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Background: A model that can predict treatment response for a patient with specific baseline characteristics would help decision-making in personalized medicine. The aim of the study is to develop such a model in the treatment of rheumatoid arthritis (RA) patients who receive certolizumab (CTZ) plus methotrexate (MTX) therapy, using individual participant data meta-analysis (IPD-MA)., Methods: We will search Cochrane CENTRAL, PubMed, and Scopus as well as clinical trial registries, drug regulatory agency reports, and the pharmaceutical company websites from their inception onwards to obtain randomized controlled trials (RCTs) investigating CTZ plus MTX compared with MTX alone in treating RA. We will request the individual-level data of these trials from an independent platform (http://vivli.org). The primary outcome is efficacy defined as achieving either remission (based on ACR-EULAR Boolean or index-based remission definition) or low disease activity (based on either of the validated composite disease activity measures). The secondary outcomes include ACR50 (50% improvement based on ACR core set variables) and adverse events. We will use a two-stage approach to develop the prediction model. First, we will construct a risk model for the outcomes via logistic regression to estimate the baseline risk scores. We will include baseline demographic, clinical, and biochemical features as covariates for this model. Next, we will develop a meta-regression model for treatment effects, in which the stage 1 risk score will be used both as a prognostic factor and as an effect modifier. We will calculate the probability of having the outcome for a new patient based on the model, which will allow estimation of the absolute and relative treatment effect. We will use R for our analyses, except for the second stage which will be performed in a Bayesian setting using R2Jags., Discussion: This is a study protocol for developing a model to predict treatment response for RA patients receiving CTZ plus MTX in comparison with MTX alone, using a two-stage approach based on IPD-MA. The study will use a new modeling approach, which aims at retaining the statistical power. The model may help clinicians individualize treatment for particular patients., Systematic Review Registration: PROSPERO registration number pending (ID#157595).

Recent evidence suggests that a substantial portion of complex disease risk alleles modify gene expression in a cell-specific manner. To identify candidate causal genes and biological pathways of immune-related complex diseases, we conducted expression quantitative trait loci (eQTL) analysis on five subsets of immune cells (CD4 + T cells, CD8 + T cells, B cells, natural killer (NK) cells and monocytes) and unfractionated peripheral blood from 105 healthy Japanese volunteers. We developed a three-step analytical pipeline comprising (i) prediction of individual gene expression using our eQTL database and public epigenomic data, (ii) gene-level association analysis and (iii) prediction of cell-specific pathway activity by integrating the direction of eQTL effects. By applying this pipeline to rheumatoid arthritis data sets, we identified candidate causal genes and a cytokine pathway (upregulation of tumor necrosis factor (TNF) in CD4 + T cells). Our approach is an efficient way to characterize the polygenic contributions and potential biological mechanisms of complex diseases.

Objectives: Rheumatoid arthritis (RA) is the most common immune-related inflammatory arthritis affecting 0.5-1.0% of the population. Both genetic and environmental factors are associated with RA onset. Twin studies reported from European countries estimated heritability of RA as 53-65%. However, no twin studies have been reported from Asian countries., Methods: We collected a total of 43 monozygotic and 43 dizygotic twins from 7550 patients with RA and obtained information of RA status in the twin pairs. We characterized RA patients whose twin siblings had RA. Variance components analysis was performed to estimate heritability in RA in the Japanese population., Results: Four and one patients with RA over 43 monozygotic (9.3%) and 43 dizygotic twins (2.3%), respectively, had twin siblings with RA. All the patients whose information of sero-positivity was available and whose twin siblings had RA were sero-positive. Heritability of RA was estimated as 62.2% and 56.1% in the AE and ACE models, respectively. The estimated heritability was comparable with those reported in the Europeans, compatible with previous overlap of RA susceptibility genes between Japanese and European populations., Conclusions: Heritability of RA onset in the Japanese population is estimated to be comparable with that in European populations.

Despite the progress in human leukocyte antigen (HLA) causal variant mapping, independent localization of major histocompatibility complex (MHC) risk from classical HLA genes is challenging. Here, we conducted a large-scale MHC fine-mapping analysis of rheumatoid arthritis (RA) in a Japanese population (6,244 RA cases and 23,731 controls) population by using HLA imputation, followed by a multi-ethnic validation study including east Asian and European populations (n = 7,097 and 23,149, respectively). Our study identified an independent risk of a synonymous mutation at HLA-DOA, a non-classical HLA gene, on anti-citrullinated protein autoantibody (ACPA)-positive RA risk (p = 1.4 × 10(-9)), which demonstrated a cis-expression quantitative trait loci (cis-eQTL) effect on HLA-DOA expression. Trans-ethnic comparison revealed different linkage disequilibrium (LD) patterns in HLA-DOA and HLA-DRB1, explaining the observed HLA-DOA variant risk heterogeneity among ethnicities, which was most evident in the Japanese population. Although previous HLA fine-mapping studies have identified amino acid polymorphisms of the classical HLA genes as driving genetic susceptibility to disease, our study additionally identifies the dosage contribution of a non-classical HLA gene to disease etiology. Our study contributes to the understanding of HLA immunology in human diseases and suggests the value of incorporating additional ancestry in MHC fine-mapping., (Copyright © 2016. Published by Elsevier Inc.)

Background: Peptidylarginine deiminase type 4 (PADI4) has been identified as a susceptibility gene for rheumatoid arthritis (RA) by genome-wide association studies. PADI4 is highly expressed in the bone marrow, macrophages, neutrophils, and monocytes. Peptidyl citrulline is an interesting molecule in RA because it is a target antigen for anti-citrullinated peptide antibodies, and only PADs (translated proteins from PADI genes) can provide peptidyl citrulline via the modification of protein substrates. The aim of this study was to evaluate the importance of the PADI4 gene in the progression of RA., Methods: We generated Padi4 knockout (Padi4(-/-)) DBA1J mice. The Padi4(-/-) DBA1J and wild-type mice were immunized with bovine type II collagen (CII) to develop collagen-induced arthritis (CIA). The expression of various inflammatory cytokines and Padi genes in immune cells was detected by the real-time TaqMan assay. Cytokine concentrations in sera were measured by enzyme-linked immunosorbent assays. Localization of the PAD4 and PAD2 proteins was indicated by immunohistochemistry., Results: We demonstrated that the clinical disease score was significantly decreased in the Padi4(-/-) mice and Padi4 expression was induced by CII immunization. In the Padi4(-/-) mice, serum anti-type II collagen (CII) immunoglobulin M (IgM), IgG, and inflammatory cytokine levels were significantly decreased compared with those in the wild-type mice. Padi2 expression was induced in the immune cells of the Padi4(-/-) mice as a compensation for the defect in Padi4., Conclusions: Padi4 affected disease severity in the CIA mice and was involved in the enhancement of the collagen-initiated inflammatory responses.

