Gut microbial signatures and their functions in Behcet's uveitis and Vogt-Koyanagi-Harada disease.

BACKGROUND: A number of public metagenomic studies reveal an association between the gut microbiome and various immune-mediated diseases including Behcet's uveitis (BU) and Vogt-Koyanagi-Harada disease (VKH). Integrated-analysis and subsequent validation of these results could be a potentially powerful way to understand the microbial signatures and their functions in these two uveitis entities. METHODS: We integrated the sequencing data of our previous metagenomic studies on two major uveitis entities, BU and VKH as well as four other publicly available immune-mediated diseases datasets, including Ankylosing Spondylitis (AS), Rheumatoid Arthritis (RA), Crohn's disease (CD) and Ulcerative Colitis (UC). Alpha-diversity and beta-diversity analysis were used to compare the gut microbiome signatures between both uveitis entities and other immune-mediated diseases and healthy controls. Amino acid homology between microbial proteins and a uveitogenic peptide of the interphotoreceptor retinoid-binding protein (IRBP)161-180 was investigated using a similarity search in the NCBI protein BLAST program (BLASTP). Enzyme-linked Immunosorbent Assay (ELISA) was performed to evaluate the cross-reactive responses of experimental autoimmune uveitis (EAU)-derived lymphocytes and BU patients-derived peripheral blood mononuclear cells (PBMCs) against homologous peptides. The area under the curve (AUC) analysis was used to test the sensitivity and specificity of gut microbial biomarkers. RESULTS: Depleted Dorea, Blautia, Coprococcus, Erysipelotrichaceae and Lachnospiraceae as well as enriched Bilophila and Stenotrophomonas were identified in BU patients. An enriched Alistipes along with a lower level of Dorea were observed in VKH patients. A peptide antigen (SteTDR) encoded by BU specifically enriched Stenotrophomonas was identified to share homology with IRBP161-180. In vitro experiments showed that lymphocytes from EAU or PBMCs from BU patients reacted to this peptide antigen as shown by the production of IFN-γ and IL-17. Addition of the SteTDR peptide to the classical IRBP immunization protocol exacerbated EAU severity. Gut microbial marker profiles consisted of 24 species and 32 species respectively differentiated BU and VKH from each other as well as from the other four immune-mediated diseases and healthy controls. Protein annotation identified 148 and 119 specific microbial proteins associated with BU and VKH, respectively. For metabolic function analysis, 108 and 178 metabolic pathways were shown to be associated with BU and VKH, respectively. CONCLUSIONS: Our study revealed specific gut microbial signatures and their potentially functional roles in BU and VKH pathogenesis that differ significantly from other immune-mediated diseases as well as healthy controls.

Surveillance of Liver Function in Uveitis with or without Chronic HBV Infection.

INTRODUCTION: Immunosuppressive therapy for uveitis may cause liver damage. METHODS: To investigate incidence of liver damage during uveitis treatment, we compared serological Hepatitis B core antibody (HBcAb) status with risk of liver dysfunction in all participants (n = 992), in anterior uveitis (AU) (n = 489), and combined of intermediate, posterior, or panuveitis (IPPU) patients (n = 503). The primary endpoint was incidence of elevated serum alanine aminotransferase level above 2-fold upper limits of normal within 6 months. RESULTS: The incidence rate of primary endpoint for HBcAb-negative and HBcAb-positive patients was 65 and 212 per 1,000 person years, respectively. The absolute rate difference was 147 (95% confidence interval [CI], 80-213) per 1,000 person years. HBcAb positivity was associated with a higher risk for primary endpoint in all participants (adjusted hazard ratio [aHR], 3.53; 95% CI, 1.79-6.99; p value = 2.8 × 10-4) and in IPPU (aHR, 3.80; 95% CI, 1.61-9.01; p value = 0.002). No significant association with primary endpoint was observed for HBcAb positivity in AU (aHR, 3.21; 95% CI, 0.94-10.95; p value = 0.063). AU was mainly treated with topical eye drops (74.0%), whereas IPPU cases received systemic therapy including prednisone (94.0%), cyclosporine (80.9%), or other additionally combined immunomodulatory agents (14.9%). CONCLUSION: Noninfectious uveitis cases with HBcAb positivity have an increased risk of liver damage. This association was predominantly driven by IPPU but was not significant in AU, suggesting that the association is mediated by systemic therapy.

