Affinity of phenolic compounds for transition metal ions immobilized on cation-exchange columns.

Immobilized metal ion affinity chromatography (IMAC) is useful in purification of histidine-tagged or histidine-rich proteins and peptides from a variety of hosts. However, phenolic compounds including polyphenols interfere with IMAC due to their high affinities for the transition metals immobilized on the column resins, which hampers the purification of proteins from plant-based host systems. In contrast to extensive knowledge of the mechanism of the interactions between phenolic compounds and transition metal ions in solution, an understanding of the interactions on the columns, where transition metal ions are immobilized on the resins, remains elusive. This study systematically investigated the affinity of phenolic compounds for transition metal ions by varying the number and position of phenolic hydroxyl groups (OH groups) and using different transition metals-Fe(II), Cu(II) and Ni(II)-on various IMACs, in which the columns were fabricated by equilibrating the cation-exchange column with transition metal solutions. It was found that the more OH groups the aromatic compounds have, the higher the affinity for transition metal ions; in particular, methyl gallate and pyrogallol were permanently bound to the IMAC column, which reflected coordinate bond formation with the transition metal ions. Importantly, the phenolic compounds showed no obvious affinity for the Ni(II)-IMAC column, in contrast to the Fe(II)- and Cu(II)-IMAC columns, whereas imidazole and histidine-tagged proteins showed evident binding to the Ni(II)-IMAC column. Ni(II)-IMAC should thus be especially effective in isolating histidine-tagged and histidine-rich species from phenolic compound-containing systems. These results indicate that the affinity between phenolic compounds and transition metal ions on the column is consistent with the results in solution. They also provide a comprehensive view for devising strategies to improve IMAC purification of target proteins and peptides from samples containing phenolic compounds.

Mast cells in early rheumatoid arthritis associate with disease severity and support B cell autoantibody production.

OBJECTIVES: Mast cells (MCs) are involved in the pathogenesis of rheumatoid arthritis (RA). However, their contribution remains controversial. To establish their role in RA, we analysed their presence in the synovium of treatment-naïve patients with early RA and their association and functional relationship with histological features of synovitis. METHODS: Synovial tissue was obtained by ultrasound-guided biopsy from treatment-naïve patients with early RA (n=99). Immune cells (CD3/CD20/CD138/CD68) and their relationship with CD117+MCs in synovial tissue were analysed by immunohistochemistry (IHC) and immunofluorescence (IF). The functional involvement of MCs in ectopic lymphoid structures (ELS) was investigated in vitro, by coculturing MCs with naïve B cells and anticitrullinated protein antibodies (ACPA)-producing B cell clones, and in vivo in interleukin-27 receptor alpha (IL27ra)-deficient and control mice during antigen-induced arthritis (AIA). RESULTS: High synovial MC counts are associated with local and systemic inflammation, autoantibody positivity and high disease activity. IHC/IF showed that MCs reside at the outer border of lymphoid aggregates. Furthermore, human MCs promote the activation and differentiation of naïve B cells and induce the production of ACPA, mainly via contact-dependent interactions. In AIA, synovial MC numbers increase in IL27ra deficient mice, in association with ELS and worse disease activity. CONCLUSIONS: Synovial MCs identify early RA patients with a severe clinical form of synovitis characterised by the presence of ELS.

The production and secretion of complement component C1q by human mast cells.

C1q is the initiation molecule of the classical pathway of the complement system and is produced by macrophages and immature dendritic cells. As mast cells share the same myeloid progenitor cells, we have studied whether also mast cells can produce and secrete C1q. Mast cells were generated in vitro from CD34+ progenitor cells from buffy coats or cord blood. Fully differentiated mast cells were shown by both RNA sequencing and qPCR to express C1QA, C1QB and C1QC. C1q produced by mast cells has a similar molecular make-up as serum C1q. Reconstituting C1q depleted serum with mast cell supernatant in haemolytic assays, indicated that C1q secreted by mast cells is functionally active. The level of C1q in supernatants produced under basal conditions was considerably enhanced upon stimulation with LPS, dexamethasone in combination with IFN- γ or via FcεRI triggering. Mast cells in human tissues stained positive for C1q in both healthy and in inflamed tissue. Moreover, mast cells in healthy and diseased skin appear to be the predominant C1q positive cells. Together, our data reveal that mast cells are able to produce and secrete functional active C1q and indicate mast cells as a local source of C1q in human tissue.

