Genetic Variations Of IL-12B, IL-12RΒ1, IL-12RΒ2in Behcet’s Diseaseand VKH Syndrome

Behcet’s disease (BD) is considered to be a long-term, complexinflammatory disorder which is characterized by vasculitis involvingmultiple organs. The clinical traits for BD comprise relapsed ulcerations inthe oral cavity and genitals, erythema nodosum, ulcer of the colon andrecurrent iritis. Previous reports suggest that T cells play an important rolein the pathogenesis of BD and an increased number of T cells is relatedwith disease activity. It has been shown that both Th1and Th17cells arecrucial to the development of BD.Vogt–Koyanagi–Harada (VKH) syndrome is a multisystem diseasewhich can often results in blindness. It is featured by a bilateralgranulomatous panuveitis and concomitant extraocular symptoms,including vitiligo, alopecia, poliosis, impaired auditory sense and centralnervous system lesions. An autoimmune reaction against melanocyteswhich may be triggered by infection or other factors, is thought to beinvolved in the pathogenesis of VKH disease. Earlier we showed that CD4T cells and several inflammatory cytokines play an important role in the progress of the disorder. Although the definite pathogenesis of the twodiseases has not been determined, recent studies have shown an importantrole for an immunogenetic predisposition to both diseases. Various studieshave demonstrated that several genes such as human leukocyte antigen(HLA)-B51, IL-23R, DHCR7, and TLR-2are associated with BD.Furthermore, HLA-DR4, IL-17, STAT4have been identified aspredisposing factors for VKH syndrome.Interleukins are types of cytokine signaling molecules in the immunesystem and play important roles in regulating the activities of lymphocytesthat are responsible for immunity. Interleukin-12B (IL-12B) encodes thekey subunit IL-12p40which is the common component of IL-12and IL-23.The bioactive subunit is generated mainly by activated macrophages anddendritic cells in response to pathogenic or infammatory factors. IL-12p40deficiency leads to decreased migration of dendritic cells and diminishedability to activate naive T cells. It also has considerable roles in thefunction of the Th1and Th17cells, the two significant kinds of T-helpersubsets involved in many important immunological processes. Morahan etal have revealed that differences in the degree of IL-12p40production mayhave significant impact on T cell reactivity which plays an essential role inimmune-related diseases. Recent studies indicated that a genetic aberrationof the IL-12B gene is associated with several autoimmune diseases such asankylosing spondylitis (AS) and psoriasis. Interleukin-12-receptor-β1 (IL-12Rβ1) is a receptor chain which is shared by IL-12and IL-23.IL-12Rβ1is critical for the function and development of memory CD4Tcells. The IL-12Rβ1-dependent pathway is essential for the differentiationand proliferation of human Th17cell subsets. IL-12Rβ1knockout micehave been shown to be resistant to Experimental AutoimmuneEncephalomyelitisn (EAE). Mutations in the IL-12Rβ1gene have beenshown to be associated with immune disorders. IL-12Rβ2is the othersubunit of the IL-12receptors. IL-12Rβ2is essential in Th1developmentand is also associated with IL-17expression. Furthermore, IL-12Rβ2defciency can lead to a diminished recruitment of activated T cells and anabnormal expression of inflammatory cytokines.Given the fact that these related genes (IL-12B, IL-12Rβ1andIL-12Rβ2) play an important role in Th1and Th17related immuneresponses, we wanted to verify the potential evidence of an association ofpolymorphisms in these genes with uveitis. We chose BD and VKH asrepresentative examples of uveitis in view of the fact that a sufficientlylarge sample size could be obtained. Eight SNPs of the three genes (IL-12B,IL-12Rβ1and IL-12Rβ2) were selected for our study, whereby the choicewas dictated by earlier studies showing a definite association with otherautoimmune diseases. PartⅠAssociations of the IL-12B gene polymorphisms withsusceptibility to BDPurpose: To investigate the associations of single nucleotidepolymorphisms (SNPs) of three genes (IL-12B, IL-12Rβ1and IL-12Rβ2)in Behcet’s disease (BD).Methods: A total of806BD cases and1000healthy controls wereinvolved in