Genetically predicted fasting blood glucose level plays a causal role in intraocular pressure: A Mendelian randomisation study.

BACKGROUND: This study aimed to examine possible causal associations between various components of metabolic syndrome and glaucoma-related phenotypes. METHODS: A two-sample Mendelian randomisation study was conducted with the models of inverse-variance weighted (IVW), maximum likelihood, weighted median and MR-Egger regression. We accessed data from publicly available genome-wide association studies for individual parameters of metabolic syndrome as the exposures and the data for glaucoma and its endophenotypes as the outcomes. RESULTS: Among 11 exposures and 6 outcomes examined in this Mendelian randomisation study, only fasting blood glucose level showed evidence of a causal influence on intraocular pressure. Results analysed by the IVW model suggested that each one-SD increase in genetically predicted fasting blood glucose level was significantly associated with 0.80 SD elevation in intraocular pressure (ß: 0.80, 95% CI: 0.38-1.22, p: 2.12e-4). The maximum likelihood model (ß: 0.82, 95% CI: 0.39-1.25, p: 1.616e-4) also supported a significant causal effect. The weighted median model (ß: 0.78, 95% CI: 0.17-1.39, p: 0.012) showed a nominally significant effect whereas the MR-Egger model (ß: 0.63, 95% CI: -0.32-1.59, p: 0.212) showed a consistent direction of effect but was not statistically significant. Several sensitivity analyses indicated no evidence of directional horizontal pleiotropy that would bias the result. CONCLUSIONS: This Mendelian randomisation study provides evidence for a causal role for genetically determined higher fasting blood glucose level in the development of increased intraocular pressure. This finding could be considered in the monitoring and control of intraocular pressure and may be instrumental in prevention strategies for ocular hypertension.

The haplotypes of various TNF related genes associated with scleritis in Chinese Han.

BACKGROUND: Several studies have stated that TNF-α participates in the pathogenesis of scleritis, but also in several systemic autoimmune diseases and vasculitis, of which some are associated with scleritis. Earlier GWAS and SNP studies have confirmed that multiple SNPs of TNF related genes are associated with many immune-mediated disorders. The purpose of this study was to examine the association of TNF related gene polymorphisms with scleritis in Chinese Han. A case-control study was carried out in 556 non-infectious scleritis cases and 742 normal controls. A total of 28 single-nucleotide polymorphisms (SNPs) were genotyped by the iPLEXGold genotyping assay. RESULTS: No significant correlations were seen between the individual SNPs in the TNF related genes and scleritis. Haplotype analysis showed a significantly decreased frequency of a TNFAIP3 TGT haplotype (order of SNPs: rs9494885, rs3799491, rs2230926) (Pc = 0.021, OR = 0.717, 95% CI = 0.563-0.913) and a significantly increased frequency of a TNFSF4 GT haplotype (order of SNPs: rs3850641, rs704840) (Pc = 0.004, OR = 1.691, 95% CI = 1.205-2.372) and TNFSF15 CCC haplotype (order of SNPs: rs6478106, rs3810936, rs7865494) (Pc = 0.012, OR = 1.662, 95% CI = 1.168-2.363) in patients with scleritis as compared with healthy volunteers. CONCLUSIONS: This study reveals that a TGT haplotype in TNFAIP3 may be a protective factor for the development of scleritis and that a GT haplotype in TNFSF4 and a CCC haplotype in TNFSF15 may be risk factors for scleritis in Chinese Han.

Linoleic acid inhibits in vitro function of human and murine dendritic cells, CD4+T cells and retinal pigment epithelial cells.