Identification of a novel non-coding mutation in C1qB in a Dutch child with C1q deficiency associated with recurrent infections.

INTRODUCTION: C1q deficiency is a rare genetic disorder that is strongly associated with development of systemic lupus erythematosus (SLE). Several mutations in the coding regions of the C1q genes have been described that result in stop-codons or other genetic abnormalities ultimately leading to C1q deficiency. Here we report on a Dutch boy suffering from recurrent infections with a complete C1q deficiency, without any SLE symptoms. METHODS: The presence of C1q in serum was assessed using ELISA and hemolytic assay. By western blot we examined the different C1q chains in cell lysates. We identified the mutation using deep-sequencing. By qPCR we studied the mRNA expression of C1qA, C1qB and C1qC in the PBMCs of the patient. RESULTS: Deep-sequencing revealed a homozygous mutation in the non-coding region of C1qB in the patient, whereas both parents were heterozygous. The mutation is located two nucleotides before the splice site of the second exon. In-silico analyses predict a complete abrogation of this natural splice site. Analyses of in vitro cultured cells from the patient revealed a lack of production of C1q and intracellular absence of C1qB in the presence of C1qA and C1qC peptides. Quantitative PCR analysis revealed total absence of C1qB mRNA, a reduced level of C1qA mRNA and normal levels of C1qC mRNA. CONCLUSION: In this study we report a new mutation in the non-coding region of C1qB that is associated with C1q deficiency.

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.

Bayesian inference analyses of the polygenic architecture of rheumatoid arthritis.

The genetic architectures of common, complex diseases are largely uncharacterized. We modeled the genetic architecture underlying genome-wide association study (GWAS) data for rheumatoid arthritis and developed a new method using polygenic risk-score analyses to infer the total liability-scale variance explained by associated GWAS SNPs. Using this method, we estimated that, together, thousands of SNPs from rheumatoid arthritis GWAS explain an additional 20% of disease risk (excluding known associated loci). We further tested this method on datasets for three additional diseases and obtained comparable estimates for celiac disease (43% excluding the major histocompatibility complex), myocardial infarction and coronary artery disease (48%) and type 2 diabetes (49%). Our results are consistent with simulated genetic models in which hundreds of associated loci harbor common causal variants and a smaller number of loci harbor multiple rare causal variants. These analyses suggest that GWAS will continue to be highly productive for the discovery of additional susceptibility loci for common diseases.

Meta-analysis identifies nine new loci associated with rheumatoid arthritis in the Japanese population.

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.

Use of a multiethnic approach to identify rheumatoid- arthritis-susceptibility loci, 1p36 and 17q12.

We have previously shown that rheumatoid arthritis (RA) risk alleles overlap between different ethnic groups. Here, we utilize a multiethnic approach to show that we can effectively discover RA risk alleles. Thirteen putatively associated SNPs that had not yet exceeded genome-wide significance (p < 5 × 10(-8)) in our previous RA genome-wide association study (GWAS) were analyzed in independent sample sets consisting of 4,366 cases and 17,765 controls of European, African American, and East Asian ancestry. Additionally, we conducted an overall association test across all 65,833 samples (a GWAS meta-analysis plus the replication samples). Of the 13 SNPs investigated, four were significantly below the study-wide Bonferroni corrected p value threshold (p < 0.0038) in the replication samples. Two SNPs (rs3890745 at the 1p36 locus [p = 2.3 × 10(-12)] and rs2872507 at the 17q12 locus [p = 1.7 × 10(-9)]) surpassed genome-wide significance in all 16,659 RA cases and 49,174 controls combined. We used available GWAS data to fine map these two loci in Europeans and East Asians, and we found that the same allele conferred risk in both ethnic groups. A series of bioinformatic analyses identified TNFRSF14-MMEL1 at the 1p36 locus and IKZF3-ORMDL3-GSDMB at the 17q12 locus as the genes most likely associated with RA. These findings demonstrate empirically that a multiethnic approach is an effective strategy for discovering RA risk loci, and they suggest that combining GWASs across ethnic groups represents an efficient strategy for gaining statistical power.