Anti-citrullinated peptide antibody (ACPA) is a highly specific autoantibody to rheumatoid arthritis (RA). Recent studies have revealed that periodontal disease (PD) is closely associated with RA and production of ACPA in RA. Analyses of associations between PD and ACPA production in a healthy population may deepen our understandings. Here, we analyzed a total of 9554 adult healthy subjects. ACPA and IgM-rheumatoid factor (RF) was quantified and PD status was evaluated using the number of missing teeth (MT), the Community Periodontal Index (CPI) and Loss of Attachment (LA) for these subjects. PD status was analyzed for its association with the positivity and categorical levels of ACPA and RF conditioned for covariates which were shown to be associated with PD, ACPA or RF. As a result, all of MT, CPI and LA showed suggestive or significant associations with positivity (p = 0.024, 0.0042 and 0.037, respectively) and levels of ACPA (p ≤ 0.00031), but none of the PD parameters were associated with those of RF. These association patterns were also observed when we analyzed 6206 non-smokers of the participants. The significant associations between PD parameters and positivity and levels of ACPA in healthy population support the fundamental involvement of PD with ACPA production., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Introduction: Although susceptibility genes for anti-citrullinated peptide/protein antibodies (ACPA)-positive rheumatoid arthritis (RA) have been successfully discovered by genome-wide association studies (GWAS), little is known about the genetic background of ACPA-negative RA. We intended to elucidate genetic background of ACPA-negative RA., Method: We performed a meta-analysis of GWAS comprising 670 ACPA-negative RA and 16,891 controls for 1,948,138 markers, followed by a replication study of the top 35 single nucleotide polymorphisms (SNPs) using 916 cases and 3,764 controls. Inverse-variance method was applied to assess overall effects. To assess overlap of susceptibility loci between ACPA-positive and -negative RA, odds ratios (ORs) of the 21 susceptibility markers to RA in Japanese were compared between the two subsets. In addition, SNPs were stratified by the p-values in GWAS meta-analysis for either ACPA-positive RA or ACPA-negative RA to address the question whether weakly-associated genes were also shared. The correlations between ACPA-positive RA and the subpopulations of ACPA-negative RA (rheumatoid factor (RF)-positive and RF-negative subsets) were also addressed., Results: Rs6904716 in LEMD2 of the human leukocyte antigen (HLA) locus showed a borderline association with ACPA-negative RA (overall p = 5.7 × 10(-8)), followed by rs6986423 in CSMD1 (p = 2.4 × 10(-6)) and rs17727339 in FCRL3 (p = 1.4 × 10(-5)). ACPA-negative RA showed significant correlations of ORs with ACPA-positive RA for the 21 susceptibility SNPs and non-HLA SNPs with p-values far from significance. These significant correlations with ACPA-positive RA were true for ACPA-negative RF-positive and ACPA-negative RF-negative RA. On the contrary, positive correlations were not observed between the ACPA-negative two subpopulations., Conclusion: Many of the susceptibility loci were shared between ACPA-positive and -negative RA.

Objectives: Cellular fibronectin (cFn) has been implicated in the pathogenesis of rheumatoid arthritis (RA), and we previously demonstrated the presence of citrullinated cFn in rheumatoid synovial tissues. The present study aimed to investigate whether citrullinated cFn can be detected in the plasma or synovial fluid of RA patients., Methods: Twenty-five rheumatoid arthritis synovial fluid (RASF), seven osteoarthritis synovial fluid (OASF) and 12 plasma samples from RA patients were examined. Citrullination of cFn was determined by immunoprecipitation (IP), western blotting and enzyme-linked immunosorbent assay (ELISA), in which peptidyl-citrulline within cFn was detected using a specific anti-cFn monoclonal antibody in combination with anti-modified citrulline antibody after chemical modification., Results: Levels of citrullination associated with cFn, as determined by ELISA, were significantly higher in RASF than in OASF samples. IP and western blotting detected citrullinated cFn in RASF but not in plasma samples from RA patients. Levels of total cFn were elevated in RASF compared with OASF, and 24 out of 25 RASF samples were positive for anti-CCP antibody. However, no correlation was observed between levels of citrullinated cFn and those of total cFn or anti-CCP antibody in RASF. On the other hand, a significant positive correlation was observed between the levels of matrix metalloproteinase-3 (MMP-3) and cFn citrullination in RASF., Conclusions: Citrullinated cFn appears to be produced within the affected joint and might be involved in the pathogenesis of rheumatoid synovitis.

A major challenge in human genetics is to devise a systematic strategy to integrate disease-associated variants with diverse genomic and biological data sets to provide insight into disease pathogenesis and guide drug discovery for complex traits such as rheumatoid arthritis (RA). Here we performed a genome-wide association study meta-analysis in a total of >100,000 subjects of European and Asian ancestries (29,880 RA cases and 73,758 controls), by evaluating ∼10 million single-nucleotide polymorphisms. We discovered 42 novel RA risk loci at a genome-wide level of significance, bringing the total to 101 (refs 2 - 4). We devised an in silico pipeline using established bioinformatics methods based on functional annotation, cis-acting expression quantitative trait loci and pathway analyses--as well as novel methods based on genetic overlap with human primary immunodeficiency, haematological cancer somatic mutations and knockout mouse phenotypes--to identify 98 biological candidate genes at these 101 risk loci. We demonstrate that these genes are the targets of approved therapies for RA, and further suggest that drugs approved for other indications may be repurposed for the treatment of RA. Together, this comprehensive genetic study sheds light on fundamental genes, pathways and cell types that contribute to RA pathogenesis, and provides empirical evidence that the genetics of RA can provide important information for drug discovery.

Objective. To re-evaluate the roles of HLA-DRB1 alleles in susceptibility to SLE and RA and their effects on autoantibody status in large-scale Japanese cohorts. Methods. A total of 656 SLE, 2410 RA and 911 control subjects, who were all Japanese, were genotyped for HLA-DRB1 alleles using sequence-specific oligonucleotide probes. The association of alleles with disease susceptibility was tested by logistic regression analysis and by the relative predispositional effect method. The association with autoantibody status was examined by the standard χ(2) test. Results. HLA-DRB1*15:01, *09:01, *08:02 and *04:01 were significantly associated with SLE susceptibility, while shared epitope (SE) alleles and DRB1*09:01 were associated with RA susceptibility. The compound heterozygote of DRB1*09:01/*15:01 conferred an increased risk for SLE compared with the homozygotes for DRB1*09:01 and *15:01 and was associated with earlier onset of disease, whereas the compound effect of DRB1-SE/*09:01 was not clear in RA. DRB1*09:01 was significantly associated with the appearance of anti-Sm antibody in SLE as well as ACPA in RA, while protectively associated with anti-dsDNA antibody in SLE. No significant interaction was observed between DRB1*09:01 and smoking status for the appearance of ACPA, unlike that observed in SE alleles in RA. Conclusion. We identified HLA-DRB1 alleles associated with SLE and RA in a Japanese population and demonstrated a shared susceptibility of DRB1*09:01 between the diseases as well as its effect on autoantibody production.