PURPOSE: Increased linoleic acid (LA) was observed in acute anterior uveitis (AAU) patient feces in our previous study. To investigate the immunoregulatory effect of LA, we studied the effect of LA on human and murine dendritic cells (DCs), CD4+T cells, and retinal pigment epithelial (RPE) cells in vitro. METHODS: The level of LA in feces from AAU patients and healthy individuals was measured by gas chromatography coupled with a mass spectrometer (GC-MS). The immunoregulatory effect of LA on human and murine DCs, CD4+ T cells, and RPE cells was evaluated by enzyme linked immunosorbent assay (ELISA) and flow cytometry (FCM). The effect of LA on DCs was evaluated by Tandem mass tag (TMT)-based proteomics analysis. RESULTS: Increased LA was observed in feces from AAU patients (1018.35 ± 900.01 mg/kg) as compared with healthy individuals (472.55 ± 365.49 mg/kg, p = 0.0136). LA attenuated the antigen-presenting function of human and murine DCs by decreasing the expression of CD40, the secretion of IL-6 and IL-12p70, and the ability to shift naïve T cells towards T helper type 1 (Th1) and Th17 cells. LA also inhibited the secretion of MCP-1 and IL-8 from RPE cells. Proteomics analysis showed differential expression of 28 proteins, including squalene epoxidase (SQLE), farnesyl-diphosphate farnesyltransferase 1 (FDFT1), and cytochrome P450 family 51 subfamily A member 1 (CYP51A1), in LA-treated DCs compared with controls. LA also accelerated the apoptosis of DCs from healthy individuals. CONCLUSION: LA inhibited the function of human and murine DCs, CD4+T cells, and RPE cells, regulated the expression of proteins, and promoted the apoptosis of human DCs. These results collectively suggest that LA might decrease the function of immune cells in vitro, and further studies are needed to investigate its role in the pathogenesis of AAU.

RETINAL VASCULITIS, A COMMON MANIFESTATION OF IDIOPATHIC PEDIATRIC UVEITIS?

PURPOSE: Pediatric idiopathic uveitis typically shows anterior segment involvement. Whether retinal vasculitis is an important manifestation of this disease remains unknown and was therefore the subject of this study. METHODS: This study was performed involving patients with pediatric idiopathic uveitis. Fundus fluorescein angiography was used to assess the presence of retinal vasculitis. RESULTS: A total of 1,867 patients with pediatric uveitis were seen between December 2008 and January 2018, of whom 1,364 had undergone fundus fluorescein angiography examination. Idiopathic uveitis was the most common entity, accounting for 81.2%. Among these patients with idiopathic uveitis, 79.6% had retinal vasculitis in at least one eye. After 1-year treatment with oral prednisone mostly combined with cyclosporine, 76.3% patients in the retinal vasculitis group achieved control of their ocular inflammation, which was significantly lower as compared with 85.1% in those without (P = 0.008). Retinal vasculitis was an independent predictor for a lower probability of inflammation control after 1-year treatment. Visual function (best-corrected visual acuity > 20/25 in the better seeing eye) was worse in the retinal vasculitis group than in the control group after 5 years. CONCLUSION: Almost 80% of patients with pediatric idiopathic uveitis show manifestations of retinal vasculitis, which is associated with a lower probability of inflammation control resulting in a worse visual prognosis.

Identification of Ribosomal Protein S4, Y-Linked 1 as a cyclosporin A plus corticosteroid resistance gene.

Combination of corticosteroids (CS) with cyclosporin A (CsA) is widely used in the treatment of autoimmune diseases, autoinflammatory diseases and transplantation rejection. However, some patients fail to respond or develop resistance to the combination regimen. In Vogt-Koyanagi-Harada (VKH) disease model, we performed RNA sequencing (RNA-seq) based transcriptomics, isobaric tags for relative and absolute quantification (iTRAQ) based proteomics and assays in vitro to screen and validate potential resistant molecules. We found that a total of 1697 differentially expressed genes (DEGs) and 21 differentially expressed proteins (DEPs) in CD4+ T cells between CsA & CS-resistant and -sensitive VKH patients. Ribosomal Protein S4, Y-Linked 1 (RPS4Y1) was verified to regulate the resistance of CD4+ T cells from male VKH patients to CsA & CS. Importantly, we showed that chlorambucil (CLB) could reverse the resistance by RPS4Y1 suppression. Taken together, we identify RPS4Y1 as an important CsA & CS resistance gene in VKH disease. Researchers should consider validating the resistant effect of RPS4Y1 in other autoimmune diseases or organ transplantation.