Meta-analysis of genome-wide association studies in celiac disease and rheumatoid arthritis identifies fourteen non-HLA shared loci.

Epidemiology and candidate gene studies indicate a shared genetic basis for celiac disease (CD) and rheumatoid arthritis (RA), but the extent of this sharing has not been systematically explored. Previous studies demonstrate that 6 of the established non-HLA CD and RA risk loci (out of 26 loci for each disease) are shared between both diseases. We hypothesized that there are additional shared risk alleles and that combining genome-wide association study (GWAS) data from each disease would increase power to identify these shared risk alleles. We performed a meta-analysis of two published GWAS on CD (4,533 cases and 10,750 controls) and RA (5,539 cases and 17,231 controls). After genotyping the top associated SNPs in 2,169 CD cases and 2,255 controls, and 2,845 RA cases and 4,944 controls, 8 additional SNPs demonstrated P<5 × 10(-8) in a combined analysis of all 50,266 samples, including four SNPs that have not been previously confirmed in either disease: rs10892279 near the DDX6 gene (P(combined) =  1.2 × 10(-12)), rs864537 near CD247 (P(combined) =  2.2 × 10(-11)), rs2298428 near UBE2L3 (P(combined) =  2.5 × 10(-10)), and rs11203203 near UBASH3A (P(combined) =  1.1 × 10(-8)). We also confirmed that 4 gene loci previously established in either CD or RA are associated with the other autoimmune disease at combined P<5 × 10(-8) (SH2B3, 8q24, STAT4, and TRAF1-C5). From the 14 shared gene loci, 7 SNPs showed a genome-wide significant effect on expression of one or more transcripts in the linkage disequilibrium (LD) block around the SNP. These associations implicate antigen presentation and T-cell activation as a shared mechanism of disease pathogenesis and underscore the utility of cross-disease meta-analysis for identification of genetic risk factors with pleiotropic effects between two clinically distinct diseases.

Genome-wide association study meta-analysis identifies seven new rheumatoid arthritis risk loci.

To identify new genetic risk factors for rheumatoid arthritis, we conducted a genome-wide association study meta-analysis of 5,539 autoantibody-positive individuals with rheumatoid arthritis (cases) and 20,169 controls of European descent, followed by replication in an independent set of 6,768 rheumatoid arthritis cases and 8,806 controls. Of 34 SNPs selected for replication, 7 new rheumatoid arthritis risk alleles were identified at genome-wide significance (P < 5 x 10(-8)) in an analysis of all 41,282 samples. The associated SNPs are near genes of known immune function, including IL6ST, SPRED2, RBPJ, CCR6, IRF5 and PXK. We also refined associations at two established rheumatoid arthritis risk loci (IL2RA and CCL21) and confirmed the association at AFF3. These new associations bring the total number of confirmed rheumatoid arthritis risk loci to 31 among individuals of European ancestry. An additional 11 SNPs replicated at P < 0.05, many of which are validated autoimmune risk alleles, suggesting that most represent genuine rheumatoid arthritis risk alleles.

TRAF1/C5 polymorphism is not associated with increased mortality in rheumatoid arthritis: two large longitudinal studies.

INTRODUCTION: Recently an association between a genetic variation in TRAF1/C5 and mortality from sepsis or cancer was found in rheumatoid arthritis (RA). The most prevalent cause of death, cardiovascular disease, may have been missed in that study, since patients were enrolled at an advanced disease stage. Therefore, we used an inception cohort of RA patients to investigate the association between TRAF1/C5 and cardiovascular mortality, and replicate the findings on all-cause mortality. As TRAF1/C5 associated mortality may not be restricted to RA, we also studied a large cohort of non-RA patients. METHODS: 615 RA patients from the Leiden Early Arthritis Clinic (EAC) (mean follow-up 7.6 years) were genotyped for rs10818488. In addition 5634 persons enrolled in the PROspective Study of Pravastatin in the Elderly at Risk (mean follow-up 3.2 years) were genotyped for rs2416808 (R(2) >0.99 with rs10818488). The life/death status was determined and for the deceased persons the cause of death was ascertained. Cox proportional hazards and regression models were used to assess hazard ratios (HR) and 95% confidence intervals (CI). RESULTS: Seventy-seven RA patients died. The main death causes in RA patients were cardiovascular diseases (37.7%), cancer (28.6%) and death due to infections (9.1%). No association was observed between the rs10818488 susceptible genotype AA and cardiovascular mortality (HR 1.08 95%CI 0.54 to 2.15) and all-cause mortality (HR 0.81 95%CI 0.27 to 2.43). Similar findings were observed for rs2416808 susceptible genotype GG in the non-RA cohort (HR 0.99; 95%CI 0.79 to 1.25 and HR 0.89; 95%CI 0.64 to 1.25, respectively). CONCLUSIONS: The TRAF1/C5 region is not associated with an increased mortality risk.

