Shengping Hou

Identification of a susceptibility locus in STAT4 for Behçet's disease in Han Chinese in a genome-wide association study

OBJECTIVE To identify susceptibility loci for Behçets disease (BD) and elucidate their functional role. METHODS A genome-wide association study (GWAS) and functional studies were conducted. A total of 149 patients and 951 controls were enrolled in the initial GWAS, and 554 patients and 1,159 controls were enrolled in the replication study. Real-time polymerase chain reaction, luciferase reporter assay, and enzyme-linked immunosorbent assay were performed. RESULTS Our GWAS and replication studies identified a susceptibility locus around STAT4 (single-nucleotide polymorphisms [SNPs] rs7574070, rs7572482, and rs897200; P = 3.36 × 10(-7) to 6.20 × 10(-9) ). Increased expression of STAT4 was observed in individuals carrying the rs897200 risk genotype AA. Consistent with the idea that STAT4 regulates the production of interleukin-17 (IL-17) and interferon-γ, IL17 messenger RNA and protein levels were increased in individuals carrying the rs897200 risk genotype AA. Interestingly, the risk allele A of rs897200 creates a putative transcription factor binding site. To test whether it directly affects STAT4 transcription, an in vitro luciferase reporter gene assay was performed. Higher transcription activity was observed in individuals carrying the risk allele A, suggesting that rs897200 is likely to directly affect STAT4 expression. Additionally, 2 SNPs, rs7574070 and rs7572482, which are tightly linked with rs897200, were cis-expression quantitative trait loci (eQTL) SNPs, suggesting that SNP rs897200 is an eQTL SNP. Most importantly, the clinical disease severity score was higher in individuals with the rs897200 risk genotype AA. CONCLUSION These findings strongly suggest that STAT4 is a novel locus underlying BD. We propose a model in which up-regulation of STAT4 expression and subsequent STAT4-driven production of inflammatory cytokines, such as IL-17, constitute a potential pathway leading to BD.

IL-23R gene confers susceptibility to Behcet's disease in a Chinese Han population

Purpose IL-23 has been shown to be involved in the pathogenesis of Behcets disease (BD) through promoting IL-17 production. This study examined whether IL-23R polymorphisms were associated with susceptibility to this disease in a Chinese Han population. Methods Four single-nucleotide polymorphisms (SNP), rs7517847, rs11209032, rs 1343151 and rs17375018 were genotyped in 338 BD patients and 407 age, sex and ethnically matched healthy controls using a PCR restriction fragment length polymorphism assay. Results A significantly increased prevalence of the homozygous rs17375018 GG genotype and G allele was found in BD patients compared with controls (corrected p (pc)<0.001,odds ratio (OR) 1.86, 95% CI 1.39 to 2.49; pc<0.001, OR 1.57, 95% CI 1.25 to 1.98, respectively). The frequencies of the AA genotype and A allele of the SNP rs11209032 were significantly higher in BD patients compared with controls (pc=0.024, OR 1.69, 95% CI 1.21 to 2.35; pc<0.001, OR 1.48, 95% CI 1.21 to 1.82, respectively). In addition, the results showed a significantly decreased frequency of the AGCG haplotype in BD patients compared with controls (pc=0.0016, OR 0.59, 95% CI 0.45 to 0.77). Conclusions This study, for the first time, identified a strong association of an SNP of IL-23R, rs17375018, with BD. The results also suggested that both rs11209032 AA and rs17375018 GG of IL-23R are predisposing genotypes for BD and that the AGCG haplotype may provide protection against BD.

Interleukin-17 gene polymorphism is associated with Vogt–Koyanagi–Harada syndrome but not with Behçet's disease in a Chinese Han population

Interleukin (IL)-17 has been shown to play an important role in certain autoimmune diseases. The present study was performed to investigate the association of IL-17A and IL-17F gene polymorphisms with two autoimmune uveitis entities, Vogt-Koyanagi-Harada (VKH) syndrome and Behçets disease (BD), in a Chinese Han population. A total of 362 BD patients, 385 VKH syndrome patients, and 412 controls were genotyped for IL-17A/rs2275913 and IL-17F/rs763780 using polymerase chain reaction-restricted fragment length polymorphism. The result showed that the genotype and allele distribution of the two single nucleotide polymorphisms (SNPs) in all subjects were in Hardy-Weinberg equilibrium. A significantly decreased frequency of IL-17F/rs763780 C allele (p = 0.006, p(c) = 0.036) and an increased frequency of TT genotype (p = 0.005, p(c) = 0.030) were observed in VKH patients compared with normal controls. There was no association of the tested two SNPs with BD, even after adjusting gender ratio. Stratification analysis failed to find any association of extraocular manifestations of two uveitis entities and the tested two SNPs. The C allele and TT genotype of rs763780 in the IL-17F gene appear to be associated with protection and susceptibility to VKH syndrome. The tested two IL-17 SNPs are not found to be associated with Behçets disease.