Uveitis genetics.

Uveitis is usually considered as a vision-threatening multiple system intraocular inflammatory disease. Among uveitis, Vogt-Koyanagi-Harada (VKH) disease and Behcet's disease (BD) are common non-infectious uveitis entities. Although the exact pathogenesis of uveitis is not yet clear, it is acknowledged that the combination of a certain genetic or epigenetic factors with an imbalance in the regulation of the immune response leads to the development of this disease. HLA genes show a strong association with both VKH disease (HLA-DR4, DRB1/DQA1) and BD (HLA-B51) in multiple ethnic populations. Candidate association studies based on a pathogenesis hypothesis laid the foundation for genetic research of uveitis and identified a large number of genes associated with VKH disease or BD including SUMO4, MCP-1, and CTLA4. Genome-wide association study (GWAS) provided a powerful tool for genome-wide level analysis to explore the genetic predisposition for uveitis and revealed several genes to be associated with uveitis including IL23R/C1orf141, STAT4 and ADO/ZNF365/EGR2. Another variant type, the so called copy number variants (CNV), in IL17F, IL23A and C4A also showed an association with uveitis. Additionally, epigenetic factors such as DNA methylation and ncRNAs play important roles in the development of uveitis. The application of new technologies such as whole exome sequencing and whole genome sequencing and other epigenetic modifications such as N6-methyl-adenosine (m6A) modification of mRNAs will be helpful to discover new pathogenic risk genes for uveitis. The understanding of the genetic and epigenetic mechanisms in uveitis may provide a foundation to find novel targets and to develop new strategies in the treatment of uveitis in the near future.

Weak association of a TNFRSF1A polymorphism with Behcet's disease in Chinese Han.

The purpose of this study was to investigate whether single nucleotide polymorphisms (SNPs) of the tumor necrosis factor receptor superfamily (TNFRSF) and their ligand (TNFSF) gene are associated with susceptibility to Behcet's Disease (BD) in Chinese Han. A two-phase case-control study was performed in 1055 BD patients and 1829 healthy controls. A total of 27 SNPs was tested using MassARRAY iPLEX® technology. Data were analyzed using a Chi-square (χ2) test and Fisher's exact calibration test. The Bonferroni correction was applied for multiple testing. A statistically significant higher frequency of the A allele and a lower frequency of the G allele of rs1800692 was found in BD (Pc = 0.013, OR = 1.233, 95% CI = 1.103-1.379: Pc = 0.013, OR = 0.811, 95% CI = 0.725-0.907, respectively). Our findings indicate that TNFRSF1A might confer genetic susceptibility to BD in a Chinese Han population.

Plasma metabolomics study of Vogt-Koyanagi-Harada disease identifies potential diagnostic biomarkers.

Vogt-Koyanagi-Harada (VKH) disease is a common type of uveitis in China, but the diagnosis criteria of VKH disease is controversial. The aim of this study was to investigate potential diagnostic plasma biomarkers for VKH disease. A case-control study including 55 VKH patients (28 active patients and 27 inactive VKH patients) and 30 healthy controls in a tertiary referral center was performed. The metabolic phenotype of VKH patients showed a significant difference compared to healthy controls. Fifteen differentially expressed metabolites (DEMs) were identified between active VKH patients and healthy controls and nine DEMs were found between inactive VKH patients and healthy controls after controlling variable importance in the projection (VIP) value > 1 and false discovery rate (FDR) < 0.05. D-mannose, stearic acid and L-lysine were shown to be potential diagnostic biomarkers which can discriminate active VKH patients from healthy controls with a diagnostic performance with AUC = 0.965, 0.936 and 0.910 respectively in independent diagnosis and an AUC = 0.999 when combined. Sarcosine was recognized as an independent potential biomarker which could distinguish inactive VKH patients from healthy controls. This study reveals a significant difference of plasma metabolic phenotype and identifies diagnostic biomarkers for VKH disease. Changes in the metabolic profile may provide clues towards the pathophysiology of VKH disease.