this study. The first stage included400BD patients,400VKHcases, and600healthy individuals. The second stage composed of the other406BD patients and another1000normal controls. Genotyping was carriedout by PCR-restriction fragment length polymorphism assay and the resultswere validated by using direct sequencing. The χ2test was performed tocompare the allele and genotype frequencies between cases and healthycontrols.Results:1. In the first stage of this case-control study, eight SNPs of the three IL-12related genes (IL-12B, IL-12Rβ1and IL-12Rβ2) were genotypedsuccessfully in400BD patients and600healthy controls. The obtaineddata of healthy subjects were in agreement with the Hardy-Weinbergequilibrium.2. Frequencies of the rs3212227/IL-12B genotype CC and C allele were significantly higher in the BD patients (pc=0.009, OR1.8,95%CI1.3to2.4; pc=0.024, OR1.3,95%CI1.1to1.6, respectively) compared with thenormal controls.3. There was no significant association between BD and the other testedSNPs (four of IL-12B, one of IL-12Rβ1and two of IL-12Rβ2).4. To confirm the result, a second set of patients was used to replicate theassociated SNP rs3212227/IL-12B using another406BD samples andanother set of1000healthy controls. In the second stage, the frequencies ofthers3212227/IL-12B genotype CC and C allele were also significantlyhigher in BD cases (pc=3.5×104, OR=1.8,95%CI1.4to2.4; pc=0.002,OR=1.4,95%CI1.2to1.6, respectively) than that observed in the controlgroup.5. Combining the data of the stage1and stage2study, showed a consistentassociation of genotype CC and C allele of rs3212227/IL-12B with BD (pccomb=6.3×107, OR=1.8,95%CI1.5to2.2; pccomb=2.0×105, OR=1.3,95%CI1.2to1.5, respectively).6. Since several organs may be affected by BD, a further study wasundertaken to explore the association of these SNPs with the clinicalmanifestations in the BD cases. No associations between tested genepolymorphisms with the separate features of BD could be detected.Conclusions: The results prove the association of genotype CC and Callele of rs3212227/IL-12B with BD in a Chinese Han population, IL-12B gene confers susceptibility to BD.PartⅡ Associations of the IL-12B gene polymorphisms withsusceptibility to VKHPurpose: To investigate the associations of single nucleotidepolymorphisms (SNPs) of three genes (IL-12B, IL-12Rβ1and IL-12Rβ2)in Vogt-Koyanagi-Harada (VKH) syndrome.Methods: A total of820VKH patients and1600healthy controls wereinvolved in this study. The first stage included400VKH cases and600healthy individuals. The second stage composed of the other420VKHcases and another1000normal controls. Genotyping was carried out byPCR-restriction fragment length polymorphism assay and the results werevalidated by using direct sequencing. The χ2test was performed tocompare the allele and genotype frequencies between cases and healthycontrols.Results:1. In the first stage study we included400VKH patients and600normalcontrols. The patients and controls were genotyped for the eight SNPsmentioned above. The obtained data of healthy subjects were in agreementwith the Hardy-Weinberg equilibrium.2. In the first stage study, significantly lower frequencies of the C allelefrequency of rs3212227/IL-12B was significantly increased in VKH patients (pc=0.012, OR1.3,95%CI1.1to1.6).3. No statistically significant association was found between VKH and theother seven SNPs tested.4. Based on the result above, we performed the second phase for anotherset of420VKH cases and1000healthy individuals. Consistent with theresult in the first stage, the C allele frequency of rs3212227/IL-12B wasmarkedly increased in VKH patients as compared to normal subjects (pc=0.046, OR1.3,95%CI1.1to1.6).5. Subsequently, we analyzed the combined data of all the cases andcontrols. The data confirmed the C allele of rs3212227/IL-12B as the riskfactor for VKH (pc=2.5×105, OR1.3,95%CI1.2to1.5).6. In addition, no associations could be detected between the separate VKHdisease characteristics and the investigated SNPs.Conclusions: Our study revealed that significantly the C allele frequencyof rs3212227/IL-12B as the risk factor for VKH (pc=2.5×105, OR1.3,95%CI1.2to1.5). IL-12B gene is involved in the susceptibility to VKHsyndrome in a Chinese Han population.