JAK2 and STAT3 Polymorphisms in a Han Chinese Population with Behçet's Disease

Abstract

Purpose.: Janus kinase 2 (JAK2) and signal transducer and activator of transcription 3 (STAT3) polymorphisms have been demonstrated as a common risk factor for a number of autoimmune diseases. The aim of this study was to investigate the association of JAK2 and STAT3 polymorphisms with Behçet's disease (BD) in a Han Chinese population.

Methods.: A case-control study was performed in 503 Chinese patients with BD and 615 healthy controls. The genotypes of three single-nucleotide polymorphisms (SNPs) (rs10758669, rs7857730, rs10119004) in the JAK2 and four SNPs (rs6503695, rs744166, rs2293152, and rs12948909) in the STAT3 gene were analyzed using polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP). In all, 10% of the samples were sequenced to validate the result of PCR-RFLP. The χ² test was performed to compare allele and genotype distributions and Bonferroni correction was applied for multiple comparisons.

Results.: A deviation from the Hardy–Weinberg equilibrium was not found in all controls tested. A significantly increased frequency of the GG genotype of the STAT3 rs2293152 was observed in patients with BD (Bonferroni-corrected P value = 0.021). None of the tested SNPs of JAK2 was associated with BD. Stratification analysis according to oral ulceration, genital ulceration, skin lesions, and arthritis for BD did not reveal an association.

Conclusions.: These results suggest that a STAT3 genetic polymorphism is associated with the susceptibility to BD.

Behçet's disease (BD) is a chronic, multisystemic, and nongranulomatous inflammatory disorder. The major clinical manifestations of BD include recurrent oral and genital ulceration, uveitis, and erythematic multiforme. ¹ The HLA-B51 gene shows the strongest association with this disease in a variety of ethnic groups, including Iranians, Koreans, Arabs, and Greeks. ^{2 –4} Additionally, genes encoding TNF-α, IL-1α, IL-1β, and IL-12 have also been associated with susceptibility to BD. ^{5 –8} Previous studies from our group have shown that polymorphisms for genes encoding small ubiquitin-like modifier 4 (SUMO4), STAT4, and interleukin (IL)-23R are associated with the susceptibility for BD. ^{9 –11} These findings indicate that genetic factors may play an important role in the development of BD. Furthermore, an autoinflammatory response has been presumed to be implicated in the pathogenesis of BD. 

It has been demonstrated that both Th1 and Th17 cells are involved in BD. Janus kinase 2 (JAK2) and signal transducer and activator of transcription 3 (STAT3) axis have been reported to be critical for Th1 and Th17 cell differentiation, ¹² suggesting an important role for these proteins in the immune response. Recent studies have shown that polymorphisms rs10758669 ^13,14 in JAK2 and rs744166, ^13,15 rs12948909, ¹⁵ and rs2293152 ^16,17 in STAT3 were associated with the susceptibility to inflammatory bowel disease. Furthermore, rs10119004 and rs7857730 in JAK2 and rs6503695 in STAT3 were shown to play an indirect role in the susceptibility to ankylosing spondylitis in a Han Chinese population. ^18,19 Previous studies have shown that the same gene variants could be involved in a common pathway of pathogenesis in autoimmune diseases. ²⁰ Based on these findings, the question was raised whether the variants of JAK2 and STAT3 were associated with the susceptibility to BD. In the study presented here, we analyzed the association of seven single-nucleotide polymorphisms (SNPs) of the JAK2 and STAT3 gene with BD in a Han Chinese population. 

Materials and Methods

Patients and Controls

The study group comprised 615 healthy subjects (age: 34.3 ± 13.8 years; female: 155 [25.2%]; male: 460 [74.8%]) and 503 patients with BD (age 36.5 ± 12.7 years; female: 107 [21.3%]; male: 396 [78.7%]), referred to the Zhongshan Ophthalmic Center, Sun Yat-sen University, and the First Affiliated Hospital of Chongqing Medical University (P.R. China) between 2005 and 2011. Patients with BD and controls were unrelated. All control subjects were matched ethnically and geographically with the patients. No statistical difference was observed between patients and controls in the distribution of age and sex (P = 0.56, P = 0.84, respectively). The diagnosis of BD was made according to the criteria of the International Study Group for BD ²¹ and all patients had intraocular inflammation. Bilateral intraocular inflammation was observed in 395 patients (78.5%). The intraocular inflammation seen in these patients included anterior uveitis, posterior uveitis, and panuveitis. The most frequent type of uveitis was panuveitis, 76.3% in males and 48.2% in females. Anterior uveitis was observed more frequently in females than that in males (17.7% vs. 3.5%). None of the controls had ocular disease. The patients and controls gave their written informed consent for this study. The study was approved by the local institutional ethics committee and adhered to the tenets of the Declaration of Helsinki on human trials. 