Rheumatoid arthritis (RA) is a joint-destructive autoimmune disease. Three composite indices evaluating the same 28 joints are commonly used for the evaluation of RA activity. However, the relationship between, and the frequency of, the joint involvements are still not fully understood. Here, we obtained and analyzed 17,311 assessments for 28 joints in 1,314 patients with RA from 2005 to 2011 from electronic clinical chart templates stored in the KURAMA (Kyoto University Rheumatoid Arthritis Management Alliance) database. Affected rates for swelling and tenderness were assessed for each of the 28 joints and compared between two different sets of RA patients. Correlations of joint symptoms were analyzed for swellings and tenderness using kappa coefficient and eigen vectors by principal component analysis. As a result, we found that joint affected rates greatly varied from joint to joint both for tenderness and swelling for the two sets. Right wrist joint is the most affected joint of the 28 joints. Tenderness and swellings are well correlated in the same joints except for the shoulder joints. Patients with RA tended to demonstrate right-dominant joint involvement and joint destruction. We also found that RA synovitis could be classified into three categories of joints in the correlation analyses: large joints with wrist joints, PIP joints, and MCP joints. Clustering analysis based on distribution of synovitis revealed that patients with RA could be classified into six subgroups. We confirmed the symmetric joint involvement in RA. Our results suggested that RA synovitis can be classified into subgroups and that several different mechanisms may underlie the pathophysiology in RA synovitis.

Genetic studies identified multiple genes and polymorphisms that increase risk to develop rheumatoid arthritis. Genomic approach is characterized with its integrative style using mathematical and statistical models. Its main targets include (1)combinatorial effect of multiple genetic and environmental factors, (2)heterogeneity of pathological states and its individuality, and (3)their chronological heterogeneity. Genomic approach will clarify pathophysiology of various diseases along with the progresses in molecular biology and other researches on individual molecules.

Rheumatoid arthritis is an autoimmune disease with a complex etiology, leading to inflammation of synovial tissue and joint destruction. Through a genome-wide association study (GWAS) and two replication studies in the Japanese population (7,907 cases and 35,362 controls), we identified two gene loci associated with rheumatoid arthritis susceptibility (NFKBIE at 6p21.1, rs2233434, odds ratio (OR) = 1.20, P = 1.3 × 10(-15); RTKN2 at 10q21.2, rs3125734, OR = 1.20, P = 4.6 × 10(-9)). In addition to two functional non-synonymous SNPs in NFKBIE, we identified candidate causal SNPs with regulatory potential in NFKBIE and RTKN2 gene regions by integrating in silico analysis using public genome databases and subsequent in vitro analysis. Both of these genes are known to regulate the NF-κB pathway, and the risk alleles of the genes were implicated in the enhancement of NF-κB activity in our analyses. These results suggest that the NF-κB pathway plays a role in pathogenesis and would be a rational target for treatment of rheumatoid arthritis., Competing Interests: The authors have declared that no competing interests exist.

Rheumatoid arthritis is a common autoimmune disease characterized by chronic inflammation. We report a meta-analysis of genome-wide association studies (GWAS) in a Japanese population including 4,074 individuals with rheumatoid arthritis (cases) and 16,891 controls, followed by a replication in 5,277 rheumatoid arthritis cases and 21,684 controls. Our study identified nine loci newly associated with rheumatoid arthritis at a threshold of P < 5.0 × 10(-8), including B3GNT2, ANXA3, CSF2, CD83, NFKBIE, ARID5B, PDE2A-ARAP1, PLD4 and PTPN2. ANXA3 was also associated with susceptibility to systemic lupus erythematosus (P = 0.0040), and B3GNT2 and ARID5B were associated with Graves' disease (P = 3.5 × 10(-4) and 2.9 × 10(-4), respectively). We conducted a multi-ancestry comparative analysis with a previous meta-analysis in individuals of European descent (5,539 rheumatoid arthritis cases and 20,169 controls). This provided evidence of shared genetic risks of rheumatoid arthritis between the populations.

Rheumatoid arthritis (RA) is a common systemic autoimmune disease and its onset and prognosis are controlled by genetic, immunological, and environmental factors. The HLA locus, particularly HLA-DRB1, is its strongest genetic risk determinant across ethnicities. Several other genes, including PTPN22 and PADI4, show modest association with RA. However, they cover only a part of its genetic components and their relative contribution is different between populations. To identify novel genetic determinants, we took a candidate gene approach in a trans-ethnic manner. After critical selection of 169 genes based on their immunological function, we performed SNP discovery of these genes by the resequencing of exons and surrounding areas using European and Japanese DNAs. We then generated a panel of 1,509 SNPs for case-control association study in both populations. The DerSimonian-Laird test for meta-analysis, using the combined results of the two populations, identified rs7551957 at the 5'-flanking region of the low-affinity Fc-gamma receptor IIa (FCGR2A) gene as the strongest candidate for the association (p = 8.6 × 10(-5), odds ratio = 1.58 with 95%CI 1.25-1.99). Suggestive signals were also obtained for three SNPs in the dihydropyrimidine dehydrogenase (DPYD) gene (rs6685859; p = 1.3 × 10(-4), rs7550959; p = 1.5 × 10(-4) and rs7531138; p = 1.7 × 10(-4)) and an intronic SNP, rs2269310, of the erythrocytic spectrin beta (SPTB) gene (p = 7.9 × 10(-4)).

HLA-DRB1, especially the shared epitope (SE), is strongly associated with rheumatoid arthritis (RA). However, recent studies have shown that SE is at most weakly associated with RA without anti-citrullinated peptide/protein antibody (ACPA). We have recently reported that ACPA-negative RA is associated with specific HLA-DRB1 alleles and diplotypes. Here, we attempted to detect genetically different subsets of ACPA-negative RA by classifying ACPA-negative RA patients into two groups based on their positivity for rheumatoid factor (RF). HLA-DRB1 genotyping data for totally 954 ACPA-negative RA patients and 2,008 healthy individuals in two independent sets were used. HLA-DRB1 allele and diplotype frequencies were compared among the ACPA-negative RF-positive RA patients, ACPA-negative RF-negative RA patients, and controls in each set. Combined results were also analyzed. A similar analysis was performed in 685 ACPA-positive RA patients classified according to their RF positivity. As a result, HLA-DRB1*04:05 and *09:01 showed strong associations with ACPA-negative RF-positive RA in the combined analysis (p = 8.8×10(-6) and 0.0011, OR: 1.57 (1.28-1.91) and 1.37 (1.13-1.65), respectively). We also found that HLA-DR14 and the HLA-DR8 homozygote were associated with ACPA-negative RF-negative RA (p = 0.00022 and 0.00013, OR: 1.52 (1.21-1.89) and 3.08 (1.68-5.64), respectively). These association tendencies were found in each set. On the contrary, we could not detect any significant differences between ACPA-positive RA subsets. As a conclusion, ACPA-negative RA includes two genetically distinct subsets according to RF positivity in Japan, which display different associations with HLA-DRB1. ACPA-negative RF-positive RA is strongly associated with HLA-DRB1*04:05 and *09:01. ACPA-negative RF-negative RA is associated with DR14 and the HLA-DR8 homozygote.

