| Rheumatoid arthritis (RA [MIM 180300]) is a common autoimmune disease characterized by chronic, destructive, and debilitating arthritis. It is associated with considerable disability and early mortality. The etiology of RA is unknown, but it is thought to be a complex disease with gene-gene and gene-environment interaction. The heritability of RA has been estimated to be in the order of 60% by twin concordance data for RA, suggesting a substantial contribution from genetic factors. Although the well-known susceptibility gene is the HLA DRB1 gene, it accounts for only approximately one-third of total genetic effects. Non-HLA susceptibility genes contribute a major portion of genetic susceptibility to RA and remain to be elucidated. The human genome project and the internaional HapMap Project facilitate the quest for genes that influence susceptibility to disease. Identification RA susceptibility gene will lead to substantial advances in our understanding of disease and, in turn, to improvements in diagnostic accuracy, prognostic precision, and the range and targeting of available therapeutic options.To identify RA susceptibility gene in Han Chinese populations, a total of 2274 subjects were recruited, including 963 RA patients and 1311 healthy controls. Genomic DNA from each subject was extracted from whole blood and clinic information of each subject such as gender, age, ethnicity, the history of RA, onset time, anti-CCP antibody level, RF level, X-rays and DAS28 (Disease Activity Score in 28 joints) were collected to build four databases, RA DNA database, RA patient's clinic information database, healthy controls DNA database, healthy controls'clinic information database. We used individually matched case-control studies in each part of this project and genotyped a total of 36 single-nucleotide polymorphisms (SNPs) in 11 genes involved in the differentiation of Th1 and Th17 and other RA susceptibility locus in Caucasian previous reported. Hardy–Weinberg equilibrium (HWE) test was undertaken using a Pearson x2 goodness-of-fit test. Association of SNPs with RA or subsets of RA, or clinic phenotype was analyzed by Pearson Chi-Square test and logistic regression analysis using SPSS 13.0 (SPSS, Chicago, USA). P values less than 0.05 were considered significant. Odds ratios and their 95% confidence intervals were also determined for each SNP.The main results of this project were shown as follow.As to STAT4 involved in Th1 differentiation, rs11889341 was selected as a tagSNP of the RA susceptibility haplotype composed of rs11889341, rs7574865, rs8179673 and rs10181656 previous reported in other populations. In Han Chinese population, there was no evidence of a significant association between rs11889341 and RA. The heterozygous CT genotype was associated with RA in female group (P = 0.027, OR 1.31, 95% CI: 1.03-1.65). No association was found in male group and in any subsets of RA based on sex, RF and anti-CCP antibody. However, in the Han Chinese with RA disease, STAT4 was associated with the presence of RF. The minor allele had a gene-dosage RA protect effect. There was a significantly decreased frequency of the minor T allele (P = 0.024, OR 0.73, 95% CI: 0.56-0.95) and TT genotype (P = 0.013, OR 0.49; 95% CI: 0.28-0.86) in the rheumatoid factor (RF) positive subgroup compared with RF negative subgroup.IL-12Rβ2 involved in Th1 differentiation was located close to IL-23R involved in Th17 differentiation on chromosome 1q. SERBP1 gene lies very close to IL-12Rβ2 and in linkage disequilibrium with IL-12Rβ2. Therefore, the 268kb region covering these three genes from HapMap HCB data were analyzed and selected 24 tag SNPs to represent this region. The tagSNPs were genotyped in a test cohort of 380 Chengdu citizens, then replicated the findings of the first stage in another independent validation cohort of 1514 Chongqing citizens, using the SNPstream Ultra High Throughput Genotyping system. The results showed that these SNPs in this region on 1q had no association with RA, or any subset of RA classified by RF. However, the result of stratification analysis for anti-CCP antibody in validation cohort was consistent with the one in test cohort.In the association analysis of anti-CCP+RA, the result of allele analysis showed that all of the minor alleles of rs1569922, rs7539625, rs11465817, rs10889677 in IL-23R were associated with anti-CCP+RA (P<0.05) and all of them were risk alleles for this subset. The Han Chinese with the minor allele T of rs1569922 had 1.23 time increased risk to anti-CCP+RA compared to those with allele C (OR=1.23, 95%CI: 1.05-1.44). The Han Chinese bearing the minor allele G of rs7539625 had 1.16 times increased risk to anti-CCP+RA compared