Gerrie Stoeken-Rijsbergen

Value of anti-modified citrullinated vimentin and third-generation anti-cyclic citrullinated peptide compared with second-generation anti-cyclic citrullinated peptide and rheumatoid factor in predicting disease outcome in undifferentiated arthritis and rheumatoid arthritis.

OBJECTIVE Autoantibodies such as rheumatoid factor (RF) and anti-citrullinated protein autoantibodies (ACPAs) determined by testing with second-generation anti-cyclic citrullinated peptide (anti-CCP-2) are frequently measured in clinical practice because of their association with disease outcome in undifferentiated arthritis (UA) and rheumatoid arthritis (RA). Recently, 2 new ACPA tests were developed: third-generation anti-CCP (anti-CCP-3) and anti-modified citrullinated vimentin (anti-MCV) autoantibody tests. To facilitate the decision on which autoantibody to test in daily practice, this study evaluated the capability of these autoantibodies and combinations of them to predict 3 outcome measures: progression from UA to RA, the rate of joint destruction in RA, and the chance of achieving sustained disease-modifying antirheumatic drug (DMARD)-free remission in RA. METHODS Patients with UA (n=625) were studied for whether UA progressed to RA after 1 year. Patients with RA (n=687) were studied for whether sustained DMARD-free remission was achieved and for the rate of joint destruction during a median followup of 5 years. Positive predictive values (PPVs) for RA development and for associations with the disease course in RA were compared between single tests (anti-CCP-2, anti-CCP-3, anti-MCV, and RF) and between combinations of these tests. RESULTS Among the single tests performed in patients with UA, anti-CCP-2 tended to have the highest PPV for RA development (67.1%), but the 95% confidence intervals of the other tests overlapped. Among the single tests in patients with RA, all 4 tests showed comparable associations with the rate of joint destruction and with the achievement of remission. In both ACPA-positive and ACPA-negative RA, the presence of RF was not associated with more joint destruction. For all outcome measures, performing combinations of 2 or 3 autoantibody tests did not increase the predictive accuracy compared with performing a single test. CONCLUSION For clinical practice, a single autoantibody test is sufficient for risk estimation in UA and RA.

Confirmation of STAT4, IL2/IL21, and CTLA4 polymorphisms in rheumatoid arthritis.

OBJECTIVE Recent advances have led to novel identification of genetic polymorphisms that are associated with susceptibility to rheumatoid arthritis (RA). Currently, 5 loci (HLA, PTPN22, TRAF1/C5, TNFAIP3, and STAT4) have been consistently reported, whereas others have been observed less systematically. The aim of the present study was to independently replicate 3 recently described RA susceptibility loci, STAT4, IL2/IL21, and CTLA4, in a large Dutch case-control cohort, and to perform a meta-analysis of all published studies to date and investigate the relevance of the findings in clinically well-defined subgroups of RA patients with or without autoantibodies. METHODS The STAT4, IL2/IL21, and CTLA4 gene polymorphisms (rs7574865, rs6822844, and rs3087243, respectively) were genotyped in 877 RA patients and 866 healthy individuals. A meta-analysis of all published studies of disease association with these polymorphisms was performed using the Mantel-Haenszel fixed-effects method. RESULTS An association of STAT4, IL2/IL21, and CTLA4 with RA was detected in Dutch patients (odds ratio [OR] 1.19 [P=0.031], OR 0.84 [P=0.051], and OR 0.87 [P=0.041], respectively). Results from the meta-analysis confirmed an association of all 3 polymorphisms with RA in Caucasians (OR 1.24 [P=1.66x10(-11)], OR 0.78 [P=5.6x10(-5)], and OR 0.91 [P=1.8x10(-3)], respectively). The meta-analysis also revealed that STAT4 predisposed to disease development equally in patients with autoantibodies and those without autoantibodies, and that CTLA4 enhanced the development of anti-citrullinated protein antibody (ACPA)-positive RA as compared with ACPA-negative RA. CONCLUSION Our results replicate and firmly establish the association of STAT4 and CTLA4 with RA and provide highly suggestive evidence for IL2/IL21 loci as a risk factor for RA. Given the strong statistical power of our meta-analysis to confirm a true-positive association, these findings provide considerable support for the involvement of CTLA4 in distinct subsets of RA patients.

