul, Republic of Korea Department of Internal Medicine and Division of Gastroenterology, Keimyung University School of Medicine, Daegu, Republic of Korea Brain Korea 21 Project for Medical Science, Yonsei University, Seoul, Republic of Korea
a r t i c l e
i n f o
Article history: Received 2 December 2011 Accepted 16 March 2012 Keywords: Behcet's disease IL17A IL23R STAT4 Polymorphism
a b s t r a c t Aims: Although polymorphisms in IL23R have recently been proposed to predispose to Behcet's disease (BD), associations between IL23R polymorphisms and intestinal BD have yet to be elucidated. We therefore performed a study to evaluate whether IL17A, IL23R, and STAT4 polymorphisms are associated with susceptibility to intestinal BD in the Korean population. Main methods: Single nucleotide polymorphisms (SNP) in the IL17A, IL23R, and STAT4 genes were analyzed using DNA sequencing, denaturing high performance liquid chromatography, and TaqMan genotyping assays. Key findings: Individual polymorphism analysis revealed that the TT genotype of IL17A rs8193036 (odds ratio (OR) 2.10, 95% confidence interval (CI) (1.12–3.92), p = 0.021), and GG + GT genotype of IL23R rs1884444 (OR 1.92, 95% CI (1.03–3.57), p = 0.034) was associated with the development of intestinal BD. When these two genotypes were combined, the risk of BD increased compared to that of patients with no-risk or one-risk genotype (OR 2.21, 95% CI (1.13–4.34), p = 0.021). Furthermore, statistically significant gene– gene interactions were observed between G149R in IL23R vs. rs11685878 in STAT4, rs2275913 in IL17A vs. rs7574865 in STAT4, and rs11889341 in STAT4 vs. rs2275913 in IL17A. The haplotypes of IL17A had a positive association with intestinal BD risks, whereas those of IL23R were protective for disease development. Significance: Our results indicate that the interaction of specific IL17A, IL23R, and STAT4 SNPs modulate susceptibility to intestinal BD in the Korean population, suggesting that the IL-17/23 axis plays a significant role in disease pathogenesis. © 2012 Elsevier Inc. All rights reserved.
Introduction Behcet's disease (BD) is a multi-systemic, chronic relapsing inflammatory disorder characterized by recurrent ulcerations of the oral cavity and genitalia as well as ocular inflammation. Other organ involvement such as the skin, joints, blood vessels, gastrointestinal tract, and central nervous system may occur concurrently. This disease is predominantly found in East Asia and in the Mediterranean countries and is relatively uncommon in the United States and Northern Europe (Dilsen, 1996; James, 1979). Although epidemiologic studies have shown that the development of the disease might be attributable to both genetic and environmental factors, the exact etiology has yet to be fully identified (Sakane et al., 1999). The disease can be classified
⁎ Corresponding authors at: Department of Internal Medicine, Yonsei University College of Medicine, 134 Shinchon-dong, Seodaemun-gu, Seoul, Republic of Korea. Tel.: +82 2 2228 1990; fax: +82 2 393 6884. E-mail addresses: [email protected] (S.W. Kim), [email protected] (J.H. Cheon). 0024-3205/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2012.03.017
as “intestinal BD” when patients with BD present with gastrointestinal symptoms and show typical intestinal ulcerations (Baba et al., 1976; Kasahara et al., 1981). While the prevalence of intestinal BD varies from 3 to 16% of all patients with BD by area, it appears to be more prevalent in East Asian countries (Kobayashi et al., 2007). The most frequent clinical symptom of intestinal BD is abdominal pain, followed by bleeding and diarrhea (Cheon et al., 2009). The most common site of involvement in the gastrointestinal tract is the ileocecal area, and lesions typically appear as ovoid-shaped deep ulcers with discrete borders (Lee et al., 2001; Sakane et al., 1999). Because of its scarcity and the lack of clinical evidence, it has been a great challenge for clinicians to make an accurate diagnosis of intestinal BD. Given the chronologic delay in systemic manifestations of BD, we have argued that the patients who do not fully satisfy the diagnostic criteria of systemic BD should be included in the category of intestinal BD patients when typical intestinal lesions are identified, and we have reported previously regarding this issue (Cheon et al., 2009; Shin et al., 2010). The cytokines IL-23 and IL-17 are essential inflammatory mediators of various autoimmune pathologies such as multiple sclerosis,