Association of apoptosis genes in PDCD1 but not PDCD1LG2, FAS, and FASLG with pediatric idiopathic uveitis in Han Chinese.

BACKGROUND: Previous studies have shown that aberrant T lymphocyte apoptosis is involved in the pathogenesis of uveitis. Genetic variants of apoptotic pathway-related factors (including PDCD1, PDCD1LG2, FAS, and FASLG) may affect apoptosis and in turn predict susceptibility to autoimmune disease. This has not yet been studied in pediatric idiopathic uveitis (PIU) and juvenile idiopathic arthritis (JIA)-associated uveitis and was therefore the subject of the study presented here. METHODS: Fourteen single-nucleotide polymorphisms (SNPs) of several apoptosis-related pathway genes were analyzed in 1238 PIU patients, 128 JIA-associated uveitis patients and 1114 healthy controls using the iPLEX Gold Assay and MassARRAY platform. RESULTS: A lower frequency of the PDCD1/rs6710479 CC genotype in PIU patients was found when compared to controls (Pc = 3.42 × 10-3). A higher frequency of the PDCD1/rs7421861 A allele (Pc = 4.85 × 10-3) was observed in PIU patients as compared with controls. Stratification analysis showed a positive association of band keratopathy with the PDCD1/rs7565639 CT genotype (Pc = 1.05 × 10-2) and a negative association of this parameter with the PDCD1/rs7565639 C allele (Pc = 3.76 × 10-2). CONCLUSIONS: This study revealed that rs6710479 and rs7421861 in the PDCD1 gene confer susceptibility to PIU in Han Chinese. A stratified analysis showed that PDCD1/rs7565639 is associated with band keratopathy in PIU patients.

A novel mutation of PANK4 causes autosomal dominant congenital posterior cataract.

Though many mutations have been identified to be associated with the occurrence of congenital cataract, pathogenic loci in some affected families are still unknown. Clinical data and genomic DNA were collected from a four-generation Chinese family. Candidate mutations were independently verified for cosegregation in the whole pedigree. Linkage analysis showed that the disease-causing mutation was located between 1p36.21 and 1p36.33. Analysis of the whole-exome sequencing data combined with linkage analysis identified a novel pathogenic variant (g.2451906C>T) at intron 4 of Pantothenate kinase 4 (PANK4 protein, PANK4 gene) in 1p36.32|606162. This variant showed complete cosegregation with the phenotype in the pedigree. The mutation was not detected in 106 normal controls nor in 40 sporadic congenital cataract patients. The mutation was demonstrated to significantly reduce the expression of the PANK4 protein level in the blood of cataract patients than that in normal individuals by ELISA. Pank4-/- mice showed a cataract phenotype with increased numbers of apoptotic lens epithelial cells, fiber cell aggregation, and significant mRNA variation of crystallin family members. Thus, the association of a new entity of an autosomal dominant cataract with mutations in PANK4, which influences cell proliferation, apoptosis of lens epithelial cells, crystallin abnormalities, and fiber cell derangement, subsequently induces cataract.

UVEOGENE: An SNP database for investigations on genetic factors associated with uveitis and their relationship with other systemic autoimmune diseases.

Uveitis is an intraocular inflammatory disease which can lead to serious visual impairment. Genetic factors have been shown to be involved in its development. However, few databases have focused on the information of associations between single nucleotide polymorphisms (SNPs) and uveitis. To discover the exact genetic background of uveitis, we developed an SNP database specific for uveitis, "UVEOGENE," which includes 370 genes and 918 SNPs covering 14 uveitis entities and 40 populations from 286 PubMed English-language papers. Stratification analyses by gender, HLA status, and different clinical features were also extracted from the publications. As a result, 371 associations were judged as "statistically significant." These associations were also shared with Global Variome shared Leiden Open Variation Database (LOVD) (https://databases.lovd.nl/shared/genes). Based on these associations, we investigated the genetic relationship among three widely studied uveitis entities including Behcet's disease (BD), Vogt-Koyanagi-Harada (VKH) disease, and acute anterior uveitis (AAU). Furthermore, "UVEOGENE" can be used as a reliable and informative resource to identify similarities as well as differences in the genetic susceptibility among uveitis and other autoimmune diseases. UVEOGENE is freely accessible at http://www.uvogene.com.