Analysis of Genetic Polymorphisms of JAK2 and STAT3

Genomic DNA was isolated from blood leukocytes by using a commercial DNA blood mini kit (Qiamp Mini Kit; Qiagen Inc., Valencia, CA). The extracted DNA was stored at −20°C until used. Amplification of the target DNA in the JAK2 and STAT3 genes was carried out by PCR using appropriate primers (Table 1). Genotyping of each SNP was carried out by PCR restriction fragment length polymorphism (PCR-RFLP) methods. PCR products of each SNP were digested with specific restriction enzymes (Table 1) in a 10 μL reaction volume overnight. Digestion products were visualized on agarose gels of appropriate concentration and stained with a commercial nucleic acid stain (GoldView; SBS Genetech, Beijing, China). Genotypes were blindly scored, and analysis of all ambiguous samples was repeated. Moreover, the assay validity of PCR-RFLP was double-checked on 10% of the samples using direct sequencing (Invitrogen Biotechnology Co., Guangzhou, China). 

Table 1.

 

Primers and Restriction Enzymes Used for RFLP Analysis

Gene	SNP	Primers	T m (°C)	Enzyme
JAK2	rs7857730	5′-CCCCTTTATTGTAATTTTGAGTTCAT-3′	37	HIN1II
		5′-TGAAAACGCCTTCCTTAGAGTC-3′
JAK2	rs10758669	5′-GAGACAAGGACATGCTGAAGTGC-3′	37	HPYCH4V
		5′-ACAGACAAGCAAGCAATGTTTTTTTT-3′
JAK2	rs10119004	5′-ATATTGTCATTTTTAACTGCGCTACT-3′	65	BSENI
		5′-TGTAGGTAGTATAATGAAGCCCAAA-3′
STAT3	rs2293152	5′-CACAAAGGGCCTCTGGCTTC-3′	55	KPN2I
		5′-CATTCCCACATCTCTGCTCCCT-3′
STAT3	rs6503695	5′-GAGCATATTAAAGTGAAGAAAATAAT-3′	65	TASI
		5′-GGGATGTAGATCCAAACTAGAT-3′
STAT3	rs744166	5′-GCTGTAATGTCTTGAGGGAATCAAGC-3′	37	HINDIII
		5′-TATTCAGATGGCGGTCACATGC-3′
STAT3	rs12948909	5′-GGATCTAAAAGAGCCCTACTCTCCA-3′	65	TASI
		5′-TCACCCTATTGTGCTACTGAACGT-3′

Statistical Analysis

Genotype frequencies were estimated by direct counting. Allele and genotype frequencies were compared between patients and controls by a χ² test (SPSS version 13.0; SPSS Inc., Chicago, IL). To assess whether any of the clinical features were associated with gene polymorphisms, allele and genotype frequencies were analyzed between patients with certain clinical features and without these clinical features by χ² test. The P-values were corrected (Pc) with the Bonferroni correction by multiplying with the number of analyses performed. The Pc < 0.05 was considered as significant. 

Results

The BD group (n = 503) and the unrelated healthy controls (n = 615) were of similar age and sex. Allele and genotype frequencies of the analyzed samples of three SNPs of JAK2 and four SNPs of STAT3 polymorphisms are depicted in Table 2. No deviation from the Hardy–Weinberg equilibrium was observed in the distribution of genotypes in the control group. 

Table 2.

 