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rheumatoid arthritis (RA), psoriasis, and Crohn's disease (CD). IL-23 is produced mainly by antigen-presenting dendritic cells and macrophages. IL-23 drives and promotes the development of a unique T-helper cell population (Th17 cells) that produces IL-17. These IL-23-driven Th17 cells are highly pathogenic and elicit IL-17-dependent inflammation in autoimmune diseases (Langrish et al., 2005). This effect of IL-23 is mediated through the IL-23 receptor (IL-23R), which is a heterodimer of the IL-12RB1 subunit. IL-17 is a pleiotropic inflammatory cytokine that plays a pivotal role in a variety of pathologic conditions by inducing numerous inflammatory molecules and the recruitment of neutrophils (Kolls and Linden, 2004). IL-17 expression is increased in patients with CD, ulcerative colitis (UC), RA, asthma, and BD compared to healthy individuals (Chabaud et al., 1999; Fujino et al., 2003; Hamzaoui et al., 2002; Linden, 2001; Lubberts et al., 2001; Zhang et al., 2006). In addition, serum IL-17A levels increase in active BD patients, suggesting that Th17 and IL-17 pathway has an important role in acute attacks of the disease (Ekinci et al., 2010). The signal transducer and activator of transcription 4 (STAT4) protein regulates the immune response by transmitting signals activated in response to several cytokines such as IFN, IL-12, and IL-23 (Watford et al., 2004). Furthermore, STAT4 is essential for the expansion of Th17 cells activated by IL-23, which perpetuates chronic inflammation in adaptive and innate immune responses and contributes to the development of many autoimmune diseases (Mathur et al., 2007). Taken together, it can be postulated that interactions among IL17, IL23R, and STAT4 variants may be involved in the pathogenesis of autoimmune diseases such as BD. Recently, genome-wide association (GWA) studies have revealed that variants in IL10 and IL23R-IL12RB2 are associated with BD (Mizuki et al., 2010; Remmers et al., 2010). Other studies have also identified a strong relationship between polymorphisms of IL23R and IL17 and BD (Jang et al., 2008; Jiang et al., 2010). However, to the best of our knowledge, no studies on genetic variants in intestinal BD have been conducted to date. Therefore, our aim in this study was to evaluate the association between polymorphisms of IL17A, IL23R, and STAT4 genes and susceptibility to intestinal BD in the Korean population. Furthermore, we assessed the relationships between these genetic variants and disease phenotypes.
as age, gender, disease location, extraintestinal manifestations (EIM), subtypes of systemic BD, steroid dependency or refractoriness, use of immunosuppressants, and need for surgical intervention were obtained by reviewing the patients’ medical records. Informed consent was obtained from each subject, and this study was approved by the ethics review committee of the Institutional Review Board of Yonsei University College of Medicine. Demographic and clinical characteristics In this study, 141 patients who had ulcerations typical of intestinal BD and 259 normal healthy controls were enrolled. Demographic and clinical characteristics of the subjects are shown in Table 1. The mean age of patients at diagnosis was 38.1 ± 11.5 years and the gender ratio (male:female) was 1:1.07. The most common EIM was oral ulcers (113, 80.1%), followed by skin lesions (60, 42.6%), and the distribution of subtypes of systemic BD according to the Japanese classification system was as follows: 7 complete (5.0%), 60 incomplete (42.6%), and 42 suspected (29.8%) subtypes. Seven patients were assigned to the oral ulcer + gastrointestinal (GI) ulcer group while 25 patients were assigned to the GI ulcer-only group (17.7%). Twenty-one (14.9%) patients were refractory to or dependent on corticosteroids and 44 (31.2%) took immunosuppressants. Surgical management was necessary in 44 (31.2%) patients. Genotyping Considering ethnic differences in the disease, especially between Eastern and Western countries, we decided to scan the entire exons of IL23R and intron/exon junction with the promoter region of IL17A instead of using common tag SNPs. As a result, we found 9 genetic variants in IL17A and 4 in IL23R. For STAT4, tag SNPs that were reported to be strongly related to autoimmune diseases were used (Kobayashi et al., 2008; Korman et al., 2008; Lee et al., 2007). In
Table 1 Demographic and clinical characteristics of patients with intestinal Behcet's disease.