Epigenome-wide association study identifies Behçet's disease-associated methylation loci in Han Chinese.

OBJECTIVE: The aetiology of Behçet's disease (BD), known as a systemic vasculitis, is not completely understood. Increasing evidence suggests that aberrant DNA methylation may contribute to the pathogenesis of BD. The aim of this epigenome-wide association study was to identify BD-associated methylation loci in Han Chinese. METHODS: Genome-wide DNA methylation profiles were compared between 60 BD patients and 60 healthy controls using the Infinium Human Methylation 450 K Beadchip. BD-associated methylation loci were validated in 100 BD patients and 100 healthy controls by pyrosequencing. Gene expression and cytokine production was quantified by real-time PCR and ELISA. RESULTS: A total of 4332 differentially methylated CpG sites were associated with BD. Five differentially methylated CpG sites (cg03546163, cg25114611, cg20228731, cg23261343 and cg14290576) revealed a significant hypomethylation status across four different genes (FKBP5, FLJ43663, RUNX2 and NFIL3) and were validated by pyrosequencing. Validation results showed that the most significant locus was located in the 5'UTR of FKBP5 (cg03546163, P = 3.81E-13). Four CpG sites with an aberrant methylation status, including cg03546163, cg25114611, cg23261343 and cg14290576, may serve as a diagnostic marker for BD (area under the receiver operating curve curve = 83.95%, 95% CI 78.20, 89.70%). A significantly inverse correlation was found between the degree of methylation at cg03546163 as well as cg25114611 and FKBP5 mRNA expression. Treatment with a demethylation agent, 5-Aza-2'-deoxycytidine resulted in an increase of FKBP5 mRNA expression and a stimulated IL-1ß production. CONCLUSION: Our findings suggest that aberrant DNA methylation, independently of previously known genetic variants, plays a vital role in the pathogenesis of BD. TRIAL REGISTRATION: Chinese Clinical Trial Registry, chictr.org.cn, ChiCTR-CCC-12002184.

Comparison of Clinical Features and Visual Outcome between Sympathetic Ophthalmia and Vogt-Koyanagi-Harada Disease in Chinese Patients.

PURPOSE: To characterize the clinical features of sympathetic ophthalmia (SO) and compare SO and Vogt-Koyanagi-Harada (VKH) disease in Chinese patients. DESIGN: Retrospective case series. PARTICIPANTS: A total of 131 consecutive SO and 500 VKH disease patients randomly selected from among those referred to our uveitis center from April 2008 through June 2018. METHODS: History, extraocular and ocular findings, best-corrected visual acuity (BCVA), auxiliary examination findings, complications, and therapeutic effects were analyzed retrospectively in SO and VKH disease patients. MAIN OUTCOME MEASURES: Visual outcome, extraocular and ocular findings, and therapeutic effects. RESULTS: Sympathetic ophthalmia manifested as posterior uveitis (68.8%) within 2 weeks and equal involvement of anterior and posterior segment (44.4%), respectively, was observed between 2 weeks and 2 months after disease onset. Two months after disease onset, SO patients showed sunset glow fundus (51.2%) and granulomatous anterior uveitis (27.3%). Vogt-Koyanagi-Harada disease patients mainly showed posterior uveitis (100%), anterior segment involvement (92.4%) associated with posterior uveitis (84.9%), and granulomatous anterior uveitis (97.4%) accompanying sunset glow fundus (91.5%) in the 3 periods mentioned above. The frequencies of extraocular manifestations were lower in SO patients (24.4%) as compared with VKH disease patients (84.8%; P < 0.001). Best-corrected visual acuity of SO patients improved from 0.68±0.86 logarithm of the minimum angle of resolution (logMAR) to 0.47±0.78 logMAR (P = 0.01), and BCVA of VKH disease patients improved from 0.67±0.79 logMAR to 0.24±0.53 logMAR (P < 0.001) at 12 months of follow-up. A worse BCVA was noted in SO patients compared with VKH disease patients after treatment (P = 0.003). Kaplan-Meier survival analysis showed that the risk of loss of useful vision in SO patients was significantly higher than that of VKH disease patients (P < 0.001). CONCLUSIONS: Chinese SO and VKH disease patients have a different evolutionary process. The frequency of extraocular manifestations in SO patients is much lower as compared with VKH disease patients. Visual outcome is worse in SO as compared with VKH disease.