Evidence for interaction between 5-hydroxytryptamine (serotonin) receptor 2A and MHC type II molecules in the development of rheumatoid arthritis.

It has repeatedly been suggested that the development of complex diseases can be elucidated by gene-gene interactions. Recently, we found that HTR2A, a member of the serotonin receptor family, is associated with rheumatoid arthritis (RA). This study was aimed to investigate the possibility of a gene-gene interaction between HTR2A and the major genetic risk factor for RA, HLA-DRB1 shared epitope (SE) alleles. We studied 4095 RA cases and 3223 controls from three different populations - from Sweden, the United States and the Netherlands - to test for interaction between the protective HTR2A haplotype and HLA-DRB1 SE alleles. Further, we analyzed mRNA and/or protein expression of HTR2A and HLA-DR in biopsy samples and in synovial fibroblasts from RA patients. The interaction was defined as departure from additivity of effects using attributable proportion due to interaction. First, we could demonstrate and further replicate an interaction between a protective haplotype in HTR2A and HLA-DRB1 SE alleles regarding risk of developing autoantibody-positive RA. Second, we could show that both genes are constitutively expressed in fibroblasts from synovial tissue of RA patients, and, by double immunofluorescence staining, we demonstrated that these two proteins are colocalized in these cells. In conclusion, our data demonstrate a statistical interaction between HTR2A and HLA-DRB1 SE alleles and colocalization of the product of these two genes in inflamed synovial tissue, which suggest a possible biological relationship between these two proteins. This finding may lead to the development of treatment based on enhancing the protective features of 5-HT2A in individuals with a certain HLA genotype.

The TRAF1-C5 region on chromosome 9q33 is associated with multiple autoimmune diseases.

OBJECTIVES: The TRAF1-C5 locus has recently been identified as a genetic risk factor for rheumatoid arthritis (RA). Since genetic risk factors tend to overlap with several autoimmune diseases, a study was undertaken to investigate whether this region is associated with type 1 diabetes (TID), celiac disease (CD), systemic sclerosis (SSc) and systemic lupus erythematosus (SLE). METHODS: The most consistently associated SNP, rs10818488, was genotyped in a total of 735 patients with T1D, 1049 with CD, 367 with SSc, 746 with SLE and 3494 ethnically- and geographically-matched healthy individuals. The replication sample set consisted of 99 patients with T1D, 272 with SLE and 482 healthy individuals from Crete. RESULTS: A significant association was detected between the rs10818488 A allele and T1D (OR 1.14, p=0.027) and SLE (OR 1.16, p=0.016), which was replicated in 99 patients with T1D, 272 with SLE and 482 controls from Crete (OR 1.64, p=0.002; OR 1.43, p=0.002, respectively). Joint analysis of all patients with T1D (N=961) and all patients with SLE (N=1018) compared with 3976 healthy individuals yielded an allelic common OR of 1.19 (p=0.002) and 1.22 (p=2.6 x 10(-4)), respectively. However, combining our dataset with the T1D sample set from the WTCCC resulted in a non-significant association (OR 1.06, p=0.087). In contrast, previously unpublished results from the SLEGEN study showed a significant association of the same allele (OR 1.19, p=0.0038) with an overall effect of 1.22 (p=1.02 x 10(-6)) in a total of 1577 patients with SLE and 4215 healthy individuals. CONCLUSION: A significant association was found for the TRAF1-C5 locus in SLE, implying that this region lies in a pathway relevant to multiple autoimmune diseases.