Materials and methods Subject population A total of 400 unrelated Korean subjects (intestinal BD, 141; healthy controls, 259) were enrolled consecutively in this study. All patients were diagnosed at Severance Hospital, Yonsei University, Seoul, Korea. The diagnosis of intestinal BD was based on a combination of clinical criteria for the diagnosis of systemic BD and the identification of intestinal ulcerations by endoscopy or radiographic imaging (Baba et al., 1976; Kobayashi et al., 2007; Lee et al., 2001). To be included in this study, patients had to have intestinal ulcerations typical of intestinal BD: b5 in number, oval in shape, deep with discrete margins, and located in the ileocecal area (Lee et al., 2001). Any patient strongly suggested of having CD, UC, intestinal tuberculosis, or a malignancy through radiographical, endoscopic, or pathological studies was excluded. For CD, in particular, which may be difficult to differentiate from intestinal BD, longitudinal, irregular/ geographic, or cobblestone shaped ulcers and diffuse or segmental distribution of the lesions were considered the typical colonoscopic features of CD (Lee et al., 2009). Patients with concomitant autoimmune diseases including systemic lupus erythematosus, RA, primary Sjogren's syndrome, type 1 diabetes, systemic sclerosis, or asthma were also excluded. The traditional criteria of the BD Research Committee of Japan, revised in 1987, were applied to detect systemic manifestations (Mizushima, 1988). Patients who lacked extraintestinal symptoms that fulfilled the Japanese criteria of BD but had typical intestinal ulcerations were enrolled in this study. Demographic and clinical information such
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Age at diagnosis (years) Sex (M:F) Extraintestinal manifestations (%) Eyes Skin Oral ulcers Genital ulcers Joints Disease location (%) Ileum Ileocecal valve Colon Steroid dependent or refractory Use of immunosuppressants Surgical management Subtypes of systemic BDa Complete Incomplete Suspected Oral ulcer + GI ulcers GI ulcers only Follow-up duration (months)
Intestinal BD (n = 141)
Controls (n = 259)
38.08 ± 11.47 68:73
34.1 ± 10.5 152:107
25 (17.7) 60 (42.6) 113 (80.1) 43 (30.5) 47 (33.3) 71 48 26 21 44 44
(50.4) (34.0) (18.4) (14.9) (31.2) (31.2)
7 (5.0) 60 (42.6) 42 (29.8) 7 (5.0) 25 (17.7) 95.50 ± 60.74
Immunosuppressants: azathioprine, 5-mercaptopurine, cyclosporine, and infliximab. Classification according to the diagnostic criteria of the Research Committee of Japan. BD, Behcet's disease; GI, gastrointestinal. Complete, 4 major symptoms; incomplete, 3 major or 2 major + 2 minor symptoms; suspected, symptoms unsatisfying incomplete criteria. a Complete, incomplete, and suspected subtypes of systemic Behcet's disease were classified according to the diagnostic criteria of the Research Committee of Japan consisting of 4 major (oral ulceration, skin lesion, eye lesion, and genital ulcers) and 5 minor symptoms (arthritis, intestinal ulcers, vascular disease, neuropsychiatric disorders, and epididymitis).
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Table 2 Genotypes of cases and controls and their association with the risk of intestinal BD. Genes
Genotypes
IL17A
rs4711998 AA AG GG rs8193036 CC CT TT TT vs. CC + CT rs9791323 AA AC CC rs3819024 AA AG GG rs2275913 GG GA AA − 168 (novel)⁎ CC GC − 159 (novel)⁎
IL23R
STAT4
TT TA rs8193037 GG GA AA − 112 (novel)⁎ GG GA rs1884444 GG GT TT TT vs. GG + GT V134D (novel)† TT TA G149R (novel)† GG GA IVS4 + 17 (novel)† CC CT rs11889341 CC CT TT rs7574865 GG GT TT rs8179673 TT CT CC rs6752770 AA AG GG rs10168266 GG AG AA rs10181656 CC CG GG rs11685878 CC CT TT
Cases, n (%) Controls
OR
p value 0.130
68 (48.2) 57 (40.4) 16 (11.3)
145 (56.0) 98 (37.8) 16 (6.2)
64 (45.4) 54 (38.3) 23 (16.3)
128 (49.4) 109 (42.1) 22 (8.5)
0.068
2.10 (1.12–3.92) 0.021 0.240 120 (85.1) 20 (14.2) 1 (0.7)
230 (88.8) 29 (11.2) 0 (0)
46 (32.6) 67 (47.5) 28 (19.9)
74 (28.6) 130 (50.2) 55 (21.2)
52 (36.9) 62 (44.0) 27 (19.1)
81 (31.3) 119 (46.0) 59 (22.8)
141 (100) 0 (0)
258 (99.6) 1 (0.4)
140(99.3) 1 (0.7)
257 (99.2) 2 (0.8)
106 (75.7) 33 (23.4) 2 (1.4)
196 (75.7) 59 (22.8) 4 (1.5)
140 (99.3) 1 (0.7)
254 (98.10 5 (1.9)
68 (48.2) 58 (41.1) 15 (10.6)
103 (39.8) 108 (41.7) 48 (18.5)
addition, we have previously reported the association of these STAT4 variants with IBD (Moon et al., 2010). Genomic DNA was isolated from whole blood samples using commercially available kits (Qiagen, Hilden, Germany). DNA sequence analysis of the promoter region of IL17A, including exon 1 (~1.2 kb), was carried out, and identification of variants in other exons of IL17A and IL23R, including splice junctions, was performed by denaturing high performance liquid chromatography (DHPLC). Primer sequences are provided in Supplementary Table 1. All variants detected by DHPLC analysis were confirmed by subsequent full DNA sequencing. Genotyping for the STAT4 variants (rs11889341, rs7574865, rs8179673, rs6752770, rs10168266, rs10181656, and rs11685878) was performed using TaqMan technology (Applied Biosystems, Foster City, California, USA) as described previously (Moon et al., 2010).