Association of TLR2 Gene Polymorphisms with Presumed Viral-Induced Anterior Uveitis in male Han Chinese.

The purpose of this study was to investigate whether single nucleotide polymorphisms (SNPs) of TLR2, TLR3, TLR4 and TLR9 genes are associated with susceptibility to presumed viral-induced anterior uveitis (PVIAU) and Posner-Schlossman syndrome (PSS). A case-control study was performed in 205 PVIAU patients and 1007 healthy controls. A total of 15 SNPs were genotyped by MassARRAY platform and iPLEX Gold Assay. Data were analyzed using a Chi-square (χ2) test and Fisher's exact calibration test. Two hundred and three PSS patients served as an extra control to investigate whether there were similar genetic factors between PVIAU and PSS in the context of these tested SNPs in TLR genes. The results showed that the frequency of TLR2/rs7656411 GG genotype and G allele were significantly higher in PVIAU patients as compared with healthy controls (P = 1.10 × 10-4, corrected P value [Pc] = 4.93 × 10-3, odds ratio [OR] = 1.848; P = 3.57 × 10-4, Pc = 1.07 × 10-2, OR = 1.478, respectively). Gender stratification analysis showed a significantly increased frequency of the G allele in male patients (P = 7.46 × 10-5, Pc = 2.24 × 10-3, OR = 1.894). A weak correlation was found between two SNPs (rs3804100 and rs5743705) of the TLR2 gene with PSS. However, after Bonferroni correction, statistical significance was lost. This study shows that the polymorphisms of TLR2/rs7656411 are positively associated with PVIAU in male Chinese patients. PVIAU and PSS have a different genetic background in the context of the tested SNPs.

Dynamic DNA Methylation Changes of Tbx21 and Rorc during Experimental Autoimmune Uveitis in Mice.

The key transcription factors of T helper cell subpopulations, including T-bet, GATA3, RORγt, and Foxp3 are involved in various autoimmune diseases. Whether methylation of these master transcription factors is associated with the development of experimental autoimmune uveitis (EAU) and the possible epigenetic regulatory mechanisms involved has however not yet been addressed. In our study, significant methylation changes in both Tbx21 and Rorc were observed in one CpG site in the retinas of EAU mice. Two CpG sites of Tbx21 and one CpG site of Rorc showed significant dynamic methylation changes in the RPE-choroid complex during EAU. The mRNA expressions of Tbx21 and Rorc in both the retinas and RPE-choroid complexes correlated with the methylation changes at the various time points during EAU development. The methylation changes were associated with the production of the Th1/Th17 cells' signature cytokines, IFN-γ and IL-17. Dynamic changes in mRNA expression of DNA methyltransferases (DNMT1) were also noted, which may be related to the observed methylation changes of these genes. The present study provides evidence that DNA methylation of Tbx21 and Rorc may be associated with the development of EAU. DNMT1 activation may have an important effect on regulating DNA methylation dynamics.

Identification of susceptibility SNPs in IL10 and IL23R-IL12RB2 for Behçet's disease in Han Chinese.