Background: HLA-DRB1 is associated with rheumatoid arthritis (RA). However, it has recently been suggested that HLA-DRB1 is only associated with patients with RA who have anticitrullinated peptide/protein antibodies (ACPA), which are specific to RA., Objective: To elucidate whether specific HLA-DR alleles are associated with ACPA-negative RA development., Methods: HLA-DRB1 typing was carried out in 368 Japanese ACPA-negative patients with RA and 1508 healthy volunteers as the first set, followed by HLA-DRB1 typing of 501 cases and 500 controls as the second set. The HLA-DRB1 allele frequency and diplotype frequency were compared in each group, and the results of the two studies were combined to detect HLA-DRB1 alleles or diplotypes associated with ACPA-negative RA., Results: HLA-DRB1*12:01 was identified as a novel susceptibility allele for ACPA-negative RA (p=0.000088, OR=1.72, 95% CI 1.31 to 2.26). HLA-DRB1*04:05 and *14:03 showed moderate associations with ACPA-negative RA (p=0.0063, OR=1.26, 95% CI 1.07 to 1.49 and p=0.0043, OR=1.81, 95% CI 1.20 to 2.73, respectively). The shared epitope was weakly associated with ACPA-negative RA, but no dosage effect was detected (p=0.016, OR=1.17, 95% CI 1.03 to 1.34). A combination of HLA-DRB1*12:01 and DRB1*09:01 showed a strong association with susceptibility to ACPA-negative RA (p=0.00013, OR=3.62, 95% CI 1.79 to 7.30). Homozygosity for HLA-DR8 was significantly associated with ACPA-negative RA (p=0.0070, OR=2.16, 95% CI 1.22 to 3.82). It was also found that HLA-DRB1*15:02 and *13:02 were protective against ACPA-negative RA (p=0.00010, OR=0.68, 95% CI 0.56 to 0.83 and p=0.00059, OR=0.66, 95% CI 0.52 to 0.84, respectively)., Conclusions: In this large-scale association study multiple alleles and diplotypes were found to be associated with susceptibility to, or protection against, ACPA-negative RA.

Rheumatoid arthritis (RA) is a typical complex trait and the major cause of chronic inflammation worldwide. Although multiple genetic loci have been shown for their association with the onset of RA, they cover only a part of its genetic components and are largely ethnicity-specific. To identify novel genetic factors related to the predisposition and prognosis of RA in Japanese, we conducted a large-scale genome-wide association (GWA) study. We performed a GWA analysis by scanning the genome of 1247 RA cases and 1486 controls for 277 420 single nucleotide polymorphisms (SNPs), followed by replication analysis using two independent sample sets consisting of 1865 cases and 1623 controls, and 2303 cases and 3380 controls. We identified two SNPs, rs2075876 and rs760426, in intron of the autoimmune regulator AIRE gene at chromosome 21q22 that showed strong associations with the disease (P= 3.6 × 10(-9) and P= 4.4 × 10(-8), respectively). Rs1800250, in exon7 of AIRE, was in strong linkage disequilibrium (r(2)= 0.94) with rs2075876 and introduced an amino acid alteration (S278R) in the SAND domain of the AIRE protein. In silico analysis showed the decreased transcription of AIRE by the risk allele of rs2075876 compared with the alternative allele (P= 6.8 × 10(-5)). No correlation was observed between the rs2075876 genotype and quantitative traits reflecting the progression of RA. As AIRE is a key molecule which regulates the expression and presentation of self-antigens in thymic negative selection, its downregulation by genetic polymorphisms may result in the survival of auto-reactive T cells to trigger auto-inflammation in RA.

Objective: To elucidate the differential role of peptidyl arginine deiminase 4 (PADI4) polymorphism in rheumatoid arthritis (RA) between Asian and European populations, possible gene-environmental interactions among the PADI4 polymorphism, sex and smoking status were analysed., Methods: Three independent sets of case-control samples were genotyped for single-nucleotide polymorphisms in PADI4; Japanese samples (first set, 1019 RA patients, 907 controls; second set, 999 RA patients, 1128 controls) using TaqMan assays and Dutch samples (635 RA patients, 391 controls) using Sequenom MassARRAY platform. The association of PADI4 with RA susceptibility was evaluated by smoking status and sex in contingency tables and logistic regression models., Results: In the first set of Japanese samples, PADI4 polymorphism (rs1748033) showed a greater risk in men (OR(allele) 1.39; 95% CI 1.10 to 1.76; p(trend)=0.0054) than in women and in ever-smokers (OR(allele) 1.25; 95% CI 1.02 to 1.53; p(trend)=0.032) than in never-smokers. Moreover, the highest risk was seen in male ever-smokers (OR(allele) 1.46; 95% CI 1.12 to 1.90; p(trend)=0.0047). Similar trends were observed in the second set of Japanese samples as well as in Dutch samples., Conclusion: PADI4 polymorphism highly predisposes male smokers to RA, and the genetic heterogeneity observed between Asian and European populations may be partly explained by differences in smoking prevalence among men.

Rheumatoid arthritis (RA) is a major cause of adult chronic inflammatory arthritis and a typical complex trait. Although several genetic determinants have been identified, they account for only a part of the genetic susceptibility. We conducted a genome-wide association study of RA in Japanese using 225,079 SNPs genotyped in 990 cases and 1,236 controls from two independent collections (658 cases and 934 controls in collection1; 332 cases and 302 controls in collection2), followed by replication studies in two additional collections (874 cases and 855 controls in collection3; 1,264 cases and 948 controls in collection4). SNPs showing p<0.005 in the first two collections and p<10(-4) by meta-analysis were further genotyped in the latter two collections. A novel risk variant, rs2000811, in intron2 of the myelin basic protein (MBP) at chromosome 18q23 showed strong association with RA (p = 2.7×10(-8), OR 1.23, 95% CI: 1.14-1.32). The transcription of MBP was significantly elevated with the risk allele compared to the alternative allele (p<0.001). We also established by immunohistochemistry that MBP was expressed in the synovial lining layer of RA patients, the main target of inflammation in the disease. Circulating autoantibody against MBP derived from human brain was quantified by ELISA between patients with RA, other connective tissue diseases and healthy controls. As a result, the titer of anti-MBP antibody was markedly higher in plasma of RA patients compared to healthy controls (p<0.001) and patients with other connective tissue disorders (p<0.001). ELISA experiment using citrullinated recombinant MBP revealed that a large fraction of anti-MBP antibody in RA patients recognized citrullinated MBP. This is the first report of a genetic study in RA implicating MBP as a potential autoantigen and its involvement in pathogenesis of the disease.