Summary of Seven SNPs of JAK2 and STAT3 with Behçet's Disease

Gene	SNP	Genotype/Allele	BD (n = 503)	Controls (n = 615)	P Value	Pc* Value	OR (95% CI)
JAK2	rs7857730	GG	131 (26.0%)	152 (24.7%)	0.611	NS	1.073 (0.818–1.406)
		GT	247 (49.1%)	298 (48.5%)	0.829	NS	1.026 (0.811–1.299)
		TT	125 (24.9%)	165 (26.8%)	0.453	NS	0.902 (0.689–1.181)
		G	509 (50.6%)	602 (48.9%)	0.436	NS	1.068 (0.904–1.262)
		T	497 (49.4%)	628 (51.1%)
JAK2	rs10758669	AA	234 (46.5%)	268 (43.6%)	0.325	NS	1.126 (0.889–1.427)
		AC	217 (43.1%)	271 (44.1%)	0.757	NS	0.963 (0.759–1.221)
		CC	52 (10.3%)	76 (12.4%)	0.291	NS	0.818 (0.562–1.189)
		A	685 (68.1%)	807 (65.6%)	0.215	NS	1.118 (0.937–1.335)
		C	321 (31.9%)	423 (34.4%)
JAK2	rs10119004	GG	149 (29.6%)	161 (26.2%)	0.201	NS	1.187 (0.913–1.543)
		GA	205 (40.8%)	290 (47.2%)	0.032	NS	0.771 (0.608–0.978)
		AA	149 (29.6%)	164 (26.7%)	0.274	NS	1.157 (0.891–1.504)
		G	503 (50.0%)	612 (49.8%)	0.909	NS	0.990 (0.827–1.289)
		A	503 (50.0%)	618 (50.2%)
STAT3	rs2293152	GG	99 (19.7%)	77 (12.5%)	0.001	0.021	1.712 (1.238–2.369)
		CG	228 (45.3%)	302 (49.1%)	0.208	NS	0.859 (0.678–1.088)
		CC	176 (35.0%)	236 (38.4%)	0.243	NS	0.864 (0.677–1.104)
		G	426 (42.3%)	456 (37.1%)	0.011	NS	1.247 (1.051–1.478)
		C	580 (57.7%)	774 (62.9%)
STAT3	rs6503695	CC	101 (20.1%)	130 (21.1%)	0.664	NS	0.937 (0.700–1.255)
		CT	236 (46.9%)	295 (48.0%)	0.727	NS	0.959 (0.757–1.214)
		TT	166 (33.0%)	190 (30.9%)	0.452	NS	1.102 (0.856–1.418)
		C	438 (43.5%)	555 (45.1%)	0.453	NS	0.937 (0.793–1.109)
		T	568 (56.5%)	675 (54.9%)
STAT3	rs744166	CC	99 (19.7%)	122 (19.8%)	0.948	NS	0.990 (0.737–1.331)
		CT	235 (46.7%)	323 (52.5%)	0.054	NS	0.793 (0.626–1.004)
		TT	169 (33.6%)	170 (27.6%)	0.031	NS	1.324 (1.025–1.711)
		C	433 (43.0%)	567 (46.1%)	0.148	NS	0.883 (0.747–1.045)
		T	573 (57.0%)	663 (53.9%)
STAT3	rs12948909	TT	308 (61.2%)	354 (57.6%)	0.154	NS	1.165 (0.916–1.481)
		CT	173 (34.4%)	231 (37.6%)	0.273	NS	0.871 (0.681–1.114)
		CC	22 (4.4%)	30 (4.9%)	0.690	NS	0.892 (0.508–1.566)
		T	789 (78.4%)	939 (76.3%)	0.241	NS	1.127 (0.923–1.376)
		C	217 (21.6%)	291 (23.7%)

*  Bonferroni-corrected P value. NS, not significant.

Concerning the distribution of allele and genotype, the frequency of the GG genotype of STAT3 rs2293152 was significantly higher in patients with BD than that in healthy controls (P = 0.001, Pc = 0.021, odds ratio [OR] = 1.712). The frequency of the G allele of the rs2293152 site was higher in patients than that in healthy controls (P = 0.011). However, the difference ceased to be significant after Bonferroni correction (Pc = 0.077, OR = 0.802). An increased frequency of the STAT3 rs744166 TT genotype (P = 0.031) and a decreased frequency of the JAK2 rs10119004 GA genotype (P = 0.032) were observed in patients with BD compared with healthy controls. However, it did not remain significant after Bonferroni correction (Pc = 0.651, OR = 1.324; and Pc = 0.672, OR = 0.771, respectively) (Table 2). No other significant differences were observed in the distribution of other alleles and genotypes between patients with BD and controls in the remaining four SNPs. 

Since the patients with BD showed different clinical features, we further analyzed the relationship between the tested SNPs and various clinical parameters, such as oral or genital ulcers, multiform skin lesions, and arthritis (Table 3). No significant difference was noted between any of the mentioned clinical characteristics and the investigated SNPs. 

Table 3.

 

Clinical Characteristics of BD Patients with Uveitis

Phenotype	Total	%
Uveitis	503	100
Oral ulcer	503	100
Genital ulcer	199	39.5
Skin lesions	239	47.5
Arthritis	134	26.7

Discussion

In this study we investigated the association of JAK2 and STAT3 polymorphisms with BD in a Han Chinese population. Our study identified an association between rs2293152 in STAT3 and BD. 