Association of the autoimmunity locus 4q27 with juvenile idiopathic arthritis

OBJECTIVE Juvenile idiopathic arthritis (JIA) is characterized by chronic arthritis and an autoimmune etiology. In several autoimmune diseases, including rheumatoid arthritis (RA), an association with the 4q27 locus has been reported. We undertook this study to investigate the possible role of the 4q27 locus in JIA. METHODS A case-control association study was conducted, with a total of 655 Caucasian JIA patients and 791 healthy controls divided into 2 independent sample sets. The rs6822844 marker in the 4q27 locus was genotyped. RESULTS In the first and larger sample set, a 5% decrease in T allele frequency was observed in patients compared with controls (allelic odds ratio [OR] 0.72 [95% confidence interval 0.55-0.95], P = 0.019), and in the second set, a 3% decrease was observed (allelic OR 0.81 [95% confidence interval 0.61-1.09], P = 0.169). The combined data set generated an OR of 0.76 (95% confidence interval 0.62-0.93, P = 7.08 x 10(-3)). When the different JIA subtypes were analyzed individually, significant decreases were seen in the subtypes with a polyarticular course of disease (extended oligoarthritis [P = 0.019] and rheumatoid factor-negative polyarthritis [P = 0.038]). CONCLUSION Our findings suggest that the 4q27 locus, previously reported to be associated with RA, type 1 diabetes mellitus, celiac disease, and psoriatic arthritis, is also associated with susceptibility to JIA.

Identification of a genetic variant for joint damage progression in autoantibody-positive rheumatoid arthritis

Background Joint destruction is a hallmark of autoantibody-positive rheumatoid arthritis (RA), though the severity is highly variable between patients. The processes underlying these interindividual differences are incompletely understood. Methods We performed a genome-wide association study on the radiological progression rate in 384 autoantibody-positive patients with RA. In stage-II 1557 X-rays of 301 Dutch autoantibody-positive patients with RA were studied and in stage-III 861 X-rays of 742 North American autoantibody-positive patients with RA. Sperm-Associated Antigen 16 (SPAG16) expression in RA synovium and fibroblast-like synoviocytes (FLS) was examined using Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR) and immunohistochemistry. FLS secrete metalloproteinases that degrade cartilage and bone. SPAG16 genotypes were related to matrix metalloproteinase (MMP)-3 and MMP-1 expression by FLS in vitro and MMP-3 production ex vivo. Results A cluster of single nucleotide polymorphisms (SNPs) at 2q34, located at SPAG16, associated with the radiological progression rate; rs7607479 reached genome-wide significance. A protective role of rs7607479 was replicated in European and North American patients with RA. Per minor allele, patients had a 0.78-fold (95% CI 0.67 to 0.91) progression rate over 7 years. mRNA and protein expression of SPAG16 in RA synovium and FLS was verified. FLS carrying the minor allele secreted less MMP-3 (p=1.60×10−2). Furthermore, patients with RA carrying the minor allele had lower serum levels of MMP-3 (p=4.28×10−2). In a multivariate analysis on rs7607479 and MMP-3, only MMP-3 associated with progression (p=2.77×10−4), suggesting that the association between SPAG16-rs7607479 and joint damage is mediated via an effect on MMP-3 secretion. Conclusions Genetic and functional analyses indicate that SPAG16 influences MMP-3 regulation and protects against joint destruction in autoantibody-positive RA. These findings could enhance risk stratification in autoantibody-positive RA.