Objectives: ACPA is a highly specific marker for RA. It was recently reported that ACPA can be used to classify RA into two disease subsets, ACPA-positive and ACPA-negative RA. ACPA-positive RA was found to be associated with the HLA-DR shared epitope (SE), but ACPA negative was not. However, the suspicion remained that this result was caused by the ACPA-negative RA subset containing patients with non-RA diseases. We examined whether this is the case even when possible non-RA ACPA-negative RA patients were excluded by selecting only patients with bone erosion., Methods: We genotyped HLA-DRB1 alleles for 574 ACPA-positive RA, 185 ACPA-negative RA (including 97 erosive RA) and 1508 healthy donors. We also tested whether HLA-DR SE is associated with RF-negative or ANA-negative RA., Results: ACPA-negative RA with apparent bone erosion was not associated with SE, supporting the idea that ACPA-negative RA is genetically distinct from ACPA-positive RA. We also tested whether these subsets are based on autoantibody-producing activity. In accordance with the ACPA-negative RA subset, the RF-negative RA subset showed a clearly distinct pattern of association with SE from the RF-positive RA. In contrast, ANA-negative as well as ANA-positive RA was similarly associated with SE, suggesting that the subsets distinguished by ACPA are not based simply on differences in autoantibody production., Conclusions: ACPA-negative erosive RA is genetically distinct from ACPA-positive RA.

In Drosophila, Eiger, a tumor necrosis factor α (TNFα) superfamily ligand, induces cell death by activating the c-Jun N-terminal kinase (JNK) pathway. Here, we report that overexpression of Plenty of SH3s (POSH) suppresses Eiger-induced cell death and produces highly deformed tissues. These results imply that high levels of POSH protect tissues from cell death. In humans, rheumatoid arthritis synovial fibroblasts (RASF) are generally resistant to apoptosis. We show that POSH is expressed at relatively high levels in RASF, and its reduction by RNAi sensitizes these cells to Fas-mediated apoptosis. Thus, we demonstrate that POSH promotes cell survival in Drosophila and in human RASF., (Copyright © 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Rheumatoid arthritis is a common autoimmune disease with a complex genetic etiology. Here, through a genome-wide association study of rheumatoid arthritis, we identified a polymorphism in CCR6, the gene encoding chemokine (C-C motif) receptor 6 (a surface marker for Th17 cells) at 6q27, that was associated with rheumatoid arthritis susceptibility and was validated in two independent replication cohorts from Japan (rs3093024, a total of 7,069 individuals with rheumatoid arthritis (cases) and 20,727 controls, overall odds ratio = 1.19, P = 7.7 x 10(-19)). We identified a triallelic dinucleotide polymorphism of CCR6 (CCR6DNP) in strong linkage disequilibrium with rs3093024 that showed effects on gene transcription. The CCR6DNP genotype was correlated with the expression level of CCR6 and was associated with the presence of interleukin-17 (IL-17) in the sera of subjects with rheumatoid arthritis. Moreover, CCR6DNP was associated with susceptibility to Graves' and Crohn's diseases. These results suggest that CCR6 is critically involved in IL-17-driven autoimmunity in human diseases.

Autoimmune diseases such as rheumatoid arthritis and systemic lupus erythematosus are generally considered multifactorial-that is, they involve both genetic and environmental factors. Technical advances in human genetics over the past 5 years have enabled the survey of the entire human genome for disease susceptibility genes and have contributed to a greater understanding of the molecular mechanisms underlying autoimmunity. Among the genetic predisposition factors identified to date, some variants have been found to be restricted to specific ethnic groups, which might reflect migration history and the natural selection that shaped genetic variation in these populations. Other genetic factors could also have exerted different magnitudes of risk for the disease among the different populations, which might be explained by their interactions with other genetic and environmental factors. These pieces of evidence suggest that substantial heterogeneity exists in the genetics underlying autoimmunity among different ethnic populations. This Review discusses the genetic heterogeneity in autoimmunity, with a focus on rheumatoid arthritis, between Asian and European populations. In addition to the most-studied and well-characterized gene HLA-DRB1, we will also describe examples of the gene-environment interactions between PADI4 and smoking, and the gene-gene interactions between PTPN22 and FCRL3.

Objective: Genome-wide association (GWA) studies in systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) in Caucasian populations have independently identified risk variants in and near the tumor necrosis factor alpha (TNFalpha)-induced protein 3 gene (TNFAIP3), which is crucial for the regulation of TNF-mediated signaling and Toll-like receptor signaling. The aim of this study was to assess the role of TNFAIP3 in the development of SLE and RA in Japanese subjects., Methods: We selected 2 single-nucleotide polymorphisms (SNPs) from previous GWA studies. Rs2230926 is a nonsynonymous SNP in TNFAIP3 and is associated with SLE, while rs10499194 is an intergenic SNP associated with RA. We then performed 2 independent sets of SLE case-control comparisons (717 patients and 1,362 control subjects) and 3 sets of RA case-control comparisons (3,446 patients and 2,344 control subjects) using Japanese subjects. We genotyped SNPs using TaqMan assays., Results: We observed a significant association between rs2230926 and an increased risk of SLE and RA in the Japanese population (for SLE, odds ratio [OR] 1.92, 95% confidence interval [95% CI] 1.53-2.41, P = 1.9 x 10(-8); for RA, OR 1.35, 95% CI 1.18-1.56, P = 2.6 x 10(-5)). The intergenic SNP rs10499194 was also associated with SLE and RA, while the risk allele for RA in Caucasians was protective against the diseases in our population., Conclusion: We demonstrated a significant association between the nonsynonymous variant in TNFAIP3 and the risk for SLE and RA in the Japanese population. TNFAIP3, similar to STAT4 and IRF5, may be a common genetic risk factor for SLE and RA that is shared between the Caucasian and Japanese populations.