0.700
Statistical analysis 0.470
0.350
0.940
0.990
0.310
We tested all single nucleotide polymorphisms (SNPs) to determine if they were in Hardy–Weinberg equilibrium (HWE) and assessed whether differences in the genotype frequencies between cases and controls were significant using the Chi-square test or Fisher's exact test. Gene–gene interaction was analyzed using an interaction test in logistic regression models (Thornton-Wells et al., 2004). Logistic regression models were used to assess relationships between SNPs and phenotypes after adjustment for possible confounding factors (gender, age at diagnosis, disease location, total follow-up period, immunosuppressant use, and requirement for an operation). For multiple comparisons, the Bonferroni correction was applied. P values of less than 0.05 were considered significant. Statistical analysis was performed using SPSS version 14.0 (SPSS, Chicago, IL, USA). Haplotypes were analyzed using Haploview 4.1 software.
0.069
Results 0.52 (0.28–0.97) 0.034 0.680
138 (97.9) 3 (2.1)
255 (98.5) 4 (1.5)
125 (88.7) 16 (11.3)
226 (87.3) 33 (12.7)
134 (95) 7 (5)
252 (97.3) 7 (2.7)
0.630
The distributions of the genotypes of SNPs in IL17A, IL23R, and STAT4 among patients and controls are presented in Table 2. The genotype distributions of all SNPs among the controls were in HWE. IL17A rs8193036 was associated with a significantly increased risk of intestinal BD under a recessive model for the variant T allele (OR 2.10, 95% CI 1.12–3.92, p = 0.021). Among the SNPs in IL23R, rs1884444 was associated with a significantly decreased risk of intestinal BD under a recessive model for the variant T allele (OR 0.52, 95% CI 0.28–0.97, p =0.034). When this was shown as a risk genotype (OR> 1.00) for better comparison in the combined genotype analysis, the OR of the GG+GT genotype compared with the TT genotype was 1.92 (95% CI 1.03–3.57, p= 0.034). There was no significant difference in the genotype frequencies of SNPs in STAT4 between the patients and controls.
0.5
Combined effect of risk genotypes, gene–gene interaction, and haplotypes for risk of intestinal BD
0.680
0.250
0.620 58 (45.7) 57 (44.9) 12 (9.4)
117 (51.1) 93 (40.6) 19 (8.3)
55 (43.3) 55 (43.3) 17 (13.4)
106 (46.3) 100 (43.7) 23 (10.0)
55 (43.3) 55 (43.3) 17 (13.4)
105 (45.9) 101 (44.1) 23 (10.0)
0.620
76 (59.8) 42 (33.1) 9 (7.1)
135 (59.0) 84 (36.7) 10 (4.4) 0.590
57 (44.9) 57 (44.9) 13 (10.2)
114 (49.8) 97 (42.4) 18 (7.9)
55 (43.3) 55 (43.3) 17 (13.4)
105 (45.9) 101 (44.1) 23 (10.0)
41 (32.2) 58 (45.7) 28 (22.1)
82 (35.8) 114 (49.8) 33 (14.4)
Association of IL17A, IL23R, and STAT4 polymorphisms with intestinal BD susceptibility
0.630
We investigated the combined effects of genetic variants of IL17A and IL23R that were found to be associated with susceptibility to intestinal BD in individual polymorphism analyses. The IL17A rs8193036 TT and IL23R rs1884444 GG + GT genotypes were considered to be risk genotypes for intestinal BD. When subjects were divided into three groups based on the number of risk genotypes they possessed, the risk Table 2 (continued)
0.190
Genes Genotypes Cases, n (%) Controls OR p value Notes to Table 2: “Novel” indicates SNPs that have not been reported previously. The number shows the nucleotide positions of the SNP calculated from ⁎the translation start site or †amino acid position. Significant associations with p b 0.05 are shown in bold.