Genetic variants in the region of the C1q genes are associated with rheumatoid arthritis

Rodent models for arthritis implicate a role for complement in disease development and progression. In humans, complement deposition has been observed in inflamed synovia of rheumatoid arthritis (RA) patients. In this study we analysed whether genetic variants of complement component C1q predispose to RA. We genotyped single nucleotide polymorphisms (SNPs) in and around the C1q genes, C1qA, C1qB and C1qC, in a Dutch set of 845 RA cases and 1046 controls. Replication was sought in a sample set from North America (868 cases/1193 controls), and a meta‐analysis was performed in a combined samples set of 8000 cases and 23 262 controls of European descent. We determined C1q serum levels in relation to C1q genotypes. In the discovery phase, five of the 13 SNPs tested in the C1q genes showed a significant association with RA. Additional analysis of the genomic area around the C1q genes revealed that the strongest associating SNPs were confined to the C1q locus. Within the C1q locus we observed no additional signal independent of the strongest associating SNP, rs292001 [odds ratio (OR) = 0·72 (0·58–0·88), P = 0·0006]. The variants of this SNP were associated with different C1q serum levels in healthy controls (P = 0·006). Interestingly, this SNP was also associated significantly in genome‐wide association studies (GWAS) from the North American Rheumatoid Arthritis Consortium study, confirming the association with RA [OR = 0·83 (0·69–1·00), P = 0·043]. Combined analysis, including integrated data from six GWAS studies, provides support for the genetic association. Genetic variants in C1q are correlated with C1q levels and may be a risk for the development of RA.

The concentration of anticitrullinated protein antibodies in serum and synovial fluid in relation to total immunoglobulin concentrations

Background Anticitrullinated protein antibodies (ACPA) are one of the best predictors for the development of rheumatoid arthritis. Nonetheless, relatively little information is present on the absolute concentration of ACPA in relation to total immunoglobulin (Ig) concentrations. Such information would be of relevance to compare ACPA levels to other antibody levels. Here, we estimated the relative abundance of ACPA Ig in serum and synovial fluid using a quantitative approach. Methods ACPA were purified using HiTrap Streptavidin columns coupled with biotinylated cyclic citrullinated peptide (CCP2). Total Ig and anti-CCP2 isotype reactivities were measured by ELISA. Results ACPA were successfully isolated as substantial antibody amounts were eluted from sera of ACPA-positive patients and neglectable antibody amounts were eluted from sera of ACPA-negative patients. Up to 1 in 80 IgG-molecules were estimated to be ACPA. Strikingly, IgM-ACPA was most abundant in synovial fluid (with the highest enrichment in the range of one IgM-ACPA for every eight IgM-antibodies). Conclusions ACPA-IgG levels are estimated to be within the range of peak levels of protective antibody responses against recall antigens. IgM-ACPA is abundantly present in synovial fluid, suggesting the presence of a continuous ongoing autoimmune response in the synovial compartment.

The fine specificity of IgM anti-citrullinated protein antibodies (ACPA) is different from that of IgG ACPA

IntroductionThe antigen recognition pattern of immunoglobulin M (IgM) could, when directed against protein antigens, provide an indication of the antigenic moieties triggering new B cells. The half-life of IgM is short and memory B cells against T-cell-dependent protein antigens typically produce IgG and not IgM antibodies. In this study, we analyzed whether a difference exists between the fine specificity of IgM versus IgG anti-citrullinated protein antibodies (ACPAs).MethodsWe determined the fine specificity of IgM and IgG ACPAs in 113 ACPA-positive rheumatoid arthritis patients with IgM cyclic citrullinated peptide 2 (CCP2) levels above 100 AU/ml. Fine specificity was assessed by performing ELISA using citrullinated peptides derived from vimentin, fibrinogen-β, fibrinogen-α and α-enolase, as well as citrullinated proteins fibrinogen and myelin basic protein. The arginine counterparts were used as controls.ResultsRecognition of defined citrullinated antigens by IgM ACPA was confined to samples that also displayed recognition by IgG ACPA. However, the IgM ACPA response displayed a more restricted antigen recognition profile than IgG ACPA (OR = 0.26, P < 0.0001).ConclusionOur data show that several defined citrullinated antigens are recognized only by IgG ACPA, whereas others are also recognized by IgM ACPA. These observations suggest that not all citrullinated antigens are able to activate new B cells despite concurrent recognition by IgG ACPA.