to those with allele A (OR=1.16, 95%CI: 1.00-1.36). People with the minor allele C of rs11465817 had increased risk to this subset of RA compared to those with allele A (OR=1.17, 95%CI: 1.00-1.36). The Han Chinese with the minor allele C of rs10889677 had increased risk to anti-CCP+RA compared to allele A carriers (OR=1.26, 95%CI: 1.06-1.49).Genotype anlysis showed that under the dominant genetic model, the Han Chinse bearing the minor allele T of rs1569922 had 1.32 times increased risk to anti-CCP+RA compared to individuals homozygous for the C allele (P = 0.017, OR = 1.32, 95%CI: 1.05-1.65). The individuals homozygous for the minor allele of rs7539625 had 1.36 fold increased risk of disease compared to those homozygous for the major allele (P = 0.05, OR = 1.36, 95%CI: 1.00-1.84). Under the dominant genetic model, the Han Chinese with the minor allele C of rs11465817 had increased risk to anti-CCP+RA compared to those with AA genotype (P = 0.032, OR = 1.29, 95%CI: 1.02-1.63). The individuals with genotype CA of rs10889677 had 1.49 times increased risk to anti-CCP+RA compared to those with genotype AA (P = 0.0005,OR = 1.49,95%CI: 1.19-1.87). In the IL-12Rβ2 gene, the rs1495964 was associated with anti-CCP+RA and the heterozygous carriers of the rs1495964 had decreased risk compared to homozygous carriers of the major allele (P = 0.005, OR = 0.71,95%CI: 0.56-0.90).In the association analysis of anti-CCP-RA, the result of allele analysis showed there were several SNPs associated with this subset of RA. rs10889677 in the 3'UTR of IL-23R was associated with it (P = 0.006) and the minor allele C had protective effect of anti-CCP-RA (OR = 0.64,95%CI: 0.47-0.88). rs11209050 in IL-12Rβ2 was also associated with it and the minor allele A was the risk allele (P = 0.004, OR = 1.60, 95%CI: 1.17-2.18). rs3762313 in SERBP1 was associated with it and the minor allel A had protective effect of it (P = 0.002, OR = 0.59, 95%CI: 0.43-0.83).Genotype anlysis showed that four SNPs (rs10889677 in IL-23R, rs11209050 and rs1496964 in IL-12Rβ2, rs3762313 in SERBP1) were associated with anti-CCP-RA. The CA genotype of rs10889677 in IL-23R was assicated with it (P = 0.001)and the heterozygous carriers had 0.48 fold decreased risk of anti-CCP-RA comaperd to homozygous carriers of the major allele (OR = 0.48, 95%CI: 0.31-0.75). A gene-dosage risk effect of the minor allele A of rs11209050 has been suggested because individuals homozygous for A allele had higher susceptibility for anti-CCP-RA (P = 0.016, OR = 2.43, 95%CI: 1.16-5.07) than heterozygous carriers (P = 0.019, OR = 1.65, 95%CI: 1.08-2.52). The minor allele C of rs1495964 in IL-12Rβ2 was risk allele. Under the dominant genetic model, individuals with C allele of had 2.06 fold increased risk to this subset of RA compared to those with TT genotype (P = 0.01,OR = 2.06, 95%CI: 1.19-3.59). The minor allele A of rs3762313 SNP in SERBP1 had a gene-dosage protective effect of anti-CCP-RA. Compared to homozygous carriers of major allele, homozygous carriers of minor allele had lower decreased risk (P = 0.013,OR = 0.33,95%CI: 0.13-0.82) than heterozygous carriers (P = 0.018,OR = 0.60,95%CI: 0.39-0.92).Haplotype analysis showed that the haplotype TGAA composed of rs1569922,rs7539625,rs11465817 and rs10889677 in IL-23R gene was associated with anti-CCP+RA (P = 0.025). The Han Chinese with this haploype had 2.73 times increased risk to this subset of RA compared to individuals with the haplotype CAAA (OR = 2.73, 95%CI: 1.14-6.53). A halotype CAAA composed of these SNPs in IL-23R was associated with anti-CCP-RA (P = 0.04). Individuals with TGCC haplotype had decreased risk to anti-CCP-RA compared to those with CAAA haplotype (OR = 0.66, 95%CI: 0.44-0.98). The haplotype CA composed of rs1496964 and rs11209050 in IL-12Rβ2 was associated with anti-CCP-RA (P = 0.046) and individuals with this haplotype had 1.48 times increased risk to this subset of RA compared to those with TC haplotype (OR = 1.48, 95%CI: 1.01-2.18).When we put the results of the association analysis between IL-23R and RA or any subset of RA and between IL-12Rβ2 and RA or any subset of RA together, we found it very interesting that all the minor allele of all the SNPs (rs1569922, rs7539625, rs11465817, rs10889677) we genotyped in IL-23R were all associated with anti-CCP+RA and all of them were risk allele while all the SNPs (rs11209050 and rs1495964) in IL-12Rβ2 were only associated with anti-CCP-RA. The heterozygous genotype of rs10889677 in the 3'UTR of IL-23R had not only the risk effect of anti-CCP+RA but also the protective effect of anti-CCP-RA. Our findings suggest that the susceptibility genes in the subtypes of RA were different and imply that the different