Association of the 6q23 region with the rate of joint destruction in rheumatoid arthritis.

BACKGROUND: Two novel genetic polymorphisms on chromosome 6q23 are associated with susceptibility to rheumatoid arthritis (RA). Both polymorphisms (rs6920220 and rs10499194) reside in a region close to the gene encoding tumour necrosis factor alpha-induced protein 3 (TNFAIP3). TNFAIP3 is a negative regulator of NF-kappaB and is involved in inhibiting TNF-receptor-mediated signalling effects. Interestingly, the initial associations were detected in patients with longstanding RA. However, no association was found for rs10499194 in a Swedish cohort with early arthritis. This might be caused by over-representation of patients with severe disease in cohorts with longstanding RA. OBJECTIVE: To analyse the effect of the 6q23 region on the rate of joint destruction. METHODS: Five single nucleotide polymorphisms in 6q23 were genotyped in 324 Dutch patients with early RA. Genotypes were correlated with progression of radiographic joint damage for a follow-up time of 5 years. RESULTS: Two polymorphisms (rs675520 and rs9376293) were associated with severity of radiographic joint damage in patients positive for anti-citrullinated protein/peptide antibodies (ACPA). Importantly, the effects were present after correction for confounding factors such as secular trends in treatment. CONCLUSIONS: These data associate the 6q23 region with the rate of joint destruction in ACPA+ RA.

Consumo de sustancias y conductas sexuales de riesgo para la transmisión del VIH en una muestra de estudiantes universitarios / Substance use and HIV-related sexual risk behaviors in a sample of college students

Los nuevos casos de infeccion por VIH afectan principalmente a la poblacion joven, siendo las conductas sexuales de riesgo una de las principales vias de transmision. El objetivo de este estudio fue analizar las conductas sexuales de riesgo en jovenes universitarios de la provincia de Alicante y su relacion con el consumo de sustancias. La muestra estuvo compuesta por 339 estudiantes universitarios, con un rango de edad entre 18 y 29 años (M = 19.53; DE = 3.27). Los estudiantes cumplimentaron un cuestionario autoadministrado durante las clases sobre su comportamiento sexual y el consumo de sustancias. El 50% de la muestra afirmo haber tenido relaciones sexuales bajo los efectos del alcohol alguna vez, mientras que el 16.2% de la muestra ha mantenido relaciones sexuales bajo los efectos del cannabis alguna vez. Existe relacion significativa entre consumo de alcohol y uso de preservativo en el coito vaginal (ƒÔ2 = 6.85; p = .03), y tambien existe una relacion significativa entre el consumo de cannabis y uso de preservativo en el coito oral (ƒÔ2 = 8.33; p = .02)(AU)

The new cases of HIV infection mainly affect youngsters with high risk sexual behaviors one of the main routes of transmission. The aim of this study was to assess the sexual risk behaviour among university students from Alicante (Spain) and its relation with the drugtaking. The sample included 339 undergraduates aged 18-29 years (M = 19.53; SD = 3.27). The students completed a self-administered questionnaire during the class about their sexual behaviors and drugtaking. The participants reported a significative higher alcohol use than cannabis use (p < .01). Fifty per cent of the sample admitted to have been engaged in sex intercourse at least once while they were under the influence of alcohol, whereas only 16.2% of the sample admitted had sex at least once under the influence of cannabis. There is a significative relation between alcohol use and condom use at vaginal sex (ƒÔ2 = 6.85, p = .03) and between cannabis use and condom use at oral sex (ƒÔ2 = 8.33, p = .02)(AU)

Genetic variants at CD28, PRDM1 and CD2/CD58 are associated with rheumatoid arthritis risk.