E.S. Kim et al. / Life Sciences 90 (2012) 740–746
increased in a dose-dependent manner as the number of risk genotypes increased. Compared with subjects with no-risk genotype, the OR was 2.029 (95% CI 1.03–4.00, p = 0.09) for those with 1 risk genotype and 4.074 (95% CI 1.65–10.04, p = 0.004) for those with 2 risk genotypes (Fig. 1A). When participants were classified into two groups (zero to one vs. two risk genotypes), the risk of the participants with two risk genotypes was significantly greater than those with zero or one-risk genotype (OR 2.21, 95% CI 1.24–4.34, p = 0.021) (Fig. 1B). We also investigated potential gene–gene interactions between SNPs in IL23R, IL17A, and STAT4. There was a significant interaction between G149R in IL23R and rs11685878 in STAT4 (p = 0.004 in codominant model; p = 0.002 in recessive model; Table 3), rs2275913 in IL17A and rs7574865 in STAT4 (p= 0.003 in codominant model; p = 0.006 in recessive model; Table 3), and rs1188941 in STAT4 and rs2275913 in IL17A (p= 0.008 in codominant model; p = 0.021 in dominant model; Table 3). Haplotype analysis revealed that the frequencies of the GCAAGCTGG and ACCGACTGG haplotypes of IL17A were significantly higher in intestinal BD patients than in controls (OR 2.05, 95% CI 1.01–4.16, p = 0.047 and OR 12.12, 95% CI 1.39–105.77, p = 0.025, respectively) (Table 4). Meanwhile, the haplotype TTGC of IL23R was inversely related with disease susceptibility (OR 0.71, 95% CI 0.52–0.98, p = 0.038) (Table 5). Associations of IL17A, IL23R, and STAT4 genetic polymorphisms and the phenotype of intestinal BD To analyze associations between genotypes and disease phenotypes, multivariate logistic regression was performed after adjusting
Fig. 1. Combined effect of polymorphisms in the IL17A and IL23R genes on the risk of intestinal Behcet's disease. The odds ratio of each group according to the number of risk genotypes (A). The odds ratio of patients with 2 risk genotypes compared with those with 0–1 risk genotype (B). Risk genotypes: IL17A rs8193036 TT and IL23R rs1884444 GG + GT.
743
for other covariates such as gender, age at diagnosis, EIMs, disease locations, total follow-up period, use of immunosuppressants, and need for an operation. In terms of the location of GI tract involvement, STAT4 rs10168266 was associated with the involvement of the ileocecal valve area under a dominant model (AA + AG vs. GG, OR 2.309, 95% CI 1.028–5.181, p = 0.042). In addition, a dominant model of IL17A rs9791323 was inversely associated with ileocecal valve involvement (AC + CC vs. AA, OR 0.25, 95% CI 0.07–0.94, p = 0.040). Subjects with the GT + TT genotype of IL23R rs1884444 were less likely to exhibit systemic BD manifestations than those with a GG genotype (dominant model; OR 0.36, 95% CI 0.15–0.90, p = 0.028). Steroid dependency or refractoriness was more often observed in patients with the TT+ CT genotype of IL17A rs8193036 than those with the CC genotype (dominant model; OR 3.98, 95% CI 1.23–12.84, p = 0.021). However, all significance of above associations between SNPs and phenotype did not remain after correction for multiple testing (Supplementary Table 2). Discussion IL17-producing Th17 cells, a novel T cell population, play a major role in autoimmunity. Th17 cell differentiation from naïve CD4+ T cells is facilitated by various cytokines, including IL–1ß, IL-6, IL-21, and IL-23 (Aggarwal et al., 2003; Bettelli et al., 2006). A critical feature of Th17 cells is the expression of IL-17A and IL-17F. The association between IL-17F genetic polymorphisms and autoimmune diseases has been extensively evaluated in various ethnic populations (Chen et al., 2009; Paradowska-Gorycka et al., 2010; Saitoh et al., 2011). IL-17A is a powerful proinflammatory mediator that presents a number of proinflammatory