Objective: To examine the risk of anti-cyclic citrullinated peptide (anti-CCP) antibody positivity in rheumatoid arthritis (RA) patients carrying certain haplotypes in the HLA region., Methods: A total of 1,389 Japanese patients with RA were genotyped for 30 single-nucleotide polymorphisms (SNPs) in the HLA region using commercial oligonucleotide arrays (from Perlegen or Affymetrix) as well as for HLA-DRB1 alleles using a sequence-specific polymerase chain reaction method. Stepwise logistic regression was used to select from among the 30 SNPs the ones that represented a risk of anti-CCP antibody positivity. Haplotypes of the selected SNPs were inferred using an expectation-maximization algorithm. Associations of individual SNPs were evaluated with the Cochran-Armitage test for trend. DRB1 alleles and haplotypes were evaluated with the chi-square test. Heterogeneities of risks among the shared epitope (SE) and non-SE HLA-DRB1 alleles were examined using the exact test. Haplotype associations that were independent of individual HLA-DRB1 alleles were evaluated using the likelihood ratio test., Results: Significant associations were found for 9 SNPs (smallest P value being 2.4x10(-8)) and in 4 HLA-DRB1 alleles (smallest P value being 2.0x10(-10) in DRB1*0405). Stepwise logistic regression selected 4 SNPs (rs9262638, rs7775228, rs4713580, and rs9277359). Among the 16 inferred haplotypes of these 4 SNPs, 6 indicated significant associations (smallest P value being 1.9x10(-11)). Risks among SE and non-SE alleles were significantly heterogeneous (P=0.0095 and P=9.8x10(-9), respectively), indicating the importance of stratification with individual DRB1 alleles rather than SE alleles. Conditional analysis of the risk associated with individual DRB1 alleles identified a risk haplotype that was independent of DRB1 (odds ratio 2.00 [95% confidence interval 1.44-2.79], P=2.6x10(-5))., Conclusion: Heterogeneous risks of anti-CCP antibody positivity were confirmed among SE and non-SE alleles in our patient population. A risk haplotype in the HLA region that is independent of HLA-DRB1 was confirmed.

A new age has begun in the genetics of rheumatoid arthritis (RA), as genome-wide association studies scanning the human genome have been put into practical use. Among the RA-susceptibility genes identified by genetic studies, HLA-DRB1 gene appears to represent the most major determinant of genetic predisposition to RA. However, inconsistent results of the contributions of non-HLA susceptibility genes have been described, with the exception of a few genes repeatedly associated with RA-susceptibility, such as PTPN22 gene in populations of European ancestry and PADI4 gene in populations of Asian ancestry, revealing the presence of genetic heterogeneity in RA. We review herein recent advances in the genetics of RA and discuss the underlying differences among populations of European and Asian ancestries, taking as examples our previous findings for RA-susceptibility genes in the Japanese population: PADI4; FCRL3; and CD244.

Rheumatoid arthritis is a chronic autoimmune inflammatory disease with a complex genetic etiology. Members of the signaling lymphocyte activation molecule (SLAM) family carry out pivotal functions in innate immunity and in conventional lymphocytes. We identified a linkage disequilibrium block associated with rheumatoid arthritis in the chromosome 1q region containing multiple SLAM family genes. In this block, the association peaked at two functional SNPs (rs3766379 and rs6682654) in CD244 in two independent rheumatoid arthritis cohorts from Japan (P = 3.23 x 10(-8) and P = 7.45 x 10(-8)). We also identified a Japanese cohort with systemic lupus erythematosus that had a similar genotype distribution as the rheumatoid arthritis cohorts. We demonstrated that the rheumatoid arthritis-susceptible alleles of rs3766379 and rs6682654 and their haplotype increased their expression in luciferase and allele-specific transcript quantification assays. CD244 is a genetic risk factor for rheumatoid arthritis and may have a role in the autoimmune process shared by rheumatoid arthritis and systemic lupus erythematosus.

Objective: The SLC22A4 polymorphisms slc2F1 (rs2073838) and slc2F2 (rs3792876) are reported to be associated with rheumatoid arthritis (RA) in Japanese, but the associations have not been replicated. We assessed the RA susceptibility of slc2F1/F2 polymorphisms., Methods: We conducted a metaanalysis for slc2F1/F2 polymorphisms to RA susceptibility, which included the replication study of an independent Japanese population consisting of 924 cases and 940 controls. A total of 9 studies (4 Japanese studies, 5 Caucasian studies) consisting of 8076 cases and 6837 controls were included in the metaanalysis., Results: The replication study demonstrated significant associations in a Japanese population (OR 1.20, 95% CI 1.04-1.37, p = 0.0099, in the allelic mode; OR 1.29, 95% CI 1.08-1.55, p = 0.006, in the dominant mode; p = 0.011 in the trend mode). Significant ethnic diversities of allele frequencies of slc2F1/F2 polymorphisms were found (p = 8.6*10(-8)) between Caucasian and Japanese populations (0.07-0.08 and 0.30-0.32, respectively). The metaanalysis demonstrated significant associations for all studies (fixed-effect OR 1.11, 95% CI 1.05-1.18, p = 0.00084; random-effect OR 1.10, 95% CI 1.02-1.19, p = 0.017 in the allelic mode). Although subgroup analysis did not detect a significant association within Caucasian studies, significant associations were found within Japanese studies (fixed-effect and random-effect OR 1.16, 95% CI 1.07-1.25, p = 0.00012 in the allelic mode)., Conclusion: The associations in Caucasian studies were not significant. Since the significantly low frequency of the risk allele made statistical power lower in Caucasians than in Japanese, whether significant relative risks existed in Caucasian populations was inconclusive. The significant relative risks in Japanese populations were confirmed.

Large studies on the genetics of common rheumatic diseases, such as rheumatoid arthritis and systemic lupus erythematosus, have identified multiple polymorphisms related to disease susceptibility, including peptidylarginine deiminase 4 (PADI4) and protein tyrosine phosphatase N22 (PTPN22). Some of the identified genes are associated with multiple autoimmune disorders, and some seem to have unique associations with particular disease entities. Although the molecules encoded by these genes have a primary role in the molecular pathways of autoimmunity, genetic variations and contribution to disease susceptibility seem to vary between ethnic groups. In this Review, we report the findings on genes associated with rheumatoid arthritis and focus on the differences in the frequency of polymorphisms between various ethnic groups.

Rheumatoid arthritis (RA) is a complex, multifactorial disease with genetic and immunological aspects. Because RA is an autoimmune condition, dysregulation of the immune system is implied. Many linkage and association studies have also indicated that multiple genetic factors are associated with RA. Although the contribution of each genetic factor is small, the combination of these factors affects RA development. Previous studies have suggested that genetic changes affect the internal immunological environment, which results in autoimmune diseases. More recent genetic studies indicate that the HLA-DRB gene is the predominant cause of RA and that other non-HLA genes are also involved. We reported that peptidylarginine deiminase (gene name abbreviated to PADI, protein name abbreviated to PAD) type 4 is the one of the non-HLA genetic factors involved in RA via citrullination. Antibodies against citrullinated proteins/peptides are highly specific to RA, but the physiological roles of PADI gene, PAD proteins as their products and citrullinated proteins/peptides are obscure. However, levels of anticitrullinated protein antibodies are apparently also increased and were involved in the pathogenesis of autoimmune arthritis in mice with collagen-induced arthritis (CIA). These data suggested that citrullinated protein and anticitrullinated protein antibodies play important roles in the development of RA. This review summarizes the relationship between RA and citrullination, as well as the role of PADI4 genetics.