To discover new rheumatoid arthritis (RA) risk loci, we systematically examined 370 SNPs from 179 independent loci with P < 0.001 in a published meta-analysis of RA genome-wide association studies (GWAS) of 3,393 cases and 12,462 controls. We used Gene Relationships Across Implicated Loci (GRAIL), a computational method that applies statistical text mining to PubMed abstracts, to score these 179 loci for functional relationships to genes in 16 established RA disease loci. We identified 22 loci with a significant degree of functional connectivity. We genotyped 22 representative SNPs in an independent set of 7,957 cases and 11,958 matched controls. Three were convincingly validated: CD2-CD58 (rs11586238, P = 1 x 10(-6) replication, P = 1 x 10(-9) overall), CD28 (rs1980422, P = 5 x 10(-6) replication, P = 1 x 10(-9) overall) and PRDM1 (rs548234, P = 1 x 10(-5) replication, P = 2 x 10(-8) overall). An additional four were replicated (P < 0.0023): TAGAP (rs394581, P = 0.0002 replication, P = 4 x 10(-7) overall), PTPRC (rs10919563, P = 0.0003 replication, P = 7 x 10(-7) overall), TRAF6-RAG1 (rs540386, P = 0.0008 replication, P = 4 x 10(-6) overall) and FCGR2A (rs12746613, P = 0.0022 replication, P = 2 x 10(-5) overall). Many of these loci are also associated to other immunologic diseases.

Association of a single-nucleotide polymorphism in CD40 with the rate of joint destruction in rheumatoid arthritis.

OBJECTIVE: The severity of joint destruction in rheumatoid arthritis (RA) is highly variable from patient to patient and is influenced by genetic factors. Genome-wide association studies have enormously boosted the field of the genetics of RA susceptibility, but risk loci for RA severity remain poorly defined. A recent meta-analysis of genome-wide association studies identified 6 genetic regions for susceptibility to autoantibody-positive RA: CD40, KIF5A/PIP4K2C, CDK6, CCL21, PRKCQ, and MMEL1/TNFRSF14. The purpose of this study was to investigate whether these newly described genetic regions are associated with the rate of joint destruction. METHODS: RA patients enrolled in the Leiden Early Arthritis Clinic were studied (n=563). Yearly radiographs were scored using the Sharp/van der Heijde method (median followup 5 years; maximum followup 9 years). The rate of joint destruction between genotype groups was compared using a linear mixed model, correcting for age, sex, and treatment strategies. A total of 393 anti-citrullinated protein antibody (ACPA)-positive RA patients from the North American Rheumatoid Arthritis Consortium (NARAC) who had radiographic data available were used for the replication study. RESULTS: The TT and CC/CG genotypes of 2 single-nucleotide polymorphisms, rs4810485 (CD40) and rs42041 (CDK6), respectively, were associated with a higher rate of joint destruction in ACPA-positive RA patients (P=0.003 and P=0.012, respectively), with rs4810485 being significant after Bonferroni correction for multiple testing. The association of the CD40 minor allele with the rate of radiographic progression was replicated in the NARAC cohort (P=0.021). CONCLUSION: A polymorphism in the CD40 locus is associated with the rate of joint destruction in patients with ACPA-positive RA. Our findings provide one of the first non-HLA-related genetic severity factors that has been replicated.

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.

Common variants at CD40 and other loci confer risk of rheumatoid arthritis.

To identify rheumatoid arthritis risk loci in European populations, we conducted a meta-analysis of two published genome-wide association (GWA) studies totaling 3,393 cases and 12,462 controls. We genotyped 31 top-ranked SNPs not previously associated with rheumatoid arthritis in an independent replication of 3,929 autoantibody-positive rheumatoid arthritis cases and 5,807 matched controls from eight separate collections. We identified a common variant at the CD40 gene locus (rs4810485, P = 0.0032 replication, P = 8.2 x 10(-9) overall, OR = 0.87). Along with other associations near TRAF1 (refs. 2,3) and TNFAIP3 (refs. 4,5), this implies a central role for the CD40 signaling pathway in rheumatoid arthritis pathogenesis. We also identified association at the CCL21 gene locus (rs2812378, P = 0.00097 replication, P = 2.8 x 10(-7) overall), a gene involved in lymphocyte trafficking. Finally, we identified evidence of association at four additional gene loci: MMEL1-TNFRSF14 (rs3890745, P = 0.0035 replication, P = 1.1 x 10(-7) overall), CDK6 (rs42041, P = 0.010 replication, P = 4.0 x 10(-6) overall), PRKCQ (rs4750316, P = 0.0078 replication, P = 4.4 x 10(-6) overall), and KIF5A-PIP4K2C (rs1678542, P = 0.0026 replication, P = 8.8 x 10(-8) overall).