effects on intestinal epithelial cells, endothelial cells, macrophages, fibroblasts and colonic myofibroblasts with increased release of proinflammatory chemokines, cytokines, and other mediators such as prostaglandins, matrix metalloproteinases, nitric oxide synthase-2, and granulocyte colony stimulating factor (Brand, 2009). In addition, IL-17A exerts a pathologic role in intestinal inflammation through activation of nuclear factor kappa B (NF-kB) and mitogen-activated protein kinase (MAPK) pathways (Awane et al., 1999; Shalom-Barak et al., 1998). Recent GWA studies, demonstrating IL23R genes as IBD susceptibility genes, validate the important role of Th17 cells that express the IL-23R on their surface in the pathogenesis of CD (Barrett et al., 2008; Duerr et al., 2006). STAT4 is a transcription factor that transduces IL-12, IL-23, and type 1 interferonmediated signals into Th1 and Th17 differentiation, monocyte activation, and interferon-gamma production. STAT4-dependent cytokine regulation plays a critical role in the pathogenesis of autoimmune diseases. Thus, it is possible that genetic variants in genes of these mediators could lead to dysregulation of the IL-23/IL-17 pathway, thereby increasing the risk for autoimmunity. Here, we investigated the relationships of the variants of IL17A, IL23R, and STAT4 with susceptibility to intestinal BD and BD phenotypes. This is the first genetic variant study conducted on intestinal BD. We identified two individual polymorphisms (IL17A rs8193036C> T and IL23R rs1884444G > T) associated with the risk of intestinal BD. Although it is challenging to know the exact mechanisms of these polymorphisms involving intestinal BD, functional variant of rs1884444 of IL23R changing glutamine into histidine may affect the structure and the biological function or stability of IL-23R. Polymorphism rs8193036 of IL17A may give a significant influence on binding affinity of transcription factors. We previously reported that rs8193036 of IL17A was significantly related to a higher level of IL-17A mRNA expression in patients with inflammatory bowel disease and showed a substantial activity to transcription factor complexes as a kind of regulatory mechanism (Kim et al., 2011). Notably, the joint effects of these two SNPs (IL17A and IL23R) raised the risk of intestinal BD as the number of risk genotypes increased. Furthermore, significant synergistic interactions between genes were observed (G149R in IL23R and rs11685878 in STAT4; rs2275913 in IL17A and
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E.S. Kim et al. / Life Sciences 90 (2012) 740–746
Table 3 Synergistic interactions between IL23R, IL17A, and STAT4 genotypes in intestinal Behcet's disease. G149R Codominant
Dominant
rs11685878 GG
GA
CC CT TT Interactive P value
0.79 (0.28–2.21) 1.75 (0.62–4.95)
1.00 0.90 (0.53–1.54) 2.12 (1.08–4.17)
AA
0.004
Recessive
GG
GA
1.00
0.79 (0.28–2.21)
AA
GG
GA
1.00
1.22 (0.60–2.49)
1.14 (0.69–1.89) 0.99 (0.38–2.53)
2.26 (1.25–4.10)
0.00
0.900
0.002
AA
rs2275913 Codominant rs7574865
GG
GG GT TT Interactive P value
Dominant GA
AA
Recessive
GG
GA
AA
GG
1.00 1.47 (0.71–3.05) 0.77 (0.31–1.96) 1.41 (0.64–3.09) 0.80 (0.38–1.67) 1.64 (0.67–4.03) 7.52 (1.43–39.52) 1.93 (0.68–5.50) 0.20 (0.02–1.62)
1.00
1.47 (0.71–3.05) 0.77 (0.31–1.96)
GA
1.00
0.92 (0.55–1.56) 0.97 (0.51–1.85)
AA
1.80 (0.86–3.79) 0.97 (0.49–1.94) 1.07 (0.47–2.47)
6.46 (1.28–32.62) 1.66 (0.62–4.43) 0.17 (0.02–1.35)
0.003
0.12
0.006
rs11889341 Codominant
Dominant TT
CC
Recessive
rs2275913
CC
CT
GG GA AA Interactive P value
1.00 1.40 (0.68–2.87) 1.34 (0.57–3.17)
2.19 (1.01–4.74) 9.89 (1.04–94.37) 1.00 2.19 (1.01–4.74) 9.89 (1.04–94.37) 1.00 1.18 (0.57–2.47) 1.73 (0.57–5.22) 1.18 (0.47–2.96) 0.00 1.38 (0.71–2.67) 1.18 (0.60–2.33) 0.96 (0.35–2.68) 1.12 (0.52–2.40)