Antibodies against citrullinated proteins are highly specific for rheumatoid arthritis. We previously reported that functional variants of the gene encoding peptidylarginine deiminase type 4 were closely associated with RA. The purpose of this study was to investigate the citrullinated autoantigens recognized by serum samples from patients with RA. The human chondrocyte cDNA expression library was citrullinated by PADI4 and was immunoscreened with anti-modified citrulline antibodies and sera from patients with rheumatoid arthritis. One immunoreactive cDNA clone containing a 2480-base pair insert was isolated and sequence analysis revealed that the cDNA included a part of the eukaryotic translation initiation factor 4G1. Immunoreactivity against a recombinant citrullinated eIF4G1 fragment was observed with high specificity in 50.0% of RA patients. The levels of antibodies against citrullinated eIF4G1 were correlated with those of anti-CCP antibodies. Citrullinated eIF4G1 was identified as a candidate citrullinated autoantigen in RA patients. Citrullination of eIF4G1 may thus be involved in the pathogenesis of RA.

Rheumatoid arthritis (RA) is one of the most common autoimmune diseases, but its autoimmune mechanisms are not clearly understood. Recently, anti-citrullinated peptide antibodies have been specifically observed in sera of RA patients. Furthermore, we identified RA-susceptible variant in a gene encoding citrullinating enzyme, peptidylarginine deiminase type 4 (PADI4). Therefore, we hypothesized that proteins which are modified in RA synovium by PADI4 act as autoantigens. Subsequently, we obtained human collagen type I (huCI) as one of the autoantigens using a RA synoviocyte cDNA library by immunoscreening. We also investigated that the levels of anti-citrullinated huCI were significantly higher in RA patient sera than in normal control sera with high specificity (99%) and positively correlated with the levels of anti-cyclic citrullinated peptide (anti-CCP) antibodies. We concluded that huCI is a novel substrate protein of PADIs and that citrullinated huCI is a candidate autoantigen of RA.

Rheumatoid arthritis is a common autoimmune disease with a complex genetic etiology. Here we identify a SNP in the promoter region of FCRL3, a member of the Fc receptor-like family, that is associated with susceptibility to rheumatoid arthritis (odds ratio = 2.15, P = 0.00000085). This polymorphism alters the binding affinity of nuclear factor-kappaB and regulates FCRL3 expression. We observed high FCRL3 expression on B cells and augmented autoantibody production in individuals with the disease-susceptible genotype. We also found associations between the SNP and susceptibility to autoimmune thyroid disease and systemic lupus erythematosus. FCRL3 may therefore have a pivotal role in autoimmunity.

Anticitrullinated peptide antibodies seem to be one of the most clinically reliable serologic markers for rheumatoid arthritis (RA). A genetic approach revealed that one of the citrullinating enzymes has a RA-susceptible variant. Peptidyl citrullination alters the chemical character of peptides and, subsequently, their antigenicity as well. This change in antigenicity of self-peptides seems to invoke citrulline-related autoimmunity. Although the precise physiologic role of citrullination is still unknown, accumulating data indicate that citrullination has a definite role in biologic phenomena, along with other posttranslational protein modifications, such as methylation and phosphorylation. In RA synovial tissue, two of five PADI isotypes are known to be expressed, and their expression is regulated at multiple steps: transcription, translation, intracellular localization, and activation/inactivation of PADI proteins. Further investigations on citrulline and PADIs from various aspects will provide a more profound understanding of RA-related autoimmunity.

Because of the limitations of candidate gene studies and linkage analyses for common diseases, genome-wide association studies are now recognized as a powerful approach to mapping responsible genes with modest effects on various diseases. We performed whole genome case-control linkage disequilibrium (LD) mapping for rheumatoid arthritis (RA)-associated genes in Japanese subjects using single nucleotide polymorphisms (SNPs) mainly discovered in gene-containing regions. We identified RA-associated polymorphisms in two genes/loci, PADI4 and SLC22A4/A5 cluster. PADI4 catalyzes the conversion of arginine residues to citrulline in proteins. Recent reports on the high specificity of autoantibodies against citrullinated proteins to RA and the results of our study suggest that citrullination by PADI4 is a fundamental phenomenon of RA. On the other hand, the functions of SLC22A4/A5 have not been studied in detail, but SLC22A4/A5 have been reported to have multiple polymorphisms associated with several autoimmune diseases. Thus, large-scale LD mapping appears to be effective for identifying RA-associated polymorphisms.

Citrullinated proteins that are produced by enzymatic deimination of arginine residues in proteins by peptidylarginine deiminases (PADIs) are of particular interest in the pathogenesis of rheumatoid arthritis (RA). First, peptidylarginine deiminase type 4 (PADI4) gene, which codes one of the PADI enzyme isotypes, has a genetic variant that increases susceptibility to RA. The RA-susceptible variant of PADI4 seems to increase the risk of RA by increasing its enzymatic activity. Second, this post-translational protein modification unfolds proteins by loss of a positive charge in arginine residues, with a subsequent change in antigenicity of the self-proteins. Third, these citrullinated proteins are recognized by anti-citrullinated peptide antibodies that are the most RA-specific autoantibodies. Finally, the expression of the PADI enzyme, citrullination of proteins, and production of anti-citrullinated protein antibodies occur in synovium. These data suggest that citrullination of proteins by PADI is related to alteration of antigenicity of peptides and very closely linked to pathogenesis of RA autoimmunity.

Thrombin is a key factor in the stimulation of fibrin deposition, angiogenesis, proinflammatory processes, and proliferation of fibroblast-like cells. Abnormalities in these processes are primary features of rheumatoid arthritis (RA) in synovial tissues. Tissue destruction in joints causes the accumulation of large quantities of free hyaluronic acid (HA) in RA synovial fluid. The present study was conducted to investigate the effects of HA and several other glycosaminoglycans on antithrombin, a plasma inhibitor of thrombin. Various glycosaminoglycans, including HA, chondroitin sulfate, keratan sulfate, heparin, and heparan, were incubated with human antithrombin III in vitro. The residual activity of antithrombin was determined using a thrombin-specific chromogenic assay. HA concentrations ranging from 250 to 1000 mug/ml significantly blocked the ability of antithrombin to inhibit thrombin in the presence of Ca2+ or Fe3+, and chondroitin A, B and C also reduced this ability under the same conditions but to a lesser extent. Our study suggests that the high concentration of free HA in RA synovium may block antithrombin locally, thereby deregulating thrombin activity to drive the pathogenic process of RA under physiological conditions. The study also helps to explain why RA occurs and develops in joint tissue, because the inflamed RA synovium is uniquely rich in free HA along with extracellular matrix degeneration. Our findings are consistent with those of others regarding increased coagulation activity in RA synovium.