subtypes of RA might have different mechanism. Our results support the viewpoint appeared recently that RA is a heterogeneous disease which has at least two major subtypes anti-CCP+RA and anti-CCP-RA, and their mechanisms may be different. Since IL-23R expressed on Th17 binding with its ligand was required for maintaining the Th17 phenotype, the association of IL-23R with anti-CCP+RA suggests that Th17 might be invloved in the induction of anti-CCP+RA. Since IL-12Rβ2 was only expressd on activated Th1 cells, the association of IL-12Rβ2 with anti-CCP-RA suggets Th1 might be invloved in the induction of anti-CCP-RA. Here is the current argument that which one is the prominent in the induction of RA, Th1 or Th17? Our findings imply that in different subtype of RA, Th1 and Th17 may play different role in the induction of autoimmune disease.In view of the limitation of Candidate-Gene Association Studies and the advantage of whole genome-wide association studies (GWAS), we assumed that there might be common RA susceptibility gene among ethnic groups and tried to replicate the results of Caucasians GWAS in Han Chinese populations in the second part of this project. Eleve potent RA susceptibility SNPs previous reported in GWAS were genotyped in test cohort firstly and then replicated the findings of the first stage in the another validation cohort. Finially, we found three SNPs of then were associated with RA in Han Chinese populations.rs6457617 between the HLA-DQB1 and the HLA-DQA2 was associated with RA in the test cohort and replicated in the validation cohort. The association analysis of combined data showed this association very clearly. The protect effect of the minor allele C was gene does dependent. Compared to individuals with genotype TT, the Han Chinese bearing genotype CC had a lower decreased risk (P = 9.4×10-12, OR = 0.35, 95%CI: 0.26-0.47) than heterozygous carriers had (P = 1.1×10-4, OR = 0.68, 95%CI: 0.56-0.82). The allele T was RA risk allele both in the British and the Han Chinese. But the Han Chinese bearing genotype TT had a 2.86-fold increased risk compared to those with genotype CC, much lower than it in the British (OR = 5.21).rs11162922 was located in gene desert of chromosome 1. The homozygous for the minor allele G was associated with RA (P = 0.045, OR = 1.46, 95%CI: 1.01-2.12).Another SNP, rs3816587 in ANAPC4 was associated with RA under recessive genetic model (P = 0.002, OR = 0.62, 95%CI: 0.46-0.84). The distributions of allele frequencies were different between the Han Chinese and the British. In the Han Chinese, the minor allele was allele T and it had RA protect effect. The minor allele was C in the British. But the risk alleles of RA in these two populations were both the allele C. The Han Chinese bearing genotype CC had a 1.51-fold increased risk to RA compared to the Han Chinese bearing genotype TT, very similar with the OR (1.35) in the British.As shown in the LD block map constructed with Hapmap CHB data, the rs3816587 is an htSNP of block4 in anaphase promoting complex subunit 4 (ANAPC4/APC4) gene, suggesting that the strong association detected with rs3816587 in ANAPC4 originates from ANAPC4 itself. The ANAPC4 encodes a core subunit of anaphase-promoting complex/cyclosome (APC/C), a large complex E3 ubiquitin ligase to carry out the ubiquitin chains on substrate proteins to mediate ubiquitination and target protein degradation. The well-known substrates of APC are important mitotic regulators such as cyclin B1, securin and Polo-like kinase-1 (Plk1) to control cell cycle and mitosis. APC has never been reported to be involved with autoimmune disease, however, some other E3 ligases, such as Casitas B cell lymphoma b (Cbl-b), gene related to anergy in lymphocytes (GRAIL), Cbl-b and Itch, have been shown to be key regulators of immune tolerance and suppression of autoreactive T cell development. Mutations to these E3 ligases can lead to the development of autoimmune disease. Are there any new substrates of APC except from mitotic regulators? Identification of the substrates of APC is an ongoing process but it is difficult because of its complex structure. Our findings showed APC4 was associated with the development of RA both in the British and the Han Chinese, strongly suggesting that there might be a new substrate of APC, which is invloved in the induction of RA. This year, Dr. Yamano developed an in vitro system which enables the identification of new substrates and investigation of the molecular mechanisms by which the APC/C recognizes its substrates. With the improvement of the method, a new substrate of APC invloved in autoimmune will be identified in the future. |
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