| Uveitis is one of the common eye diseases leading to blindness accompanied by systemic autoimmune disease or inflammatory disease. Behcet’s disease (BD) and Vogt-Koyanagi-Harada (VKH) syndrome are two important uveitis entities in Asia. BD is considered as an autoinflammatory disease involving multiple organ systems and manifesting with recurrent oral ulcers, genital ulcers, nongranulomatous uveitis, and skin lesions, and is often associated with disorders such as arthritis, intestinal ulcers, and central nervous system lesions. VKH syndrome is recognized as an autoimmune disease accompanied by a bilateral granulomatous uveitis, but also involving other organs containing melanocyte target autoantigens. The complement system is crucial in innate immunity. The activation of the complement system occurs along three routes named alternative pathway, classical pathway and lectin pathway. The sequential activation of the three pathways collectively leads to complement C3 activation and C5 cleavage and finally to the membrane attack complex (MAC) to be generated on the cell surface. Many studies have confirmed that complement components play a critical role in the development of some autoimmune diseases including experimental autoimmune uveitis (EAU). Whether the copy number variations (CNVs) and SNPs of complement C3, C5 to C9 are associated with BD and VKH syndrome has not yet been reported and was therefore the subject of the study reported here. Through this study, a further knowledge concerning the genetic susceptibility for Behcet disease and VKH syndrome would provide a new targets and strategies for uveitis.Part I Association of complement genes copy number variation with Behcet’s disease and Vogt-Koyanagi-Harada syndromePurpose:The complement is involved in many immune-mediated diseases. This study was performed to investigate CNVs of complement C3, C5 to C9 in Behcet’s disease and Vogt-Koyanagi-Harada syndrome.Methods:A total of seven CNVs including C3, C5, C6, C7, C8A, C8B and C9 were analyzed in 382 patients with BD,383 patients with VKH syndrome and 573 healthy controls in the first stage. The other independent 682 patients with BD,676 patients with VKH syndrome and 1601 healthy individuals were examined in the second stage. The copy number and mRNA expression were examined by real-time PCR. To compare the differences between patients and controls, χ2 test (SPSS version 17.0) were applied to analyze the frequencies of CNVs. Cytokines production of IL-17, IFN-γ, IL-10, IL-8, TNF-α, IL-6, IL-1β and MCP-1 in PBMCs culture supernatants was measured by ELISA.Results:The copy number variation of C3, and C5 to C9 in patients with BD, patients with VKH and controls was calculated in three stages. When the statistical difference was significant at the first stage between patients and normal controls (P<0.05), we repeated the analysis in a second (replication) cohort to confirm the results. At the first stage, the frequency of exceeding 2 copies of C3 was increased in BD (Pc=5.5×10-3, OR=3.4) and VKH (Pc=0.018, OR=3.1) as compared with controls. We accept the similar result of C3 CNVs between patients and controls at the second stage. At the third stage, we combined the data of the two cohorts and confirmed that the frequency of >2 copies of C3 was significantly increased in both BD and VKH (Pc=5.3×10-9, OR=3.0; Pc=6.4×10-8, OR=2.8, respectively). The frequency of>2 copies of C5 was increased in BD compared with controls in both the first and second cohort. Taken together, the combined data identified that more than 2 copies of C5 was a risk factor for BD (Pc=1.1×10-8, OR=3.4). The results showed that mRNA expression of C3 and C5 in individuals having >2 copies was higher than in persons having less than 2 copies or 2 copies (P=0.015, P=0.018; Pc= 0.039, Pc= 0.0006, respectively). The production of IL-17 and IFN-γ was increased in individuals carrying more than 2 copies of the C3 gene as compared with those with less than 2 copies (Pc= 0.003, Pc= 0.002) or 2 copies (Pc= 0.01, Pc= 0.001). The IL-17 produced in high C5 copy number group was significantly increased as compared to the other two groups (<2 copies (P=0.0002) and 2 copies (P=0.002).Conclusion:Our study revealed that C3 CNVs is associated with BD and VKH syndrome. Whereas C5 CNVs is only associated with BD.Part Ⅱ Association of complement genes polymorphisms with Behcet’s disease and Vogt-Koyanagi-Harada syndromePurpose:This study was conducted to investigate the polymorphisms of complement C3 and C5 in Behcet’s disease and Vogt-Koyanagi-Harada syndrome.Methods:A total of 10 SNPs of C3 and C5 were genotyped in 376 Behcet’s disease patients,373 VKH syndrome patients and 562 healthy controls at the first stage in this study. The other independent 572 patients with Behcet’s disease and 807 healthy individuals were examined at the second stage. Genotyping was determined by PCR-RFLP or TaqMan assay. The mRNA expression was examined by real-time PCR.χ2 test (SPSS version 17.0) were applied to analyze the frequencies of alleles and genotypes between patients and controls. Cytokines production of IL-17, IFN-γ, IL-10, IL-8, TNF-α, IL-6, IL-1β and MCP-1 in PBMCs culture supernatants was measured by ELISA.Results:The polymorphisms of C3 and C5 in patients with BD, patients with VKH and controls were calculated in three stages. When the statistical difference was significant at the first stage between patients and normal controls (P<0.05), we repeated the analysis in a second (replication) cohort to confirm the results. At the first stage, the frequency of C3 rs408290 GG genotype was increased in BD as compared with controls (Pc=8.6×10-3, OR= 2.07,95% CI=1.38-3.10). The frequency of GG genotype and CG genotype of C3 rs408290 was increased in BD as compared with controls at the second stage (Pc=3.7×10-4, OR= 2.92,95% CI=1.76-4.83; Pc =1.5×10-3, OR= 0.61,95% CI=0.48-0.78. In the third analysis stage, we combined the data of the two cohorts and confirmed that the frequency of GG genotype and CG genotype of C3 rs408290 was significantly increased in BD (Pc=1.5×10-7, Pc= 4.9×10-4, respectively). Similarly to C3 rs408290 GG genotype, the frequency of GG genotype of C5 rs2269067 was increased in BD compared with controls in both the first and second cohort. Taken together, the combined data identified that the frequency of C5 rs2269067 GG genotype was a risk factor for BD (Pc=1.3×10-8, OR =1.73,95% CI=1.45-2.06). The results showed that mRNA expression of GG genotype individuals of C3 rs408290 and C5 rs2269067 was higher than in persons with CG genotype or CC genotype (Pc= 8.5×10-5, Pc= 2.3×10-5; Pc= 0.015, Pc= 0.001, respectively). The production of IL-17 and IFN-y was increased in GG genotype individuals of the C3 rs408290 as compared with those with CG genotype (Pc= 8.5×10-5, Pc= 2.3×10-5) or CC genotype (Pc= 0.015, Pc= 0.001). The production of IL-17 in C5 rs2269067 GG cases was higher than in CG genotypes and CC genotypes (P=0.021, P=0.006, respectively).Conclusion:Our study revealed that the polymorphisms of C3 and C5 are associated with BD. |
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