Recently we reported that SLC22A4 and RUNX1 are associated with rheumatoid arthritis (RA). SLC22A4 is an organic cation transporter with unknown physiological function, and RUNX1 is a hematological transcriptional regulator that has been shown to be responsible for acute myelogenic leukemia. It is suggested that the association of RUNX1 with RA is due to its regulation of expression of SLC22A4. Because the physiological function of SLC22A4 is still unclear, further investigation is needed into how SLC22A4 affects RA susceptibility. Although the association of RUNX1 with RA was identified as a regulatory factor of SLC22A4, it is possible that RUNX1 is a key molecule in autoimmunity, as it has been reported to be associated with systemic lupus erythematosus and psoriasis, two other autoimmune diseases.

Objective: To examine the entire HLA region for loci (other than the DRB1 locus) associated with rheumatoid arthritis (RA) susceptibility, by typing HLA-DRB1 alleles and multiple single-nucleotide polymorphisms (SNPs) in the Japanese population., Methods: The HLA-DRB1 alleles and 88 SNPs distributed over the HLA gene complex were genotyped, for 828 patients with RA and 1,032 control subjects. The data were evaluated for linkage disequilibrium, and case-control associations were analyzed in 2 ways, in the presence or absence of the disease-susceptibility DRB1 allele, to detect loci independent of the DRB1 allele., Results: HLA-DRB1 alleles *0405, *0401, *0901, *0101, *1401, *1602, *0403, and *1405 were significantly associated with RA in the Japanese population. The smallest P value (P = 1.4 x 10(-27)) was observed in association with an intronic SNP of the NOTCH4 gene, which was due to strong linkage disequilibrium with the HLA-DRB1 allele. A strong association that was independent of HLA-DRB1 shared epitope alleles was observed in 2 SNPs: one in the intron of the MICA gene, the other in the intron of the HLA-DQB2 gene. Their association with RA, independent of HLA-DRB1 shared epitope alleles, was suggestive (P = 0.0024 [corrected P (P(corr)) = 0.068, and P = 0.00037 [P(corr) = 0.012], respectively)., Conclusion: These findings suggest that 1 or more other loci besides the HLA-DRB1 or other DRB1 (non-shared epitope, non-*0901) alleles are involved in RA susceptibility/protection.

Rheumatoid arthritis is a common inflammatory disease with complex genetic components. We investigated the genetic contribution of the cytokine gene cluster in chromosome 5q31 to susceptibility to rheumatoid arthritis in the Japanese population by case-control linkage disequilibrium (LD) mapping using single nucleotide polymorphisms (SNPs). Here we report that there is significant association between rheumatoid arthritis and the organic cation transporter gene SLC22A4 (P = 0.000034). We show that expression of SLC22A4 is specific to hematological and immunological tissues and that SLC22A4 is also highly expressed in the inflammatory joints of mice with collagen-induced arthritis. A SNP affects the transcriptional efficiency of SLC22A4 in vitro, owing to an allelic difference in affinity to Runt-related transcription factor 1 (RUNX1), a transcriptional regulator in the hematopoietic system. A SNP in RUNX1 is also strongly associated with rheumatoid arthritis (P = 0.00035). Our data indicate that the regulation of SLC22A4 expression by RUNX1 is associated with susceptibility to rheumatoid arthritis, which may represent an example of an epistatic effect of two genes on this disorder.

Recent studies using linkage disequilibrium and SNPs uncovered a rheumatoid arthritis (RA)-susceptible haplotype in the gene encoding peptidylarginine deiminase (PADI) type 4. This gene is one of four known PADI genes that encode enzymes to change arginine into citrulline in proteins. Post-translational modifications of proteins, including peptidyl citrullination, are related to autoimmunity, and peptidyl citrulline is a known target of one of the most RA-specific autoantibodies. Further research on PADI4, its citrullination of native peptides, subsequent breakdown of tolerance, and the role of these peptides in the development of RA, is expected to bring a better understanding of autoimmunity and arthritis, and advancements in the medical care of RA.

Individuals with rheumatoid arthritis frequently have autoantibodies to citrullinated peptides, suggesting the involvement of the peptidylarginine deiminases citrullinating enzymes (encoded by PADI genes) in rheumatoid arthritis. Previous linkage studies have shown that a susceptibility locus for rheumatoid arthritis includes four PADI genes but did not establish which PADI gene confers susceptibility to rheumatoid arthritis. We used a case-control linkage disequilibrium study to show that PADI type 4 is a susceptibility locus for rheumatoid arthritis (P = 0.000008). PADI4 was expressed in hematological and rheumatoid arthritis synovial tissues. We also identified a haplotype of PADI4 associated with susceptibility to rheumatoid arthritis that affected stability of transcripts and was associated with levels of antibody to citrullinated peptide in sera from individuals with rheumatoid arthritis. Our results imply that the PADI4 haplotype associated with susceptibility to rheumatoid arthritis increases production of citrullinated peptides acting as autoantigens, resulting in heightened risk of developing the disease.

The aim of this study was to prepare a DNA immunoadsorbent for the specific, extracorporeal removal of anti-DNA antibodies from the blood of patients with systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA). Two kinds of cellulose beads were applied as a carrier. Calf thymus DNA was covalently coupled to the carrier using the epichlorohydrin method. Efforts were focused on optimization of conditions for activation and coupling, trying to couple as much DNA as possible to a certain amount of carrier. It was found that the activation level increased with the increase of NaOH concentration and the amount of epichlorohydrin used. The mole of epichlorohydrin must be in excess of that of NaOH because excess NaOH could react further with the epoxy groups in the beads resulting in a decrease of activation level. High activation level could be obtained in a medium of 3.0 M NaOH. The DNA coupling was found to be mainly temperature and pH dependent. Using 0.1 M Tris-HCl buffer, pH 8 at a temperature of 50-90 degrees C, more than 3 mg of DNA could be coupled to 1 ml of wet beads. Prolonging the coupling reaction under 50 degrees C to 72 h resulted in the same coupling capacity as that obtained under 90 degrees C. To evaluate the adsorption ability for anti-DNA of this immunoadsorbent, batch and circulation tests were applied using SLE patient plasma. The immunoadsorbents showed excellent adsorption capacity, especially the cellulose with smaller size (200-300 microm). The incubation of 20 ml of patient's plasma with 1 ml of adsorbent resulted in an 80% decline in the anti-DNA antibody level. In the circulation tests, 30 ml of plasma was circulated through a column containing 3 ml of adsorbent. The maximum decline in anti-DNA level, 80%, was obtained after 60 min. Such high adsorption capacity and high adsorption rate suggest this immunoadsorbent may be used for treatment. For comparison, 1,4-butanediol diglycidyl ether activation method and other DNA sources were tested with the same protocol.