MicroRNA-146a and Ets-1 gene polymorphisms in ocular Behçet's disease and Vogt–Koyanagi–Harada syndrome

Aim MicroRNA-146a (miR-146a) is involved in certain immune-mediated diseases. Transcription factor Ets-1 strongly affects miR-146a promoter activity and directly regulates miR-146a expression. This study was performed to investigate the association of miR-146a and Ets-1 gene polymorphisms with Behçets disease (BD) and Vogt–Koyanagi–Harada (VKH) disease in a Chinese Han population. Methods A total of 809 patients with BD, 613 patients with VKH and 1132 normal controls were genotyped for miR-146a/rs2910164, rs57095329 and rs6864584, Ets-1/rs1128334 and rs10893872 using a PCR restriction fragment length polymorphism assay. miR-146a expression was examined in peripheral blood mononuclear cells (PBMCs) by real-time PCR. Cytokine production by PBMCs was measured by ELISA. Results A significantly decreased frequency of the homozygous rs2910164 CC genotype and C allele was observed in patients with BD compared with controls (pca=1.24×10−5, OR 0.61; pca=1.33×10−4, OR 0.75, respectively). MiR-146a expression in GG cases was 2.45-fold and 1.99-fold higher, respectively, than that in CC cases and GC cases. There was no association of the other four single nucleotide polymorphisms (SNPs) with BD. There was also no association of these five SNPs with its main clinical features. No associations were found with the five SNPs tested or with its clinical manifestations in VKH disease. Interleukin (IL)-17, tumour necrosis factor (TNF)α and IL-1β production from rs2910164 CC cases was markedly lower than that in GG cases. No effect of genotype was observed on IL-6 and monocyte chemoattractant protein (MCP)-1 production and IL-8 expression was slightly higher in CC cases. Conclusions Our study identified a strong association of rs2910164 of miR-146a with BD in a Chinese population and decreased expression of miR-146a and certain proinflammatory cytokines in individuals carrying the CC genotype.

Genome-wide association analysis of Vogt-Koyanagi-Harada syndrome identifies two new susceptibility loci at 1p31.2 and 10q21.3

To identify new genetic risk factors for Vogt-Koyanagi-Harada (VKH) syndrome, we conducted a genome-wide association study of 2,208,258 SNPs in 774 cases and 2,009 controls with follow-up in a collection of 415 cases and 2,006 controls and a further collection of 349 cases and 1,588 controls from a Han Chinese population. We identified three loci associated with VKH syndrome susceptibility (IL23R-C1orf141, rs117633859, Pcombined = 3.42 × 10−21, odds ratio (OR) = 1.82; ADO-ZNF365-EGR2, rs442309, Pcombined = 2.97 × 10−11, OR = 1.37; and HLA-DRB1/DQA1, rs3021304, Pcombined = 1.26 × 10−118, OR = 2.97). The five non-HLA genes were all expressed in human iris tissue. IL23R was also expressed in the ciliary body, and EGR2 was expressed in the ciliary body and choroid. The risk G allele of rs117633859 in the promoter region of IL23R exhibited low transcriptional activation in a cell-based reporter assay and was associated with diminished IL23R mRNA expression in human peripheral blood mononuclear cells.

STAT4 polymorphism in a Chinese Han population with Vogt-Koyanagi-Harada syndrome and Behçet's disease.

This study investigated the association of rs7574865 polymorphism in STAT4 with Vogt-Koyanagi-Harada (VKH) syndrome and Behçets disease (BD) in a Chinese Han population. Genotyping of rs7574865 polymorphism in the STAT4 gene was performed using polymerase chain reaction restriction fragment length polymorphisms in 379 VKH patients, 366 BD patients, and 414 controls. Of the samples, 20% were sequenced to validate polymerase chain reaction restriction fragment length polymorphism results. A binary logistic regression analysis was used to assess the influence of the gender on the association of STAT4 polymorphism with BD. A significantly increased frequency of TT genotype of the STAT4 rs7574865 was observed in VKH patients (p = 0.013). GT genotypic frequency was significantly lower in BD patients than in controls (p = 0.003) However the significance of rs7574865 was lost in all tested BD patients when adjusted for gender (p = 0.775). A significantly lower frequency of GT genotype and a significantly higher frequency of GG genotype was found in male BD patients compared with male controls (p = 0.000458 and p = 0.009, respectively). Stratification analysis according to tinnitus, alopecia, poliosis, headache, and vitiligo for VKH syndrome and oral ulceration, genital ulceration, skin lesions and arthritis for BD failed to find any association between the tested single nucleotide polymorphism and any of the extraocular findings. Our results suggest that TT genotype of rs7574865 may be a susceptible factor for VKH syndrome in a Chinese Han population, and that GG genotype of this SNP may confer susceptibility in male BD patients.

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

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 Behçets 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 χ(2) 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.