BD has a distinctive geographic distribution. It is one of the common autoinflammatory diseases and one of the most commonly seen uveitis entities (16.5%) in China. ²² BD is quite common in countries along the ancient “Silk Route” such as China, Turkey, and Japan. ^7,22,23 Previous studies showed that this disease occasionally displays a familial aggregation pattern. ^24,25 Recently, several immune response-related genes have been shown to be associated with BD. ¹⁵ These data suggest that genetic factors may play an important role in the pathogenesis of this disease. 

The JAK2–STAT3 pathway is a signaling target of a multitude of cytokines that are thought to play significant biological roles in immune-mediated diseases. This pathway is essential in Th1 cell differentiation and proliferation. Additionally, it is also important for the development of Th17 cells. ¹² Both Th1 and Th17 cells play critical roles in immune-mediated diseases such as BD. ²⁶ The JAK2 and STAT3 signaling pathway may thus exert its influence on immune-mediated diseases, including BD, by modulating the Th1 and Th17 response. Recently, genetics polymorphisms of JAK2 and STAT3 have been investigated for their association with a number of autoimmune diseases including Crohn's disease and ulcerative colitis. ^{20,27 –32} All these results prompted us to examine whether the polymorphisms of the JAK2 and STAT3 genes could possibly also contribute to the development of BD in a Han Chinese population. To ensure the results, we made the following efforts. First, we strictly selected the patients with BD according to the criteria of the International Study Group. If there was any doubt, patients were excluded from the study. Second, unrelated healthy individuals were selected from the same geographic regions as the patients with BD. The patients and controls were age, sex, and ethnically matched. Third, 10% of the samples were randomly chosen and analyzed by direct sequencing in an attempt to validate the RFLP method used in this study. 

Our result showed that the frequency of the rs2293152 GG genotype was significantly increased in patients with BD, indicating its predisposing role in this disease. Japanese patients with Crohn's disease on the other hand showed a significant association with the C allele and the CC genotype. ¹⁷ Our result showed that none of the SNPs of JAK2 were associated with susceptibility to BD. This finding is in disagreement with findings reported for other autoimmune diseases ^13,18 such as Crohn's disease and ankylosing spondylitis and indicates that predisposition between JAK2 and immune-mediated disease may be different. The association between BD and the G allele of rs2293152 is not as significant as with the homozygous GG genotype. The reason for this finding is not yet clear but could be explained by the fact that it represents the recessive allele for this gene. To exclude that the association between rs2293152 of STAT3 and BD was due to an association with other genes in its vicinity, we performed a linkage disequilibrium analysis using the HapMap SNP database. This analysis showed that genetic polymorphisms in the genes encoding STAT5A and PTRF, which are located near rs2293152 of STAT3, were not in linkage disequilibrium with this locus. 

Like other studies on the association of gene polymorphisms with BD, there were some limitations in our study. Because BD usually affects various systems, the patients recruited from an ophthalmic department may represent a subpopulation of this disease. Therefore, the SNPs identified in this study may be associated only with uveitis in BD. Studies on patients with BD from other medical departments, such as dermatology and stomatology, are needed to confirm the present results. On the other hand, the tested patients with BD came solely from a Han Chinese population. Studies on multiethnic populations and larger samples may definitely clarify the association of STAT3 with BD. In addition, the biological function of rs2293152, a strongly associated SNP demonstrated in our study, is not yet known and needs to be investigated further. 

In conclusion, this study suggests that the GG genotype of rs2293152 may be a risk factor involved in the hereditary predisposition to BD. 

Footnotes

 Supported in part by the Project of Medical Science and Technology of Chongqing, Key Project of Health Bureau of Chongqing, Chongqing Key Laboratory of Ophthalmology Grant CSTC 2008CA5003, Project of Health Bureau of Chongqing Grant 20092397, National Natural Science Foundation Project Grant 81100657, Natural Science Foundation Major International (Regional) Joint Research Project Grant 30910103912, and a grant from the Program for the Training of a Hundred Outstanding Science and Technology Leaders of Chongqing Municipality and Fund for PAR-European Union Scholars Program.

Footnotes

 Disclosure: K. Hu, None; S. Hou, None; Z. Jiang, None; A. Kijlstra, None; P. Yang, None

The authors thank technician Hongyang Zhou for assistance in collection of blood samples from patients and controls in the Zhongshan Ophthalmic Center, and all patients and controls enrolled in the present study.