The major risk alleles of age-related macular degeneration (AMD) in CFH do not play a major role in rheumatoid arthritis (RA)

Because activation of the alternative pathway (AP) of the complement system is an important aspect of both age‐related macular degeneration (AMD) and rheumatoid arthritis (RA), we wished to address the question whether genetic risk factors of the AP inhibitor complement factor H (CFH) for AMD would also be risk factors for RA. For this purpose we genotyped single nucleotide polymorphisms (SNPs) in a Dutch set of RA patients and controls. Similarly, a meta‐analysis using a Spanish cohort of RA as well as six large genome‐wide association studies (GWAS) studies was performed. For these SNPs we analysed more than 6000 patients and 20 000 controls. The CFH variants, I62V, Y402H, IVS1 and IVS10, known to associate strongly with AMD, did not show a significant association with the risk of developing RA despite a strong statistical power to detect such differences. In conclusion, the major risk alleles of AMD in CFH do not have a similar effect on developing RA.

The interaction between HLA SE alleles and smoking and its contribution to autoimmunity against several citrullinated antigens

Objective Recent data suggest that a gene-environment interaction exists between smoking and the HLA SE alleles in shaping the autoimmune reaction towards specific citrullinated antigens. Here, we determined the effects of HLA SE alleles and TE (tobacco exposure) on the immune response against various citrullinated antigens. These associations were analysed in the anticitrullinated peptide antibody (ACPA) - positive stratum to control for the possibility that found associations are explained by the known interaction between HLA SE alleles and TE on the ACPA status. Methods The reactivity of 661 RA patients against several citrullinated antigens from vimentin, fibrinogen, enolase and myelin basic protein was determined by ELISA. The effects of the HLA SE alleles and TE were assessed by logistic regression analysis. Biologic interaction was analysed by investigating if the effects of the risk factors combined exhibited departure from additivity. Results A significant interaction between TE and HLA SE alleles appeared present for the presence of ACPA as reported previously. When these interaction effects were studied for several ACPA ‘fine-specificities’ significant interactions were noted for several citrullinated peptides. However these interactions were not present after stratification for ACPA status, indicating that the interaction between TE and HLA SE alleles does not influence autoimmunity towards specific citrullinated antigens, but rather to ACPA development. Conclusion Our data indicate that the gene-environment interaction between HLA SE alleles and smoking does not shape the reactivity of the ACPA response.

Genetic variants of C1q are a risk for rheumatoid arthritis

Background and objectives The recognition molecule of the classical pathway of complement activation, C1q, is next to its role in complement activation also implicated in protection against autoimmunity. Complete genetic deficiency of C1q predisposes strongly to autoimmunity, culminating in a Systemic Lupus Erythematosus-like disease. In this study the authors analysed whether genetic variants of C1q would predispose to rheumatoid arthritis (RA). Materials and methods For this purpose the authors genotyped single-nucleotide polymorphisms (SNPs) in C1q in a Dutch set of RA patients and controls. The authors obtained replication in the North American RA Cohort (NARAC) and performed a meta-analysis on data from several cohorts. Results In the discovery phase, the authors tested 13 SNPs in the C1q genes, of which 5 showed a significant association with RA. A calculation for global significance revealed a significant association for the C1q gene as a whole, which was replicated in the NARAC cohort. Further meta-analyses on several cohorts displayed a similar trend. Finally the authors screened the C1q locus in order to fine map the association, and found a set of four SNPs that associate with RA and associate with C1q serum levels in healthy individuals. Conclusions Genetic variants in C1q, some of which associate with C1q levels, are a risk for the development of RA.