BACKGROUND: Although previous genome-wide association studies in various cohorts have identified several susceptibility loci underlying Behçet's disease (BD), this has not yet led to a breakthrough in the management of BD. OBJECTIVE: This study aimed to further investigate the association of 26 candidate single nucleotide polymorphisms with previous genome-wide association studies-identified nearly positive P values (5.0 × 10-8 < P < 1.0 × 10-5) in Chinese Han patients with BD. METHODS: A case-control association study was performed in 1206 patients with BD and 2475 healthy controls. Genotyping was performed using iPLEX Gold genotyping assay. Gene expression and cytokine production was quantified by real-time PCR and ELISA. RESULTS: The results showed that significantly higher frequencies of the IL23R-IL12RB2/rs924080 TT genotype (P = 2.03 × 10-8; odds ratio [OR] = 1.50), IL23R-IL12RB2/rs12141431 CC genotype (P = 2.18 × 10-8; OR = 1.53), IL10/rs1800871 TT genotype (P = 5.88 × 10-8; OR = 1.47), and IL10/rs3024490 TT genotype (P = 2.80 × 10-5; OR = 1.34) were found in BD. Functional experiments showed an increased IL23R expression and IL-17 production in rs12141431/CC genotype carriers compared with GG genotype carriers. A decreased IL10 expression and IL-10 production was observed in rs3024490/TT genotype carriers as compared with GG genotype carriers. CONCLUSIONS: Our findings not only confirmed the association of IL10/rs1800871 and IL23R-IL12RB2/rs924080 with BD but also identified 2 susceptibility single nucleotide polymorphisms in IL10 and IL23R-IL12RB2 (rs3024490 and rs12141431) with BD in Han Chinese.

Association of a NOS3 gene polymorphism with Behçet's disease but not with Vogt-Koyanagi-Harada syndrome in Han Chinese.

PURPOSE: Previous studies have identified that nitric oxide synthase (NOS) genes are associated with several immune-mediated diseases. This study aimed to investigate whether NOS2 and NOS3 gene polymorphisms are associated with Behçet's disease (BD) and Vogt-Koyanagi-Harada (VKH) syndrome in a Han Chinese population. METHODS: An association analysis of NOS2/rs4795067, NOS3/rs1799983 and NOS3/rs1800779 was performed in 733 patients with BD, 800 patients with VKH syndrome, and 1,359 controls using PCR restriction fragment length polymorphism (PCR-RFLP) assay. Statistical analysis was performed with the chi-square test followed by the Bonferroni correction. RESULTS: The result showed a decreased frequency of the NOS3/rs1799983 GG genotype and an increased frequency of NOS3/rs1799983 GT genotype in the patients with BD (Bonferroni correction test [Pc]=0.02, odds ratio [OR]=0.74; Pc=2.1×10(-3), OR=1.57, respectively). No significant association was found between rs1799983 and VKH syndrome. NOS2/ rs4795067 and NOS3/rs1800779 were not associated with either BD or VKH syndrome. CONCLUSIONS: Our findings suggest that a NOS3/rs1799983polymorphism is associated with susceptibility to BD in Han Chinese.

FAS Gene Copy Numbers are Associated with Susceptibility to Behçet Disease and VKH Syndrome in Han Chinese.

Previous studies have identified that disturbed apoptosis was involved in the pathogenesis of Behçet disease (BD) and Vogt-Koyanagi-Harada (VKH) syndrome. This study aims to investigate whether copy number variations of apoptosis-related genes, including FAS, CASPASE8, CASPASE3, and BCL2, are associated with BD and VKH syndrome in Han Chinese. A two-stage association study was performed in 1,014 BD patients, 1,051 VKH syndrome patients, and 2,076 healthy controls. TaqMan(®) Copy Number Assays and real-time PCR were performed. The first-stage study showed that increased frequency of high FAS copy number (>2) was found in BD (P = 1.05 × 10(-3) ) and VKH syndrome (P = 2.56 × 10(-3) ). Replication and combined study confirmed the association of high copy number (>2) of FAS with BD (P = 3.35 × 10(-8) ) and VKH syndrome (P = 9.77 × 10(-8) ). A significant upregulated mRNA expression of FAS was observed in anti-CD3/CD28 antibodies-stimulated CD4(+) T cells from individuals carrying a high gene copy number (>2) as compared to normal diploid 2 copy number carriers (P = 0.004). Moreover, the mRNA expression of FAS both in active patients with BD and VKH syndrome was significantly higher than that in controls (P = 0.001 and P = 0.007, respectively). Our findings suggest that a high copy number of FAS gene confers risk for BD and VKH syndrome.