Increased Expression of IL-22 Is Associated with Disease Activity in Behcet’s Disease

Objective Interleukin (IL)-22 has been reported to be involved in the development of autoimmune diseases. This study aimed to analyze the expression and potential role of IL-22 in the pathogenesis of Behcet’s disease (BD). Methods The levels of IL-22 in patient sera or supernatants of cultured peripheral blood mononuclear cells (PBMCs) and CD4+T cells were detected by enzyme-linked immunosorbent assay (ELISA). Flow cytometry was used to evaluate the frequency of IL-22–producing CD4+ T cells. IL-22 mRNA from erythema nodosum skin lesions was examined using real time quantitative RT-PCR. Results BD patients with active uveitis showed a significantly higher expression of IL-22 in the supernatants of stimulated PBMCs and CD4+T cells compared with BD patients without active uveitis and normal controls. An increased frequency of IL-22-producing CD4+T cells was also found in BD patients with active uveitis. IL-22 mRNA expression was elevated in erythema nodosum skin lesions. In BD patients, a high IL-22 level in the supernatant of stimulated PBMCs correlated with the presence of retinal vasculitis and erythema nodosum. Conclusions IL-22 was associated with disease activity in BD and correlated with the presence of small vessel inflammation, suggesting that it may be involved in its pathogenesis.

Upregulation of interleukin 21 and promotion of interleukin 17 production in chronic or recurrent Vogt-Koyanagi-Harada disease.

OBJECTIVES To analyze the expression and potential role of interleukin (IL) 21 in the pathogenesis of Vogt-Koyanagi-Harada (VKH) disease. METHODS Blood samples were obtained from patients with VKH disease and from healthy control subjects. Serum IL-21 level and IL-21 messenger RNA (mRNA) expression by peripheral blood mononuclear cells (PBMCs) were determined by enzyme-linked immunosorbent assay and by reverse transcriptase-polymerase chain reaction, respectively. Interleukin 17 and interferon γ levels in the supernatants of PBMCs and CD4(+) T cells cultured with anti-CD3 and anti-CD28 antibodies in the presence or absence of recombinant IL-21 were detected by enzyme-linked immunosorbent assay. RESULTS The results showed a significantly increased serum IL-21 level, as well as higher IL-21 mRNA expression by PBMCs, in patients having chronic or recurrent active VKH disease compared with patients having inactive VKH disease and with controls. In vitro experiments showed that recombinant IL-21 significantly increased IL-17 production by PBMCs and by CD4(+) T cells from patients and from controls. However, recombinant IL-21 did not affect interferon γ expression by PBMCs or by CD4(+) T cells. CONCLUSION Interleukin 21 may be involved in the pathogenesis of chronic or recurrent VKH disease, possibly by promoting IL-17 secretion. CLINICAL RELEVANCE Findings from the present study suggest that IL-21 may be a potential target in the development of therapy for VKH disease.

Association of TLR2 gene polymorphisms with ocular Behcet's disease in a Chinese Han population.

PURPOSE TLR2, TLR4, TLR8, and TLR9 have been reported to be associated with several autoimmune diseases. The current study aimed to explore whether singe nucleotide polymorphisms (SNPs) of these four genes were associated with ocular Behçets disease (BD), Vogt-Koyanagi-Harada (VKH) syndrome, acute anterior uveitis (AAU) with or without ankylosing spondylitis (AS), or pediatric uveitis in Han Chinese. METHODS Genotyping was performed by PCR-restriction fragment length polymorphism. The first stage study comprised 400 ocular BD patients, 400 VKH syndrome patients, 400 AAU ± AS patients, 400 pediatric uveitis patients and 600 healthy subjects. The second stage included 438 ocular BD patients and 1000 healthy subjects. Allele and genotype frequencies were compared between patients and controls using the χ(2) test. Real-time PCR was used to detect mRNA expression from PBMCs obtained from healthy controls. Levels of TNF-α, IL-6, IL-10, and IL-1beta in culture supernatants were measured by ELISA. RESULTS In the first stage study, only the frequencies of the rs2289318/TLR2 genotype A and C allele and rs3804099/TLR2 genotype CT were significantly higher in ocular BD patients (Pc = 0.048; Pc = 0.008; Pc = 0.005, respectively) compared with controls. The second stage and combined studies confirmed the association (Pc = 0.001; Pc = 6.89E-06, Pc = 2.426E-06, respectively). TLR2 mRNA expression in PBMCs was increased in healthy carriers of the CC genotype of rs2289318/TLR2 and TT genotype of rs3804099/TLR2 following stimulation with peptidoglycan (PGN; P = 0.028; P = 0.004, respectively). No effect of the various TLR2 rs2289318 and rs3804099 genotypes on the release of TNF-α, IL-6, IL-10, and IL-1beta could be detected. CONCLUSIONS This study provides evidence that the TLR2 gene is involved in the susceptibility to ocular BD.