0.008
CT
TT
CC
0.021
CT
TT
1.29 (0.77–2.16) 2.07 (0.83–5.13) 0.98 (0.43–2.26) 0.00
0.015
Significant associations with p b 0.05 are shown in bold. BD, Behcet's disease
rs7574865 in STAT4; rs11889341 in STAT4 and rs2275913 in IL17A). Altogether, these results highlight the importance of combined analysis of polymorphisms for risk prediction rather than analysis of each SNP individually and also suggest that interactions of variants of different genes in the same pathway may modulate susceptibility to intestinal BD. Increasing evidence has suggested that IL23R polymorphisms are associated with a number of inflammatory and immune-linked diseases including inflammatory bowel disease, psoriasis, ankylosing spondylitis, and BD (Cargill et al., 2007; Duerr et al., 2006; Remmers et al., 2010; Rueda et al., 2008). Two recent genome-wide association studies of BD identified a significant association between two IL23R polymorphisms (rs924080 and rs1495965) and disease susceptibility (Mizuki et al., 2010; Remmers et al., 2010). In the present study, the locus rs1884444G > T of the IL23R gene protected the development of intestinal BD. These discrepancies in the results concerning the role of genetic variants in IL23R for disease risk are interesting. For instance, some SNPs of IL23R are associated with a greater risk for
CD (Newman et al., 2009; Okazaki et al., 2008), whereas others appear to protect against the development of CD, which are similar to our results (Dubinsky et al., 2007; Glas et al., 2007; Taylor et al., 2008). Schmechel et al. (2008) reported that the level of IL-22 that correlates with the disease activity was higher in carriers of CD riskincreasing IL23R variants than in those of CD risk-decreasing IL23R variants, suggesting that IL23R genotypes influence IL-22 serum expression. Regarding the IL17 gene, while a recent Chinese study failed to find an association between IL17A SNP rs2275913 and BD susceptibility (Shu et al., 2010), we found a significant relationship between an IL17A variant and the intestinal BD risk and synergistic interaction between rs2275913 of IL17A and rs7574865 of STAT4. Interestingly, the IL17A gene and the HLA B51, TNFα, and SUMO4 genes that are known to increase the BD risk are located on the same chromosome (chromosome 6), suggesting an association between IL17A variants and disease susceptibility (Hou et al., 2008; Remmers et al., 2010; Verity et al., 1999). As the evidence of IL17A genetic variants having a potential risk of the autoimmune disease development, our very
Table 4 Association of IL17A haplotypes with intestinal Behcet's disease. rs4711998
rs8193036
rs9791323
rs3819024
rs2275913
− 168
− 159
rs8193037
− 112
Freq
OR (95% CI)
p value
A G A A A G A A G A A G A A
C T C T C C C T C C T C C T
A A A A A A A C A A A A C A
G A A A A A G A G A A A G G
A G G G G G G G A A G G A A
C C C C C C C C C C C C C C
T T T T T T T T T T T T T T
G G A G G G G G G G A A G G
G G G G G G G G G G G G G G
0.3307 0.1328 0.0807 0.0705 0.0704 0.0555 0.045 0.0366 0.0232 0.0209 0.019 0.0168 0.0114 0.0102
1 1.45 (0.88–2.39) 0.92 (0.50–1.69) 1.53 (0.83–2.84) 0.46 (0.21–1.02) 2.05 (1.01–4.16) 0.59 (0.24–1.42) 0.93 (0.39–2.22) 0.54 (0.12–2.44) 0.14 (0.02–1.12) 1.61 (0.46–5.69) 1.12 (0.28–4.55) 12.12 (1.39–105.77) 0.65 (0.11–3.83)
0.14 0.79 0.17 0.056 0.047 0.24 0.86 0.42 0.065 0.46 0.87 0.025 0.64
Significant associations with p b 0.05 are shown in bold.
E.S. Kim et al. / Life Sciences 90 (2012) 740–746
745
Table 5 Association of IL23R haplotypes with intestinal Behcet's disease. rs1884444
E4.V134D
E4.G149R
E4.IVS4 + 17
Freq
OR (95% CI)
p value
G T G T
T T T T
G G A A
C C C C
0.5883 0.3277 0.0343 0.0247
1 0.71 (0.52–0.98) 0.78 (0.30–2.00) 0.76 (0.24–2.39)
0.038 0.6 0.64
Significant associations with p b 0.05 are shown in bold.
recent study investigating the association of genetic variants of IL23R and IL17A with inflammatory bowel disease identified the relationship of rs2275913 of IL17A with the risk of UC (Kim et al., 2011). Although we were not able to find any association of SNPs of STAT4 with the risk of intestinal BD in the individual polymorphism analysis, they were found to exert a significant synergistic effect both with IL17A and IL23R in gene–gene interaction analysis. There are several studies suggesting the association of STAT4 SNP rs7574865, which showed synergistic interaction with rs2275913 of IL17A in our study, with autoimmune diseases. The Chinese study identified a significant association between STAT4 SNP rs7574865 and the BD risk (Hu et al., 2010), and another study suggested that this variant was associated with colonic CD (Glas et al., 2010). Inconsistent results among different genetic studies on BD susceptibility may be due to differences in patient characteristics because of the different clinical settings of each study. Because of the wide range of BD systemic manifestations, patients with BD may visit various clinics such as dermatology, rheumatology, ophthalmology, or gastroenterology clinics based on their main symptoms. Therefore, subjects may be recruited from different clinical settings, leading to heterogeneity in the severity of symptoms in the different studies. We previously reported that a significant number of patients with typical intestinal lesions who did not fully satisfy the diagnostic criteria of systemic BD at the time of initial presentation presented with systemic manifestations at the end of the follow-up period (Cheon et al., 2009; Shin et al., 2010). Based on these studies, we included patients with typical intestinal lesions irrespective of systemic manifestations in our study, in contrast to prior BD studies, which could perhaps explain the differences in our findings compared to the studies mentioned above. An alternative explanation could be ethnic differences between the diverse populations examined in the above studies. Intriguingly, functional variants in the IL23R gene, which were identified as susceptibility loci in Caucasian IBD patients, showed no association with IBD in Asian populationbased studies (Bin et al., 2009; Yamazaki et al., 2007). Likewise, although GWA studies identified an association between SNPs in IL23R and the risk of BD in Turkish and Japanese cohorts, no significant association was found in a Korean cohort (Mizuki et al., 2010; Remmers et al., 2010). Importantly, combined effect analysis of multiple genetic variants of IL23R and IL17A resulted in better detection of high-risk individuals susceptible to intestinal BD than analysis of a single polymorphism. Compared to patients with no-risk genotypes, patients with one or two risk genotypes had an intestinal BD risk 2.03-fold or 4.07-fold greater, respectively. Furthermore, gene–gene interaction analysis revealed evidence for epistatic interactions among IL17A, IL23R, and STAT4 genes, indicating that the IL-17/23 axis plays a significant role in the pathogenesis of intestinal BD. Although significant associations appeared to be observed between SNPs in the IL17A, IL23R, and STAT4 genes and disease phenotypes, statistical significance failed to remain after correction for multiple testing. Further studies with a larger sample population than that examined in our study are warranted to clarify this issue. We acknowledge the small sample size as a significant limitation of the current study. In comparison to results of genome-wide association studies, the p values obtained were not very impressive, which is mainly attributed to the limited sample size of the present study. However, given that Behcet's disease is rarer than CD at least in
Western European populations, we assumed that the number of cases of this study might be acceptable. Regarding the diagnostic criteria of systemic BD, one might think that the International Study Group of BD (ISGBD) criteria should be used because they have been widely accepted and used among clinicians worldwide since its description. However, we decided to use the Japanese criteria instead of the ISGBD criteria for the following several reasons. First, the ISGBD criteria failed to identify patients if they did not show preceding oral ulcerations. It has been reported that 3% of 914 patients with definite BD who lacked oral ulcerations were excluded in the original survey for the ISGBD criteria (1990). Second, in Korea, the rate of positive pathergy test, which is one of the major factors included in ISGBD criteria, has been reported to be lower than 40% (Chang and Cheon, 2002). Third, Korea and Japan have numerous similarities in terms of ethnic and geographical backgrounds. Fourth, the Japanese criteria have a gradual diagnostic system that can reflect a temporal change better than the ISGBD criteria during the follow-up period. Finally, the gastrointestinal manifestation is included in the Japanese criteria but not in the ISGBD criteria. Conclusions Our results indicate that the joint effect of SNPs of IL17A, IL23R, and STAT4 may modulate susceptibility to intestinal BD in the Korean population, suggesting a potential significance of the IL-17/23 axis in the pathogenesis of intestinal BD. Conflict of interest statement The authors declare no conflict of interest.
Acknowledgements This research was supported by a Korea Research Foundation Grant funded by the Korean Government (MOEHRD, Basic Research Promotion Fund) (KRF-2008-331-E00105), a grant from the Korea Healthcare Technology R&D Project (A080588), the Ministry for Health, Welfare & Family Affairs, Republic of Korea, a grant from the 2009 Good Health R&D Project, Ministry of Health and Welfare, Republic of Korea (A084943), and the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (grant number 2009-0069149). Appendix A. Supplementary data Supplementary data to this article can be found online at doi:10. 1016/j.lfs.2012.03.017. References Aggarwal S, Ghilardi N, Xie MH, de Sauvage FJ, Gurney AL. Interleukin-23 promotes a distinct CD4 T cell activation state characterized by the production of interleukin17. J Biol Chem 2003;278:1910–4. Awane M, Andres PG, Li DJ, Reinecker HC. NF-kappa B-inducing kinase is a common mediator of IL-17-, TNF-alpha-, and IL-1 beta-induced chemokine promoter activation in intestinal epithelial cells. J Immunol 1999;162:5337–44.
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